WO2023044171A1 - Inhibitors of cysteine proteases and methods of use thereof - Google Patents

Inhibitors of cysteine proteases and methods of use thereof Download PDF

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Publication number
WO2023044171A1
WO2023044171A1 PCT/US2022/044163 US2022044163W WO2023044171A1 WO 2023044171 A1 WO2023044171 A1 WO 2023044171A1 US 2022044163 W US2022044163 W US 2022044163W WO 2023044171 A1 WO2023044171 A1 WO 2023044171A1
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substitute sheet
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group
compound
dimethyl
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PCT/US2022/044163
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French (fr)
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Lee D. Arnold
Walter Keung
Dange Vijay Kumar
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Pardes Biosciences, Inc.
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Publication of WO2023044171A1 publication Critical patent/WO2023044171A1/en

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    • C07D513/04—Ortho-condensed systems

Definitions

  • Coronaviridae family of viruses are enveloped, single-stranded, positive-sense RNA viruses and include 141 species classified into four genera according to their phylogenetic relationships: a-, P-, y-, and 6-coronavirus.
  • Coronaviruses are zoonotic viruses that infect a variety of animals from whales to birds, bats, cats, and humans. Typically, CoV infection results in mild to moderate respiratory tract infections; however, some CoV species are extremely virulent and can result in widespread fatality.
  • Severe acute respiratory syndrome coronavirus (SARS-CoV) is a human CoV responsible for the first pandemic of the 21 st century, infecting over 8,000 people with a 10% mortality rate.
  • Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in November 2012 and had since infected over 1,600 people in 26 countries with a 36% mortality rate. More recently, COVID-19 (SARS CoV2) coronaviruses have raised a global pandemic since first identified in late 2019. Therefore, it is important to identify coronavirus drug targets that can be utilized for the development of broadspectrum anti-coronaviral therapeutics to combat infections of existing and emerging coronaviruses.
  • All CoVs express a >800 kDa replicase polyprotein that contains either two or three cysteine proteases, the papain-like protease(s) (PLPpro, nsp3, or PLP1 and PLP2) and the SC- like protease (3CLpro, nsp5, or Mpro).
  • PLPpro papain-like protease
  • SC- like protease 3CLpro, nsp5, or Mpro.
  • These proteases process the CoV replicase polyprotein by cleaving it into 16 non-structural proteins, which are responsible for a variety of aspects of CoV replication.
  • the CoV 3CLpro responsible for processing 11 cleavage sites of within the replicase polyprotein and is essential for CoV replication, making it a highly valuable target for therapeutic development.
  • the overall active site architecture and substrate recognition pockets are structurally conserved across CoV 3CLpros, increasing its attractiveness as a target for the ⁇ f broad-spectrum anti-CoV therapeutics.
  • compositions comprising at least one disclosed compound and a pharmaceutically acceptable carrier.
  • an antiviral compound comprising a warhead (e.g., a nitrile warhead) covalently bound to a 3C or 3 CL protease inhibitor, wherein the antiviral compound covalently binds to a Cys residue of the protease, and wherein the antiviral compound is active against one or more viruses.
  • a warhead e.g., a nitrile warhead
  • the antiviral compound covalently bounds to a Cys residue of the protease
  • the antiviral compound is active against one or more viruses.
  • R 1 is selected from the group consisting of -CH(R n )-NHR 12 , a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R 1 may optionally be substituted by one, two, or three substituents each selected from R 5 ;
  • SUBSTITUTE SHEET RULE 26 is select — > o — o > > .yl, -CJh-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
  • R 12 is selected from the group consisting of -C(O)R B , -C(O)OR B , -C(O)NHR B , -R B , - NHC(O)R B , -NHC(O)OR B , -NHC(O)NHR B , and -NHR B ; wherein R 12 or R B may optionally be substituted by one or more substituents each independently selected from R 13 ;
  • R B is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
  • R 2 is hydrogen or Ci-Csalkyl
  • R 3a is hydrogen or Ci-Csalkyl
  • R 3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R 3 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ; or R 3 is -Si(CH3)3; or R 2 and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R 6 ; or R 3a and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic carbocyclyl which may optional
  • R 4a is hydrogen or Ci-C3alkyl optionally substituted by one or more halogens
  • R 4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R 4 may optionally be substituted by one, two, or three substituents each independently selected from R 7 ; or
  • R 4a and R 4 together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R 8 ;
  • R 5 ’ R 6 , R 7 ’ R 8 and R 13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R 56 )2, -N(R 56 )C(O)R 56 , -C(O)N(R 56 )2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C 9 H2iSi-, Ci-Cealkyl, C 2 -C 6 alkynyl, Ci-C 6 alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- ealkoxy, C3-Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may
  • SUBSTITUTE SHEET RULE 26 optionally be sub sth - selected from the group consisting of halogen, hydroxyl, Ci-Csalkyl, C2-Cealkynyl, Ci-Cealkoxy, and -CF3;
  • R 56 is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens; and R a is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
  • the protease inhibitory compound of Formula I is represented by Formula I': Formula I' or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
  • R 1 is selected from the group consisting of -CH(R n )-NHR 12 , a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R 1 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ;
  • R 11 is selected from the group consisting of branched Ci-Cealkyl, -CFF-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
  • R 12 is selected from the group consisting of -NHC(O)R B , -NHC(O)OR B , -NHC(O)NHR B , and -NHR B ; wherein R 12 or R B may optionally be substituted by one or more substituents each independently selected from R 13 ;
  • R B is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
  • R 2 is hydrogen or Ci-Csalkyl
  • R 3a is hydrogen or Ci-Csalkyl
  • R 3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R 3 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ; or R 3 is -Si(CH3)3; or R 2 and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R 6 ; or R 3a and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered
  • R 4a is hydrogen or Ci-Csalkyl optionally substituted by one or more halogens
  • R 4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R 4 may optionally be substituted by one, two, or three substituents each independently selected from R 7 ; or
  • R 4a and R 4 together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R 8 ;
  • R 5 ’ R 6 , R 7 ’ R 8 and R 13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R 56 )2, -N(R 56 )C(O)R 56 , -C(O)N(R 56 )2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C 9 H2iSi-, Ci-Cealkyl, C 2 -C 6 alkynyl, Ci-C 6 alkoxy, C3- Cecycloalkyl, Cs-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, Cs-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents
  • R 56 is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens
  • R a is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
  • the protease inhibitory compound of Formula I is represented by Formula I": Formula I" or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
  • R 1 is selected from the group consisting of -CH(R n )-NHR 12 , a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R 1 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ;
  • R 11 is selected from the group consisting of branched Ci-Cealkyl, -CFb-phenyl, -CH2- nanhthvl. and -CH2-(8-10 membered bicyclic heteroaryl);
  • R 12 is selects > o — o , -C(O)NHR B , and -R B ; wherein R 12 or R B may optionally be substituted by one or more substituents each independently selected from R 13 ;
  • R B is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
  • R 2 is hydrogen or Ci-Csalkyl
  • R 3a is hydrogen or Ci-Csalkyl
  • R 3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R 3 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ; or R 3 is -Si(CH3)3; or R 2 and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R 6 ; or R 3a and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic carbocyclyl which may optional
  • R 4a is hydrogen or Ci-C3alkyl optionally substituted by one or more halogens
  • R 4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R 4 may optionally be substituted by one, two, or three substituents each independently selected from R 7 ; or
  • R 4a and R 4 together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R 8 ;
  • R 5 ’ R 6 , R 7 ’ R 8 and R 13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R 56 )2, -N(R 56 )C(O)R 56 , -C(O)N(R 56 )2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C 9 H2iSi-, Ci-Cealkyl, C 2 -C 6 alkynyl, Ci-C 6 alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, C3-Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents
  • R a is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
  • R 100 , R 200 , R 300 , R 400a and R 400 are as defined herein.
  • methods of ameliorating or treating a viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of any of the compounds described herein.
  • kits for inhibiting transmission of a virus comprising administering a therapeutically effective amount of a compound described herein to a patient suffering from the virus, and/or contacting an effective amount of a compound described herein with a virally infected cell.
  • treating includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder and the like, including a reduction of viral shedding in asymptomatic individuals and prophylaxis of exposed individuals, independent of symptoms.
  • alkyl refers to a saturated straight or branched hydrocarbon.
  • exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as Ci-ealkyl, Ci-4alkyl, and Ci- salkyl, respectively.
  • Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2 -m ethyl- 1 -butyl, 3-methyl-2-butyl, 2 -m ethyl- 1 -pentyl, 3 -methyl- 1 -pentyl, 4- m ethyl- 1 -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l-butyl, 2-ethyl-l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.
  • alkenyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond.
  • alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as Ci-Csalkenyl, C2-Cealkenyl, and C3-C4alkenyl, respectively.
  • alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.
  • alkynyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond.
  • exemplary alkynyl groups include, but are not limited to, straight or branched groups of 2-6, or 3-6 carbon atoms, referred to herein as C2-ealkynyl, and C3-ealkynyl, respectively.
  • exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, etc.
  • alkoxy refers to a straight or branched alkyl group attached to oxygen (alkyl-O-).
  • alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci-Csalkoxy, Ci-Cealkoxy, and C2- Cealkoxy, respectively.
  • Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.
  • alkoxy as used herein may also refer to a cycloalkyl group ygen (cycloalkyl-O-).
  • SUBSTITUTE SHEET RULE 26 [00020] The tei ... > - die e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 % electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-14 aryl”).
  • an aryl group has six ring carbon atoms (“Ce aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“Ci4 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene.
  • aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
  • Examples of representative substituted aryls include the following wherein one of R 56 and R 57 may be hydrogen and at least one of R 56 and R 57 is each independently selected from Ci-Cs alkyl, Ci-Cs haloalkyl, 4-10 membered heterocyclyl, alkanoyl, Ci-Cs alkoxy, heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR 58 COR 59 , NR 58 SOR 59 NR 58 SO 2 R 59 , COOalkyl, COOaryl, CONR 58 R 59 , CONR 58 OR 59 , NR 58 R 59 , SO 2 NR 58 R 59 , S-alkyl, SOalkyl, SChalkyl, Saryl, SOaryl, SCharyl; or R 56 and R
  • R 60 and R 61 are each independently hydrogen, Ci-Cs alkyl, C1-C4 haloalkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, Ce-Cio aryl, substituted Ce-Cio aryl, 5-10 membered heteroaryl, or substituted 5-10 membered heteroaryl.
  • carbonyl refers to the radical -C(O)-.
  • cyano refers to the radical -CN.
  • the tei -tician - o > sin refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3-6, or 4-6 carbons, referred to herein as C3-Ciocycloalkyl, C3-6cycloalkyl or C4-6cycloalkyl, respectively.
  • Exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl or cyclopropyl.
  • halo or halogen as used herein refer to F, Cl, Br, or I.
  • haloalkyl refers to an alkyl radical in which the alkyl group is substituted with one or more halogens.
  • Typical haloalkyl groups include, but are not limited to, trifluoromethyl (i.e. CF3), difluoromethyl, fluoromethyl, chloromethyl, dichloromethyl, dibromoethyl, tribromomethyl, tetrafluoroethyl, and the like.
  • Exemplary haloalkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms substituted with a halogen (i.e. Cl, F, Br and I), referred to herein as Ci- ehaloalkyl, Ci-4 haloalkyl, and Ci-3haloalkyl, respectively.
  • hetero when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g,. heteroaryl, cycloalkenyl, e.g. cycloheteroalkenyl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
  • alkyl e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g,. heteroaryl, cycloalkenyl, e.g. cycloheteroalkenyl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
  • heteroaryl or “heteroaromatic group” as used herein refers to an aromatic 5-10 membered ring system containing one or more heteroatoms, for example one to three heteroatoms, such as nitrogen, oxygen, and sulfur.
  • the term may also be used to refer to a 5-7 membered monocyclic heteroaryl or an 8-10 membered bicyclic heteroaryl. Where possible, said heteroaryl ring may be linked to the adjacent radical though carbon or nitrogen.
  • heteroaryl rings include but are not limited to furan, thiophene, pyrrole, pyrrol opyri dine, indole, thiazole, oxazole, isothiazole, isoxazole, imidazole, benzoimidazole, imidazopyridine, pyrazole, triazole, pyridine or pyrimidine, etc.
  • each Z is selected from carbonyl, N, NR 65 , O, and S; and R 65 is each independently hydrogen, Ci-Cs alkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, Ce-Cio aryl, or 5-10 membered heteroaryl.
  • heterocyclyl refers to saturated or partially unsaturated 4-10 membered ring structures, whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen.
  • the term may also be used to refer to 4-10 membered saturated or partially unsaturated ring structures that are bridged, fused or spirocyclic ring structures, whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur.
  • heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran, dihydrofuran, dihydropyran, tetrahydropyran, etc.
  • the heterocycle is a spiro heterocycle (e.g., 2,8-diazaspiro[4.5]decane).
  • the heterocycle is a bridged heterocycle (e.g., octahydro- 1H-4, 7- methanoisoindole).
  • Spiro heterocyclyl refers to a polycyclic heterocyclyl with rings connected through one common atom (called a spiro atom), wherein the rings have one or more heteroatoms selected from the group consisting of N, O, and S(O) m (wherein m is an integer of 0 to 2) as ring atoms.
  • Representative examples of heterocyclyl include, for example:
  • “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
  • compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • composition refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
  • “Individual,” “patient,” or “subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
  • the compounds of the disclosure can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
  • “Modulation” includes antagonism (e.g., inhibition), agonism, partial antagonism and/or partial agonism.
  • the term “therapeutically effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system or animal, (e.g. mammal or human) that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • the compounds of the disclosure are administered in therapeutically effective amounts to treat a disease.
  • a therapeutically effective amount of a compound is the quantity required to achieve a desired therapeutic and/or
  • SUBSTITUTE SHEET RULE 26 [00037]
  • the tei ... flick — i refers to salts of acidic or basic groups that may be present in compounds used in the compositions.
  • Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, -toluenesulfonate and pamoate (i.e., l,l'-methylene
  • Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations.
  • Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
  • Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids.
  • the compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt.
  • the compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers.
  • stereoisomers when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols “(+),” “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.
  • the present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated “( ⁇ )” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.
  • the compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond.
  • the symbol — denotes a bond that may be a single, double or triple bond as described herein.
  • Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance " ” * ” itandards. Unless otherwise specified, structures depicting double bonds
  • SUBSTITUTE SHEET RULE 26 encompass both the > > arbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond.
  • Compounds of the disclosure may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring.
  • the arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards.
  • structures depicting carbocyclic or heterocyclic rings encompass both “Z” and “E” isomers.
  • Substituents around a carbocyclic or heterocyclic rings may also be referred to as “cis” or “trans”, where the term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”
  • Stereoselective syntheses a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art.
  • Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009.
  • the compound is amorphous.
  • the compound is a single polymorph.
  • the compound is a mixture of polymorphs.
  • the compound is in a crystalline form.
  • the disclosure also embraces isotopically labeled compounds of the disclosure which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 C1, respectively.
  • a compound of the disclosure may have one or more H atom replaced with deuterium.
  • isotopically-labeled disclosed compounds are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3 H) and carbon-14 (z.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (z.e., 2 H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
  • Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.
  • prodrug refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255).
  • a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (Ci-s)alkyl, (C2-i2)alkylcarbonyloxymethyl, l-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1 -methyl- l-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxy carbonyloxymethyl having from 3 to 6 carbon atoms, 1 -(alkoxy carbonyloxy)ethyl having rbon atoms, 1 -methyl- 1 -(alkoxy carbonyloxy)ethyl having from 5 to 8 carbon
  • SUBSTITUTE SHEET RULE 26 atoms, N-(alkoxyca. j > j o > > , l-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3 -phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(Ci-2)alkylamino(C2-3)alkyl (such as P- dimethylaminoethyl), carbamoyl-(Ci-2)alkyl, N,N-di(Ci-2)alkylcarbamoyl-(Ci-2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-3)alkyl.
  • a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (Ci-6)alkylcarbonyloxymethyl, l-((Ci-6)alkylcarbonyloxy)ethyl, 1 -methyl- l-((Ci- 6)alkylcarbonyloxy)ethyl (Ci-6)alkoxycarbonyloxymethyl, N-(Ci-6)alkoxycarbonylaminomethyl, succinoyl, (Ci-e)alkylcarbonyl, a-amino(Ci-4)alkylcarbonyl, arylalkylcarbonyl and a- aminoalkylcarbonyl, or a-aminoalkylcarbonyl-a-aminoalkylcarbonyl, where each a- aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(O)
  • a prodrug can be formed, for example, by creation of an amide or carbamate, an N-alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine.
  • a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can metabolically cleaved to generate a bioactive primary or secondary amine.
  • warhead refers to a functional group present on a compound wherein that functional group is capable of reversibly or irreversibly participating in a reaction with a protein, e.g., 3C or 3CL protease (e.g., with a cysteine on the protease such as Cys 145).
  • Warheads may, for example, form covalent bonds with the protein, or may create stable transition states, or be a reversible or an irreversible alkylating agent.
  • the warhead moiety can be a functional group on an inhibitor that can participate in a bond-forming reaction, wherein a new covalent bond is formed between a portion of the warhead and a donor, for example an amino acid residue of a protein.
  • the warhead is an electrophile and the “donor” is a nucleophile such as the side chain of a cysteine residue.
  • a warhead may include a nitrile group.
  • nitriles may be reversible covalent warheads for cysteine protease inhibition.
  • the mechanism of action may involve aformation of reversible covalent bond between the nitrile and the active cysteine to idate adduct. Reaction of cysteine of glutathione or other proteins is generally
  • contemplated compounds herein may be a reversible or an irreversible inhibitor. It will be appreciated to one of skilled in the art that the compounds disclosed herein that include the warheads above also contemplate the precursors to those compounds, for example, where a cyano moiety involved in a warheads may be replaced with e.g., a halo moiety.
  • inhibitor refers to a compound that binds to and /or inhibits a target protease with measurable affinity.
  • reversible or "reversible inhibitor” as used herein refers to a protease inhibitor that associates with a protease in such a way as to inhibit the activity of the protease while the protease and inhibitor are bound, but does not associate with a protease in such a way as to inhibit the activity of the protease when the protease and inhibitor are no longer bound.
  • Reversible inhibitors can effect inhibition by competing with substrate for binding to the active site of the protease (competitive reversible inhibitor), or by associating with the protease bound to its substrate in a way to make the complex inactive (uncompetitive reversible inhibitor), or by associating with the protease and/or protease-substrate complex in a way that inhibits the activity of either and/or both.
  • the term “irreversible” or “irreversible inhibitor” refers to an inhibitor (i.e. a compound) that is able to be covalently bonded to a target protease in a substantially non-reversible manner.
  • An irreversible inhibitor will remain substantially bound to the target protease once covalent bond formation has occurred.
  • Irreversible inhibitors usually display time dependency, whereby the degree of inhibition increases with the time with which the inhibitor is in contact with the enzyme.
  • an irreversible inhibitor will remain substantially bound to target protease once covalent bond formation has occurred and will remain bound for a time period that is longer than the life of the protein.
  • the disclosure is directed to, in part, compounds that inhibit a viral protease.
  • viral proteases include, but not limited to, Cathepsin K, coronavirus main protease ise 3, Calpain 1, and Cathepsin S. Accordingly, in various embodiments, a
  • the SUBSTITUTE SHEET RULE 26 compound of the pre v . o . > JI) is a viral protease inhibitor, wherein the viral protease is selected from the group consisting of Cathepsin K, coronavirus main protease (Mpro), Caspase 3, Calpain 1, and Cathepsin S.
  • the viral protease is a coronavirus main protease (Mpro).
  • the viral protease is Cathepsin K.
  • the viral protease is Caspase 3.
  • the viral protease is Calpain 1.
  • the viral protease is Cathepsin S.
  • R 1 is selected from the group consisting of -CH(R n )-NHR 12 , a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R 1 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ;
  • R 11 is selected from the group consisting of branched Ci-Cealkyl, -CFh-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
  • R 12 is selected from the group consisting of -NHC(O)R B , -NHC(O)OR B , -NHC(O)NHR B , and -NHR B ; wherein R 12 or R B may optionally be substituted by one or more substituents each independently selected from R 13 ;
  • R B is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
  • R 2 is hydrogen or Ci-Csalkyl
  • R 3a is hydrogen or Ci-Csalkyl
  • R 3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R 3 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ; or R 3 is -Si(CH3)3; or R 2 and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered snirocvclic heterocyclyl which may optionally be substituted by one, two or three substituents each r J selected from R 6 ;
  • SUBSTITUTE SHEET RULE 26 or R 3a and R , > may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic carbocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R 6 ;
  • R 4a is hydrogen or Ci-Csalkyl optionally substituted by one or more halogens
  • R 4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, and a 4-10 membered heterocyclyl; wherein R 4 may optionally be substituted by one, two, or three substituents each independently selected from R 7 ; or
  • R 4a and R 4 together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R 8 ;
  • R 5 ’ R 6 , R 7 ’ R 8 and R 13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R 56 )2, -N(R 56 )C(O)R 56 , -C(O)N(R 56 )2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C 9 H2iSi-, Ci-Cealkyl, C 2 -C 6 alkynyl, Ci-C 6 alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, C3-Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents
  • R 56 is hydrogen or Ci-C3alkyl optionally substituted by one, two or three halogens
  • R a is hydrogen or Ci-C3alkyl optionally substituted by one, two or three halogens.
  • R 1 is -CH(R 12 )-NHR 12 or a 9-10 membered bicyclic heteroaryl having a ring nitrogen; wherein R 1 or R 11 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ;
  • R 11 is selected from the group consisting of branched Ci-Cealkyl, -Cfh-phenyl, -CH2- no Ji+hm onri -CH2-(8-10 membered bicyclic heteroaryl);
  • R 12 is -C(O) > - - - - - j > y be substituted by one or more halogens;
  • R 2 is hydrogen or Ci-Csalkyl
  • R 3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R 3 may optionally be substituted by one, two, or three substituents each independently selected from R 5 ; or R 3 is -Si(CH3)3; or R 2 and R 3 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R 6 ;
  • R 4a is hydrogen or Ci-C3alkyl optionally substituted by one or more halogens
  • R 4 is 5-10 membered heteroaryl optionally substituted by one, two, or three substituents each independently selected from R 7 ; or
  • R 4a and R 4 together with the carbon to which they are attached, may be joined together to form an 8-10 membered partially unsaturated bicyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R 8 ;
  • R 5 ’ R 6 , R 7 and R 8 are independently selected for each occurrence from the group consisting of halogen, hydroxyl, cyano, -N(R 56 )2, -N(R 56 )C(O)R 56 , -C(O)N(R 56 )2, -CF3, Ci-Cealkyl, Ci- Cealkoxy, Cs-Cecycloalkyl, and phenyl; wherein Ci-Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, and phenyl may optionally be substituted by one or more substituents each independently selected from the group consisting of halogen and hydroxyl; and
  • R 56 is hydrogen or Ci-C3alkyl optionally substituted by one, two or three halogens.
  • R 2 and R 3 together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl.
  • a compound disclosed herein e.g., a compound of Formula I, may be represented by: selected from 0, 1 and 2; and n is selected from 0, 1, 2 and 3.
  • I may be represented by: wherein m is 0, 1 or 2; n is 0, 1, 2, or 3; and t is 0, 1 or 2.
  • R 6 when present is independently selected for each occurrence from the group consisting of, for example, fluoro, chloro and -CH3.
  • R 2 and R 3 together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
  • R 2 is hydrogen.
  • a compound disclosed herein e.g., a compound of Formula I, may be represented by: wherein t is 0, 1 or 2.
  • R 3 is selected from the group consisting of:
  • R 11 is selected, for example, from the group consisting of:
  • a compound disclosed herein e.g., a compound of Formula I
  • a compound of Formula I may be represented by: wherein m is 0, 1 or 2; and n is 0, 1, 2 or 3, and R 6 when present is independently selected for each occurrence from the group consisting of fluoro, chloro and -CHs.
  • RTM is hydrogen.
  • R 4 may be selected, for example, from the group consisting of:
  • X is O, S, or NR a ;
  • R 7 is independently selected for each occurrence from the group consisting of chloro, fluoro, bromo, -CN, -CCH, -CH3, -CH2F, -CHF2, -CF3, cyclopropyl, -OH, -OCH3 and -OCFs;
  • R 77 and R 78 are independently selected, for each occurrence, from the group consisting of hydrogen, chloro, fluoro, bromo, -CN, -CCH, -CH3, -CH2F, -CHF2, CF3, -OCH3, -OCF3 and cyclopropyl; and
  • R a is selected from the group consisting of hydrogen, Ci-C3alkyl, C3-Cecycloalkyl, and phenyl, wherein Ci-C3alkyl, C3-Cecycloalkyl, and phenyl may optionally be substituted by one or more halogens.
  • R 4 may be selected from the group consisting of
  • R 4 is selected from the group consisting of:
  • R 4a and R 4 together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of, for example:
  • R 8 and R 9 are independently selected for each occurrence from the group consisting of chloro, fluoro, -CH3, -CH2F, -CHF2, -CF3, -OH and -OCH3; or two geminal R 9 form oxo.
  • R 4a and R 4 together with the carbon to which they are attached, join together to form:
  • R 1 is -CH(R n )-NHR 12 .
  • R 1 is a 9-10 membered bicyclic heteroaryl having a ring nitrogen.
  • R 1 is a a 5-6 membered monocyclic heteroaryl having a ring nitrogen.
  • R 1 is optionally substituted thiazolyl, pyrrolyl, oxadiazolyl, pyrazinyl, or isoindolyl.
  • R 1 is selected from the group consisting of
  • SUBSTITUTE SHEET RULE 26 is is sele o — o > C(O)-NR a -Ci-Cealkyl, -
  • R 10 may optionally be substituted on an available carbon by one or more substituents each independently selected from the group of halogen, hydroxy, -CF3, -NR a R b , -C(O)NR a R b , and Ci-Csalkoxy;
  • R 15 is selected from the group consisting of:
  • R 20 is hydrogen
  • R 30 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R 30 may optionally be substituted by one, two or three substituents each independently selected from R 33 ; or R 30 -Si(CH3)3;
  • R 33 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R 20 and R 30 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
  • R 40a and R 40 together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of:
  • R 10 is -C(O)- or -CH2-;
  • R 20 is hydrogen;
  • R 30 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R 30 may optionally be substituted by one, two or three substituents each independently selected from R 33 ; or R 30 -Si(CH3)3;
  • R 33 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or
  • R 20 and R 30 together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected
  • R 40 is selected from the group consisting of: selected for each occurrence from the group consisting of chloro, fluoro, -CH 3 , -CH 2 F, -CHF 2 , - CF 3 , -OH and -OCH 3 ;
  • R 700 is independently selected for each occurrence from the group consisting of -CH 3 , -CH 2 F, -CHF 2 and -CF 3 ;
  • R aa is selected from the group consisting of hydrogen, Ci-C 3 alkyl, and C 3 -Cecycloalkyl, wherein Ci-C 3 alkyl and C 3 -Cecycloalkyl may optionally be substituted by one or more halogens; or R 40a and R 40 , together with the carbon to which they are attached, join together to form an 8-10 membered partially unsaturated bicyclic heterocyclyl selected from the group consisting of: wherein R 80 is independently selected for each occurrence from the group consisting of chloro, fluoro, -
  • R 100 is independently selected for each occurrence from the group consisting of halogen and Ci-Csalkoxy;
  • R 200 is hydrogen or Ci-Csalkyl
  • R 300 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R 300 may optionally be substituted by one, two or three substituents each independently selected from R 330 ; or R 300 -Si(CH3)3;
  • R 330 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R 200 and R 300 , together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
  • R 400a is hydrogen
  • R 400 is selected from the group consisting of:
  • the disclosed compound is a compound identified in Table 1, Table 2, and Table 3 below or a pharmaceutically acceptable salt thereof.
  • SUBSTITUTE SHEET RULE 26 [00080] In some embodiments, described herein is a compound of the following formula: or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
  • R 104 is selected from the group consisting of Ci-Cealkyl, wherein R 105 is Ci-Cealkyl, and n is 1, 2, or 3;
  • R 101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, C 3 -Cecycloalkyl, and C 3 -Cecycloalkenyl, wherein R 101 may optionally be substituted by one, two, three, or four substituents each independently selected from R D ;
  • R D is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C 3 - Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
  • R m is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C 3 - Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF 3 .
  • the compound is a compound of the following formula: Formula X-Ib-1, or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
  • R 104 is selected from the group consisting of Ci-Cealkyl, wherein R 105 is Ci-Cealkyl, and n is 1, 2, or 3;
  • R 101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, C3-Cecycloalkyl, and C3-Cecycloalkenyl, wherein R 101 may optionally be substituted by one, two, three, or four substituents each independently selected from R D ;
  • R D is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
  • R m is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C3- Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3.
  • the compound is a compound of the following formula: Formula X-Ic, or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
  • R 104 is selected from the group consisting of Ci-Cealkyl, wherein R 105 is Ci-Cealkyl, and n is 1, 2, or 3;
  • R 101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R 101 may optionally be substituted by one, two, three, or four substituents each independently selected from R D ; and
  • R D is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo.
  • R 104 is Ci-Cealkyl. In some embodiments, R 104 is , some embodiments, R 104 is
  • the disclosed compound is a compound selected from the group consisting of:
  • the disclosed compound is a compound selected from the group consisting of:
  • R 104 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and C3- Cecycloalkenyl, wherein R 101 may optionally be substituted by one, two, three, or four substituents each independently selected from R D ;
  • R D is selected from the group consisting of
  • SUBSTITUTE SHEET RULE 26 halogen, Ci-Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
  • R m is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and Cs-Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3;
  • R 103 is 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, wherein R 103 may optionally be substituted by one, two, or three substituents each independently selected from R E ; and
  • R E is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2-Cealkyny
  • R 104 is Ci-Cealkyl. In some embodiments, R 104 is , some embodiments, R 104 is
  • R 101 may optionally be substituted by one, two, three, or four substituents each independently selected from R D ;
  • R D is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, O-Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
  • R m is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and Cs-Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3;
  • R 103 is 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, wherein R 103 may optionally be substituted by one, two, or three substituents each independently selected from R E ; and R E is, for each
  • Formula X is represented by:
  • Cealkyl or Cs-Cecycloalkyl, and R 105 is Ci-Cealkyl, wherein the R 101 is optionally substituted by one -CH3 or cyclopropyl.
  • n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
  • R 101 is selected from the group consisting of:
  • R 101 is selected from the group consisting of:
  • R 105 is -CH3.
  • R 103 is selected from the group consisting of:
  • R D is selected from the group consisting of methoxy, halo, - CH3, and cyano.
  • R D is Ci-Cealkyl, wherein the Ci-Cealkyl is optionally substituted by one, two or three halos.
  • one of the carbons in the alkyl or alkenyl in the R 101 may optionally be replaced by an oxygen.
  • the compound of Formula X is a compound identified in
  • Formula XI is represented by:
  • Cealkyl or Cs-Cecycloalkyl, and R 105 is Ci-Cealkyl, wherein the R 101 is optionally substituted by one -CH3 or cyclopropyl.
  • n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
  • R 101 is selected from the group consisting of:
  • R 101 is selected from the group consisting of: ,
  • R 106 and R 106a together with the carbon to which they are attached, are joined together to form a 12 membered partially unsaturated tricyclic heterocyclyl ring.
  • R F is selected from the group consisting of methoxy, halo, - CH3, and cyano.
  • R F is Ci-Cealkyl, wherein the Ci-Cealkyl is optionally substituted by one, two or three halos.
  • one of the carbons in the alkyl or alkenyl in the R 101 may optionally be replaced by an oxygen.
  • the compound of Formula XI is a compound identified in
  • the diastereomers can then be separated by any conventional means such as crystallization or chromatography, and the desired enantiomer recovered (such as by treatment with an acid in the instance where the diastereomer is a salt).
  • a racemic mixture of esters can be resolved by kinetic hydrolysis using a variety of biocatalysts (for example, see Patel Stereoselective Biocatalysts, Marcel Decker; New York 2000).
  • a broad spectrum, covalent 3 CL or 3C protease antiviral compound comprising a nitrile warhead covalently bound to a 3 CL protease inhibitor, wherein the antiviral compound covalently binds to Cys on the protease, and wherein the antiviral compound is active against multiple viruses.
  • the broad spectrum covalent
  • the broad spectrum covalent compound of Formula I, II, or III, wherein the compound is active against caliciviruses, picornaviruses and coronaviruses.
  • compounds of Formula I, II, or III may also comprise one or more isotopic substitutions.
  • hydrogen may be 2 H (D or deuterium) or 3 H (T or tritium); carbon may be, for example, 13 C or 14 C; oxygen may be, for example, 18 O; nitrogen may be, for example, 15 N, and the like.
  • a particular isotope (e.g., 3 H, 13 C, 14 C, 18 O, or 15 N) can represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the total isotopic abundance of an element that occupies a specific site of the compound.
  • Another aspect of the disclosure provides methods of treating patients suffering from a viral infection, e.g., a coronaviral infection.
  • the disclosure provides a method of treating the below medical indications comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein, such as a compound of Formula I, II, or III.
  • the disclosure provides a method of ameliorating or treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the compounds described herein.
  • the viral infection is from a virus selected from the group consisting of an RNA virus, a DNA virus, a coronavirus, an astrovirus, a papillomavirus, a pneumovirus, a picornavirus, an influenza virus, an adenovirus, a cytomegalovirus, a polyomavirus, a poxvirus, a flavivirus, an alphavirus, an ebola virus, a morbillivirus, an enterovirus, an orthopneumovirus, a
  • the viral infection is a coronavirus infection.
  • the viral infection is a coronavirus selected from the group consisting of: 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS- CoV), and SARS-CoV-2 (COVID-19).
  • the viral infection is SARS-CoV-2.
  • the viral infection is from a virus selected from the group consisting of caliciviruses, MD145, murine norovirus, vesicular exanthema of swine virus, abbit hemorrhagic disease virus, porcine teschovirus, bovine coronavirus, feline infectious peritonitis virus, EV-68 virus, EV-71 virus, poliovirus, norovirus, human rhinovirus (HRV), hepatitis A virus (HAV) and foot-and-mouth disease virus (FMDV).
  • caliciviruses MD145, murine norovirus, vesicular exanthema of swine virus, abbit hemorrhagic disease virus, porcine teschovirus, bovine coronavirus, feline infectious peritonitis virus, EV-68 virus, EV-71 virus, poliovirus, norovirus, human rhinovirus (HRV), hepatitis A virus (HAV) and foot-and-mouth disease virus (FMDV).
  • the viral infection is an arenovirus infection.
  • the arenovirus is selected from the group consisting of: Junin virus, Lassa virus, Lujo virus, Machupo virus, and Sabia virus.
  • the viral infection is an influenza infection.
  • the influenza is influenza H1N1, H3N2 or H5N1.
  • Another aspect of the disclosure provides methods of treating patients suffering from a viral infection, e.g., a noroviral infection.
  • the disclosure provides a method of treating a viral infection from a norovirus in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the compounds described herein.
  • a method of inhibiting transmission of a virus comprising administering a therapeutically effective amount of a compound described herein to a patient suffering from the virus, and/or contacting an effective amount of a compound described herein with a virally infected cell.
  • the method further comprises administering another therapeutic.
  • the method further comprises administering an additional anti-viral therapeutic.
  • the anti-viral therapeutic is selected from the group consisting of ribavirin, favipiravir, ST-193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil,
  • the another therapeutic is selected from the group consisting of protease inhibitors, fusion inhibitors, M2 proton channel blockers, polymerase inhibitors, 6- endonuclease inhibitors, neuraminidase inhibitors, reverse transcriptase inhibitor, aciclovir, acyclovir, protease inhibitors, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, docosanol, edoxudine, entry inhibitors, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, inosine, integrase inhibitor, interferons, lopinavir, loviride, moroxydine, nexavir, nucleoside analogues, penciclo
  • the additional anti-viral therapeutic is selected from the group consisting of lamivudine, an interferon alpha, a VAP anti-idiotypic antibody, enfuvirtide, amantadine, rimantadine, pleconaril, aciclovir, zidovudine, fomivirsen, a morpholino, a protease inhibitor, double-stranded RNA activated caspase oligomerizer (DRACO), rifampicin, zanamivir, oseltamivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir,
  • Contemplated patients include not only humans, but other animals such as companion animals (e.g. dogs, cats), domestic animals (e.g. cow, swine), and wild animals (e.g. monkeys, bats, snakes).
  • companion animals e.g. dogs, cats
  • domestic animals e.g. cow, swine
  • wild animals e.g. monkeys, bats, snakes.
  • described herein is a method of ameliorating or treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula
  • Other contemplated methods of treatment include method of treating or ameliorating a virus infection condition or co-morbidity, by administering a compound disclosed herein to a subject.
  • Exemplary co-morbidities include lung diseases, cardiac disorders, endocrine disorders, respiratory disorders, hepatic disorders, skeletal disorders, psychiatric disorders, metabolic disorders, and reproductive disorders.
  • the viral infection is from a virus selected from the group consisting of an RNA virus, a DNA virus, a coronavirus, a papillomavirus, a pneumovirus, a picornavirus, an influenza virus, an adenovirus, a cytomegalovirus, a polyomavirus, a poxvirus, a flavivirus, an alphavirus, an ebola virus, a morbillivirus, an enterovirus, an orthopneumovirus, a lentivirus, arenovirus, a herpes virus, and a hepatovirus.
  • the viral infection is a coronavirus infection.
  • the viral infection is a coronavirus selected from the group consisting of: 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS- CoV), and SARS-CoV-2 (CO VID-19).
  • the viral infection is SARS-CoV-
  • the viral infection is an arenovirus infection.
  • the arenovirus is selected from the group consisting of: Junin virus, Lassa virus, Lujo virus, Machupo virus, and Sabia virus.
  • the viral infection is an influenza infection.
  • the influenza is influenza H1N1, H3N2 or H5N1.
  • the viral infection is a respiratory viral infection.
  • the viral infection is an upper respiratory viral infection or a lower respiratory viral infection.
  • the method further comprises administering another therapeutic.
  • the virus is selected from the group consisting of a retrovirus (e.g., human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), human T-cell lymphotropic virus (HTLV)-l, HTLV-2, HTLV-3, HTLV-4), Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, a herpes simplex virus (HSV) (e.g., HSV-1, HSV-2, varicella zoster virus, cytomegalovirus), an adenovirus, an orthomyxovirus (e.g., influenza virus A, influenza virus B, influenza virus C, influenza virus D, thogotovirus), a flavivirus (e.g., dengue virus, Zika virus), West Nile virus, Rift Valley fever virus, an arenavirus, Crimean- Congo hemorrhagic fever virus
  • a retrovirus e.g., human immunodeficiency virus
  • Louis encephalitis virus Japanese encephalitis virus, a tick-borne encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, a rabies virus, a polyomavirus (e.g., JC virus, BK virus), an alphavirus, and a rubivirus (e.g., rubella virus).
  • a polyomavirus e.g., JC virus, BK virus
  • an alphavirus e.g., rubella virus
  • the disease or disorder is a viral infection, e.g., a disease or disorder selected from the group consisting of acquired immune deficiency syndrome (AIDS), HTLV-1 associated myelopathy/tropical spastic paraparesis, Ebola virus disease, hepatitis A, hepatitis B, hepatitis C, herpes, herpes zoster, acute varicella, mononucleosis, respiratory infections, pneumonia, influenza, dengue fever, encephalitis (e.g., Japanese encephalitis, St.
  • AIDS acquired immune deficiency syndrome
  • HTLV-1 associated myelopathy/tropical spastic paraparesis Ebola virus disease
  • hepatitis A hepatitis B
  • hepatitis C herpes
  • herpes herpes zoster
  • the virus is an RNA virus (having a genome that is composed of RNA).
  • RNA viruses may be single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA).
  • RNA viruses have high mutation rates compared to DNA viruses, as RNA
  • the RNA virus is a positive-strand RNA virus (e.g., a SARS-CoV virus, polio virus, Coxsackie virus, Enterovirus, Human rhino virus, Foot/Mouth disease virus, encephalomyocarditis virus, Dengue virus, Zika virus, Hepatitis C virus, or New Castle Disease virus).
  • a positive-strand RNA virus e.g., a SARS-CoV virus, polio virus, Coxsackie virus, Enterovirus, Human rhino virus, Foot/Mouth disease virus, encephalomyocarditis virus, Dengue virus, Zika virus, Hepatitis C virus, or New Castle Disease virus.
  • RNA viruses are classified by the type of genome (double-stranded, negative (-), or positive (+) single-stranded). Double-stranded RNA viruses contain a number of different RNA molecules, each coding for one or more viral proteins.
  • Positive-sense ssRNA viruses utilize their genome directly as mRNA; ribosomes within the host cell translate mRNA into a single protein that is then modified to form the various proteins needed for viral replication.
  • One such protein is RNA-dependent RNA polymerase (RNA replicase), which copies the viral RNA in order to form a double-stranded, replicative form.
  • Negative-sense ssRNA viruses have their genome copied by an RNA replicase enzyme to produce positive-sense RNA for replication.
  • the virus comprises an RNA replicase enzyme.
  • the resultant positive-sense RNA then acts as viral mRNA and is translated by the host ribosomes.
  • the virus is a dsRNA virus.
  • the virus is a negative ssRNA virus.
  • the virus is a positive ssRNA virus.
  • the positive ssRNA virus is a coronavirus.
  • SARS-CoV2 also sometimes referred to as the novel coronavirus of 2019 or 2019- nCoV, is a positive-sense single-stranded RNA virus.
  • SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins.
  • the N protein holds the RNA genome together; the S, E, and M proteins form the viral envelope.
  • Spike allows the virus to attach to the membrane of a host cell, such as the ACE2 receptor in human cells (Kruse R.L. (2020), Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China (version 2). FlOOOResearch, 9:72).
  • SARS-CoV2 is the highly contagious, causative viral agent of coronavirus disease 2019 (COVID19), a global pandemic.
  • the virus is a DNA virus (having a genome that is composed of DNA).
  • DNA viruses include, without limitation, parvoviruses (e.g., adeno- associated viruses), adenoviruses, asfarviruses, herpesviruses (e.g., herpes simplex virus 1 and 2
  • SUBSTITUTE SHEET RULE 26 HSV-1 and HSV-2
  • Epstein-Barr virus EBV
  • CMV cytomegalovirus
  • papillomoviruses e.g., HPV
  • polyomaviruses e.g., simian vacuolating virus 40 (SV40)
  • poxviruses e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus.
  • RNA viruses include, without limitation, bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, influenza virus type C), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)) and toroviruses.
  • bunyaviruses e.g., hantavirus
  • coronaviruses e.g., flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus)
  • the methods described herein may inhibit viral replication transmission, replication, assembly, or release, or minimize expression of viral proteins.
  • described herein is a method of inhibiting transmission of a virus, a method of inhibiting viral replication, a method of minimizing expression of viral proteins, or a method of inhibiting virus release, comprising administering a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, to a patient suffering from the virus, and/or contacting an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof, with a virally infected cell.
  • a method of treating a respiratory disorder in a subject in need thereof comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula I, II, or III described herein) or a pharmaceutically acceptable salt thereof.
  • the respiratory disorder is selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, fibrosis, chronic asthma, acute asthma, lung disease secondary to environmental exposures, acute lung infection, chronic lung infection, al antitrypsin disease, cystic fibrosis and an autoimmune disease.
  • COPD chronic obstructive pulmonary disease
  • asthma chronic obstructive pulmonary disease
  • fibrosis chronic asthma
  • chronic asthma chronic asthma
  • acute asthma acute asthma
  • lung disease secondary to environmental exposures acute lung infection
  • cystic fibrosis and an autoimmune disease.
  • the respiratory disorder is associated with a heart attack.
  • a method of treating a disorder associated with cathepsin comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula I, II, or III described herein) or a pharmaceutically acceptable salt thereof.
  • a compound described herein e.g., a compound of Formula I, II, or III described herein
  • the disorder is a cathepsin dependent condition or disease.
  • SUBSTITUTE SHEET RULE 26 disorder is selected from the group consisting of breast cancer, pycnodysostosis, glioblastoma, osteosclerosis, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, osteogenesis imperfecta, atherosclerosis, obesity, glaucoma, chronic obstructive pulmonary disease, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
  • Compounds described herein can be administered in combination with one or more additional therapeutic agents to treat a disorder described herein, such as an infection by a pathogen described herein, e.g., a virus, fungus, or protozoan.
  • a pathogen described herein e.g., a virus, fungus, or protozoan.
  • contemplated herein are both a fixed composition comprising a disclosed compound and another therapeutic agent such as disclosed herein, and methods of administering, separately a disclosed compound and a disclosed therapeutic.
  • a pharmaceutical composition comprising a compound described herein, e.g., a compound of Formula I, II, or III as defined herein, one or more additional therapeutic agents, and a pharmaceutically acceptable excipient.
  • a compound of Formula I, II, or III as defined herein and one additional therapeutic agent is administered.
  • a disclosed compound as defined herein and two additional therapeutic agents are administered.
  • a disclosed compound as defined herein and three additional therapeutic agents are administered.
  • Combination therapy can be achieved by administering two or more therapeutic agents, each of which is formulated and administered separately.
  • a compound of Formula I, II, or III as defined herein and an additional therapeutic agent can be formulated and administered separately.
  • Combination therapy can also be achieved by administering two or more therapeutic agents in a single formulation, for example a pharmaceutical composition comprising a compound of Formula I, II, or III as one therapeutic agent and one or more additional therapeutic agents such as an antibiotic, a viral protease inhibitor, or an anti-viral nucleoside anti-metabolite.
  • a compound of Formula I, II, or III as defined herein and an additional therapeutic agent can be administered in a single formulation.
  • Other combinations are also encompassed by combination therapy. While the two or more agents in the combination therapy can be administered simultaneously, they need not be. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents)
  • SUBSTITUTE SHEET RULE 26 by minutes, hours, days, or weeks.
  • the two or more agents can be administered within minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or weeks of each other. In some cases even longer intervals are possible. While in many cases it is desirable that the two or more agents used in a combination therapy be present in within the patient's body at the same time, this need not be so.
  • Combination therapy can also include two or more administrations of one or more of the agents used in the combination using different sequencing of the component agents. For example, if agent X and agent Y are used in a combination, one could administer them sequentially in any combination one or more times, e.g., in the order X-Y-X, X-X-Y, Y-X-Y, Y- Y-X, X-X-Y- Y, etc.
  • the one or more additional therapeutic agents that may be administered in combination with a compound provided herein can be an antibiotic, a viral protease inhibitor, an anti-viral anti-metabolite, a lysosomotropic agent, a M2 proton channel blocker, a polymerase inhibitor (e.g., EIDD-2801, which is also known as MOLNUPIRAVIR), a neuraminidase inhibitor, a reverse transcriptase inhibitor, a viral entry inhibitor, an integrase inhibitor, interferons (e.g., types I, II, and III), or a nucleoside analogue.
  • EIDD-2801 which is also known as MOLNUPIRAVIR
  • a neuraminidase inhibitor e.g., a reverse transcriptase inhibitor
  • a viral entry inhibitor e.g., an integrase inhibitor
  • interferons e.g., types I, II, and III
  • the one or more additional therapeutic agents that may be administered in combination wiht a compounds provided herein can be a steroid (e.g., corticosteroids, such as bethamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, dexamethasone; mineralcorticoid such as fludrocortisone; glucocorticoids, such as hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone; vitamin D such as dihydrotachysterol; androgens such as apoptone, oxandrolone, oxabolone, testosterone, nandrolone (also known as anabolic steroids), oestrogens such as diethylstilbestrol, progestins such as danazol, norethindrone, medroxypro
  • the one or more additional therapeutic agent is Cathepsin L.
  • the one or more additional therapeutic agent is dehydrodidemnin B (also known as Plitidepsin or APLIDIN) or Zotatifin (eFT226).
  • methods described herein further comprise administering an additional anti-viral therapeutic.
  • the anti-viral therapeutic is selected from the group consisting of ribavirin, favipiravir, ST- 193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, par
  • the another therapeutic is selected from the group consisting of protease inhibitors (e.g., nafamostat, camostat, gabexate, epsilon-aminocapronic acid and aprotinin), fusion inhibitors (e.g., BMY-27709, CL 61917, and CL 62554), M2 proton channel blockers (e.g., amantadine and rimantadine), polymerase inhibitors (e.g., 2-deoxy-2'fluoroguanosides (2'-fluoroGuo), 6- endonuclease inhibitors (e.g., L- 735,822 and flutamide) neuraminidase inhibitors (e.g., zanamivir (Relenza), oseltamivir, peramivir and ABT-675 (A-315675), reverse transcriptase inhibitor (e.g., abacavir, adefovir, delavir
  • SUBSTITUTE SHEET RULE 26 (e.g., types I, II, and III), lopinavir, loviride, moroxydine, nexavir, nucleoside analogues (e.g., aciclovir), penciclovir, pleconaril, podophyllotoxin, ribavirin, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zodovudine.
  • types I, II, and III lopinavir, loviride, moroxydine, nexavir, nucleoside analogues (e.g., aciclovir), penciclovir, pleconaril, podophyllotoxin, ribavirin, tipranavir, trifluridine, trizivir, tromantadine, truvada, val
  • the additional anti-viral therapeutic is selected from the group consisting of lamivudine, an interferon alpha, a VAP anti-idiotypic antibody, enfuvirtide, amantadine, rimantadine, pleconaril, aciclovir, zidovudine, fomivirsen, a morpholino, a protease inhibitor, double-stranded RNA activated caspase oligomerizer (DRACO), rifampicin, zanamivir, oseltamivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir
  • the another therapeutic is selected from the group consisting of quinine (optionally in combination with clindamycin), chloroquine, amodiaquine, artemisinin and its derivatives (e.g., artemether, artesunate, dihydroartemisinin, arteether), doxycycline, pyrimethamine, mefloquine, halofantrine, hydroxychloroquine, eflornithine, nitazoxanide, ornidazole, paromomycin, pentamidine, primaquine, pyrimethamine, proguanil (optionally in combination with atovaquone), a sulfonamide (e.g., sulfadoxine, sulfamethoxypyridazine), tafenoquine, tinidazole and a PPT1 inhibitor (including Lys05 and DC661).
  • quinine optionally in combination with clindamycin
  • the another therapeutic is an antibiotic.
  • the antibiotic is a penicillin antibiotic, a quinolone antibiotic, a tetracycline antibiotic, a macrolide antibiotic, a lincosamide antibiotic, a cephalosporin antibiotic, or an RNA synthetase inhibitor.
  • the antibiotic is selected from the group consisting of azithromycin, vancomycin, metronidazole, gentamicin, colistin, fidaxomicin, telavancin, oritavancin, dalbavancin, daptomycin, cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, ceftobiprole, cipro, Levaquin, floxin, tequin, avelox, norflox, tetracycline, minocycline, oxytetracycline, doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,
  • SUBSTITUTE SHEET RULE 26 methicillin, ertapenem, doripenem, imipenem/cilastatin, meropenem, amikacin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefoxotin, and streptomycin.
  • the antibiotic is azithromycin.
  • the one or more additional therapeutic agents that may be administered in combination with a compound provided herein can be selected from the group consisting of ribavirin, favipiravir, ST-193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, sime
  • the compounds described herein may be used in combination with one or more other agents which may be useful in the prevention or treatment of respiratory disease, inflammatory disease, autoimmune disease, for example; anti-histamines, corticosteroids, (e.g., fluticasone propionate, fluticasone furoate, beclomethasone dipropionate, budesonide, ciclesonide, mometasone furoate, triamcinolone, flunisolide), NSAIDs, leukotriene modulators (e.g., montelukast, zafirlukast.pranlukast), tryptase inhibitors, IKK2 inhibitors, p38 inhibitors, Syk inhibitors, protease inhibitors such as elastase inhibitors, integrin antagonists (e.g., beta-2 integrin antagonists),
  • SUBSTITUTE SHEET RULE 26 monoclonal antibody therapy such as anti-lgE, anti- TNF, anti-IL-5, anti-IL-6, anti-IL-12, anti- IL-1 and similar agents; cytokine receptor therapies e.g. etanercept and similar agents; antigen non-specific immunotherapies (e.g.
  • cytokines/chemokines interferon or other cytokines/chemokines, chemokine receptor modulators such as CCR3, CCR4 or CXCR2 antagonists, other cytokine/chemokine agonists or antagonists, TLR agonists and similar agents), suitable anti-infective agents including antibiotic agents, antifungal agents, anthelmintic agents, antimalarial agents, antiprotozoal agents and antituberculosis agents.
  • the additional therapeutic agents can be kinase inhibitors including but not limited to erlotinib, gefitinib, neratinib, afatinib, osimertinib, lapatanib, crizotinib, brigatinib, ceritinib, alectinib, lorlatinib, everolimus, temsirolimus, abemaciclib, LEE011, palbociclib, cabozantinib, sunitinib, pazopanib, sorafenib, regorafenib, sunitinib, axitinib, dasatinib, imatinib, nilotinib, ponatinib, idelalisib, ibrutinib, Loxo 292, larotrectinib, and quizartinib.
  • kinase inhibitors including but not limited to erlotinib,
  • the additional therapeutic agents can be therapeutic anti-viral vaccines.
  • the additional therapeutic agents can be immunomodulatory agents including but not limited to anti-PD-lor anti-PDL-1 therapeutics including pembrolizumab, nivolumab, atezolizumab, durvalumab, BMS-936559, or avelumab, anti-TIM3 (anti-HAVcr2) therapeutics including but not limited to TSR-022 or MBG453, anti-LAG3 therapeutics including but not limited to relatlimab, LAG525, or TSR-033, anti-4-lBB (anti- CD37, anti-TNERSL9), CD40 agonist therapeutics including but not limited to SGN-40, CP- 870,893 or R07009789, anti-CD47 therapeutics including but not limited to Hu5L9-G4, anti- CD20 therapeutics, anti-CD38 therapeutics, SUNG agonists including but not limited to ADU- S100, MK-1454, ASA404, or amidobenzimidazoles, anthracy c
  • the additional therapeutic agent is a p2-adrenoreceptor agonist including, but not limited to, vilanterol, salmeterol, salbutamol. formoterol, salmefamol, fenoterol carmoterol, etanterol,
  • SUBSTITUTE SHEET RULE 26 naminterol, clenbuterol, pirbuterol.flerbuterol, reproterol, bambuterol, indacaterol, terbutaline and salts thereof, for example the xinafoate (1 -hydroxy-2- naphthalenecarboxylate) salt of salmeterol, the sulphate salt of salbutamol or the fumarate salt of formoterol.
  • the additional therapeutic agent is an anticholinergic agent, including, but not limited to, umeclidinium (for example, as the bromide), ipratropium (for example, as the bromide), oxitropium (for example, as the bromide) and tiotropium (for example, as the bromide).
  • umeclidinium for example, as the bromide
  • ipratropium for example, as the bromide
  • oxitropium for example, as the bromide
  • tiotropium for example, as the bromide
  • the disclosure provides a method of treating the above medical indications comprising administering a subject in need thereof a therapeutically effective amount of a compound described herein, such as a disclosed compound.
  • boosting amount or “boosting dose” is the amount of a compound needed to improve the pharmacokinetics of a second compound (or increase availability or exposure).
  • the boosting amount or boosting dose may improve the pharmacokinetics (or increase availability or exposure) of the second compound to a level to therapeutic levels in a subject.
  • the disclosure provides for a disclosed compound to be administered together with an antiviral therapeutic such as disclosed herein, and e.g., thereby boosting the dose of the anti-viral therapeutic or therapeutics.
  • an antiviral therapeutic such as disclosed herein
  • Such a boost combination may be used, e.g., as prophylactic or therapeutic treatment of a viral infection in a subject in need thereof.
  • the protease inhibitor is a compound described herein (e.g. a compound of Formula I, II, or III).
  • compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
  • These formulations include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) rectal, vaginal, or aerosol administration, although the most suitable form of administration in any given case will
  • compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration.
  • compositions of this disclosure may be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains one or more of the compound of the disclosure, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease.
  • the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the disclosure, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stea
  • the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for
  • SUBSTITUTE SHEET RULE 26 example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.
  • the compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl a
  • Suspensions in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
  • suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
  • Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • compositions and compounds of the present disclosure may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • a non-aqueous (e.g., fluorocarbon propellant) suspension could be used.
  • Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
  • compositions of this disclosure suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate and cyclodextrins.
  • Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants
  • enteral pharmaceutical formulations including a disclosed compound and an enteric material; and a pharmaceutically acceptable carrier or excipient thereof.
  • Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, and that are predominantly soluble in intestinal fluids at specific pHs.
  • the small intestine is the part of the gastrointestinal tract (gut) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum.
  • the pH of the duodenum is about 5.5
  • the pH of the jejunum is about 6.5
  • the pH of the distal ileum is about 7.5.
  • enteric materials are not soluble, for example, until a pH of about 5.0, of about 5.2, of about 5.4, of about 5.6, of about 5.8, of about 6.0, of about 6.2, of about 6.4, of about 6.6, of about 6.8, of about 7.0, of about 7.2, of about 7.4, of about 7.6, of about 7.8, of about 8.0, of about 8.2, of about 8.4, of about 8.6, of about 8.8, of about 9.0, of about 9.2, of about 9.4, of about 9.6, of about 9.8, or of about 10.0.
  • Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate,
  • CAP cellulose acetate phthalate
  • HPMCP hydroxypropyl methylcellulose phthalate
  • PVAP polyvinyl acetate phthalate
  • HPMCAS hydroxypropyl methylcellulose acetate succinate
  • trimellitate hydroxypropyl methylcellulose succinate
  • cellulose acetate succinate cellulose acetate hexahydrophthalate
  • cellulose propionate phthalate cellulose acetate maleate
  • SUBSTITUTE SHEET RULE 26 cellulose acetate butyrate, cellulose acetate propionate, copolymer of methylmethacrylic acid and methyl methacrylate, copolymer of methyl acrylate, methylmethacrylate and methacrylic acid, copolymer of methylvinyl ether and maleic anhydride (Gantrez ES series), ethyl methyacrylate- methylmethacrylate-chlorotrimethylammonium ethyl acrylate copolymer, natural resins such as zein, shellac and copal collophorium, and several commercially available enteric dispersion systems (e. g.
  • kits for use by a e.g. a consumer in need of 3 CL inhibitor include a suitable dosage form such as those described above and instructions describing the method of using such dosage form to mediate, reduce or prevent inflammation.
  • the instructions would direct the consumer or medical personnel to administer the dosage form according to administration modes known to those skilled in the art.
  • kits could advantageously be packaged and sold in single or multiple kit units.
  • An example of such a kit is a so-called blister pack.
  • Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material.
  • the packaging process recesses are formed in the plastic foil.
  • the recesses have the size and shape of the tablets or capsules to be packed.
  • the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed.
  • the tablets or capsules are sealed in the recesses between the plastic foil and the sheet.
  • the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
  • a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested.
  • a memory aid is a calendar printed on the card, e.g., as follows “First Week, Monday, Tuesday, . . . etc. . . . Second Week, Monday, Tuesday, . . . “ etc.
  • a “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day.
  • a daily dose of a first compound can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa.
  • the memory aid should reflect this.
  • a subject or patient can further have viral infection- or virus-related co-morbidities, i.e., diseases and other adverse health conditions associated with, exacerbated by, or precipitated by being infected by a virus.
  • viral infection- or virus-related co-morbidities i.e., diseases and other adverse health conditions associated with, exacerbated by, or precipitated by being infected by a virus.
  • Contemplated herein are disclosed compounds in combination with at least one other agent that has previously been shown to treat these virus-related conditions.
  • NMR spectra are recorded at ambient temperature using e.g., a Varian Unity Inova (400MHz) spectrometer with a triple resonance 5mm probe for Example compounds, and either a Bruker Avance DRX (400MHz) spectrometer or a Bruker Avance DPX (300MHz) spectrometer for Intermediate compounds. Chemical shifts are expressed in ppm relative to tetramethylsilane.
  • Scheme 1 illustrates an exemplary preparation of A-A.
  • a Pinner reaction of amine A-1 with a reagent such as methanol affords amino ester A-2.
  • A-2 is then coupled with carboxyxlic acid B-l in the presence of reagents such as l-ethyl-3-(3- dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine to afford the amide product A- 3.
  • reagents such as l-ethyl-3-(3- dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine
  • Removal of the carbamate protecting group in A-3 provides amine A-4, which is coupled with carboxylic acid B-2 to afford amide A-5.
  • the carboxylic acid group A-5 is converted to an amide (A-6), which is then converted to nitrile A-A in the presence of, for example, Burgess reagent.
  • examples of A c and A cc each independently include a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or A c and A cc , together with the atoms they are attached to, may form a substituted or unsubstituted carbocycle, a substituted or unsubstituted monocyclic heterocycle, or a substituted or unsubstituted bicyclic heterocycle;
  • examples of R AA and R AB each independently include a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a
  • SUBSTITUTE SHEET RULE 26 attached to, form a ring, wherein the ring may be a substitued or unsubstituted heterocycle; and examples of R AD include a substituted or unsubstituted bicyclic heteroaryl moiety.
  • Scheme 2 illustrates an exemplary preparation of C-C.
  • Carboxylic acid C-l is coupled with amine D-l in the presence of couple reagents such as benzotriazol- 1- yloxytripyrrolidinophosphonium hexafluorophosphate and tri ethylamine to afford amide C-2.
  • the ester in C-2 is then converted to its correspending amide (C-3) in the presence of, for example, NH3 and methanol. Removal of the carbamate protecting group in C-3 provides amine C-4, which is then converted to trifluoracetamide C-5. Dehydration of the primary amide in C-5 in the presence of, for example, Burgess Reagent, yields nitrile C-C.
  • examples of R DA and DB each independently include a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or R DA and DB , together with the atoms they are attached to, form a ring, wherein the ring may be a substitued or unsubstituted heterocycle;
  • examples of R E include a substituted or unsubstituted alkyl;
  • examples of R DC and DD each independently include a
  • SUBSTITUTE SHEET RULE 26 hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or R DC and DD , together with the atoms they are attached to, may form a substituted or unsubstituted carbocycle, a substituted or unsubstituted monocyclic heterocycle, or a substituted or unsubstituted bicyclic heterocycle; and examples of R DE include a substituted or unsubstituted alkyl.
  • Step 2 (2S)-2-(tert-butoxycarbonylamino)-3-(l-fluorocyclopropyl)propanoic acid
  • Step 3 tert-butyl N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]carbamate
  • Step 5 N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo- ethyl] -5 -cyclopropyl- 1 H-pyrrole-2-carboxamide
  • Step 2 tert-butyl (lR,2S,5S)-2-[[cyano(4-isoquinolyl)methyl]carbamoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-3-carboxylate
  • Step 4 (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • reaction solution was dried over N2 to give a residue.
  • the residue was purified by prep-HPLC (neutral condition: column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water (NHd4CO3)-ACN]; B%: 40%-70%, 10 min) to give desired compound (100 mg, purity 98%) as a white solid.
  • T3P (1.89 g, 2.96 mmol, 1.76 mL, 50% purity, 2 eq) in DCM (12.5 mL) was added in one portion at -30 °C. The mixture was stirred at -30 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with DCM 40 mL (20 mL * 2). The combined organic layers was concentrated under reduced pressure to give a residue. Then crude product was triturated with ethyl acetate at 25 °C for 30 min.
  • Step 1 2-((4-methoxybenzyl)amino)-2-(4-(trifluoromethyl)pyridin-3-yl)acetonitrile
  • Step 3 methyl 2-amino-2-(4-(trifluoromethyl)pyridin-3-yl)acetate
  • Step 4 methyl 2-((S)-3-cyclopropyl-2-(7-fluoro-lH-indole-2-carboxamido)propanamido)-2-(4- (trifluoromethyl)pyridin-3-yl)acetate
  • Step 5 N-((2S)-l-((2-amino-2-oxo-l-(4-(trifluoromethyl)pyri din-3 -yl)ethyl)amino)-3 - cyclopropyl- 1 -oxopropan-2-yl)-7-fluoro- 1 H-indole-2-carboxamide
  • Step 6 N-((2S)-l-((cyano(4-(trifluoromethyl)pyridin-3-yl)methyl)amino)-3-cyclopropyl-l- oxopropan-2-yl)-7-fluoro- 1 H-indole-2-carboxamide
  • Step 2 N-[2-[[cyano(4-isoquinolyl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro- lH-indole-2-carboxamide
  • reaction solution was quenched with H2O (15 mL), and extracted with DCM 45 mL (15 mL * 3). The organic layers were combined, washed with citric acid (20 mL), sat. aq. NaHCCh (20 mL), brine (20 mL), dried over Na2SO 4 and concentrated under reduced pressure to give a residue.
  • Step 1 tert-butyl (lR,2S,5S)-2-[[2-methoxy-2-oxo-l-(3-pyridyl)ethyl]carbamoyl]-6,6-dimethyl- 3-azabicyclo[3.1 ,0]hexane-3-carboxylate
  • Step 2 methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3- pyridyl)acetate
  • Step 3 methyl 2-[[(lR,2S,5S)-3-(7-fhroro-lH-indole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3-pyridyl)acetate
  • Step 4 (lR,2S,5S)-N-[2-amino-2-oxo-l-(3-pyridyl)ethyl]-3-(7-fluoro-lH-indole-2-carbonyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 5 (lR,2S,5S)-N-[cyano(3-pyridyl)methyl]-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 2 tert-butyl (lR,2S,5S)-2-[[l-(4-isoquinolyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-3-carboxylate
  • Step 3 methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(4- isoquinolyl)acetate
  • Step 4 methyl 2-[[(lR,2S,5S)-3-(7-fhroro-lH-indole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(4-isoquinolyl)acetate
  • Step 5 (lR,2S,5S)-N-[2-amino-l-(4-isoquinolyl)-2-oxo-ethyl]-3-(7-fluoro-lH-indole-2- carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 6 (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 2 N-[(lS)-2-[[cyano(l,6-naphthyridin-8-yl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo- ethyl]-7-fluoro-lH-indole-2-carboxamide
  • Step 3 N-[(lS)-2-[[cyano(l,6-naphthyridin-8-yl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo- ethyl]-7-fluoro-lH-indole-2-carboxamide
  • Step 2 N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro- 1 H-indole-2-carboxamide
  • the resluting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by H2O 20 mL, and then extracted with DCM 45 mL (15 mL * 3). The combined organic layers were washed with brine 20 mL (20 mL * 1),
  • Step 3 N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro- 1 H-indole-2-carboxamide
  • Step 4 N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]- 7-fluoro-lH-indole-2-carboxamide
  • N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo- ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 1, 150 mg) was further separated by SFC (condition: column: DAICEL CHIRALPAK AD(250mm*30mm,10um);mobile phase: [0.1%NH 3 H 2 O ETOH];B%: 54%-54%,12min) to give N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl- ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 3, 30 mg, 71.35 umol, 5.29% yield, 100% purity) as a white solid.
  • Step 2 methyl (2S)-2-[[(lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3-pyridyl)acetate
  • Step 3 tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[(lS)-2-amino-2-oxo-l-(3-pyridyl)ethyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
  • Step 4 (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[(lS)-2-amino-2-oxo-l-(3- pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 5 (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[(lS)-2-amino-2-oxo-l-(3- pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 6 (lR,2S,5S)-N-[(S)-cyano(3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
  • Step 1 methyl 2-[[(lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-[4-(trifluoromethyl)-3- pyridyl]acetate
  • Step 3 (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[2-amino-2-oxo-l-[4- (trifluoromethyl)-3-pyridyl]ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 4 tetrahydrofuran-3-yl N-[(lS)-l-[(lR,2S,5S)-2-[[(lS)-l-cyano-2-[(6R)-5-oxo-4- azaspiro[2.4]heptan-6-yl]ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]- 2, 2-dimethyl-propyl] carbamate
  • the resluting mixture was stirred at 0 °C for 1 h, then was added TFAA (145.77 mg, 694.06 umol, 96.54 uL, 1.5 eq) in DCM (1 mL) at 0 °C, stirred at 0 °C for 1 h, and then TFAA (145.77 mg, 694.06 umol, 96.54 uL, 1.5 eq) in DCM (1 mL) was added
  • Step 5 (lR,2S,5S)-N-[cyano-[4-(trifhioromethyl)-3-pyridyl]methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 1 tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(4-isoquinolyl)methyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
  • Step 2 (lR,2S,5S)-3 - [(2S)-2-amino-3 ,3 -dimethyl-butanoy 1] -N- [cyano(4-isoquinolyl)methyl] - 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 3 (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 1 tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(2,6-naphthyridin-4-yl)methyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
  • Step 2 (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(2,6-naphthyridin-4- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 3 ( 1 R,2S , 5 S)-N - [cyano(2,6-naphthyridin-4-y l)methy 1] -3 - [(2S)-3 , 3 -dimethyl-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
  • Step 1 tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
  • Step 2 (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-(l-cyano-l-pyrazin-2-yl-ethyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 3 (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 4 (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 1 tert-butylN-[(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(5-fluoro-3-pyridyl)ethyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
  • Step 2 (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[l-cyano-l-(5-fluoro-3- pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 3 (lR,2S,5S)-N-[l-cyano-l-(5-fhioro-3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 4 (lR,2S,5S)-N-[l-cyano-l-(5-fluoro-3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
  • Step 1 2-amino-2-(5-chloro-3 -pyridyl) propanenitrile
  • Step 2 tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-(5-chloro-3-pyridyl)-l-cyano-ethyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
  • Step 3 (1R, 2S, 5S)-3-[(2S)-2-amino-3, 3-dimethyl-butanoyl]-N-[l-(5-chloro-3-pyridyl)-l- cyano-ethyl]-6, 6-dimethyl-3 -azabicyclo [3.1.0] hexane-2-carboxamide

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Abstract

The disclosure provides compounds with nitrile warheads and their use in treating medical diseases or disorders, such as viral infections. Pharmaceutical compositions and methods of making various compounds with nitrile warheads are provided. The compounds are contemplated to inhibit proteases, such as the 3C, CL- or 3CL-like protease.

Description

INHIBITORS < > F USE THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims the benefit of, and priority to, U.S.S.N. 63/246,001, filed September 20, 2021; U.S.S.N. 63/288,206, filed December 10, 2021; U.S.S.N. 63/392,847, filed July 27, 2022; and U.S.S.N. 63/392,849, filed July 27, 2022; the contents of each of which are incorporated herein by reference.
BACKGROUND
[0002] The Coronaviridae family of viruses are enveloped, single-stranded, positive-sense RNA viruses and include 141 species classified into four genera according to their phylogenetic relationships: a-, P-, y-, and 6-coronavirus. Coronaviruses (CoVs) are zoonotic viruses that infect a variety of animals from whales to birds, bats, cats, and humans. Typically, CoV infection results in mild to moderate respiratory tract infections; however, some CoV species are extremely virulent and can result in widespread fatality. Severe acute respiratory syndrome coronavirus (SARS-CoV) is a human CoV responsible for the first pandemic of the 21st century, infecting over 8,000 people with a 10% mortality rate. Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in November 2012 and had since infected over 1,600 people in 26 countries with a 36% mortality rate. More recently, COVID-19 (SARS CoV2) coronaviruses have raised a global pandemic since first identified in late 2019. Therefore, it is important to identify coronavirus drug targets that can be utilized for the development of broadspectrum anti-coronaviral therapeutics to combat infections of existing and emerging coronaviruses.
[0003] All CoVs express a >800 kDa replicase polyprotein that contains either two or three cysteine proteases, the papain-like protease(s) (PLPpro, nsp3, or PLP1 and PLP2) and the SC- like protease (3CLpro, nsp5, or Mpro). These proteases process the CoV replicase polyprotein by cleaving it into 16 non-structural proteins, which are responsible for a variety of aspects of CoV replication. The CoV 3CLprois responsible for processing 11 cleavage sites of within the replicase polyprotein and is essential for CoV replication, making it a highly valuable target for therapeutic development. The overall active site architecture and substrate recognition pockets are structurally conserved across CoV 3CLpros, increasing its attractiveness as a target for the □f broad-spectrum anti-CoV therapeutics. Moreover, high sequence conservation
1
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 in the vicinity of act o - - - - us subclasses make them an excellent target for the development of broad-spectrum therapeutics for coronavirus infections. Accordingly, the development of CoV 3CLpro inhibitors is a promising path for the treatment of respiratory tract infections and related diseases.
[0004] Numerous studies on targeting the immediate zoonotic reservoirs of coronaviruses with small molecule inhibitors have helped inform structure-based design strategies aimed at creating molecular scaffolds that may aid in the development of therapeutic against coronaviral infection; however, small molecule antiviral agents or effective commercially available broadspectrum therapeutics have not yet been identified. There is a critical need for the development of broad-spectrum CoV therapeutics to overcome the challenges of traditional anti-CoV therapeutic development, as broad-spectrum therapeutics can be rapidly implemented upon zoonotic disease outbreak.
SUMMARY
[0005] The disclosure is directed in part, to viral protease inhibitors. Also disclosed herein are pharmaceutical compositions comprising at least one disclosed compound and a pharmaceutically acceptable carrier.
[0006] In an embodiment, disclosed herein is an antiviral compound, comprising a warhead (e.g., a nitrile warhead) covalently bound to a 3C or 3 CL protease inhibitor, wherein the antiviral compound covalently binds to a Cys residue of the protease, and wherein the antiviral compound is active against one or more viruses.
[0007] For example, disclosed herein is a protease inhibitory compound represented by Formula I:
Figure imgf000003_0001
Formula I or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R1 is selected from the group consisting of -CH(Rn)-NHR12, a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R1 may optionally be substituted by one, two, or three substituents each selected from R5;
2 IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 R11 is select — > o — o > > .yl, -CJh-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
R12 is selected from the group consisting of -C(O)RB, -C(O)ORB, -C(O)NHRB, -RB, - NHC(O)RB, -NHC(O)ORB, -NHC(O)NHRB, and -NHRB; wherein R12 or RB may optionally be substituted by one or more substituents each independently selected from R13;
RB is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
R2 is hydrogen or Ci-Csalkyl;
R3ais hydrogen or Ci-Csalkyl;
R3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R3 may optionally be substituted by one, two, or three substituents each independently selected from R5; or R3 is -Si(CH3)3; or R2 and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6; or R3a and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic carbocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6;
R4ais hydrogen or Ci-C3alkyl optionally substituted by one or more halogens;
R4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R4 may optionally be substituted by one, two, or three substituents each independently selected from R7; or
R4a and R4, together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R8;
R5’ R6, R7’ R8 and R13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R56)2, -N(R56)C(O)R56, -C(O)N(R56)2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C9H2iSi-, Ci-Cealkyl, C2-C6alkynyl, Ci-C6alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- ealkoxy, C3-Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may
3
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 optionally be sub sth - selected from the group consisting of halogen, hydroxyl, Ci-Csalkyl, C2-Cealkynyl, Ci-Cealkoxy, and -CF3;
R56 is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens; and Rais hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
[0008] In some embodiments, the protease inhibitory compound of Formula I is represented by Formula I':
Figure imgf000005_0001
Formula I' or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R1 is selected from the group consisting of -CH(Rn)-NHR12, a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R1 may optionally be substituted by one, two, or three substituents each independently selected from R5;
R11 is selected from the group consisting of branched Ci-Cealkyl, -CFF-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
R12 is selected from the group consisting of -NHC(O)RB, -NHC(O)ORB, -NHC(O)NHRB, and -NHRB; wherein R12 or RB may optionally be substituted by one or more substituents each independently selected from R13;
RB is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
R2 is hydrogen or Ci-Csalkyl;
R3ais hydrogen or Ci-Csalkyl;
R3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R3 may optionally be substituted by one, two, or three substituents each independently selected from R5; or R3 is -Si(CH3)3; or R2 and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6; or R3a and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 spirocyclic carbocyc.? , three substituents each independently selected from R6;
R4ais hydrogen or Ci-Csalkyl optionally substituted by one or more halogens;
R4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R4 may optionally be substituted by one, two, or three substituents each independently selected from R7; or
R4a and R4, together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R8;
R5’ R6, R7’ R8 and R13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R56)2, -N(R56)C(O)R56, -C(O)N(R56)2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C9H2iSi-, Ci-Cealkyl, C2-C6alkynyl, Ci-C6alkoxy, C3- Cecycloalkyl, Cs-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, Cs-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl, Ci-Csalkyl, C2-Cealkynyl, Ci-Cealkoxy, and -CF3;
R56 is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens; and
Rais hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
[0009] In some embodiments, the protease inhibitory compound of Formula I is represented by Formula I":
Figure imgf000006_0001
Formula I" or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R1 is selected from the group consisting of -CH(Rn)-NHR12, a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R1 may optionally be substituted by one, two, or three substituents each independently selected from R5;
R11 is selected from the group consisting of branched Ci-Cealkyl, -CFb-phenyl, -CH2- nanhthvl. and -CH2-(8-10 membered bicyclic heteroaryl);
5 IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 R12 is selects > o — o , -C(O)NHRB, and -RB; wherein R12 or RB may optionally be substituted by one or more substituents each independently selected from R13;
RB is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
R2 is hydrogen or Ci-Csalkyl;
R3ais hydrogen or Ci-Csalkyl;
R3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R3 may optionally be substituted by one, two, or three substituents each independently selected from R5; or R3 is -Si(CH3)3; or R2 and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6; or R3a and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic carbocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6;
R4ais hydrogen or Ci-C3alkyl optionally substituted by one or more halogens;
R4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R4 may optionally be substituted by one, two, or three substituents each independently selected from R7; or
R4a and R4, together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R8;
R5’ R6, R7’ R8 and R13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R56)2, -N(R56)C(O)R56, -C(O)N(R56)2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C9H2iSi-, Ci-Cealkyl, C2-C6alkynyl, Ci-C6alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, C3-Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents each independently selected from the group lalogen, hydroxyl, Ci-C3alkyl, C2-Cealkynyl, Ci-Cealkoxy, and -CF3;
6
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 R56 is hydrog - - , • three halogens; and
Rais hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
[00010] In another aspect, provided herein is a protease inhibitory compound represented by Formula II:
Figure imgf000008_0001
or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein R10, R15, R20, R30, R40a and R40 are as defined herein.
[00011] In a further aspect, provided herein is a compound represented by Formula III:
Figure imgf000008_0002
Formula III
[00012] or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein R100, R200, R300, R400a and R400 are as defined herein. Also provided herein are methods of ameliorating or treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the compounds described herein.
[00013] In some embodiments, provided herein are methods of inhibiting transmission of a virus, a method of inhibiting viral replication, a method of minimizing expression of viral proteins, or a method of inhibiting virus release, comprising administering a therapeutically effective amount of a compound described herein to a patient suffering from the virus, and/or contacting an effective amount of a compound described herein with a virally infected cell.
DETAILED DESCRIPTION
[00014] The features and other details of the disclosure will now be more particularly described. Before further description of the present disclosure, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and as understood by a person of skill in the art.
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 Unless defined othe „ve the same meaning as commonly understood by a person of ordinary skill in the art.
Definitions
[00015] The term “treating” includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder and the like, including a reduction of viral shedding in asymptomatic individuals and prophylaxis of exposed individuals, independent of symptoms.
[00016] The term “alkyl” as used herein refers to a saturated straight or branched hydrocarbon. Exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as Ci-ealkyl, Ci-4alkyl, and Ci- salkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2 -m ethyl- 1 -butyl, 3-methyl-2-butyl, 2 -m ethyl- 1 -pentyl, 3 -methyl- 1 -pentyl, 4- m ethyl- 1 -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l-butyl, 2-ethyl-l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.
[00017] The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Exemplary alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as Ci-Csalkenyl, C2-Cealkenyl, and C3-C4alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.
[00018] The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Exemplary alkynyl groups include, but are not limited to, straight or branched groups of 2-6, or 3-6 carbon atoms, referred to herein as C2-ealkynyl, and C3-ealkynyl, respectively. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, etc.
[00019] The term “alkoxy” as used herein refers to a straight or branched alkyl group attached to oxygen (alkyl-O-). Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci-Csalkoxy, Ci-Cealkoxy, and C2- Cealkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc. The term “alkoxy” as used herein may also refer to a cycloalkyl group ygen (cycloalkyl-O-).
8
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SUBSTITUTE SHEET RULE 26 [00020] The tei ... > - die e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 % electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Ce aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“Cio aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“Ci4 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene. Particularly aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Examples of representative substituted aryls include the following
Figure imgf000010_0001
wherein one of R56 and R57 may be hydrogen and at least one of R56 and R57 is each independently selected from Ci-Cs alkyl, Ci-Cs haloalkyl, 4-10 membered heterocyclyl, alkanoyl, Ci-Cs alkoxy, heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR58COR59, NR58SOR59NR58SO2R59, COOalkyl, COOaryl, CONR58R59, CONR58OR59, NR58R59, SO2NR58R59, S-alkyl, SOalkyl, SChalkyl, Saryl, SOaryl, SCharyl; or R56 and R57 may be joined to form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionally containing one or more heteroatoms selected from the group N, O, or S. R60 and R61 are each independently hydrogen, Ci-Cs alkyl, C1-C4 haloalkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, Ce-Cio aryl, substituted Ce-Cio aryl, 5-10 membered heteroaryl, or substituted 5-10 membered heteroaryl.
[00021] The term “carbonyl” as used herein refers to the radical -C(O)-.
Che term “cyano” as used herein refers to the radical -CN.
9 IPTS/l20253274.1
SUBSTITUTE SHEET RULE 26 [00023] The tei - „ - o > sin refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3-6, or 4-6 carbons, referred to herein as C3-Ciocycloalkyl, C3-6cycloalkyl or C4-6cycloalkyl, respectively. Exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl or cyclopropyl.
[00024] The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.
[00025] The terms “haloalkyl” as used herein refers to an alkyl radical in which the alkyl group is substituted with one or more halogens. Typical haloalkyl groups include, but are not limited to, trifluoromethyl (i.e. CF3), difluoromethyl, fluoromethyl, chloromethyl, dichloromethyl, dibromoethyl, tribromomethyl, tetrafluoroethyl, and the like. Exemplary haloalkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms substituted with a halogen (i.e. Cl, F, Br and I), referred to herein as Ci- ehaloalkyl, Ci-4 haloalkyl, and Ci-3haloalkyl, respectively.
[00026] The term “hetero” when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g,. heteroaryl, cycloalkenyl, e.g. cycloheteroalkenyl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
[00027] The terms “heteroaryl” or “heteroaromatic group” as used herein refers to an aromatic 5-10 membered ring system containing one or more heteroatoms, for example one to three heteroatoms, such as nitrogen, oxygen, and sulfur. The term may also be used to refer to a 5-7 membered monocyclic heteroaryl or an 8-10 membered bicyclic heteroaryl. Where possible, said heteroaryl ring may be linked to the adjacent radical though carbon or nitrogen. Examples of heteroaryl rings include but are not limited to furan, thiophene, pyrrole, pyrrol opyri dine, indole, thiazole, oxazole, isothiazole, isoxazole, imidazole, benzoimidazole, imidazopyridine, pyrazole, triazole, pyridine or pyrimidine, etc.
[00028] Examples of representative heteroaryls include the following:
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000012_0001
wherein each Z is selected from carbonyl, N, NR65, O, and S; and R65 is each independently hydrogen, Ci-Cs alkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, Ce-Cio aryl, or 5-10 membered heteroaryl.
[00029] The terms “heterocyclyl,” “heterocycle,” or “heterocyclic group” are art-recognized and refer to saturated or partially unsaturated 4-10 membered ring structures, whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen. The term may also be used to refer to 4-10 membered saturated or partially unsaturated ring structures that are bridged, fused or spirocyclic ring structures, whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Examples of heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran, dihydrofuran, dihydropyran, tetrahydropyran, etc. In some embodiments, the heterocycle is a spiro heterocycle (e.g., 2,8-diazaspiro[4.5]decane). In some embodiments, the heterocycle is a bridged heterocycle (e.g., octahydro- 1H-4, 7- methanoisoindole). "Spiro heterocyclyl," or “spiro heterocycle” refers to a polycyclic heterocyclyl with rings connected through one common atom (called a spiro atom), wherein the rings have one or more heteroatoms selected from the group consisting of N, O, and S(O)m (wherein m is an integer of 0 to 2) as ring atoms. Representative examples of heterocyclyl include, for example:
Figure imgf000012_0002
11
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00030] The terms “hydroxy” and “hydroxyl” as used herein refers to the radical -OH.
[00031] The term “oxo” as used herein refers to the radical =0.
[00032] “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
[00033] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
[00034] The term “pharmaceutical composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.
[00035] “Individual,” “patient,” or “subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. The compounds of the disclosure can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like). “Modulation” includes antagonism (e.g., inhibition), agonism, partial antagonism and/or partial agonism.
[00036] In the present specification, the term “therapeutically effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system or animal, (e.g. mammal or human) that is being sought by the researcher, veterinarian, medical doctor or other clinician. The compounds of the disclosure are administered in therapeutically effective amounts to treat a disease. Alternatively, a therapeutically effective amount of a compound is the quantity required to achieve a desired therapeutic and/or
Figure imgf000013_0001
12
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00037] The tei ... „ — i refers to salts of acidic or basic groups that may be present in compounds used in the compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, -toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3- naphthoate)) salts. Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids. The compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt.
[00038] The compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers. The term “stereoisomers” when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols “(+),”
Figure imgf000014_0001
“R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.
[00039] The compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. The symbol — denotes a bond that may be a single, double or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance " ” * ” itandards. Unless otherwise specified, structures depicting double bonds
13
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 encompass both the > > arbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond.
[00040] Compounds of the disclosure may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both “Z” and “E” isomers. Substituents around a carbocyclic or heterocyclic rings may also be referred to as “cis” or “trans”, where the term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”
[00041] Individual enantiomers and diastereomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective syntheses, a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art. Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009.
[00042] The compounds disclosed herein can exist in solvated as well as unsolvated forms eutically acceptable solvents such as water, ethanol, and the like, and it is intended
14
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 that the disclosure e > nbodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form.
[00043] The disclosure also embraces isotopically labeled compounds of the disclosure which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 170, 31P, 32P, 35S, 18F, and 36C1, respectively. For example, a compound of the disclosure may have one or more H atom replaced with deuterium.
[00044] Certain isotopically-labeled disclosed compounds (e.g., those labeled with 3H and 14C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3H) and carbon-14 (z.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (z.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.
[00045] The term “prodrug” refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). For example, if a compound of the disclosure or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (Ci-s)alkyl, (C2-i2)alkylcarbonyloxymethyl, l-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1 -methyl- l-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxy carbonyloxymethyl having from 3 to 6 carbon atoms, 1 -(alkoxy carbonyloxy)ethyl having rbon atoms, 1 -methyl- 1 -(alkoxy carbonyloxy)ethyl having from 5 to 8 carbon
15
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 atoms, N-(alkoxyca. j > j o > > , l-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3 -phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(Ci-2)alkylamino(C2-3)alkyl (such as P- dimethylaminoethyl), carbamoyl-(Ci-2)alkyl, N,N-di(Ci-2)alkylcarbamoyl-(Ci-2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-3)alkyl.
[00046] Similarly, if a compound of the disclosure contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (Ci-6)alkylcarbonyloxymethyl, l-((Ci-6)alkylcarbonyloxy)ethyl, 1 -methyl- l-((Ci- 6)alkylcarbonyloxy)ethyl (Ci-6)alkoxycarbonyloxymethyl, N-(Ci-6)alkoxycarbonylaminomethyl, succinoyl, (Ci-e)alkylcarbonyl, a-amino(Ci-4)alkylcarbonyl, arylalkylcarbonyl and a- aminoalkylcarbonyl, or a-aminoalkylcarbonyl-a-aminoalkylcarbonyl, where each a- aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)2, -P(O)(O(Ci-e)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).
[00047] If a compound of the disclosure incorporates an amine functional group, a prodrug can be formed, for example, by creation of an amide or carbamate, an N-alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine. In addition, a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can metabolically cleaved to generate a bioactive primary or secondary amine. For examples, see Simplicio, et al., Molecules 2008, 13, 519 and references therein.
[00048] The term "warhead" or "warhead group" as used herein refers to a functional group present on a compound wherein that functional group is capable of reversibly or irreversibly participating in a reaction with a protein, e.g., 3C or 3CL protease (e.g., with a cysteine on the protease such as Cys 145). Warheads may, for example, form covalent bonds with the protein, or may create stable transition states, or be a reversible or an irreversible alkylating agent. For example, the warhead moiety can be a functional group on an inhibitor that can participate in a bond-forming reaction, wherein a new covalent bond is formed between a portion of the warhead and a donor, for example an amino acid residue of a protein. In embodiments, the warhead is an electrophile and the “donor” is a nucleophile such as the side chain of a cysteine residue. As provided herein, a warhead may include a nitrile group. For example, nitriles may be reversible covalent warheads for cysteine protease inhibition. For example, where the mechanism of action may involve aformation of reversible covalent bond between the nitrile and the active cysteine to idate adduct. Reaction of cysteine of glutathione or other proteins is generally
16
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 reversible, while the o - .rreversibly forms a thiazolidine adduct. It can be appreciated that contemplated compounds herein may be a reversible or an irreversible inhibitor. It will be appreciated to one of skilled in the art that the compounds disclosed herein that include the warheads above also contemplate the precursors to those compounds, for example, where a cyano moiety involved in a warheads may be replaced with e.g., a halo moiety.
[00049] It will be appreciated to one of skilled in the art that the compounds disclosed herein can also irreversibly bind, or may otherwise inhibit e.g., a virus protein via any other mechanism of action.
[00050] The term "inhibitor" as used herein refers to a compound that binds to and /or inhibits a target protease with measurable affinity.
[00051] The term “reversible” or "reversible inhibitor" as used herein refers to a protease inhibitor that associates with a protease in such a way as to inhibit the activity of the protease while the protease and inhibitor are bound, but does not associate with a protease in such a way as to inhibit the activity of the protease when the protease and inhibitor are no longer bound. Reversible inhibitors can effect inhibition by competing with substrate for binding to the active site of the protease (competitive reversible inhibitor), or by associating with the protease bound to its substrate in a way to make the complex inactive (uncompetitive reversible inhibitor), or by associating with the protease and/or protease-substrate complex in a way that inhibits the activity of either and/or both.
[00052] As used herein, the term “irreversible” or “irreversible inhibitor” refers to an inhibitor (i.e. a compound) that is able to be covalently bonded to a target protease in a substantially non-reversible manner. An irreversible inhibitor will remain substantially bound to the target protease once covalent bond formation has occurred. Irreversible inhibitors usually display time dependency, whereby the degree of inhibition increases with the time with which the inhibitor is in contact with the enzyme. In certain embodiments, an irreversible inhibitor will remain substantially bound to target protease once covalent bond formation has occurred and will remain bound for a time period that is longer than the life of the protein.
I. Viral Protease Inhibitor Compounds
[00053] The disclosure is directed to, in part, compounds that inhibit a viral protease.
Examples of viral proteases include, but not limited to, Cathepsin K, coronavirus main protease ise 3, Calpain 1, and Cathepsin S. Accordingly, in various embodiments, a
17
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 compound of the pre v .o. > JI) is a viral protease inhibitor, wherein the viral protease is selected from the group consisting of Cathepsin K, coronavirus main protease (Mpro), Caspase 3, Calpain 1, and Cathepsin S. In certain embodiments, the viral protease is a coronavirus main protease (Mpro). In some embodiments, the viral protease is Cathepsin K. In some embodiments, the viral protease is Caspase 3. In some embodiments, the viral protease is Calpain 1. In some embodiments, the viral protease is Cathepsin S.
[00054] For example, disclosed herein is a protease inhibitory compound represented by Formula I:
Figure imgf000019_0001
Formula I or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R1 is selected from the group consisting of -CH(Rn)-NHR12, a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R1 may optionally be substituted by one, two, or three substituents each independently selected from R5;
R11 is selected from the group consisting of branched Ci-Cealkyl, -CFh-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
R12 is selected from the group consisting of -NHC(O)RB, -NHC(O)ORB, -NHC(O)NHRB, and -NHRB; wherein R12 or RB may optionally be substituted by one or more substituents each independently selected from R13;
RB is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
R2 is hydrogen or Ci-Csalkyl;
R3ais hydrogen or Ci-Csalkyl;
R3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R3 may optionally be substituted by one, two, or three substituents each independently selected from R5; or R3 is -Si(CH3)3; or R2 and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered snirocvclic heterocyclyl which may optionally be substituted by one, two or three substituents each r J selected from R6;
18
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 or R3a and R , > , may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic carbocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6;
R4ais hydrogen or Ci-Csalkyl optionally substituted by one or more halogens;
R4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, and a 4-10 membered heterocyclyl; wherein R4 may optionally be substituted by one, two, or three substituents each independently selected from R7; or
R4a and R4, together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R8;
R5’ R6, R7’ R8 and R13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R56)2, -N(R56)C(O)R56, -C(O)N(R56)2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C9H2iSi-, Ci-Cealkyl, C2-C6alkynyl, Ci-C6alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, C3-Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl, Ci-C3alkyl, C2-Cealkynyl, Ci-Cealkoxy, and -CF3;
R56 is hydrogen or Ci-C3alkyl optionally substituted by one, two or three halogens; and
Rais hydrogen or Ci-C3alkyl optionally substituted by one, two or three halogens.
[00055] For example, disclosed herein is a protease inhibitory compound represented by Formula I:
Figure imgf000020_0001
Formula I or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:
R1 is -CH(R12)-NHR12 or a 9-10 membered bicyclic heteroaryl having a ring nitrogen; wherein R1 or R11 may optionally be substituted by one, two, or three substituents each independently selected from R5;
R11 is selected from the group consisting of branched Ci-Cealkyl, -Cfh-phenyl, -CH2- no Ji+hm onri -CH2-(8-10 membered bicyclic heteroaryl);
19
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 R12 is -C(O) > - - - - - j > y be substituted by one or more halogens;
R2 is hydrogen or Ci-Csalkyl;
R3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R3 may optionally be substituted by one, two, or three substituents each independently selected from R5; or R3 is -Si(CH3)3; or R2 and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6;
R4ais hydrogen or Ci-C3alkyl optionally substituted by one or more halogens;
R4 is 5-10 membered heteroaryl optionally substituted by one, two, or three substituents each independently selected from R7; or
R4a and R4, together with the carbon to which they are attached, may be joined together to form an 8-10 membered partially unsaturated bicyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R8;
R5’ R6, R7 and R8 are independently selected for each occurrence from the group consisting of halogen, hydroxyl, cyano, -N(R56)2, -N(R56)C(O)R56, -C(O)N(R56)2, -CF3, Ci-Cealkyl, Ci- Cealkoxy, Cs-Cecycloalkyl, and phenyl; wherein Ci-Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, and phenyl may optionally be substituted by one or more substituents each independently selected from the group consisting of halogen and hydroxyl; and
R56 is hydrogen or Ci-C3alkyl optionally substituted by one, two or three halogens.
[00056] In some embodiments, R2 and R3, together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl.
[00057] For example, in some embodiments a compound disclosed herein, e.g., a compound of Formula I, may be represented by:
Figure imgf000021_0001
selected from 0, 1 and 2; and n is selected from 0, 1, 2 and 3.
20
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00058] In othe. - , , .o., „ compound of Formula
I, may be represented by:
Figure imgf000022_0001
wherein m is 0, 1 or 2; n is 0, 1, 2, or 3; and t is 0, 1 or 2.
[00059] In certain embodiments, R6 when present is independently selected for each occurrence from the group consisting of, for example, fluoro, chloro and -CH3.
[00060] In some embodiments, for example, R2 and R3, together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
Figure imgf000022_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00061] In othe. - , .. > , > which they are attached, join together to form a 4-6 membered monocyclic or 5-10 membered spirocyclic carbocyclyl selected from the group consisting of:
Figure imgf000023_0001
[00062] In some embodiments, R2 is hydrogen. In other embodiments, a compound disclosed herein, e.g., a compound of Formula I, may be represented by:
Figure imgf000023_0002
wherein t is 0, 1 or 2.
[00063] In certain embodiments, R3 is selected from the group consisting of:
Figure imgf000023_0003
22
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00064] In certa - - , > — — - itly selected for each occurrence from the group consisting of, for example, fluoro, chloro, bromo, -CFL, -CF3, isopropyl, tert-butyl, cyclopropyl and -OCH3.
[00065] In further embodiments R11 is selected, for example, from the group consisting of:
Figure imgf000024_0001
[00066] For example, in some embodiments a compound disclosed herein, e.g., a compound of Formula I, may be represented by:
Figure imgf000024_0002
wherein m is 0, 1 or 2; and n is 0, 1, 2 or 3, and R6 when present is independently selected for each occurrence from the group consisting of fluoro, chloro and -CHs.
23
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00067] In som - , .. > - - - its, R™ is hydrogen. In still other embodiments, R4 may be selected, for example, from the group consisting of:
Figure imgf000025_0001
24
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000026_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 wherein:
X is O, S, or NRa;
R7 is independently selected for each occurrence from the group consisting of chloro, fluoro, bromo, -CN, -CCH, -CH3, -CH2F, -CHF2, -CF3, cyclopropyl, -OH, -OCH3 and -OCFs;
R77 and R78 are independently selected, for each occurrence, from the group consisting of hydrogen, chloro, fluoro, bromo, -CN, -CCH, -CH3, -CH2F, -CHF2, CF3, -OCH3, -OCF3 and cyclopropyl; and
Ra is selected from the group consisting of hydrogen, Ci-C3alkyl, C3-Cecycloalkyl, and phenyl, wherein Ci-C3alkyl, C3-Cecycloalkyl, and phenyl may optionally be substituted by one or more halogens.
[00068] For example, in some embodiments R4 may be selected from the group consisting
Figure imgf000027_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000028_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000029_0001
28
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000030_0001
29
IPTS/120253274.1
SUBSTITUTE SHEET ( RULE 26)
Figure imgf000031_0001
30
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000032_0001
[00069] In some embodiments,
Figure imgf000032_0002
, wherein two R80 may be joined, together with the atoms to which they are attached, to form a 5-6 membered heterocyclyl or 5-6 membered heteroaryl.
[00070] In some embodiments, R4 is selected from the group consisting of:
Figure imgf000032_0003
[00071] In certain embodiments, R4a and R4, together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of, for example:
31
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000033_0001
wherein R8 and R9 are independently selected for each occurrence from the group consisting of chloro, fluoro, -CH3, -CH2F, -CHF2, -CF3, -OH and -OCH3; or two geminal R9 form oxo.
[00072] For example, in some embodiments R4a and R4, together with the carbon to which they are attached, join together to form:
Figure imgf000033_0002
32
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000034_0001
[00073] In some embodiments, R1 is -CH(Rn)-NHR12. In some embodiments, R1 is a 9-10 membered bicyclic heteroaryl having a ring nitrogen. In some embodiments, R1 is a a 5-6 membered monocyclic heteroaryl having a ring nitrogen. . In some embodiments, R1 is optionally substituted thiazolyl, pyrrolyl, oxadiazolyl, pyrazinyl, or isoindolyl.
[00074] In some embodiments, R1 is selected from the group consisting of
Figure imgf000034_0002
Figure imgf000034_0003
[00075] Also disclosed herein is a compound represented by Formula II:
Figure imgf000034_0004
Formula II or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
33
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 R10 is is sele o — o > C(O)-NRa-Ci-Cealkyl, -
C(O)-O-Ci-Cealkyl, -C(0)-0-CH2-phenyl, and Ci-Cealkyl, wherein R10 may optionally be substituted on an available carbon by one or more substituents each independently selected from the group of halogen, hydroxy, -CF3, -NRaRb, -C(O)NRaRb, and Ci-Csalkoxy;
R15 is selected from the group consisting of:
Figure imgf000035_0001
R20 is hydrogen;
R30 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R30 may optionally be substituted by one, two or three substituents each independently selected from R33; or R30 -Si(CH3)3;
R33 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R20 and R30, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
Figure imgf000035_0002
34
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000036_0001
35
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000037_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000038_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000039_0001
38
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000040_0001
39
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000041_0001
or R40a and R40, together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of:
Figure imgf000041_0002
40
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000042_0001
[00076] For example, disclosed herein is a compound represented by Formula II:
Figure imgf000042_0002
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R10 is -C(O)- or -CH2-; R20 is hydrogen; R30 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R30 may optionally be substituted by one, two or three substituents each independently selected from R33; or R30 -Si(CH3)3; R33 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R20 and R30, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected
Figure imgf000042_0003
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000043_0001
hydrogen Ci-C3alkyl; R40 is selected from the group consisting of:
Figure imgf000043_0002
selected for each occurrence from the group consisting of chloro, fluoro, -CH3, -CH2F, -CHF2, - CF3, -OH and -OCH3; R700 is independently selected for each occurrence from the group consisting of -CH3, -CH2F, -CHF2 and -CF3; and Raa is selected from the group consisting of hydrogen, Ci-C3alkyl, and C3-Cecycloalkyl, wherein Ci-C3alkyl and C3-Cecycloalkyl may optionally be substituted by one or more halogens; or R40a and R40, together with the carbon to which they are attached, join together to form an 8-10 membered partially unsaturated bicyclic heterocyclyl selected from the group consisting of:
Figure imgf000043_0003
wherein R80 is independently selected for each occurrence from the group consisting of chloro, fluoro, -CH3, -CH2F, -CHF2, -CF3, -OH and -OCH3.
[00077] Further disclosed herein is a compound represented by Formula III:
Figure imgf000043_0004
42
IPTS/l20253274.1
SUBSTITUTE SHEET RULE 26 or a pharmaceutical. „ — , , > uereof, wherein:
R100 is independently selected for each occurrence from the group consisting of halogen and Ci-Csalkoxy;
R200 is hydrogen or Ci-Csalkyl;
R300 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R300 may optionally be substituted by one, two or three substituents each independently selected from R330; or R300 -Si(CH3)3;
R330 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R200 and R300, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
Figure imgf000044_0001
R400a is hydrogen; and
R400 is selected from the group consisting of:
Figure imgf000044_0002
43
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000045_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000046_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000047_0001
46
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 IPTS/120253274.1 47
SUBSTITUTE SHEET (RULE 26)
Figure imgf000049_0001
or j^4ooa anj R4OO, tOgether with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group
48
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000050_0001
[00078] In some embodiments, the disclosed compound is a compound identified in Table 1, Table 2, and Table 3 below or a pharmaceutically acceptable salt thereof.
Table 1. Exemplary compounds.
Figure imgf000050_0002
Figure imgf000050_0003
49
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000051_0001
Figure imgf000051_0002
50
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000052_0001
Figure imgf000052_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000053_0001
Figure imgf000053_0002
52
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000054_0001
Figure imgf000054_0002
53
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000055_0001
Figure imgf000055_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000056_0002
Figure imgf000056_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000057_0001
Figure imgf000057_0002
56
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000058_0001
Figure imgf000058_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000059_0001
Figure imgf000059_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000060_0002
Figure imgf000060_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000061_0001
Figure imgf000061_0002
60
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000062_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000063_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000064_0001
Table 2. Exemplary compounds.
Figure imgf000064_0002
Figure imgf000064_0003
63
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000065_0001
Figure imgf000065_0002
64
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000066_0002
Figure imgf000066_0001
65
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000067_0002
Figure imgf000067_0001
66
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000068_0001
Figure imgf000068_0002
67
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000069_0002
Figure imgf000069_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000070_0002
Figure imgf000070_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 Table 3. Exemplary - r
Figure imgf000071_0001
Figure imgf000071_0002
70
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000072_0002
Figure imgf000072_0001
71
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000073_0002
Figure imgf000073_0001
72
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000074_0001
Figure imgf000074_0002
73
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000075_0001
Figure imgf000075_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000076_0001
Figure imgf000076_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000077_0001
Figure imgf000077_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000078_0002
Figure imgf000078_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000079_0001
78
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000080_0001
Figure imgf000080_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000081_0002
Figure imgf000081_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000082_0001
Figure imgf000082_0002
81
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000083_0002
Figure imgf000083_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000084_0001
Figure imgf000084_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000085_0002
Figure imgf000085_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000086_0001
Figure imgf000086_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000087_0001
Figure imgf000087_0002
SUBSTITUTE SHEET RULE 26
Figure imgf000088_0001
Figure imgf000088_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000089_0002
Figure imgf000089_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000090_0001
Figure imgf000090_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000091_0002
Figure imgf000091_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000092_0001
Figure imgf000092_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000093_0001
Figure imgf000093_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000094_0001
Figure imgf000094_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000095_0002
Figure imgf000095_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000096_0001
Figure imgf000096_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000097_0001
Figure imgf000097_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000098_0001
Figure imgf000098_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000099_0001
Figure imgf000099_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 ketNo. PARB-015WO
Figure imgf000100_0002
Figure imgf000100_0001
99
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000101_0001
Figure imgf000101_0002
100
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000102_0002
Figure imgf000102_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000103_0001
Figure imgf000103_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000104_0001
Figure imgf000104_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000105_0002
Figure imgf000105_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000106_0001
Figure imgf000106_0002
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26
Figure imgf000107_0002
Figure imgf000107_0001
IPTS/120253274.1
SUBSTITUTE SHEET RULE 26 [00080] In some embodiments, described herein is a compound of the following formula:
Figure imgf000108_0001
or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
R104 is selected from the group consisting of Ci-Cealkyl,
Figure imgf000108_0002
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3;
R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, C3-Cecycloalkyl, and C3-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD;
RD is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
R102 is -NRm(C=O)Rm; and
Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C3- Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3.
[00081] In some embodiments, the compound is a compound of the following formula:
Figure imgf000108_0003
Formula X-Ib-1, or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
107
SUBSTITUTE SHEET RULE 26 R104 is selected from the group consisting of Ci-Cealkyl,
Figure imgf000109_0001
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3;
R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, C3-Cecycloalkyl, and C3-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD;
RD is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
R102 is -NRm(C=O)Rm; and
Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C3- Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3.
[00082] In some embodiments, the compound is a compound of the following formula:
Figure imgf000109_0002
Formula X-Ic, or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
R104 is selected from the group consisting of Ci-Cealkyl,
Figure imgf000109_0003
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3;
R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD; and
RD is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo.
108
SUBSTITUTE SHEET RULE 26 [00083] In some embodiments, R104 is Ci-Cealkyl. In some embodiments, R104 is
Figure imgf000110_0001
, some embodiments, R104 is
Figure imgf000110_0003
[00084] In some embodiments, the disclosed compound is a compound selected from the group consisting of:
Figure imgf000110_0002
109
SUBSTITUTE SHEET RULE 26 [00085] In some embodiments, the disclosed compound is a compound selected from the group consisting of:
Figure imgf000111_0001
[00086] Also provided herein is a protease inhibitory compound represented by Formula X':
Figure imgf000111_0002
Formula X',
[00087] or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein: R104
Figure imgf000111_0003
is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and C3- Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD; RD is selected from the group consisting of
110
SUBSTITUTE SHEET RULE 26 halogen, Ci-Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo; R102 is -NRm(C=O)Rni; Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and Cs-Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3; R103 is 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, wherein R103 may optionally be substituted by one, two, or three substituents each independently selected from RE; and RE is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2-Cealkynyl, Ci-Cealkoxy, and Ci-
Cealkoxy-OCFF. In some embodiments, R104 is Ci-Cealkyl. In some embodiments, R104 is
Figure imgf000112_0001
, some embodiments, R104 is
Figure imgf000112_0004
[00088] Also provided herein is a protease inhibitory compound represented by Formula X:
Figure imgf000112_0002
Formula X, or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein: R104 is
Figure imgf000112_0003
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3; R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl,
111
SUBSTITUTE SHEET RULE 26 wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD; RD is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, O-Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo; R102 is -NRm(C=O)Rni; Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and Cs-Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3; R103 is 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, wherein R103 may optionally be substituted by one, two, or three substituents each independently selected from RE; and RE is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2-Cealkynyl, Ci-Cealkoxy, and Ci-Cealkoxy-OCFF.
[00089] In some embodiments, Formula X is represented by:
Figure imgf000113_0001
[00090] In some embodiments,
Figure imgf000113_0002
Cealkyl or Cs-Cecycloalkyl, and R105 is Ci-Cealkyl, wherein the R101 is optionally substituted by one -CH3 or cyclopropyl.
112
SUBSTITUTE SHEET RULE 26
Figure imgf000114_0001
[00092] In some embodiments, the
Figure imgf000114_0002
selected from the group consisting of
Figure imgf000114_0003
Figure imgf000114_0004
[00094] In some embodiments,
Figure imgf000114_0005
selected from
Figure imgf000114_0006
some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
[00095] In some embodiments, R101 is selected from the group consisting of:
Figure imgf000114_0007
113
SUBSTITUTE SHEET RULE 26 [00096] In some embodiments, R101 is selected from the group consisting of:
Figure imgf000115_0001
[00097] In some embodiments, R105 is -CH3.
[00098] In some embodiments, R102 is -NH(C=O)CF3.
[00099] In some embodiments,
Figure imgf000115_0002
[000100] In some embodiments, R103 is selected from the group consisting of:
Figure imgf000115_0003
Figure imgf000115_0004
[000101] In some embodmients, RD is selected from the group consisting of methoxy, halo, - CH3, and cyano. In some embodmients, RD is Ci-Cealkyl, wherein the Ci-Cealkyl is optionally substituted by one, two or three halos. In some embodiments, one of the carbons in the alkyl or alkenyl in the R101 may optionally be replaced by an oxygen.
114
SUBSTITUTE SHEET RULE 26 [000102] In some embodiments, the compound of Formula X is a compound identified in
Table 3a.
Table 3a. Exemplary compounds.
Figure imgf000116_0001
Figure imgf000116_0002
115
SUBSTITUTE SHEET RULE 26
Figure imgf000117_0002
Figure imgf000117_0001
116
SUBSTITUTE SHEET RULE 26
Figure imgf000118_0001
Figure imgf000118_0002
SUBSTITUTE SHEET RULE 26
Figure imgf000119_0001
Figure imgf000119_0002
SUBSTITUTE SHEET RULE 26
Figure imgf000120_0001
Figure imgf000120_0002
119
SUBSTITUTE SHEET RULE 26
Figure imgf000121_0001
Figure imgf000121_0002
120
SUBSTITUTE SHEET RULE 26 [000103] In another aspect, disclosed herein is a protease inhibitory compound represented by Formula XI':
Figure imgf000122_0001
Formula XI', or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein: R104 is Ci-Cealkyl,
Figure imgf000122_0002
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3; R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RF; RF is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, cyano, and O-Cecycloalkyl, wherein the alkyl may optionally be substituted by one or more halo; R102 is -NRm(C=O)Rni; Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and Cs-Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3; R106 and R106a, together with the carbon to which they are attached, are joined together to form an 10-14 membered partially unsaturated tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from RH; and RH is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2-Cealkynyl, and Ci-Cealkoxy. In some embodiments, R104 is Ci-Cealkyl. In some embodiments,
Figure imgf000122_0003
some embodiments,
Figure imgf000122_0004
some embodiments, R104 is selected from
Figure imgf000122_0005
some embodiments, R104
121
SUBSTITUTE SHEET RULE 26
Figure imgf000123_0003
[000104] In another aspect, disclosed herein is a protease inhibitory compound represented by Formula XI:
Figure imgf000123_0001
Formula XI, or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein: R104 is
Figure imgf000123_0002
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3; R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RF; RF is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, cyano, and O-Cecycloalkyl, wherein the alkyl may optionally be substituted by one or more halo; R102 is -NRm(C=O)Rni; Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and Cs-Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3; R106 and R106a, together with the carbon to which they are attached, are joined together to form an 10-14 membered partially unsaturated tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from RH; and RH is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2-Cealkynyl, and Ci-Cealkoxy.
122
SUBSTITUTE SHEET RULE 26 [000105] In some embodiments, Formula XI is represented by:
Figure imgf000124_0001
[000106] In some embodiments,
Figure imgf000124_0002
Cealkyl or Cs-Cecycloalkyl, and R105 is Ci-Cealkyl, wherein the R101 is optionally substituted by one -CH3 or cyclopropyl.
[000107] In some embodiments, the
Figure imgf000124_0003
selected from the group consisting of
Figure imgf000124_0004
123
SUBSTITUTE SHEET RULE 26
Figure imgf000125_0007
[000109] In some embodiments,
Figure imgf000125_0001
Figure imgf000125_0002
[000110] In some embodiments, the
Figure imgf000125_0003
selected from
Figure imgf000125_0004
some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.
[000111] In some embodiments, R101 is selected from the group consisting of:
Figure imgf000125_0005
[000112] In some embodiments, R101 is selected from the group consisting of:
Figure imgf000125_0006
,
[000116] In some embodiments, R106 and R106a, together with the carbon to which they are attached, are joined together to form a 12 membered partially unsaturated tricyclic heterocyclyl ring.
124
SUBSTITUTE SHEET RULE 26 [000117] In some embodiments, the heterocyclyl ring is represented
Figure imgf000126_0001
[000118] In some embodmients, RF is selected from the group consisting of methoxy, halo, - CH3, and cyano. In some embodmients, RF is Ci-Cealkyl, wherein the Ci-Cealkyl is optionally substituted by one, two or three halos. In some embodiments, one of the carbons in the alkyl or alkenyl in the R101 may optionally be replaced by an oxygen.
[000119] In some embodiments, the compound of Formula XI is a compound identified in
Table 3b
Table 3b. Exemplary compounds.
Figure imgf000126_0002
Figure imgf000126_0003
[000120] Procedures for making compounds described herein are provided in the examples below. In the reactions described below, it may be necessary to protect reactive functional groups (such as hydroxyl, amino, thio or carboxyl groups) to avoid their unwanted participation in the reactions. The incorporation of such groups, and the methods required to introduce and remove them are known to those skilled in the art (for example, see Greene, Wuts, Protective Groups in Organic Synthesis. 2nd Ed. (1999)). The deprotection step may be the final step in the synthesis such that the removal of protecting groups affords compounds of Formula I, II, or III as
125
SUBSTITUTE SHEET RULE 26 disclosed herein. Starting materials used in the following scheme can be purchased or prepared by methods described in the chemical literature, or by adaptations thereof, using methods known by those skilled in the art. The order in which the steps are performed can vary depending on the groups introduced and the reagents used, but would be apparent to those skilled in the art.
[000121] Compounds of any of Formula I, II, or III depicted below, or any of the intermediates described in the schemes above, can be further derivatised by using one or more standard synthetic methods known to those skilled in the art. Such methods can involve substitution, oxidation or reduction reactions. These methods can also be used to obtain or modify compounds of Formula I, II, or III or any preceding intermediates by modifying, introducing or removing appropriate functional groups.
[000122] Where it is desired to obtain a particular enantiomer of a compound of Formula I, II, or III this may be produced from a corresponding mixture of enantiomers by employing any suitable conventional procedure for resolving enantiomers known to those skilled in the art. For example, diastereomeric derivatives (such as salts) can be produced by reaction of a mixture of enantiomers of a compound of Formula I, II, or III (such a racemate) and an appropriate chiral compound (such as a chiral base). The diastereomers can then be separated by any conventional means such as crystallization or chromatography, and the desired enantiomer recovered (such as by treatment with an acid in the instance where the diastereomer is a salt). Alternatively, a racemic mixture of esters can be resolved by kinetic hydrolysis using a variety of biocatalysts (for example, see Patel Stereoselective Biocatalysts, Marcel Decker; New York 2000).
[000123] In another resolution process a racemate of compounds of Formula I, II, or III can be separated using chiral High Performance Liquid Chromatography. Alternatively, a particular enantiomer can be obtained by using an appropriate chiral intermediate in one of the processes described above. Chromatography, recrystallisation and other conventional separation procedures may also be used with intermediates or final products where it is desired to obtain a particular geometric isomer of the disclosure.
[000124] Also provided herein is a broad spectrum, covalent 3 CL or 3C protease antiviral compound, comprising a nitrile warhead covalently bound to a 3 CL protease inhibitor, wherein the antiviral compound covalently binds to Cys on the protease, and wherein the antiviral compound is active against multiple viruses. In some embodiments, the broad spectrum covalent
126
SUBSTITUTE SHEET RULE 26 compound of Formula I, II, or III, wherein the compound is active against caliciviruses, picornaviruses and coronaviruses. In some embodiments, the broad spectrum covalent compound of Formula I, II, or III, wherein the compound is active against caliciviruses, picornaviruses and coronaviruses. In some embodiments, the broad spectrum covalent compound of Formula I, II, or III, wherein the compound is active against caliciviruses, picornaviruses and coronaviruses.
Alternative Embodiments
[0001] In an alternative embodiment, compounds of Formula I, II, or III may also comprise one or more isotopic substitutions. For example, hydrogen may be 2H (D or deuterium) or 3H (T or tritium); carbon may be, for example, 13C or 14C; oxygen may be, for example, 18O; nitrogen may be, for example, 15N, and the like. In other embodiments, a particular isotope (e.g., 3H, 13C, 14C, 18O, or 15N) can represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the total isotopic abundance of an element that occupies a specific site of the compound.
II. Methods
[000125] Another aspect of the disclosure provides methods of treating patients suffering from a viral infection, e.g., a coronaviral infection. In particular, in certain embodiments, the disclosure provides a method of treating the below medical indications comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein, such as a compound of Formula I, II, or III.
[000126] In certain embodiments, the disclosure provides a method of ameliorating or treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the compounds described herein. In some embodiments, the viral infection is from a virus selected from the group consisting of an RNA virus, a DNA virus, a coronavirus, an astrovirus, a papillomavirus, a pneumovirus, a picornavirus, an influenza virus, an adenovirus, a cytomegalovirus, a polyomavirus, a poxvirus, a flavivirus, an alphavirus, an ebola virus, a morbillivirus, an enterovirus, an orthopneumovirus, a
127
SUBSTITUTE SHEET RULE 26 lentivirus, arenavirus, a herpes virus, and a hepatovirus. In certain embodiments, the viral infection is a coronavirus infection. In some embodiments, the viral infection is a coronavirus selected from the group consisting of: 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS- CoV), and SARS-CoV-2 (COVID-19). In embodiments, the viral infection is SARS-CoV-2.
[000127] In some embodiments, the viral infection is from a virus selected from the group consisting of caliciviruses, MD145, murine norovirus, vesicular exanthema of swine virus, abbit hemorrhagic disease virus, porcine teschovirus, bovine coronavirus, feline infectious peritonitis virus, EV-68 virus, EV-71 virus, poliovirus, norovirus, human rhinovirus (HRV), hepatitis A virus (HAV) and foot-and-mouth disease virus (FMDV).
[000128] In embodiments, the viral infection is an arenovirus infection. In some embodiments, the arenovirus is selected from the group consisting of: Junin virus, Lassa virus, Lujo virus, Machupo virus, and Sabia virus. In some embodiments, the viral infection is an influenza infection. In some embodiments, the influenza is influenza H1N1, H3N2 or H5N1.
[000129] Another aspect of the disclosure provides methods of treating patients suffering from a viral infection, e.g., a noroviral infection. In some embodiments, the disclosure provides a method of treating a viral infection from a norovirus in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the compounds described herein.
[000130] Also provided herein, in certain embodiments, is a method of inhibiting transmission of a virus, a method of inhibiting viral replication, a method of minimizing expression of viral proteins, or a method of inhibiting virus release, comprising administering a therapeutically effective amount of a compound described herein to a patient suffering from the virus, and/or contacting an effective amount of a compound described herein with a virally infected cell. In some embodiments, the method further comprises administering another therapeutic. In some embodiments, the method further comprises administering an additional anti-viral therapeutic. In embodiments, the anti-viral therapeutic is selected from the group consisting of ribavirin, favipiravir, ST-193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil,
128
SUBSTITUTE SHEET RULE 26 tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX-355, valacyclovir, VIR- 576, and zalcitabine. In some embodiments, the another therapeutic is selected from the group consisting of protease inhibitors, fusion inhibitors, M2 proton channel blockers, polymerase inhibitors, 6- endonuclease inhibitors, neuraminidase inhibitors, reverse transcriptase inhibitor, aciclovir, acyclovir, protease inhibitors, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, docosanol, edoxudine, entry inhibitors, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, inosine, integrase inhibitor, interferons, lopinavir, loviride, moroxydine, nexavir, nucleoside analogues, penciclovir, pleconaril, podophyllotoxin, ribavirin, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zodovudine. In embodiments, the additional anti-viral therapeutic is selected from the group consisting of lamivudine, an interferon alpha, a VAP anti-idiotypic antibody, enfuvirtide, amantadine, rimantadine, pleconaril, aciclovir, zidovudine, fomivirsen, a morpholino, a protease inhibitor, double-stranded RNA activated caspase oligomerizer (DRACO), rifampicin, zanamivir, oseltamivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX-355, valacyclovir, VIR- 576, and zalcitabine.
129
SUBSTITUTE SHEET RULE 26 [000131] Contemplated patients include not only humans, but other animals such as companion animals (e.g. dogs, cats), domestic animals (e.g. cow, swine), and wild animals (e.g. monkeys, bats, snakes).
[000132] Accordingly, in one embodiment, described herein is a method of ameliorating or treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula
1, II, or III described herein) or a pharmaceutically acceptable salt thereof.
[000133] Other contemplated methods of treatment include method of treating or ameliorating a virus infection condition or co-morbidity, by administering a compound disclosed herein to a subject.
[000134] Exemplary co-morbidities include lung diseases, cardiac disorders, endocrine disorders, respiratory disorders, hepatic disorders, skeletal disorders, psychiatric disorders, metabolic disorders, and reproductive disorders.
[000135] In some embodiments, the viral infection is from a virus selected from the group consisting of an RNA virus, a DNA virus, a coronavirus, a papillomavirus, a pneumovirus, a picornavirus, an influenza virus, an adenovirus, a cytomegalovirus, a polyomavirus, a poxvirus, a flavivirus, an alphavirus, an ebola virus, a morbillivirus, an enterovirus, an orthopneumovirus, a lentivirus, arenovirus, a herpes virus, and a hepatovirus. In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the viral infection is a coronavirus selected from the group consisting of: 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS- CoV), and SARS-CoV-2 (CO VID-19). In some embodiments, the viral infection is SARS-CoV-
2. In some embodiments, the viral infection is an arenovirus infection. In some embodiments, the arenovirus is selected from the group consisting of: Junin virus, Lassa virus, Lujo virus, Machupo virus, and Sabia virus. In some embodiments, the viral infection is an influenza infection. In some embodiments, the influenza is influenza H1N1, H3N2 or H5N1. In some embodiments, the viral infection is a respiratory viral infection. In some embodiments, the viral infection is an upper respiratory viral infection or a lower respiratory viral infection. In some embodiments, the method further comprises administering another therapeutic.
130
SUBSTITUTE SHEET RULE 26 [000136] In certain embodiments, the virus is selected from the group consisting of a retrovirus (e.g., human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), human T-cell lymphotropic virus (HTLV)-l, HTLV-2, HTLV-3, HTLV-4), Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, a herpes simplex virus (HSV) (e.g., HSV-1, HSV-2, varicella zoster virus, cytomegalovirus), an adenovirus, an orthomyxovirus (e.g., influenza virus A, influenza virus B, influenza virus C, influenza virus D, thogotovirus), a flavivirus (e.g., dengue virus, Zika virus), West Nile virus, Rift Valley fever virus, an arenavirus, Crimean- Congo hemorrhagic fever virus, an echovirus, a rhinovirus, coxsackie virus, a coronavirus (e.g., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronavirus disease 2019 (COVID-19), a respiratory syncytial virus, a mumps virus, a rotavirus, measles virus, rubella virus, a parvovirus (e.g., an adeno-associated virus), a vaccinia virus, a variola virus, a molluscum virus, bovine leukemia virus, bovine diarrhea virus, a poliovirus, St. Louis encephalitis virus, Japanese encephalitis virus, a tick-borne encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, a rabies virus, a polyomavirus (e.g., JC virus, BK virus), an alphavirus, and a rubivirus (e.g., rubella virus).
[000137] In certain embodiments, the disease or disorder is a viral infection, e.g., a disease or disorder selected from the group consisting of acquired immune deficiency syndrome (AIDS), HTLV-1 associated myelopathy/tropical spastic paraparesis, Ebola virus disease, hepatitis A, hepatitis B, hepatitis C, herpes, herpes zoster, acute varicella, mononucleosis, respiratory infections, pneumonia, influenza, dengue fever, encephalitis (e.g., Japanese encephalitis, St. Louis encephalitis, or tick-borne encephalitis such as Powassan encephalitis), West Nile fever, Rift Valley fever, Crimean-Congo hemorrhagic fever, Kyasanur Forest disease, Yellow fever, Zika fever, aseptic meningitis, myocarditis, common cold, lung infections, molloscum contagiosum, enzootic bovine leucosis, coronavirus disease 2019 (COVID- 19), mumps, gastroenteritis, measles, rubella, slapped-cheek disease, smallpox, warts (e.g., genital warts), molluscum contagiosum, polio, rabies, and pityriasis rosea.
[000138] In some embodiments, the virus is an RNA virus (having a genome that is composed of RNA). RNA viruses may be single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA). RNA viruses have high mutation rates compared to DNA viruses, as RNA
131
SUBSTITUTE SHEET RULE 26 polymerase lacks proofreading capability (see Steinhauer DA, Holland J J (1987). "Rapid evolution ofRNA viruses". Annu. Rev. Microbiol. 41: 409 -33). In some embodiments, the RNA virus is a positive-strand RNA virus (e.g., a SARS-CoV virus, polio virus, Coxsackie virus, Enterovirus, Human rhino virus, Foot/Mouth disease virus, encephalomyocarditis virus, Dengue virus, Zika virus, Hepatitis C virus, or New Castle Disease virus).
[000139] RNA viruses are classified by the type of genome (double-stranded, negative (-), or positive (+) single-stranded). Double-stranded RNA viruses contain a number of different RNA molecules, each coding for one or more viral proteins. Positive-sense ssRNA viruses utilize their genome directly as mRNA; ribosomes within the host cell translate mRNA into a single protein that is then modified to form the various proteins needed for viral replication. One such protein is RNA-dependent RNA polymerase (RNA replicase), which copies the viral RNA in order to form a double-stranded, replicative form. Negative-sense ssRNA viruses have their genome copied by an RNA replicase enzyme to produce positive-sense RNA for replication. Therefore, the virus comprises an RNA replicase enzyme. The resultant positive-sense RNA then acts as viral mRNA and is translated by the host ribosomes. In some embodiments, the virus is a dsRNA virus. In some embodiments, the virus is a negative ssRNA virus. In some embodiments, the virus is a positive ssRNA virus. In some embodiments, the positive ssRNA virus is a coronavirus.
[000140] SARS-CoV2, also sometimes referred to as the novel coronavirus of 2019 or 2019- nCoV, is a positive-sense single-stranded RNA virus. SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein holds the RNA genome together; the S, E, and M proteins form the viral envelope. Spike allows the virus to attach to the membrane of a host cell, such as the ACE2 receptor in human cells (Kruse R.L. (2020), Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China (version 2). FlOOOResearch, 9:72). SARS-CoV2 is the highly contagious, causative viral agent of coronavirus disease 2019 (COVID19), a global pandemic.
[000141] In some embodiments, the virus is a DNA virus (having a genome that is composed of DNA). Exemplary DNA viruses include, without limitation, parvoviruses (e.g., adeno- associated viruses), adenoviruses, asfarviruses, herpesviruses (e.g., herpes simplex virus 1 and 2
132
SUBSTITUTE SHEET RULE 26 (HSV-1 and HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV)), papillomoviruses (e.g., HPV), polyomaviruses (e.g., simian vacuolating virus 40 (SV40)), and poxviruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus). Exemplary RNA viruses include, without limitation, bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, influenza virus type C), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)) and toroviruses.
[000142] The methods described herein may inhibit viral replication transmission, replication, assembly, or release, or minimize expression of viral proteins. In one embodiment, described herein is a method of inhibiting transmission of a virus, a method of inhibiting viral replication, a method of minimizing expression of viral proteins, or a method of inhibiting virus release, comprising administering a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, to a patient suffering from the virus, and/or contacting an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof, with a virally infected cell.
[000143] Also described herein is a method of treating a respiratory disorder in a subject in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula I, II, or III described herein) or a pharmaceutically acceptable salt thereof. In embodiments, the respiratory disorder is selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, fibrosis, chronic asthma, acute asthma, lung disease secondary to environmental exposures, acute lung infection, chronic lung infection, al antitrypsin disease, cystic fibrosis and an autoimmune disease. In some embodiments, the respiratory disorder is associated with a heart attack.
[000144] Also described herein is a method of treating a disorder associated with cathepsin (e.g. Cathepsin K) in a subject in need thereof, comprising administering to the patient a therapeutically effective amount of a compound described herein (e.g., a compound of Formula I, II, or III described herein) or a pharmaceutically acceptable salt thereof. In some embodiments, the disorder is a cathepsin dependent condition or disease. In embodiments, the
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SUBSTITUTE SHEET RULE 26 disorder is selected from the group consisting of breast cancer, pycnodysostosis, glioblastoma, osteosclerosis, osteoporosis, glucocorticoid induced osteoporosis, Paget's disease, abnormally increased bone turnover, periodontal disease, tooth loss, bone fractures, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, osteogenesis imperfecta, atherosclerosis, obesity, glaucoma, chronic obstructive pulmonary disease, metastatic bone disease, hypercalcemia of malignancy, and multiple myeloma.
[000145] Compounds described herein, e.g., a compound of Formula I, II, or III as defined herein, can be administered in combination with one or more additional therapeutic agents to treat a disorder described herein, such as an infection by a pathogen described herein, e.g., a virus, fungus, or protozoan. For clarity, contemplated herein are both a fixed composition comprising a disclosed compound and another therapeutic agent such as disclosed herein, and methods of administering, separately a disclosed compound and a disclosed therapeutic. For example, provided in the present disclosure is a pharmaceutical composition comprising a compound described herein, e.g., a compound of Formula I, II, or III as defined herein, one or more additional therapeutic agents, and a pharmaceutically acceptable excipient. In some embodiments, a compound of Formula I, II, or III as defined herein and one additional therapeutic agent is administered. In some embodiments, a disclosed compound as defined herein and two additional therapeutic agents are administered. In some embodiments, a disclosed compound as defined herein and three additional therapeutic agents are administered.
Combination therapy can be achieved by administering two or more therapeutic agents, each of which is formulated and administered separately. For example, a compound of Formula I, II, or III as defined herein and an additional therapeutic agent can be formulated and administered separately. Combination therapy can also be achieved by administering two or more therapeutic agents in a single formulation, for example a pharmaceutical composition comprising a compound of Formula I, II, or III as one therapeutic agent and one or more additional therapeutic agents such as an antibiotic, a viral protease inhibitor, or an anti-viral nucleoside anti-metabolite. For example, a compound of Formula I, II, or III as defined herein and an additional therapeutic agent can be administered in a single formulation. Other combinations are also encompassed by combination therapy. While the two or more agents in the combination therapy can be administered simultaneously, they need not be. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents)
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SUBSTITUTE SHEET RULE 26 by minutes, hours, days, or weeks. Thus, the two or more agents can be administered within minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or weeks of each other. In some cases even longer intervals are possible. While in many cases it is desirable that the two or more agents used in a combination therapy be present in within the patient's body at the same time, this need not be so.
[000146] Combination therapy can also include two or more administrations of one or more of the agents used in the combination using different sequencing of the component agents. For example, if agent X and agent Y are used in a combination, one could administer them sequentially in any combination one or more times, e.g., in the order X-Y-X, X-X-Y, Y-X-Y, Y- Y-X, X-X-Y- Y, etc.
[000147] In some embodiments, the one or more additional therapeutic agents that may be administered in combination with a compound provided herein can be an antibiotic, a viral protease inhibitor, an anti-viral anti-metabolite, a lysosomotropic agent, a M2 proton channel blocker, a polymerase inhibitor (e.g., EIDD-2801, which is also known as MOLNUPIRAVIR), a neuraminidase inhibitor, a reverse transcriptase inhibitor, a viral entry inhibitor, an integrase inhibitor, interferons (e.g., types I, II, and III), or a nucleoside analogue. In some embodiments, the one or more additional therapeutic agents that may be administered in combination wiht a compounds provided herein can be a steroid (e.g., corticosteroids, such as bethamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, dexamethasone; mineralcorticoid such as fludrocortisone; glucocorticoids, such as hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone; vitamin D such as dihydrotachysterol; androgens such as apoptone, oxandrolone, oxabolone, testosterone, nandrolone (also known as anabolic steroids), oestrogens such as diethylstilbestrol, progestins such as danazol, norethindrone, medroxyprogesterone acetate, 17-Hydroxyprogesterone caproate; and progestins such as mifepristone and gestrinone) or an immunomodulator (e.g., 6-Mercaptopurine, 6MP, Alferon N, anakinra, Arcalyst, Avonex, AVOSTARTGRIP, Bafiertam, Berinert, Betaseron, BG- 12, Cl esterase inhibitor recombinant, Cl inhibitor human, Cinryze, Copaxone, dimethyl fumarate, diroximel fumarate, ecallantide, emapalumab, emapalumab-lzsg, Extavia, fingolimod, Firazyr, Gamifant, Gilenya, glatiramer, Glatopa, Haegarda, icatibant, Infergen, interferon alfa n3,
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SUBSTITUTE SHEET RULE 26 interferon alfacon 1, interferon beta la, interferon beta lb, Kalbitor, Kineret, mercaptopurine, monomethyl fumarate, peginterferon beta- la, Plegridy, Purinethol, Purixan, Rebif, Rebif Rebidose, remestemcel-L, rilonacept, ropeginterferon alfa 2b, Ruconest, Ryoncil, siltuximab, sutimlimab, Sylvant, Tecfidera, and Vumerity). In some embodiments, the one or more additional therapeutic agent is Cathepsin L. In some embodiments, the one or more additional therapeutic agent is dehydrodidemnin B (also known as Plitidepsin or APLIDIN) or Zotatifin (eFT226).
[000148] In some embodiments, methods described herein further comprise administering an additional anti-viral therapeutic. In some embodiments, the anti-viral therapeutic is selected from the group consisting of ribavirin, favipiravir, ST- 193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX- 355, valacyclovir, VIR- 576, and zalcitabine. In some embodiments, the another therapeutic is selected from the group consisting of protease inhibitors (e.g., nafamostat, camostat, gabexate, epsilon-aminocapronic acid and aprotinin), fusion inhibitors (e.g., BMY-27709, CL 61917, and CL 62554), M2 proton channel blockers (e.g., amantadine and rimantadine), polymerase inhibitors (e.g., 2-deoxy-2'fluoroguanosides (2'-fluoroGuo), 6- endonuclease inhibitors (e.g., L- 735,822 and flutamide) neuraminidase inhibitors (e.g., zanamivir (Relenza), oseltamivir, peramivir and ABT-675 (A-315675), reverse transcriptase inhibitor (e.g., abacavir, adefovir, delavirdine, didanosine, efavirenz, emtricitabine, lamivudine, nevirapine, stavudine, tenofovir, tenofovir disoproxil, and zalcitabine), acyclovir, acyclovir, protease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir), arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, docosanol, edoxudine, entry inhibitors (e.g., enfuvirtide and maraviroc), entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, inosine, integrase inhibitor (e.g., raltegravir), interferons
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SUBSTITUTE SHEET RULE 26 (e.g., types I, II, and III), lopinavir, loviride, moroxydine, nexavir, nucleoside analogues (e.g., aciclovir), penciclovir, pleconaril, podophyllotoxin, ribavirin, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zodovudine. In some embodiments, the additional anti-viral therapeutic is selected from the group consisting of lamivudine, an interferon alpha, a VAP anti-idiotypic antibody, enfuvirtide, amantadine, rimantadine, pleconaril, aciclovir, zidovudine, fomivirsen, a morpholino, a protease inhibitor, double-stranded RNA activated caspase oligomerizer (DRACO), rifampicin, zanamivir, oseltamivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX-355, valacyclovir, VIR- 576, and zalcitabine. In some embodiments, the another therapeutic is selected from the group consisting of quinine (optionally in combination with clindamycin), chloroquine, amodiaquine, artemisinin and its derivatives (e.g., artemether, artesunate, dihydroartemisinin, arteether), doxycycline, pyrimethamine, mefloquine, halofantrine, hydroxychloroquine, eflornithine, nitazoxanide, ornidazole, paromomycin, pentamidine, primaquine, pyrimethamine, proguanil (optionally in combination with atovaquone), a sulfonamide (e.g., sulfadoxine, sulfamethoxypyridazine), tafenoquine, tinidazole and a PPT1 inhibitor (including Lys05 and DC661). In some embodiments, the another therapeutic is an antibiotic. In some embodiments, the antibiotic is a penicillin antibiotic, a quinolone antibiotic, a tetracycline antibiotic, a macrolide antibiotic, a lincosamide antibiotic, a cephalosporin antibiotic, or an RNA synthetase inhibitor. In some embodiments, the antibiotic is selected from the group consisting of azithromycin, vancomycin, metronidazole, gentamicin, colistin, fidaxomicin, telavancin, oritavancin, dalbavancin, daptomycin, cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, ceftobiprole, cipro, Levaquin, floxin, tequin, avelox, norflox, tetracycline, minocycline, oxytetracycline, doxycycline, amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,
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SUBSTITUTE SHEET RULE 26 methicillin, ertapenem, doripenem, imipenem/cilastatin, meropenem, amikacin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefoxotin, and streptomycin. In some embodiments, the antibiotic is azithromycin.
[000149] In some embodiments, the one or more additional therapeutic agents that may be administered in combination with a compound provided herein can be selected from the group consisting of ribavirin, favipiravir, ST-193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX-355, valacyclovir, VIR- 576, and zalcitabine.
[000150] In some embodiments, the compounds described herein (e.g. a compound of Formula I, II, or III) and pharmaceutically acceptable salts thereof may be used in combination with one or more other agents which may be useful in the prevention or treatment of respiratory disease, inflammatory disease, autoimmune disease, for example; anti-histamines, corticosteroids, (e.g., fluticasone propionate, fluticasone furoate, beclomethasone dipropionate, budesonide, ciclesonide, mometasone furoate, triamcinolone, flunisolide), NSAIDs, leukotriene modulators (e.g., montelukast, zafirlukast.pranlukast), tryptase inhibitors, IKK2 inhibitors, p38 inhibitors, Syk inhibitors, protease inhibitors such as elastase inhibitors, integrin antagonists (e.g., beta-2 integrin antagonists), adenosine A2a agonists, mediator release inhibitors such as sodium chromoglycate, 5-lipoxygenase inhibitors (zyflo), DPI antagonists, DP2 antagonists, PI3K delta inhibitors, ITK inhibitors, LP (lysophosphatidic) inhibitors or FLAP (5-lipoxygenase activating protein) inhibitors (e.g., sodium 3-(3-(tert-butylthio)-l -(4-(6- ethoxypyri din-3 - yl)benzyl)-5-((5-ethylpyridin-2-yl)methoxy)-l H-indol-2-yl)-2,2- dimethylpropanoate), bronchodilators (e.g.. muscarinic antagonists, beta-2 agonists), methotrexate, and similar agents;
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SUBSTITUTE SHEET RULE 26 monoclonal antibody therapy such as anti-lgE, anti- TNF, anti-IL-5, anti-IL-6, anti-IL-12, anti- IL-1 and similar agents; cytokine receptor therapies e.g. etanercept and similar agents; antigen non-specific immunotherapies (e.g. interferon or other cytokines/chemokines, chemokine receptor modulators such as CCR3, CCR4 or CXCR2 antagonists, other cytokine/chemokine agonists or antagonists, TLR agonists and similar agents), suitable anti-infective agents including antibiotic agents, antifungal agents, anthelmintic agents, antimalarial agents, antiprotozoal agents and antituberculosis agents.
[000151] In some embodiments, the additional therapeutic agents can be kinase inhibitors including but not limited to erlotinib, gefitinib, neratinib, afatinib, osimertinib, lapatanib, crizotinib, brigatinib, ceritinib, alectinib, lorlatinib, everolimus, temsirolimus, abemaciclib, LEE011, palbociclib, cabozantinib, sunitinib, pazopanib, sorafenib, regorafenib, sunitinib, axitinib, dasatinib, imatinib, nilotinib, ponatinib, idelalisib, ibrutinib, Loxo 292, larotrectinib, and quizartinib.
[000152] In some embodiments, the additional therapeutic agents can be therapeutic anti-viral vaccines.
[000153] In some embodiments, the additional therapeutic agents can be immunomodulatory agents including but not limited to anti-PD-lor anti-PDL-1 therapeutics including pembrolizumab, nivolumab, atezolizumab, durvalumab, BMS-936559, or avelumab, anti-TIM3 (anti-HAVcr2) therapeutics including but not limited to TSR-022 or MBG453, anti-LAG3 therapeutics including but not limited to relatlimab, LAG525, or TSR-033, anti-4-lBB (anti- CD37, anti-TNERSL9), CD40 agonist therapeutics including but not limited to SGN-40, CP- 870,893 or R07009789, anti-CD47 therapeutics including but not limited to Hu5L9-G4, anti- CD20 therapeutics, anti-CD38 therapeutics, SUNG agonists including but not limited to ADU- S100, MK-1454, ASA404, or amidobenzimidazoles, anthracy clines including but not limited to doxorubicin or mitoxanthrone, hypomethylating agents including but not limited to azacytidine or decitabine, other immunomodulatory therapeutics including but not limited to epidermal growth factor inhibitors, statins, metformin, angiotensin receptor blockers, thalidomide, lenalidomide, pomalidomide, prednisone, or dexamethasone. In some embodiments, the additional therapeutic agent is a p2-adrenoreceptor agonist including, but not limited to, vilanterol, salmeterol, salbutamol. formoterol, salmefamol, fenoterol carmoterol, etanterol,
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SUBSTITUTE SHEET RULE 26 naminterol, clenbuterol, pirbuterol.flerbuterol, reproterol, bambuterol, indacaterol, terbutaline and salts thereof, for example the xinafoate (1 -hydroxy-2- naphthalenecarboxylate) salt of salmeterol, the sulphate salt of salbutamol or the fumarate salt of formoterol. In some embodiments, the additional therapeutic agent is an anticholinergic agent, including, but not limited to, umeclidinium (for example, as the bromide), ipratropium (for example, as the bromide), oxitropium (for example, as the bromide) and tiotropium (for example, as the bromide).
[000154] In particular, in certain embodiments, the disclosure provides a method of treating the above medical indications comprising administering a subject in need thereof a therapeutically effective amount of a compound described herein, such as a disclosed compound.
[000155] The term "boosting amount" or "boosting dose" is the amount of a compound needed to improve the pharmacokinetics of a second compound (or increase availability or exposure). The boosting amount or boosting dose may improve the pharmacokinetics (or increase availability or exposure) of the second compound to a level to therapeutic levels in a subject.
[000156] In one embodiment, the disclosure provides for a disclosed compound to be administered together with an antiviral therapeutic such as disclosed herein, and e.g., thereby boosting the dose of the anti-viral therapeutic or therapeutics. Such a boost combination may be used, e.g., as prophylactic or therapeutic treatment of a viral infection in a subject in need thereof. In one embodiment, the protease inhibitor is a compound described herein (e.g. a compound of Formula I, II, or III).
III. Pharmaceutical Compositions and Kits
[000157] Another aspect of the disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) rectal, vaginal, or aerosol administration, although the most suitable form of administration in any given case will
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SUBSTITUTE SHEET RULE 26 depend on the degree and severity of the condition being treated and on the nature of the particular compound being used. For example, disclosed compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration.
[000158] Exemplary pharmaceutical compositions of this disclosure may be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains one or more of the compound of the disclosure, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease.
[000159] For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the disclosure, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
[000160] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for
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SUBSTITUTE SHEET RULE 26 example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
[000161] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.
[000162] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.
[000163] Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
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SUBSTITUTE SHEET RULE 26 [000164] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.
[000165] Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
[000166] The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
[000167] Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
[000168] Compositions and compounds of the present disclosure may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
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SUBSTITUTE SHEET RULE 26 [000169] Pharmaceutical compositions of this disclosure suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
[000170] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants
[000171] In another aspect, the disclosure provides enteral pharmaceutical formulations including a disclosed compound and an enteric material; and a pharmaceutically acceptable carrier or excipient thereof. Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, and that are predominantly soluble in intestinal fluids at specific pHs. The small intestine is the part of the gastrointestinal tract (gut) between the stomach and the large intestine, and includes the duodenum, jejunum, and ileum. The pH of the duodenum is about 5.5, the pH of the jejunum is about 6.5 and the pH of the distal ileum is about 7.5. Accordingly, enteric materials are not soluble, for example, until a pH of about 5.0, of about 5.2, of about 5.4, of about 5.6, of about 5.8, of about 6.0, of about 6.2, of about 6.4, of about 6.6, of about 6.8, of about 7.0, of about 7.2, of about 7.4, of about 7.6, of about 7.8, of about 8.0, of about 8.2, of about 8.4, of about 8.6, of about 8.8, of about 9.0, of about 9.2, of about 9.4, of about 9.6, of about 9.8, or of about 10.0. Exemplary enteric materials include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate,
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SUBSTITUTE SHEET RULE 26 cellulose acetate butyrate, cellulose acetate propionate, copolymer of methylmethacrylic acid and methyl methacrylate, copolymer of methyl acrylate, methylmethacrylate and methacrylic acid, copolymer of methylvinyl ether and maleic anhydride (Gantrez ES series), ethyl methyacrylate- methylmethacrylate-chlorotrimethylammonium ethyl acrylate copolymer, natural resins such as zein, shellac and copal collophorium, and several commercially available enteric dispersion systems (e. g. , Eudragit L30D55, Eudragit FS30D, Eudragit LI 00, Eudragit SI 00, Kollicoat EMM30D, Estacryl 30D, Coateric, and Aquateric). The solubility of each of the above materials is either known or is readily determinable in vitro. The foregoing is a list of possible materials, but one of skill in the art with the benefit of the disclosure would recognize that it is not comprehensive and that there are other enteric materials that would meet the objectives of the present disclosure.
[000172] Advantageously, the disclosure also provides kits for use by a e.g. a consumer in need of 3 CL inhibitor. Such kits include a suitable dosage form such as those described above and instructions describing the method of using such dosage form to mediate, reduce or prevent inflammation. The instructions would direct the consumer or medical personnel to administer the dosage form according to administration modes known to those skilled in the art. Such kits could advantageously be packaged and sold in single or multiple kit units. An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
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SUBSTITUTE SHEET RULE 26 [000173] It may be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested. Another example of such a memory aid is a calendar printed on the card, e.g., as follows “First Week, Monday, Tuesday, . . . etc. . . . Second Week, Monday, Tuesday, . . . “ etc. Other variations of memory aids will be readily apparent. A “daily dose” can be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of a first compound can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this.
[000174] Also contemplated herein are methods and compositions that include a second active agent, or administering a second active agent. For example, in addition to having a viral infection, a subject or patient can further have viral infection- or virus-related co-morbidities, i.e., diseases and other adverse health conditions associated with, exacerbated by, or precipitated by being infected by a virus. Contemplated herein are disclosed compounds in combination with at least one other agent that has previously been shown to treat these virus-related conditions.
EXAMPLES
[000175] The compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated. The starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials.
[000176] At least some of the compounds identified as “Intermediates” herein are contemplated as compounds of the disclosure.
146
SUBSTITUTE SHEET RULE 26 [000177] NMR spectra are recorded at ambient temperature using e.g., a Varian Unity Inova (400MHz) spectrometer with a triple resonance 5mm probe for Example compounds, and either a Bruker Avance DRX (400MHz) spectrometer or a Bruker Avance DPX (300MHz) spectrometer for Intermediate compounds. Chemical shifts are expressed in ppm relative to tetramethylsilane. The following abbreviations have been used: br = broad signal, s = singlet, d = doublet, dd = double doublet, dt = double triplet, ddd = double double doublet, t = triplet, td = triple doublet, tdd = triple double doublet, q = quartet, m = multiplet.
[000178] Abbreviations:
Figure imgf000148_0001
Figure imgf000148_0002
147
SUBSTITUTE SHEET RULE 26
Figure imgf000149_0001
Figure imgf000149_0002
General Chemistry
[000179] Exemplary compounds described herein are available by the general synthetic method illustrated in Scheme 1 or 2, including preparations of Intermediates and preparation of accompanying Examples.
Synthetic Scheme(s)
Scheme 1
148
SUBSTITUTE SHEET RULE 26
Figure imgf000150_0001
[000180] Scheme 1 illustrates an exemplary preparation of A-A. A Pinner reaction of amine A-1 with a reagent such as methanol affords amino ester A-2. A-2 is then coupled with carboxyxlic acid B-l in the presence of reagents such as l-ethyl-3-(3- dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine to afford the amide product A- 3. Removal of the carbamate protecting group in A-3 provides amine A-4, which is coupled with carboxylic acid B-2 to afford amide A-5. Finally, the carboxylic acid group A-5 is converted to an amide (A-6), which is then converted to nitrile A-A in the presence of, for example, Burgess reagent.
[000181] In Scheme 1, examples of Ac and Acc each independently include a hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or Ac and Acc, together with the atoms they are attached to, may form a substituted or unsubstituted carbocycle, a substituted or unsubstituted monocyclic heterocycle, or a substituted or unsubstituted bicyclic heterocycle; examples of RAA and RAB each independently include a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or RAA and R^, together with the atoms they are
149
SUBSTITUTE SHEET RULE 26 attached to, form a ring, wherein the ring may be a substitued or unsubstituted heterocycle; and examples of RAD include a substituted or unsubstituted bicyclic heteroaryl moiety.
Scheme 2
Figure imgf000151_0001
C-C
[000182] Scheme 2 illustrates an exemplary preparation of C-C. Carboxylic acid C-l is coupled with amine D-l in the presence of couple reagents such as benzotriazol- 1- yloxytripyrrolidinophosphonium hexafluorophosphate and tri ethylamine to afford amide C-2. The ester in C-2 is then converted to its correspending amide (C-3) in the presence of, for example, NH3 and methanol. Removal of the carbamate protecting group in C-3 provides amine C-4, which is then converted to trifluoracetamide C-5. Dehydration of the primary amide in C-5 in the presence of, for example, Burgess Reagent, yields nitrile C-C.
[000183] In Scheme 2, examples of RDA and DB each independently include a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or RDA and DB, together with the atoms they are attached to, form a ring, wherein the ring may be a substitued or unsubstituted heterocycle; examples of RE include a substituted or unsubstituted alkyl; examples of RDC and DD each independently include a
150
SUBSTITUTE SHEET RULE 26 hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted monocylic heteroaryl moiety, and a substituted or unsubstituted bicyclic heteroaryl moiety, or RDC and DD, together with the atoms they are attached to, may form a substituted or unsubstituted carbocycle, a substituted or unsubstituted monocyclic heterocycle, or a substituted or unsubstituted bicyclic heterocycle; and examples of RDE include a substituted or unsubstituted alkyl.
[000184] Compounds of Table 1, Table 2, Table 3, Table 3a, and Table 3b have been prepared following general Scheme 1 or 2.
Example 1: Synthesis of N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-5-cyclopropyl-lH-pyrrole-2-carboxamide (Compound 150)
Figure imgf000152_0001
Step 1: 5-cyclopropyl-lH-pyrrole-2-carboxylic acid
[000185] To a solution of methyl 5-cyclopropyl-lH-pyrrole-2-carboxylate (400 mg, 2.42 mmol, 1 eq) in THF (3 mL) H2O (3 mL) and MeOH (3 mL) was added LiOH.I O (304.82 mg, 7.26 mmol, 3 eq). After the addition, the resulting mixture was stirred at 60 °C for 16 h. Upon completion, the reaction mixture was adjusted pH about 3 by HC1 (IM), the resulting mixture was extracted with EA (5 mL * 3). The combined organic layers were dried over Na2SO4,
151
SUBSTITUTE SHEET RULE 26 filtered, concentrated in a vacuum to give 5-cyclopropyl-lH-pyrrole-2-carboxylic acid (320 mg, crude) as a yellow solid.
Step 2: (2S)-2-(tert-butoxycarbonylamino)-3-(l-fluorocyclopropyl)propanoic acid
[000186] To a solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(l- fluorocyclopropyl)propanoate (500 mg, 1.91 mmol, 1 eq) in THF (5 mL) and H2O (5 mL) was added LiOH.TbO (281.05 mg, 6.70 mmol, 3.5 eq). After the addition, the resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was adjusted pH about 3 with HC1 (IM). The resulting mixture was extracted with EA (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered, concentrated in a vacuum to give (2S)-2-(tert-butoxycarbonylamino)-3-(l- fluorocyclopropyl)propanoic acid as a white solid.
Step 3: tert-butyl N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]carbamate
[000187] To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-(l- fluorocyclopropyl)propanoic acid (400 mg, 1.62 mmol, 1 eq) and 2-amino-2-(4- isoquinolyl)acetonitrile (326.02 mg, 1.78 mmol, 1.1 eq) in DCM (5 mL) was added DMAP (592.90 mg, 4.85 mmol, 3 eq) and EDCI (620.24 mg, 3.24 mmol, 2 eq). After the addition, the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by 5 mL water. The resulting mixture was extracted with DCM (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered, concentrated in a vacuum to afford a residue, which was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to 1 :2) to give tert-butyl N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl] carbamate (380 mg, 897.17 umol, 55.46% yield, 97.38% purity) as a yellow solid. MS (ESI) m/z 413.2 [M+H]+.
Step 4: (2S)-2-amino-N-[cyano(4-isoquinolyl)methyl]-3-(l-fluorocyclopropyl)propanamide
[000188] To a solution of tert-butyl N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]carbamate (550 mg, 1.33 mmol, 1 eq) in DCM (2.5 mL) was added TFA (5.08 g, 44.57 mmol, 3.30 mL, 33.42 eq) in DCM(1 mL) dropwise. After the addition, the resulting mixture was stirred 25 °C for 0.5 h. Upon completion, the residue was
152
SUBSTITUTE SHEET RULE 26 blow-dryed with DCM (5 mL * 3) to give (2S)-2-amino-N-[cyano(4-isoquinolyl)methyl]-3-(l- fluorocyclopropyl)propanamide (380 mg, crude, TFA) as yellow solid.
Step 5: N-[(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo- ethyl] -5 -cyclopropyl- 1 H-pyrrole-2-carboxamide
[000189] To a solution of (2S)-2-amino-N-[cyano(4-isoquinolyl)methyl]-3-(l- fluorocyclopropyl)propanamide (380 mg, 891.26 umol, 1 eq, TFA) and 5-cyclopropyl-lH- pyrrole-2-carboxylic acid (148.20 mg, 980.38 umol, 1.1 eq) in DCM (5 mL) was added DMAP (326.65 mg, 2.67 mmol, 3 eq) and EDCI (341.71 mg, 1.78 mmol, 2 eq). After the addition, the resulting mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by 1 mL water. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75 * 30 mm * 3 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 23%-53%, 10 min) to give N- [(lS)-2-[[cyano(4-isoquinolyl)methyl]amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-5- cyclopropyl-lH-pyrrole-2-carboxamide (56.6 mg, 115.40 umol, 12.95% yield, 90.83% purity) as white solid. MS (ESI) m/z 446.1 [M+H]+.
[000190] 1H NMR (400 MHz, DMSO-t76) 5 = 11.41 - 11.10 (m, 1H), 9.76 - 9.53 (m, 1H), 9.49 - 9.31 (m, 1H), 8.87 - 8.60 (m, 1H), 8.31 - 8.20 (m, 1H), 8.07 - 7.99 (m, 1H), 7.99 - 7.84 (m,
2H), 7.81 - 7.73 (m, 1H), 6.89 - 6.79 (m, 1H), 6.75 - 6.66 (m, 1H), 5.68 (td, J= 2.9, 5.6 Hz, 1H),
4.80 - 4.68 (m, 1H), 2.43 - 2.21 (m, 1H), 2.11 - 2.01 (m, 1H), 1.98 - 1.84 (m, 1H), 0.92 - 0.69 (m,
5H), 0.66 - 0.46 (m, 2H), 0.44 - 0.41 (m, 1H)
Example 2: Synthesis of (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(5-cyclopropyl-lH- pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Cmpd 196)
153
SUBSTITUTE SHEET RULE 26
Figure imgf000155_0001
Step 1: 5-cyclopropyl-lH-pyrrole-2-carboxylic acid
[000191] To methyl 5-cyclopropyl-lH-pyrrole-2-carboxylate (300 mg, 1.82 mmol, 1 eq) in a mixture of THF (3 mL), MeOH (3 mb) and H2O (3 mL) was added LiOH.H2O (266.74 mg, 6.36 mmol, 3.5 eq). The mixture was stirred at 60 °C for 16 h. Upon completion, to the reaction solution was added 1 M HC1 until pH of the solution was about 4. Then the reaction solution was extrated by EtOAc 30 mL (10 mL * 3). The organic layers was combined, washed with brine and concentrated under reduced pressure to give 5-cyclopropyl-lH-pyrrole-2-carboxylic acid (280 mg, 1.49 mmol, 82.17% yield) as a yellow solid. MS (ESI) m/z 152.2 [M+H]+.
Step 2: tert-butyl (lR,2S,5S)-2-[[cyano(4-isoquinolyl)methyl]carbamoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-3-carboxylate
[000192] To a solution of (lR,2S,5S)-3-tert-butoxycarbonyl-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (400 mg, 1.57 mmol, 1 eq) and 2-amino-2-(4- isoquinolyl)acetonitrile (287.04 mg, 1.57 mmol, 1 eq) in DCM (10 mL) was added DMAP (574.21 mg, 4.70 mmol, 3 eq) and EDCI (600.69 mg, 3.13 mmol, 2 eq). The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction solution was quenched with H2O (20 mL) and extracted with DCM 60 mL (20 mL * 3). The organic layers were combined and washed with
154
SUBSTITUTE SHEET RULE 26 citric acid (20 mL), sat. aq. NaHCCh (20 mL), brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM:MeOH = 10:1) to give tert-butyl (lR,2S,5S)-2-[[cyano(4- isoquinolyl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (520 mg, 1.16 mmol, 74.19% yield, 94% purity) as a brown oil. MS (ESI) m/z 421.3 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000193] To tert-butyl (lR,2S,5S)-2-[[cyano(4-isoquinolyl)methyl]carbamoyl]-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-3-carboxylate (510 mg, 1.21 mmol, 1 eq) was added a solution of TFA (4.62 g, 40.52 mmol, 3 mL, 33.41 eq) to DCM (6 mL) in a ratio of 1:2. The mixture was stirred at 25 °C for 1 h. Upon completion, the residue was added sat. aq. NaHCCh until pH was about 8. Then the solution was extracted by DCM 80 mL (10 mL * 8). The organic layers were combined, dried over Na2SO4, and concentrated under reduced pressure to give (1R,2S,5S)-N- [cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (380 mg, crude) as a brown solid. MS (ESI) m/z 321.3 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000194] To a solution of (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (380 mg, 1.19 mmol, 1 eq) and 5-cyclopropyl-lH- pyrrole-2-carboxylic acid (179.29 mg, 1.19 mmol, 1 eq) in DCM (10 mL) was added DMAP (434.70 mg, 3.56 mmol, 3 eq) and EDCI (454.74 mg, 2.37 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction solution was dried over N2 to give a residue. The residue was purified by prep-HPLC (neutral condition: column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water (NHd4CO3)-ACN]; B%: 40%-70%, 10 min) to give desired compound (100 mg, purity 98%) as a white solid. The obtained solid was purified by prep-HPLC (acidic condition: column: Phenomenex Luna Cl 8 75 * 30 mm * 3 um; mobile phase: [water(LA)- ACN];B%: 25%-55%, 8 min) to give (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(5- cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (65 mg, 143.32 umol, 12.08% yield, 100% purity) as a white solid. MS (ESI) m/z 454.1 [M+H]+.
155
SUBSTITUTE SHEET RULE 26 [000195] 1H NMR (400MHz, DMSO-t76) 5 = 11.38 - 11.14 (m, 1H), 9.81 - 9.34 (m, 2H), 8.71 (br s, 1H), 8.25 (br d, J= 8.1 Hz, 1H), 8.13 - 7.73 (m, 3H), 6.82 (br d, J= 7.4 Hz, 1H), 6.64 - 5.90 (m, 1H), 5.84 - 5.10 (m, 1H), 4.56 - 4.31 (m, 1H), 3.98 (dt, J = 5.3, 11.1 Hz, 1H), 3.74 - 3.53 (m, 1H), 2.00 - 1.82 (m, 1H), 1.66 - 1.44 (m, 1H), 1.36 - 1.03 (m, 1H), 0.89 - 0.74 (m, 5H), 0.61 (br d, J= 6.0 Hz, 2H).
[000196] 'H NMR (400MHz, DMSO-t/e, T = 273+80 K) 5 = 11.19 - 10.66 (m, 1H), 9.53 - 9.13 (m, 2H), 8.75 (d, J= 2.4 Hz, 1H), 8.23 (d, J= 8.2 Hz, 1H), 8.05 (br d, J= 8.1 Hz, 1H), 7.89 (q, J= 8.0 Hz, 1H), 7.82 - 7.72 (m, 1H), 6.74 (d, J= 7.6 Hz, 1H), 6.50 (br s, 1H), 5.72 (br s, 1H), 4.52 (d, J= 5.1 Hz, 1H), 4.07 - 3.66 (m, 2H), 2.00 - 1.86 (m, 1H), 1.63 - 1.48 (m, 1H), 1.45 - 1.16 (m, 1H), 1.09 - 0.94 (m, 3H), 0.90 - 0.78 (m, 5H), 0.69 - 0.58 (m, 2H).
Example 3: Synthesis of N-[(lS)-2-[[cyano(3-pyridyl)methyl]amino]-l- (cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Compound 100)
Figure imgf000157_0001
Step 1: 2-amino-2-(3-pyridyl)acetonitrile
[000197] To a mixture of pyridine-3-carbaldehyde (5 g, 46.68 mmol, 4.39 mL, 1 eq) in NHa/MeOH (50 mL) was added NH4CI (4.99 g, 93.36 mmol, 2 eq). The mixture was stirred at 25 °C for 30 min, TLC showed the reaction was completion, TMSCN (11.58 g, 116.70 mmol, 14.60 mL, 2.5 eq) was added in one portion at 0 °C. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 50 mL at 0 °C, and extracted with EA 300 mL (100 mL * 3). The combined organic layers were washed with brine 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10: 1 to 1: 1). Compound 2-amino-2-(3-pyridyl)acetonitrile (4.26 g, 30.39 mmol, 65.11% yield, 95% purity) was obtained as a yellow oil. MS (ESI) m/z 134.1 [M+H]+.
156
SUBSTITUTE SHEET RULE 26 Step 2: N-[(l S)-2-[[cyano(3-pyridyl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro-lH-indole-2-carboxamide
[000198] To a mixture of (2S)-3-cyclopropyl-2-[(7-fluoro-lH-indole-2- carbonyl)amino]propanoic acid (500.0 mg, 1.48 mmol, 86% purity, 1 eq) and 2-amino-2-(3- pyridyl)acetonitrile (236.68 mg, 1.78 mmol, 1.2 eq) in DCM (25.0 mL) was added TEA (449.67 mg, 4.44 mmol, 618.53 uL, 3 eq). Then T3P (1.89 g, 2.96 mmol, 1.76 mL, 50% purity, 2 eq) in DCM (12.5 mL) was added in one portion at -30 °C. The mixture was stirred at -30 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with DCM 40 mL (20 mL * 2). The combined organic layers was concentrated under reduced pressure to give a residue. Then crude product was triturated with ethyl acetate at 25 °C for 30 min. Compound N- [(lS)-2-[[cyano(3-pyridyl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro-lH- indole-2-carboxamide (346 mg, 809.05 umol, 54.62% yield, 94.8% purity) was obtained as a white solid. MS (ESI) m/z 406.0 [M+H]+.
[000199] 1H NMR (400 MHz, DMSO- t76) 5 = 12.10 (br s, 1H), 9.71 - 9.34 (m, 1H), 8.81 - 8.44 (m, 3H), 8.03 - 7.74 (m, 1H), 7.63 - 7.40 (m, 2H), 7.32 (br s, 1H), 7.11 - 6.88 (m, 2H), 6.48 - 6.15 (m, 1H), 4.75 - 4.36 (m, 1H), 1.81 (dt, J = 8.7, 14.8 Hz, 1H), 1.60 - 1.33 (m, 1H), 0.99 - 0.67 (m, 1H), 0.52 - 0.11 (m, 4H).
Example 4: Synthesis of N-((2S)-l-((cyano(4-(trifluoromethyl)pyridin-3-yl)methyl)amino)- 3-cyclopropyl-l-oxopropan-2-yl)-7-fluoro-lH-indole-2-carboxamide (Compound 101)
157
SUBSTITUTE SHEET RULE 26
Figure imgf000159_0001
Step 1: 2-((4-methoxybenzyl)amino)-2-(4-(trifluoromethyl)pyridin-3-yl)acetonitrile
[000200] To a solution of 4-(trifluoromethyl)pyridine-3-carbaldehyde (500 mg, 2.86 mmol, 1 eq) in DCM (20 mL) was added MgSC (343.70 mg, 2.86 mmol, 1 eq) and PMBNH2 (391.70 mg, 2.86 mmol, 369.53 uL, 1 eq). The mixture was stirred at 25 °C for 3 h, then TMSCN (566.57 mg, 5.71 mmol, 714.46 uL, 2 eq) was added. The mixture was stirred at 40 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 3: 1) to give 2-[(4-methoxyphenyl)methylamino]-2-[4-(trifluoromethyl)-3-pyridyl]acetonitrile (700 mg, 2.18 mmol, 76.3% yield) as a yellow solid. MS (ESI) m/z 322.2 [M+H]+
[000201] 1H NMR (400 MHz, DMSO-t/6) 5 = 9.15 (s, 1H), 8.89 (d, J = 5.0 Hz, 1H), 7.81 (d, J= 5.1 Hz, 1H), 7.24 (d, J= 8.6 Hz, 2H), 6.88 (d, J= 8.6 Hz, 2H), 5.08 (d, J= 10.1 Hz, 1H), 4.00 - 3.82 (m, 2H), 3.80 - 3.75 (m, 1H), 3.73 (s, 3H)
Step 2: 2-amino-2-(4-(trifluoromethyl)pyridin-3-yl)acetonitrile
[000202] To a solution of 2-[(4-methoxyphenyl)methylamino]-2-[4-(trifluoromethyl)-3- pyridyl] acetonitrile (600 mg, 1.87 mmol, 1 eq) in TFA (6 mL) was added TfOH (850.00 mg, 5.66 mmol, 0.5 mL, 3.03 eq). The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was quenched by saturated aq. NaHCOi (20 mL) at 25 °C to adjust pH = 7~8,
158
SUBSTITUTE SHEET RULE 26 and extracted with ethyl acetate (10 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-[4- (trifluoromethyl)-3-pyridyl]acetonitrile (400 mg, crude) as a white solid. MS (ESI) m/z 202.2 [M+H]+
Step 3: methyl 2-amino-2-(4-(trifluoromethyl)pyridin-3-yl)acetate
[000203] A mixture of 2-amino-2-[4-(trifluoromethyl)-3-pyridyl]acetonitrile (700 mg, 3.48 mmol, 1 eq) in HCl/MeOH (4 M, 100.46 mL, 115.48 eq) was stirred at 75 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75*30mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: l%-30%,8min) to give methyl 2-amino-2-[4-(trifluoromethyl)-3- pyridyl] acetate (110 mg, 469.73 umol, 13.50% yield) as a yellow solid. MS (ESI) m/z 235.1 [M+H]+
Step 4: methyl 2-((S)-3-cyclopropyl-2-(7-fluoro-lH-indole-2-carboxamido)propanamido)-2-(4- (trifluoromethyl)pyridin-3-yl)acetate
[000204] To a solution of (2S)-3-cyclopropyl-2-[(7-fluoro-lH-indole-2- carbonyl)amino]propanoic acid (100 mg, 344.48 umol, 1 eq) in DCM (2 mL) was added methyl 2-amino-2-[4-(trifhroromethyl)-3-pyridyl]acetate (107.21 mg, 396.16 umol, 1.15 eq, HC1). To the resulting mixture was added EbN (104.57 mg, 1.03 mmol, 143.84 uL, 3 eq), followed by T3P (657.65 mg, 1.03 mmol, 614.63 uL, 50% purity, 3 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with water (10 mL) and extracted with DCM (5 mL * 2). The combined organic layers were washed with brine (8 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give methyl 2-[[(2S)-3-cyclopropyl- 2-[(7-fluoro-lH-indole-2-carbonyl)amino]propanoyl]amino]-2-[4-(trifluoromethyl)-3- pyridyl] acetate (150 mg, 296.18 umol, 85.98% yield) as a yellow solid. MS (ESI) m/z 507.2 [M+H]+
Step 5: N-((2S)-l-((2-amino-2-oxo-l-(4-(trifluoromethyl)pyri din-3 -yl)ethyl)amino)-3 - cyclopropyl- 1 -oxopropan-2-yl)-7-fluoro- 1 H-indole-2-carboxamide
[000205] A mixture of methyl 2-[[(2S)-3-cyclopropyl-2-[(7-fluoro-lH-indole-2- carbonyl)amino]propanoyl]amino]-2-[4-(trifluoromethyl)-3-pyridyl]acetate (130 mg, 256.69
159
SUBSTITUTE SHEET RULE 26 umol, 1 eq) in NHa/MeOH (7 M, 20 mL, 545.41 eq) was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved with DCM (20 mL) and concentrated under reduced pressure for three times to give N-[(lS)-2-[[2-amino-2-oxo-l-[4-(trifluoromethyl)-3-pyridyl]ethyl]amino]-l- (cyclopropyhnethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (130 mg, crude) as a yellow solid. MS (ESI) m/z 492.2 [M+H]+
Step 6: N-((2S)-l-((cyano(4-(trifluoromethyl)pyridin-3-yl)methyl)amino)-3-cyclopropyl-l- oxopropan-2-yl)-7-fluoro- 1 H-indole-2-carboxamide
[000206] To a solution of N-[(lS)-2-[[2-amino-2-oxo-l-[4-(trifluoromethyl)-3- pyridyl]ethyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (130 mg, 264.53 umol, 1 eq) in DCM (2 mL) was added Burgess reagent (1.01 g, 4.23 mmol, 16 eq). The mixture was stirred at 25 °C for 6 h. Upon completion, the reaction mixture was quenched by water (0.5 mL) at 20 °C, and the system was blow-dried with N2 to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80*40mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 25%-55%,8min) to give N-[(lS)-2-[[cyano-[4- (trifluoromethyl)-3-pyridyl]methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH- indole-2-carboxamide (40.5 mg, 85.55 umol, 32.34% yield, 100% purity) as a white solid. MS (ESI) m/z 274.1 [M+H]+
[000207] 1H NMR (400 MHz, DMSO-t76) 5 = 11.95 (br d, J= 11.6 Hz, 1H), 9.71 - 9.53 (m, 1H), 9.03 (d, J= 9.5 Hz, 1H), 8.84 (t, J= 4.1 Hz, 1H), 8.53 (dd, J= 7.6, 17.6 Hz, 1H), 7.77 (br d, J= 3.9 Hz, 1H), 7.40 - 7.31 (m, 1H), 7.20 (br d, J= 6.4 Hz, 1H), 6.91 (td, J= 3.8, 7.7 Hz, 2H), 6.23 (d, J= 7.2 Hz, 1H), 4.48 (td, J= 4.0, 7.7 Hz, 1H), 1.77 - 1.58 (m, 1H), 1.50 - 1.27 (m, 1H), 0.78 - 0.57 (m, 1H), 0.39 -0.15 (m, 4H).
Example 5: Synthesis of N-((2S)-l-((cyano(4-(trifluoromethyl)pyridin-3-yl)methyl)amino)- 3-cyclopropyl-l-oxopropan-2-yl)-7-fluoro-lH-indole-2-carboxamide (Compound 102)
Figure imgf000161_0001
160
SUBSTITUTE SHEET RULE 26 Step 1: 2-amino-2-(4-isoquinolyl)acetonitrile
[000208] To isoquinoline-4-carbaldehyde (2 g, 12.73 mmol, 1 eq) and TMSCN (1.26 g, 12.73 mmol, 1.59 mL, 1 eq) was added NH3/MeOH (7 M, 20.00 mL, 11 eq) and NH4C1 (2.04 g, 38.18 mmol, 3 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, to the reaction solution was added H2O 20 mL and extracted with EtOAc 60 mL (20 mL * 3). The organic phase was separated, dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM:MeOH = 10: 1) to give 2- amino-2-(4-isoquinolyl)acetonitrile (2.2 g, 12.01 mmol, 94.37% yield) as a yellow solid. MS (ESI) m/z 184.2 [M+H]+.
Step 2: N-[2-[[cyano(4-isoquinolyl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro- lH-indole-2-carboxamide
[000209] To a solution of 2-amino-2-(4-isoquinolyl)acetonitrile (315.12 mg, 1.72 mmol, 1 eq) and (2S)-3-cyclopropyl-2-[(7-fluoro-lH-indole-2-carbonyl)amino]propanoic acid (500 mg, 1.72 mmol, 1 eq) in DCM (10 mL) was added DMAP (630.40 mg, 5.16 mmol, 3 eq) and EDCI (659.45 mg, 3.44 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction solution was quenched with H2O (15 mL), and extracted with DCM 45 mL (15 mL * 3). The organic layers were combined, washed with citric acid (20 mL), sat. aq. NaHCCh (20 mL), brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure to give a residue. The residue was purified by /v -HPLC (column: Waters Xbridge BEH C18 250 * 50 mm * 10 um; mobile phase: [water(NH4HCO3)-ACN]; B%: 35%-55%, 10 min) to give the product, which was further separated by SFC (column: DAI CEL CHIRALPAK IC(250 mm * 30 mm, 10 um); mobile phase: [0.1%NH3H2O IP A]; B%: 55%-55%, 15 min) to give N-[2-[[cyano(4- isoquinolyl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2- carboxamide (Isomer 2, 30 mg, 65.34 umol, 3.80% yield, 99.2% purity) as a white solid. MS (ESI) m/z 456.1 [M+H]+ and N-[2-[[cyano(4-isoquinolyl)methyl]amino]-l-(cyclopropylmethyl)- 2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 3, 20 mg, 43.91 umol, 2.55% yield, 100% purity) as a white solid. MS (ESI) m/z 456.1 [M+H]+.
[000210] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.06 (br d, J= 14.8 Hz, 1H), 9.69 - 9.50 (m, 1H), 9.41 (d, J= 6.0 Hz, 1H), 8.73 (d, J= 11.3 Hz, 1H), 8.63 (dd, J= 7.7, 10.6 Hz, 1H), 8.25 (br d, J= 8.0 Hz, 1H), 8.04 (t, J= 9.3 Hz, 1H), 7.95 - 7.87 (m, 1H), 7.84 - 7.74 (m, 1H),
161
SUBSTITUTE SHEET RULE 26 7.53 - 7.39 (m, 1H), 7.30 (br s, 1H), 7.09 - 6.97 (m, 2H), 6.85 (d, J= 7.5 Hz, 1H), 4.65 - 4.48 (m, 1H), 1.91 - 1.70 (m, 1H), 1.59 - 1.35 (m, 1H), 0.87 - 0.62 (m, 1H), 0.49 - 0.35 (m, 2H), 0.33 - 0.09 (m, 1H).
[000211] Isomer 3: XH NMR (400 MHz, DMSO-t/e) 5 = 12.17 - 11.89 (m, 1H), 9.69 - 9.49 (m, 1H), 9.46 - 9.35 (m, 1H), 8.78 - 8.69 (m, 1H), 8.67 - 8.55 (m, 1H), 8.25 (br d, J= 7.9 Hz, 1H), 8.08 - 7.97 (m, 1H), 7.94 - 7.85 (m, 1H), 7.84 - 7.74 (m, 1H), 7.51 - 7.42 (m, 1H), 7.30 (d, J = 2.6 Hz, 1H), 7.07 - 6.96 (m, 2H), 6.85 (d, J= 7.5 Hz, 1H), 4.63 - 4.49 (m, 1H), 1.88 - 1.69 (m, 1H), 1.60 - 1.36 (m, 1H), 0.87 - 0.65 (m, 1H), 0.47 - 0.35 (m, 1H), 0.33 - 0.10 (m, 2H), 0.09 - 0.10 (m, 1H).
Example 6: Synthesis of (lR,2S,5S)-N-[cyano(3-pyridyl)methyl]-3-(7-fluoro-lH-indole-2- carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 104)
Figure imgf000163_0001
Step 1: tert-butyl (lR,2S,5S)-2-[[2-methoxy-2-oxo-l-(3-pyridyl)ethyl]carbamoyl]-6,6-dimethyl- 3-azabicyclo[3.1 ,0]hexane-3-carboxylate
[000212] To a solution of methyl 2-amino-2-(3-pyridyl)acetate (500.00 mg, 2.47 mmol, 1 eq, HC1) and (1R, 2S,5S)-3-tert-butoxy carbonyl-6, 6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (629.96 mg, 2.47 mmol, 1 eq) in DCM (10 mL) was added DMAP (602.90 mg, 4.93 mmol, 2 eq) and EDCI (946.02 mg, 4.93 mmol, 2 eq) at 0 °C, and the solution was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was diluted with water (50 mL) and extracted with DCM (30 mL * 3). The combined organic layers were dried over Na2SO4, filtered
162
SUBSTITUTE SHEET RULE 26 and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10:1 to 1:1) to give tert-butyl (lR,2S,5S)-2-[[2-methoxy-2-oxo-l-(3-pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-3-carboxylate (0.65 g, 1.45 mmol, 58.76% yield, 90% purity) as a yellow oil.
Step 2: methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3- pyridyl)acetate
[000213] A mixture of tert-butyl (lR,2S,5S)-2-[[2-methoxy-2-oxo-l-(3- pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-3-carboxylate (650.00 mg, 1.61 mmol, 1 eq) in HCl/MeOH (4 M, 15 mL, 37.24 eq) was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give methyl 2- [[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3-pyridyl)acetate (0.55 g, crude) as a yellow oil.
Step 3: methyl 2-[[(lR,2S,5S)-3-(7-fhroro-lH-indole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3-pyridyl)acetate
[000214] To a solution of methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carbonyl]amino]-2-(3-pyridyl)acetate (550.00 mg, 1.62 mmol, 1 eq, HC1) and 7-fluoro-lH- indole-2-carboxylic acid (347.94 mg, 1.94 mmol, 1.2 eq) in DCM (10 mL) was added DMAP (395.47 mg, 3.24 mmol, 2 eq) and EDCI (620.54 mg, 3.24 mmol, 2 eq) at 0 °C, the solution was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was diluted with water (50 mL) and extracted with DCM (30 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 1 :1) to give methyl 2- [[(lR,2S,5S)-3-(7-fhroro-lH-indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carbonyl]amino]-2-(3-pyridyl)acetate (0.5 g, 968.81 umol, 59.86% yield, 90% purity) as a yellow oil.
Step 4: (lR,2S,5S)-N-[2-amino-2-oxo-l-(3-pyridyl)ethyl]-3-(7-fluoro-lH-indole-2-carbonyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
163
SUBSTITUTE SHEET RULE 26 [000215] A mixture of methyl 2-[[(lR,2S,5S)-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3-pyridyl)acetate (0.5 g, 1.08 mmol, 1 eq) in NHa/MeOH (7 M, 20 mL, 130.06 eq) was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-N-[2-amino-2- oxo-1 -(3-pyridyl)ethyl]-3-(7-fluoro-lH-indole-2-carbonyl)-6, 6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.5 g, crude) as a yellow oil.
Step 5: (lR,2S,5S)-N-[cyano(3-pyridyl)methyl]-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000216] A mixture of (lR,2S,5S)-N-[2-amino-2-oxo-l-(3-pyridyl)ethyl]-3-(7-fluoro-lH- indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (500.00 mg, 1.11 mmol, 1 eq) in DCM (7 mL) was added burgess reagent (795.29 mg, 3.34 mmol, 3 eq), the solution was stirred at 25 °C for 3 h. Upon completion, H2O (1 mL) was added to the solution drop-wise, the mixture was dried by blowing N2. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100*30mm*10um;mobile phase: [water( NH4HCO3)- ACN];B%: 35%-65%,8min) to give (lR,2S,5S)-N-[cyano(3-pyridyl)methyl]-3-(7-fluoro-lH- indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (129.63 mg, 300.44 umol, 64.81% yield, 100% purity) as a yellow solid. MS (ESI) m/z 432.1 [M+H]+.
[000217] 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.01 (br d, J= 9.0 Hz, 1H), 9.58 (dd, J= 7.9, 19.3 Hz, 1H), 8.82 - 8.47 (m, 2H), 8.03 - 7.66 (m, 1H), 7.63 - 7.30 (m, 2H), 7.19 (br s, 1H), 7.09
- 6.93 (m, 2H), 6.65 - 6.17 (m, 1H), 4.55 (s, 1H), 4.23 (br s, 1H), 3.90 (d, J= 10.6 Hz, 1H), 1.77
- 1.51 (m, 1H), 1.47 - 1.37 (m, 1H), 1.14 - 1.02 (m, 3H), 0.99 - 0.85 (m, 3H).
164
SUBSTITUTE SHEET RULE 26 Example 7: Synthesis of (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(7-fluoro-lH-indole-
2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 106)
Figure imgf000166_0001
Step 1: methyl 2-amino-2-(4-isoquinolyl)acetate
[000218] A mixture of 2-amino-2-(4-isoquinolyl) acetonitrile (200.00 mg, 1.09 mmol, 1 eq) in HCl/MeOH (4 M, 10 mL, 36.64 eq) was stirred at 75 °C for 12 h. Upon completion, the mixture was concentrated under the reduced pressure to give the product methyl 2-amino-2-(4- isoquinolyl) acetate (370 mg, crude) as an orange solid. MS (ESI) m/z 216.1 [M+H]+
Step 2: tert-butyl (lR,2S,5S)-2-[[l-(4-isoquinolyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-3-carboxylate
[000219] To a mixture of methyl 2-amino-2-(4-isoquinolyl)acetate (900 mg, 3.56 mmol, 1 eq, HC1) in DCM (20 mL) was added (lR,2S,5S)-3-tert-butoxycarbonyl-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.25 g, 3.92 mmol, 80% purity, 1.1 eq), DMAP (1.31 g, 10.68 mmol, 3 eq), and EDCI (1.37 g, 7.12 mmol, 2 eq). The mixture was stirred at 25 °C for
2 h. Upon completion, the residue was quenched by water (20 mL) and extracted with DCM (10 mL * 3). The combined organic phase was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was
165
SUBSTITUTE SHEET RULE 26 purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 6:4 to 55:45) to give the product tert-butyl (lR,2S,5S)-2-[[l-(4-isoquinolyl)-2-methoxy-2-oxo-ethyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (1.15 g, 2.42 mmol, 68.03% yield, 95.56% purity) as a yellow solid. MS (ESI) m/z 453.2 [M+H]+
Step 3: methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(4- isoquinolyl)acetate
[000220] A mixture of tert-butyl (lR,2S,5S)-2-[[l-(4-isoquinolyl)-2-methoxy-2-oxo- ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carboxylate (1.15 g, 2.54 mmol, 1 eq} in HCl/MeOH (4 M, 15 mL, 23.66 eq was stirred at 25 °C for 1 h. Upon completion, the mixture was concentrated under reduced pressure to give methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(4-isoquinolyl)acetate (990 mg, crude, HC1) as a yellow solid. MS (ESI) m/z 353.2 [M+H]+
Step 4: methyl 2-[[(lR,2S,5S)-3-(7-fhroro-lH-indole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(4-isoquinolyl)acetate
[000221] To a solution of methyl 2-[[(lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carbonyl]amino]-2-(4-isoquinolyl)acetate (990 mg, 2.54 mmol, 1 eq, HC1) in DCM (20 mL) was added 7-fluoro-lH-indole-2-carboxylic acid (625.49 mg, 2.79 mmol, 80% purity, 1.1 eq}, DMAP (930.65 mg, 7.62 mmol, 3 eq}, and EDCI (973.57 mg, 5.08 mmol, 2 eq}. The mixture was stirred at 25 °C for 3 h. Upon completion, the reaction mixture was quenched by water (20 mL) and extracted with DCM (20 mL * 3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE:EA = 0: 1) to give the product methyl 2- [[(lR,2S,5S)-3-(7-fhroro-lH-indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carbonyl]amino]-2-(4-isoquinolyl)acetate (400 mg, 677.18 umol, 26.67% yield, 87.11% purity) as a yellow solid. MS (ESI) m/z 514.2 [M+H]+
Step 5: (lR,2S,5S)-N-[2-amino-l-(4-isoquinolyl)-2-oxo-ethyl]-3-(7-fluoro-lH-indole-2- carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000222] A mixture of methyl 2-[[(lR,2S,5S)-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carbonyl]amino]-2-(4-isoquinolyl)acetate (400 mg,
166
SUBSTITUTE SHEET RULE 26 777.38 umol, 1 eq) in NH MeOH (7 M, 15 mL, 135.07 eq) was stirred at 25 UC for 14 h. Upon completion, the mixture was concentrated under reduced pressure to give the product (lR,2S,5S)-N-[2-amino-l-(4-isoquinolyl)-2-oxo-ethyl]-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (390 mg, crude) as a yellow solid. MS (ESI) m/z 499.2 [M+H]+
Step 6: (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(7-fluoro-lH-indole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000223] To a solution of (lR,2S,5S)-N-[2-amino-l-(4-isoquinolyl)-2-oxo-ethyl]-3-(7-fluoro- lH-indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (390 mg, 780.73 umol, 1 eq) in DCM (10 mL) was added burgess reagent (558.15 mg, 2.34 mmol, 3 eq). The mixture was stirred at 25 °C for 3 h. Upon completion, the reaction was quenched by water (0.5 mL) and dried by blowing N2 to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40mm * lOum; mobile phase: [water (NH4HCO3) - ACN]; B%:35%-60%, 8min) to give the product (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]- 3-(7-fluoro-lH-indole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (102.04 mg, 209.43 umol, 26.83% yield, 98.83% purity) as a white solid. MS (ESI) m/z 481.2 [M+H]+
[000224] XH NMR (400 MHz, DMSO-t76) 5 = 12.04 - 11.91 (m, 1H), 9.64 (d, J = 7.6 Hz, 1H), 9.46 - 9.35 (m, 1H), 8.78 - 8.69 (m, 1H), 8.30 - 8.20 (m, 1H), 8.18 - 8.02 (m, 1H), 7.98 - 7.88 (m, 1H), 7.83 - 7.72 (m, 1H), 7.66 - 7.44 (m, 1H), 7.17 (dd, J= 2.8, 6.4 Hz, 1H), 7.09 - 6.92 (m, 2H), 6.89 - 6.60 (m, 1H), 4.64 - 4.52 (m, 1H), 4.22 (dt, J= 5.4, 10.0 Hz, 1H), 3.92 - 3.78 (m, 1H), 1.73 - 1.48 (m, 1H), 1.44 - 1.16 (m, 1H), 1.10 - 0.94 (m, 3H), 0.92 - 0.83 (m, 3H)
167
SUBSTITUTE SHEET RULE 26 Example 8: Synthesis of (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-(7-fluoro-lH-indole- 2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 123)
Figure imgf000169_0001
Step 1: 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile
[000225] A mixture of l,6-naphthyridine-8-carbaldehyde (2 g, 12.65 mmol, 1 eq) in NHa/MeOH (7M, 20 mL) was added NH4CI (1.35 g, 25.29 mmol, 2 eq) and stirred at 25 °C for 2 h, and then TMSCN (2.51 g, 25.29 mmol, 3.16 mL, 2 eq) was added the former mixture at 0 °C. The resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue, then diluted with H2O (50 mL) and extracted with EA and MeOH(10:l) 200 mL (25 mL * 8). The combined organic layers were concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (S1O2, petroleum etherethyl acetate = 5: 1 to EA:MeOH = 10:1) to give 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile (2 g, 9.23 mmol, 72.98% yield, 85% purity) as a black brown solid. MS (ESI) m/z 185.1 [M+H]+
Step 2: N-[(lS)-2-[[cyano(l,6-naphthyridin-8-yl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo- ethyl]-7-fluoro-lH-indole-2-carboxamide
[000226] A mixture of 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile (300 mg, 1.63 mmol, 1 eq), (2S)-3-cyclopropyl-2-[(7-fluoro-lH-indole-2-carbonyl)amino]propanoic acid (577.24 mg, 1.71 mmol, 86% purity, 1.05 eq) in DMF (10 mL) was added PyBOP (932.32 mg, 1.79 mmol, 1.1 eq) and TEA (329.61 mg, 3.26 mmol, 453.39 uL, 2 eq) at -30 °C, and the resulting mixture was stirred at -30 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (40 mL) and extracted with EA 75 mL (25 mL * 3). The combined organic layers were washed with
168
SUBSTITUTE SHEET RULE 26 brine 60 mL (30 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Welch Xtimate Cl 8 250*70mm#10um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-60%,20min) to give N-[(l S)-2-[[cyano(l,6-naphthyridin-8-yl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo- ethyl] -7-fluoro-lH-indole-2-carboxamide (380 mg, 824.15 umol, 50.60% yield, 99% purity) as a white solid. MS (ESI) m/z 457.2 [M+H]+
Step 3: N-[(lS)-2-[[cyano(l,6-naphthyridin-8-yl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo- ethyl]-7-fluoro-lH-indole-2-carboxamide
[000227] The desired compound (300 mg, purity 99%) was further separated by SFC (condition: column: DAICEL CHIRALPAK IC(250mm*30mm,10um);mobile phase: [0.1%NH3H2O IPA];B%: 55%-55%,20min) to give N-[(lS)-2-[[cyano(l,6-naphthyridin-8- yl)methyl]amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 1, 90.85 mg, 195.25 umol, 29.71% yield, 98.1% purity) as a white solid. MS (ESI) m/z 457.2 [M+H]+and N-[(lS)-2-[[cyano(l,6-naphthyridin-8-yl)methyl]amino]-l- (cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 2, 51.82 mg, 112.27 umol, 17.08% yield, 98.9% purity) as a white solid MS (ESI) m/z 457.2 [M+H]+
[000228] Isomer 1 : 1 H NMR (400 MHz, DMSO-t/r,) 5 = 12.08 (br d, J = 13.0 Hz, 1 H), 9.66 -
9.48 (m, 2H), 9.20 (dt, J= 1.7, 4.3 Hz, 1H), 9.01 - 8.89 (m, 1H), 8.74 - 8.60 (m, 2H), 7.86 - 7.76 (m, 1H), 7.52 - 7.43 (m, 1H), 7.30 (br d, J= 19.0 Hz, 1H), 7.09 - 6.97 (m, 2H), 6.91 (dd, J= 2.0, 7.8 Hz, 1H), 4.72 - 4.55 (m, 1H), 1.90 - 1.71 (m, 1H), 1.62 - 1.45 (m, 1H), 0.86 - 0.71 (m, 1H), 0.45 - -0.03 (m, 4H)
[000229] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.07 (br d, J= 13.0 Hz, 1H), 9.66 -
9.49 (m, 2H), 9.20 (dt, J= 1.7, 4.3 Hz, 1H), 9.02 - 8.91 (m, 1H), 8.75 - 8.60 (m, 2H), 7.86 - 7.76 (m, 1H), 7.52 - 7.42 (m, 1H), 7.30 (dd, J= 1.7, 18.8 Hz, 1H), 7.10 - 6.98 (m, 2H), 6.92 (dd, J = 1.7, 7.7 Hz, 1H), 4.74 - 4.56 (m, 1H), 1.92 - 1.71 (m, 1H), 1.65 - 1.44 (m, 1H), 0.91 - 0.70 (m, 1H), 0.50 - -0.05 (m, 4H).
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SUBSTITUTE SHEET RULE 26 Example 9: Synthesis of N-[(lS)-2-[[(lS)-l-cyano-2-[(6R)-5-oxo-4-azaspiro[2.4]heptan-6- yl]ethyl] amino]-l-[(l-methylcyclopropyl)methyl]-2-oxo-ethyl]-6,7-difluoro-lH-indole-2- carboxamide (Compound 125)
Figure imgf000171_0001
Step 1: 2-amino-2-pyrazin-2-yl-propanenitrile
[000230] A mixture of 1 -pyrazin-2-ylethanone (4 g, 32.75 mmol, 1 eq) in NHa/MeOH (7 M, 65.51 mL, 14 eq) was added NH4C1 (5.26 g, 98.26 mmol, 3 eq) and TMSCN (9.75 g, 98.26 mmol, 12.29 mL, 3 eq) at 0 °C and the resulting mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue, and the residue was dissolved by DCM 150 mL and filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 0:1) to give 2-amino-2-pyrazin-2-yl-propanenitrile (3.9 g, 25.01 mmol, 76.35% yield, 95% purity) as a yellow oil. MS (ESI) m/z 149.2 [M+H]+
Step 2: N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro- 1 H-indole-2-carboxamide
[000231] A mixture of 2-amino-2-pyrazin-2-yl-propanenitrile (200 mg, 1.35 mmol, 1 eq), (2S)-3-cyclopropyl-2-[(7-fluoro-lH-indole-2-carbonyl)amino]propanoic acid (391.85 mg, 1.35 mmol, 1 eq) in ACN (10 mL) was added 1 -methylimidazole (387.90 mg, 4.72 mmol, 376.60 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (757.48 mg, 2.70 mmol, 2 eq). The resluting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by H2O 20 mL, and then extracted with DCM 45 mL (15 mL * 3). The combined organic layers were washed with brine 20 mL (20 mL * 1),
170
SUBSTITUTE SHEET RULE 26 concentrated under reduced pressure to give the crude product. The crude was purified by prep- HPLC (neutral condition; column: Waters Xbridge Prep OBD Cl 8 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 25%-55%,8min) to give N-[(lS)-2-[(l-cyano-l-pyrazin-2- yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (300 mg, purity 93%) as a white solid. MS (ESI) m/z 421.2 [M+H]+
Step 3: N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7- fluoro- 1 H-indole-2-carboxamide
[000232] The desired product was further separated by SFC (condition: column: DAICEL CHIRALPAK AS(250mm*30mm,10um);mobile phase: [0.1%NH3H2O ME0H];B%: 5%- 25%,16min) to give N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2- oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 1, 150 mg) as a white solid and N-[(1S)- 2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH- indole-2-carboxamide (Isomer 2) 40 mg, 95.14 umol, 7.05% yield, 100% purity) as a white solid. MS (ESI) m/z 421.2 [M+H]+
[000233] 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.08 (br s, 1H), 9.64 (s, 1H), 8.73 (d, J= 1.0 Hz, 1H), 8.68 - 8.58 (m, 3H), 7.49 - 7.43 (m, 1H), 7.31 (br s, 1H), 7.08 - 6.96 (m, 2H), 4.67 (q, J 1H), 1.95 - 1.81 (m, 4H), 1.59 - 1.44 (m, 1H), 0.88 (br dd, J= 5.8, 7.5 Hz, 1H), 0.53 -
0.39 (m, 2H), 0.29 - 0.13 (m, 2H).
Step 4: N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]- 7-fluoro-lH-indole-2-carboxamide
[000234] N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo- ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 1, 150 mg) was further separated by SFC (condition: column: DAICEL CHIRALPAK AD(250mm*30mm,10um);mobile phase: [0.1%NH3H2O ETOH];B%: 54%-54%,12min) to give N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl- ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 3, 30 mg, 71.35 umol, 5.29% yield, 100% purity) as a white solid. MS (ESI) m/z 421.2 [M+H]+and N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-(cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro- lH-indole-2-carboxamide (Isomer 4, 34 mg, 80.87 umol, 5.99% yield, 100% purity) as a white solid. MS (ESI) m/z 421.2 [M+H]+and N-[(lS)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)amino]-l-
171
SUBSTITUTE SHEET RULE 26 (cyclopropylmethyl)-2-oxo-ethyl]-7-fluoro-lH-indole-2-carboxamide (Isomer 5, 34 mg, 80.87 umol, 5.99% yield, 100% purity) as a white solid. MS (ESI) m/z 421.2 [M+H]+
[000235] Isomer 3: 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.10 (s, 1H), 9.67 (s, 1H), 8.73 (d, J= 1.1 Hz, 1H), 8.68 - 8.59 (m, 3H), 7.49 - 7.43 (m, 1H), 7.32 (br s, 1H), 7.07 - 6.98 (m, 2H), 4.67 (q, J= 7.3 Hz, 1H), 1.96 - 1.83 (m, 4H), 1.57 - 1.46 (m, 1H), 0.94 - 0.81 (m, 1H), 0.47 (br d, J = 7.7 Hz, 2H), 0.30 - 0.15 (m, 2H)
[000236] Isomer 4: 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.09 (s, 1H), 9.51 (s, 1H), 8.70 (s, 1H), 8.68 - 8.63 (m, 2H), 8.58 (d, J= 8.0 Hz, 1H), 7.51 - 7.41 (m, 1H), 7.30 (br s, 1H), 7.09 - 6.95 (m, 2H), 4.75 - 4.65 (m, 1H), 1.89 (s, 3H), 1.79 - 1.69 (m, 1H), 1.67 - 1.56 (m, 1H), 0.88 - 0.75 (m, 1H), 0.50 - 0.34 (m, 2H), 0.29 - 0.10 (m, 2H).
[000237] Isomer 5: 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.09 (s, 1H), 9.51 (s, 1H), 8.73 - 8.62 (m, 3H), 8.59 (d, J= 7.9 Hz, 1H), 7.50 - 7.42 (m, 1H), 7.30 (br s, 1H), 7.07 - 6.97 (m, 2H), 4.80 - 4.65 (m, 1H), 1.89 (s, 3H), 1.80 - 1.69 (m, 1H), 1.67 - 1.55 (m, 1H), 0.88 - 0.75 (m, 1H), 0.49 - 0.34 (m, 2H), 0.29 - 0.10 (m, 2H).
Example 10: Synthesis of (lR,2S,5S)-N-[(S)-cyano(3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 107)
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SUBSTITUTE SHEET RULE 26
Figure imgf000174_0001
Step 1: methyl (2S)-2-amino-2-(3-pyridyl)acetate
[000238] A mixture of (2S)-2-(tert-butoxycarbonylamino)-2-(3-pyridyl)acetic acid (0.45 g, 1.78 mmol, 1 eq) in HCl/MeOH (4 M, 20 mL, 44.85 eq) (batch 2) was stirred at 70 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give methyl (2S)-2-amino-2-(3-pyridyl)acetate (720 mg, crude, HC1) as a white oil. MS (ESI) m/z 167.1 [M+H]+.
Step 2: methyl (2S)-2-[[(lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3-pyridyl)acetate
[000239] To a solution of (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (350 mg, 949.88 umol, 1 eq), methyl (2S)-2-amino-2-(3-pyridyl)acetate (230.98 mg, 1.14 mmol, 1.2 eq, HC1) and PyBOP (494.31 mg, 949.88 umol, 1 eq) in DMF (10 mL) was cooled to -30 °C, and then E N (288.36 mg, 2.85 mmol, 396.64 uL, 3 eq) in DMF (3 mL) was added drop-wise. The resluting mixture was stirred at -30 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O 60 mL at 0 °C, and then extracted with DCM (30 mL * 3). The combined organic layers
173
SUBSTITUTE SHEET RULE 26 were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, ethyl acetate: petroleum ether = 1 :0 to 0:1) to afford methyl (2S)-2-[[(lR,2S,5S)-3-[(2S)-2-(tert- butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carbonyl]amino]-2-(3-pyridyl)acetate (320 mg, 619.40 umol, 65.21% yield) as a yellow oil. MS (ESI) m/z 517.3 [M+H]+.
Step 3: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[(lS)-2-amino-2-oxo-l-(3-pyridyl)ethyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000240] A mixture of methyl (2S)-2-[[(lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)- 3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-(3- pyridyl)acetate (320 mg, 619.40 umol, 1 eq) in NFh/MeOH (7 M, 20 mL, 226.02 eq), the was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[(lS)-2-amino-2-oxo-l-(3- pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (280 mg, crude) as a white solid. MS (ESI) m/z 502.4 [M+H]+.
Step 4: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[(lS)-2-amino-2-oxo-l-(3- pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000241] A mixture of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[(lS)-2-amino-2-oxo-l-(3- pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (240 mg, 478.45 umol, 1 eq) in DCM (15 mL) was added TFA (4.62 g, 40.52 mmol, 3 mL, 84.69 eq), and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2- amino-3,3-dimethyl-butanoyl]-N-[(lS)-2-amino-2-oxo-l-(3-pyridyl)ethyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (170 mg, crude) as a yellow oil.
Step 5: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[(lS)-2-amino-2-oxo-l-(3- pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000242] To a solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[(lS)-2- amino-2-oxo-l-(3-pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (170 mg, 423.41 umol, 1 eq) in DCM (20 mL) was added drop- wise DIEA (164.17 mg, 1.27 mmol,
174
SUBSTITUTE SHEET RULE 26 221.25 uL, 3 eq), then TFAA (133.39 mg, 635.12 umol, 88.34 uL, 1.5 eq) in DCM (4 mL) at 0 °C, the mixture was stirred at 0 °C for 3 h. Upon completion, the reaction mixture was quenched by addition H2O 40 mL at 0 °C, and extracted with DCM (20 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give (lR,2S,5S)-N-[(lS)-2-amino-2-oxo-l-(3-pyridyl)ethyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide (150 mg, crude) as a yellow oil. MS (ESI) m/z 498.2 [M+H]+.
Step 6: (lR,2S,5S)-N-[(S)-cyano(3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000243] To a solution of (lR,2S,5S)-N-[(lS)-2-amino-2-oxo-l-(3-pyridyl)ethyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide (150 mg, 301.50 umol, 1 eq) in DCM (10 mL) was added burgess reagent (71.85 mg, 301.50 umol, 1 eq), and the resulting mixture was stirred at 25 °C for 3 h. Upon completion, water (1.0 mL) was added to the solution, then dried by blowing N2. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75 * 30 mm * 3 um; mobile phase: [water(NH4HCO3) - ACN]; B%: 40% - 60%, 8 mm) and SFC (column: DAI CEL CHIRALPAK IG (250mm * 30 mm, 10 um); mobile phase: [0.1% NH3H2O EtOH]; B%: 24%-24%,7min) to give (1R,2S,5S)-N- [(S)-cyano(3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (23 mg, 47.97 umol, 100% purity) as a white solid. MS (ESI) m/z 480.3 [M+H]+.
[000244] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 - 9.45 (m, 1H), 9.39 (br s, 1H), 8.70 - 8.64 (m, 1H), 8.63 - 8.56 (m, 1H), 7.93 - 7.82 (m, 1H), 7.50 - 7.42 (m, 1H), 6.42 - 6.24 (m, 1H), 4.39 (br s, 1H), 4.27 (d, J= 3.2 Hz, 1H), 3.96 - 3.85 (m, 1H), 3.70 (t, J= 11.0 Hz, 1H), 1.61 - 1.53 (m, 1H), 1.40 - 1.27 (m, 1H), 1.05 - 0.95 (m, 12H), 0.85 (d, J= 6.5 Hz, 3H).
Example 11: Synthesis of (lR,2S,5S)-N-[(S)-cyano(3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 108)
175
SUBSTITUTE SHEET RULE 26
Figure imgf000177_0001
Step 1: methyl 2-[[(lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-[4-(trifluoromethyl)-3- pyridyl]acetate
[000245] A solution of (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (300 mg, 542.79 umol, 1 eq) and methyl 2-amino-2-[4-(trifluoromethyl)-3-pyridyl]acetate (146.90 mg, 542.79 umol, 1 eq, HC1) in DCM (10 mL) was cooled to 0 °C, and then was added DMAP (198.93 mg, 1.63 mmol, 3 eq) and EDCI (208.11 mg, 1.09 mmol, 2 eq) at 0 °C. The resulting mixture was stirred at 25 °C for 3 h. Upon completion, the mixture was quenched by water (30 mL) and was extracted with DCM (10 mL * 3), then the organic phase was concentrated in vacuum and was purified by column (SiCh, PE:EA = 1 :0 to 1:1) to give methyl 2-[[(lR,2S,5S)-3-[(2S)-2-(tert- butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carbonyl]amino]-2-[4-(trifluoromethyl)-3-pyridyl]acetate (360 mg, 523.41 umol, 75.43% yield, 85% purity) as a light yellow gum. MS (ESI) m/z 585.3 [M+H]+
176
SUBSTITUTE SHEET RULE 26 Step 2: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[2-amino-2-oxo-l-[4-(trifluoromethyl)-3- pyridyl]ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate
[000246] A solution of methyl 2-[[(lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3- dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonyl]amino]-2-[4- (trifluoromethyl)-3-pyridyl]acetate (130 mg, 222.36 umol, 1 eq) in NH MeOH (7M, 10 mL) was stirred at 25 °C for 16 h. Upon completion, the mixture was concentrated in vacuum to give tertbutyl N- [( 1 S)- 1 - [( 1 R,2S, 5 S)-2- [ [2-amino-2-oxo- 1 - [4-(trifluoromethyl)-3 - pyridyl]ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (200 mg, crude, 2 batchs in parallels) as a light yellow solid. MS (ESI) m/z 570.3 [M+H]+
Step 3: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[2-amino-2-oxo-l-[4- (trifluoromethyl)-3-pyridyl]ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000247] A solution of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[2-amino-2-oxo-l-[4- (trifluoromethyl)-3-pyridyl]ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3- carbonyl] -2, 2-dimethyl-propyl] carbamate (270 mg, 474.00 umol, 1 eq) in TFA (2 mL) and DCM (4 mL) was stirred at 0 °C for 1 h. Upon completion, the mixture was dried by blowing N2 to afford (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[2-amino-2-oxo-l-[4- (trifluoromethyl)-3-pyridyl]ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (270 mg, crude, TFA) as a yellow gum. MS (ESI) m/z 470.2 [M+H]+
Step 4: tetrahydrofuran-3-yl N-[(lS)-l-[(lR,2S,5S)-2-[[(lS)-l-cyano-2-[(6R)-5-oxo-4- azaspiro[2.4]heptan-6-yl]ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]- 2, 2-dimethyl-propyl] carbamate
[000248] To a solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[2-amino- 2-oxo- l-[4-(trifluoromethyl)-3-pyridyl]ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (270 mg, 462.71 umol, 1 eq, TFA) in DCM (4 mL) was added DIEA (299.01 mg, 2.31 mmol, 402.98 uL, 5 eq) at 0 °C, and then was added TFAA (145.77 mg, 694.06 umol, 96.54 uL, 1.5 eq) in DCM (1 mL) at 0 °C. The resluting mixture was stirred at 0 °C for 1 h, then was added TFAA (145.77 mg, 694.06 umol, 96.54 uL, 1.5 eq) in DCM (1 mL) at 0 °C, stirred at 0 °C for 1 h, and then TFAA (145.77 mg, 694.06 umol, 96.54 uL, 1.5 eq) in DCM (1 mL) was added
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SUBSTITUTE SHEET RULE 26 at 0 °C. The resulting mixture was stirred at 0 °C for 1 h. Upon completion, the mixture was quenched by water (12 mL) and was extracted with DCM (5 mL * 3), then was dried by Na2SO4, filtered and concerntration in vacuum to afford (lR,2S,5S)-N-[2-amino-2-oxo-l-[4- (trifluoromethyl)-3-pyridyl]ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (260 mg, crude) as a yellow gum. MS (ESI) m/z 566.3 [M+H]+
Step 5: (lR,2S,5S)-N-[cyano-[4-(trifhioromethyl)-3-pyridyl]methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000249] To a solution of (lR,2S,5S)-N-[2-amino-2-oxo-l-[4-(trifluoromethyl)-3- pyridyl]ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (250 mg, 442.08 umol, 1 eq) in DCM (5 mL) was added Burgess reagent (316.05 mg, 1.33 mmol, 3 eq and the resulting mixture was stirred at 25 °C for 3 h. Upon completion, the mixture was quenched by water (1 mL) and was dried by blowing N2. The crude product was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%-75%, 8 min) and repurified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NHIHCO3)-ACN]; B%: 50% - 70%, 8 min) to afford (lR,2S,5S)-N-[cyano-[4- (trifluoromethyl)-3-pyridyl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (50 mg, 86.76 umol, 19.63% yield, 95% purity) as a white solid. MS (ESI) m/z 548.1 [M+H]+
[000250] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.34 - 9.59 (m, 1H), 9.46 - 9.24 (m, 1H), 9.21 - 9.03 (m, 1H), 8.99 - 8.85 (m, 1H), 7.92 - 7.81 (m, 1H), 6.28 (dd, J= 7.4, 11.2 Hz, 1H), 4.59 - 4.31 (m, 1H), 4.25 (d, J= 10.8 Hz, 1H), 3.96 - 3.83 (m, 1H), 3.75 - 3.60 (m, 1H), 1.61 - 1.51 (m, 1H), 1.34 (d, J = 7.6 Hz, 0.5H), 1.20 - 1.14 (m, 0.5H), 1.03 - 0.90 (m, 12H), 0.87 - 0.82 (m, 3H).
Example 12: Synthesis of (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 109)
178
SUBSTITUTE SHEET RULE 26
Figure imgf000180_0001
Step 1: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(4-isoquinolyl)methyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000251] To a solution of (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (402.24 mg, 1.09 mmol, 1 eq) and 2-amino-2-(4-isoquinolyl)acetonitrile (200 mg, 1.09 mmol, 1 eq) in DCM (10 mL) was added T3P (1.39 g, 2.18 mmol, 4.37 mL, 50% purity, 2 eq) and TEA (331.39 mg, 3.27 mmol, 455.84 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 20 mL at 25 °C, and then extracted with DCM 45 mL (15 mL * 3). The combined organic layers were combined, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:1) to afford tert-butyl N-[(1S)-1- [(lR,2S,5S)-2-[[cyano(4-isoquinolyl)methyl]carbamoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate (480 mg, 845.48 umol, 77.45% yield, 94% purity) as a yellow solid. MS (ESI) m/z 534.4 [M+H]+.
Step 2: (lR,2S,5S)-3 - [(2S)-2-amino-3 ,3 -dimethyl-butanoy 1] -N- [cyano(4-isoquinolyl)methyl] - 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000252] To a solution of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(4- isoquinolyl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2- dimethyl-propyl] carbamate (440 mg, 824.49 umol, 1 eq) was added TLA (3.85 g, 33.77 mmol, 2.5 mL, 40.95 eq) in DCM (5 mL). The mixture was stirred at 0 °C for 4 h. Upon completion, the
179
SUBSTITUTE SHEET RULE 26 reaction solution was dried over N2 to give (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]- N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (350 mg, 807.30 umol, 97.91% yield) as a white solid. MS (ESI) m/z 434.3 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000253] To a solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(4- isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (350 mg, 807.30 umol, 1 eq) in DCM (10 mL) was added DIPEA (313.01 mg, 2.42 mmol, 421.85 uL, 3 eq) and then to the solution was added TFAA (254.34 mg, 1.21 mmol, 168.43 uL, 1.5 eq) in DCM (5 mL). The mixture was stirred at 0 °C for 4 h. Upon completion, the reaction solution was dried over N2 to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30 mm * 10 urn; mobile phase: [water(NH4HCO3)-ACN];B%: 55%-75%, 8 min) to give (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (100 mg, 188.84 umol, 23.39% yield, 100% purity) as a white solid. MS (ESI) m/z 530.2 [M+H]+.
[000254] 'H NMR (400MHz, DMSO-t/r,) 5 = 9.62 - 9.48 (m, 1H), 9.44 - 9.32 (m, 2H), 8.72 (d, J= 5.9 Hz, 1H), 8.27 - 8.21 (m, 1H), 8.03 - 7.75 (m, 3H), 6.87 (dd, J= 1.8, 14.9 Hz, 1H), 4.39 (t, J = 7.6 Hz, 1H), 4.23 (d, J= 14.3 Hz, 1H), 3.93 (td, J= 5.1, 10.2 Hz, 1H), 3.70 (dd, J = 10.5, 14.3 Hz, 1H), 1.64 - 1.50 (m, 1H), 1.36 - 1.11 (m, 1H), 1.04 - 1.01 (m, 6H), 0.96 - 0.88 (m, 6H), 0.81 (d, J= 6.9 Hz, 3H).
Example 13: Synthesis of (lR,2S,5S)-N-[cyano(4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 110)
180
SUBSTITUTE SHEET RULE 26
Figure imgf000182_0001
Step 1: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(2,6-naphthyridin-4-yl)methyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000255] To a solution of (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (0.26 g, 705.63 umol, 1 eq) and 2-amino-2-(2,6-naphthyridin-4-yl)acetonitrile (129.97 mg, 705.63 umol, 1 eq) in DCM (5 mL) was added T3P (898.07 mg, 1.41 mmol, 839.31 uL, 50% purity, 2 eq) and then DIEA (273.59 mg, 2.12 mmol, 368.71 uL, 3 eq) was added. The resulting mixture was stirred at -30 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (8 mL) and extracted with DCM (4 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, di chloromethane: methanol = 10: 1) to give tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(2,6- naphthyridin-4-yl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2- dimethyl-propyl] carbamate (242 mg, 431.81 umol, 61.20% yield, 95.4% purity) as a yellow solid.
Step 2: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(2,6-naphthyridin-4- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000256] To a solution of tert-butyl N-[(l S)-l-[(lR,2S,5S)-2-[[cyano(2,6-naphthyridin-4- yl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (240 mg, 448.89 umol, 1 eq) in DCM (5 mL) was added TLA (3.08 g, 27.01
181
SUBSTITUTE SHEET RULE 26 mmol, 2 mL, 60.18 eq) at 25 °C, and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was poured into saturated NaHCCh (8 mL) and extracted with DCM (4 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl- butanoyl]-N-[cyano(2,6-naphthyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (190 mg, crude) as a yellow solid.
Step 3: ( 1 R,2S , 5 S)-N - [cyano(2,6-naphthyridin-4-y l)methy 1] -3 - [(2S)-3 , 3 -dimethyl-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000257] To a solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(2,6- naphthyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (190 mg, 437.25 umol, 1 eq) in DCM (8 mL) was added DIEA (169.53 mg, 1.31 mmol, 228.48 uL, 3 eq), and then TFAA (183.67 mg, 874.50 umol, 121.64 uL, 2 eq) was added drop-wise at 0 °C. The resulting mixture was stirred at 0 °C for 2 h. Upon completion, the reaction mixture was poured into saturated NaHCCh (6 mL) and extracted with DCM (3 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75 * 30 mm * 3 um; mobile phase: [water(NH4HCO3)-ACN]; B%: 30%-60%, 8 min) to give (1R,2S,5S)-N- [cyano(2,6-naphthyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (32 mg, 57.48 umol, 13.15% yield, 95.3% purity) as a yellow solid. MS (ESI) m/z 531.3 [M+H]+.
[000258] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.01 - 9.88 (m, 1H), 9.57 - 9.36 (m, 1H), 9.27 - 9.14 (m, 1H), 8.76 - 8.60 (m, 2H), 8.02 - 7.91 (m, 1H), 6.53 - 6.38 (m, 2H), 4.94 (s, 1H), 4.51 (s, 1H), 4.03 - 3.95 (m, 1H), 3.88 (br d, J= 10.8 Hz, 1H), 1.71 - 1.52 (m, 2H), 1.07 - 1.04 (m, 3H), 1.02 (s, 9H), 0.92 - 0.90 (m, 3H).
Example 14: Synthesis of (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 111)
182
SUBSTITUTE SHEET RULE 26
Figure imgf000184_0001
Step 1: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[(l-cyano-l-pyrazin-2-yl-ethyl)carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000259] To a solution of (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (696.32 mg, 1.89 mmol, 1 eq) and 2-amino-2-pyrazin-2-yl-propanenitrile (280 mg, 1.89 mmol, 1 eq) in ACN (10 mL) was added 1 -methylimidazole (543.06 mg, 6.61 mmol, 527.24 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (1.06 g, 3.78 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue, and then diluted with H2O 50 mL and extracted with ethyl acetate 75 mL (25 mL * 3). The combined organic layers were washed with brine 90 mL (30 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 5: 1 to 1:1) to give tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[(l-cyano-l-pyrazin- 2-yl-ethyl)carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (1.3 g, 1.83 mmol, 96.57% yield, 70% purity) as a white solid. MS (ESI) m/z 499.5 [M+H]+
Step 2: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-(l-cyano-l-pyrazin-2-yl-ethyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000260] A mixture of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[(l-cyano-l-pyrazin-2-yl- ethyl)carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-
183
SUBSTITUTE SHEET RULE 26 propyl] carbamate (1.08 g, 2.17 mmol, 1 eq) in DCM (14 mL) and TFA (7 mL) was stirred at 0 °C for 0.5 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-(l-cyano-l-pyrazin-2-yl-ethyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (0.863 g, crude) as a yellow oil. MS (ESI) m/z 399.5 [M+H]+
Step 3: (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000261] A mixture of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-(l-cyano-l- pyrazin-2-yl-ethyl) -6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (0.863 g, 2.17 mmol, 1 eq) in DCM (20 mL) was added DIPEA (559.78 mg, 4.33 mmol, 754.42 uL, 2 eq) and TFAA (682.27 mg, 3.25 mmol, 451.83 uL, 1.5 eq). The resulting mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 10 mL at 0 °C, and then extracted with DCM 20 mL (10 mL * 2). The combined organic layers were concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Welch Xtimate C18 250*70mm#10um;mobile phase: [water( NH4HCO3)- ACN];B%: 15%-50%,20min) to give (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (300 mg, purity 97.5%) as a white solid. MS (ESI) m/z 495.5 [M+l]+
Step 4: (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000262] The desired product was further separated by SFC (condition: column: DAICEL CHIRALPAK IC(250mm*30mm,10um);mobile phase: [0.1%NH3H2O IPA];B%: 33%- 33%,6min) to give (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (Isomer
1, 110 mg, 222.44 umol, 10.27% yield, 100% purity) as a white solid. MS (ESI) m/z 495.5 [M+l]+and (lR,2S,5S)-N-(l-cyano-l-pyrazin-2-yl-ethyl)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (Isomer
2, 160 mg, 323.55 umol, 14.94% yield, 100% purity) as a white solid. MS (ESI) m/z 495.5 [M+l]+
184
SUBSTITUTE SHEET RULE 26 [000263] Isomer 1 : 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.73 (s, 1H), 9.38 (br d, J= 6.7 Hz, 1H), 8.74 (d, J= 1.3 Hz, 1H), 8.68 - 8.59 (m, 2H), 4.42 (s, 1H), 4.35 (br d, J= 6.8 Hz, 1H), 3.87 (dd, J= 5.5, 10.3 Hz, 1H), 3.72 (d, J= 10.5 Hz, 1H), 1.84 (s, 3H), 1.62 (dd, J = 5.5, 7.5 Hz, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.06 (s, 3H), 0.92 (s, 12H)
[000264] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.51 (s, 1H), 9.34 (br s, 1H), 8.75 (s, 1H), 8.67 - 8.62 (m, 2H), 4.40 - 4.32 (m, 2H), 3.81 (dd, J = 5.4, 10.3 Hz, 1H), 3.66 (d, J = 10.5 Hz, 1H), 1.85 (s, 3H), 1.51 (dd, J= 5.4, 7.4 Hz, 1H), 1.41 (d, J= 7.6 Hz, 1H), 1.05 (s, 3H), 0.97 (s, 9H), 0.87 (s, 3H)
Example 15: Synthesis of (lR,2S,5S)-N-[l-cyano-l-(5-fhioro-3-pyridyl)ethyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 115)
Figure imgf000186_0001
Step 1: tert-butylN-[(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(5-fluoro-3-pyridyl)ethyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000265] To a solution of 2-amino-2-(5-fluoro-3-pyridyl)propanenitrile (250 mg, 1.51 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (501.95 mg, 1.36 mmol, 0.9 eq) in ACN (10 mb) was added 1 -methylimidazole (434.96 mg, 5.30 mmol, 422.29 uL, 3.5 eq and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (849.38 mg, 3.03
185
SUBSTITUTE SHEET RULE 26 mmol, 2 eq). The resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with EA 45 mL (15 mL * 3). The combined organic layers were washed with brine 20 mL (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 5: 1 to 1:1) to give tert-butyl N- [(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(5-fhioro-3-pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3-azabicy clo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate (550 mg, 1.01 mmol, 66.95% yield, 95% purity) as a yellow solid. MS (ESI) m/z 516.3 [M+H]+
Step 2: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[l-cyano-l-(5-fluoro-3- pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000266] A mixture of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(5-fluoro-3- pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (500 mg, 969.71 umol, 1 eq) in DCM (6 mL) and TFA (4.62 g, 40.52 mmol, 3.00 mL, 41.78 eq) was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl- butanoyl]-N-[l -cyano- l-(5-fluoro-3-pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (400 mg, crude) as a yellow oil. MS (ESI) m/z 416.3 [M+H]+
Step 3: (lR,2S,5S)-N-[l-cyano-l-(5-fhioro-3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000267] A mixture of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[l-cyano-l-(5- fluoro-3-pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (400 mg, 962.69 umol, 1 eq) in DCM (10 mL) was added DIEA (248.84 mg, 1.93 mmol, 335.37 uL, 2 eq), and then another mixture of TFAA (303.29 mg, 1.44 mmol, 200.86 uL, 1.5 eq) in DCM (1.5 mL) was added the former mixture and stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 5 mL at 0 °C, and then extracted with DCM 6 mL (6 mL * 1). The combined organic layers were concentrated under reduced pressure and purified by prep- HPLC (neutral condition; column: Welch Xtimate Cl 8 250*70mm#10um;mobile phase: [water( NH4HCO3)-ACN];B%: 35%-65%,20min) to give (lR,2S,5S)-N-[l-cyano-l-(5-fluoro-3- pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-
186
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, purity 94%) as a white solid. MS (ESI) m/z 512.3 [M+H]+
Step 4: (lR,2S,5S)-N-[l-cyano-l-(5-fluoro-3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000268] The desired product was further separated by SFC (condition: column: REGIS(S,S)WHELK-01(250mm*25mm,10um);mobile phase: [Neu-IPA];B%: 25%-25%,5min) to give (lR,2S,5S)-N-[l-cyano-l-(5-fluoro-3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (Isomer 1, 160 mg, 312.80 umol, 32.49% yield, 100% purity) as a white solid. MS (ESI) m/z 512.3 [M+H]+ and (lR,2S,5S)-N-[l-cyano-l-(5-fluoro-3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 2, 60 mg, 117.30 umol, 12.18% yield, 100% purity) as a white solid. MS (ESI) m/z 512.3 [M+H]+
[000269] Isomer 1 : 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.45 (s, 1H), 9.37 (br d, J= 8.0 Hz, 1H), 8.62 - 8.54 (m, 2H), 7.76 (td, J= 2.2, 9.8 Hz, 1H), 4.38 - 4.32 (m, 2H), 3.81 (dd, J= 5.4, 10.4 Hz, 1H), 3.65 (d, J= 10.4 Hz, 1H), 1.89 (s, 3H), 1.51 (dd, J= 5.4, 7.3 Hz, 1H), 1.34 (d, J = 7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.87 (s, 3H)
[000270] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.64 (s, 1H), 9.42 (br d, J= 7.9 Hz, 1H), 8.61 (s, 1H), 8.55 (d, J= 2.6 Hz, 1H), 7.70 (td, J= 2.2, 9.8 Hz, 1H), 4.40 - 4.32 (m, 2H), 3.86 (dd, J= 5.5, 10.4 Hz, 1H), 3.72 (d, J= 10.4 Hz, 1H), 1.88 (s, 3H), 1.62 (dd, J = 5.6, 7.3 Hz, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.05 (s, 3H), 0.91 (s, 12H)
187
SUBSTITUTE SHEET RULE 26 Example 16: Synthesis of (1R, 2S, 5S)-N-[l-(5-chloro-3-pyridyl)-l-cyano-ethyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 116)
Figure imgf000189_0001
Step 1 : 2-amino-2-(5-chloro-3 -pyridyl) propanenitrile
[000271] To a solution of l-(5-chloro-3-pyridyl)ethanone (250 mg, 1.61 mmol, 1 eq) in NHa/MeOH (10 mL) was added NH4CI (257.86 mg, 4.82 mmol, 3 eq), and the resulting mixture was stirred at 25 °C for 1 h. TMSCN (478.24 mg, 4.82 mmol, 603.07 uL, 3 eq) at 0 °C was added, and the mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NH MeOH. The reaction mixture was quenched by addition H2O 20 mL, and then extracted with DCM (15 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, petroleum ether: ethyl acetate = 1: 10 to 74:26) to give 2-amino-2-(5-chloro-3 -pyridyl) propanenitrile (460 mg, 1.24 mmol, 77.23% yield, 98% purity) (batch * 2) as a white solid. MS (ESI) m/z 182.0 [M+H]+.
Step 2: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-(5-chloro-3-pyridyl)-l-cyano-ethyl]carbamoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000272] A solution of 2-amino-2-(5-chloro-3-pyridyl)propanenitrile (430 mg, 2.37 mmol, 1 eq), (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-
188
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxylic acid (872.37 mg, 2.37 mmol, 1 eq) in DCM (25 mL) was added DMAP (578.48 mg, 4.74 mmol, 2 eq) and EDCI (680.80 mg, 3.55 mmol, 1.5 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 40 mL, and extracted with DCM (20 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:0 to 79:21) to give tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-(5-chloro-3- pyridyl)-l-cyano-ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2- dimethyl-propyl] carbamate (858 mg, 1.53 mmol, 64.71% yield, 95% purity) as a white solid. MS (ESI) m/z 532.3 [M+H]+.
Step 3: (1R, 2S, 5S)-3-[(2S)-2-amino-3, 3-dimethyl-butanoyl]-N-[l-(5-chloro-3-pyridyl)-l- cyano-ethyl]-6, 6-dimethyl-3 -azabicyclo [3.1.0] hexane-2-carboxamide
[000273] A solution of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-(5-chloro-3-pyridyl)-l-cyano- ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (858 mg, 1.61 mmol, 1 eq) in TFA (3 mL) and DCM (6 mL) was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by addition NaHCCh 30 mL, and then extracted with ethyl acetate (25 mL * 3). The combined organic layers were washed with brine 40 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue to give (1R, 2S, 5S)-3-[(2S)-2-amino-3, 3-dimethyl-butanoyl]-N-[l-(5-chloro-3-pyridyl)- l-cyano-ethyl]-6, 6-dimethy 1-3 -azabicyclo [3.1.0] hexane-2-carboxamide (690 mg, crude) as a white solid. MS (ESI) m/z 432.2 [M+H]+.
Step 4: (lR,2S,5S)-N-[l-(5-chloro-3-pyridyl)-l-cyano-ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000274] A solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[l-(5-chloro- 3-pyridyl)-l-cyano-ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (690 mg, 1.61 mmol, 1 eq) in DCM (8 mL) was added TFAA (542.24 mg, 2.58 mmol, 359.10 uL, 1.6 eq) and DIPEA (625.63 mg, 4.84 mmol, 843.17 uL, 3 eq) at 0 °C, and then the resulting mixture was stirred for 2 h. Upon completion, the reaction mixture was quenched by addition H2O 15 mL, and then extracted with DCM mL (20 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a
189
SUBSTITUTE SHEET RULE 26 residue. The residue was purified by prep-HPLC (column: C18 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-60%, 10 min) to give (lR,2S,5S)-N-[l-(5-chloro-3- pyridyl)-l-cyano-ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 1, 85.33 mg, 160.00 umol, 9.92% yield, 99% purity) as a white solid. MS (ESI) m/z 528.2 [M+H]+ and 1R, 2S, 5S)-N-[l-(5-chloro- 3-pyridyl)-l-cyano-ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 2, 250.42 mg, 474.31 umol, 29.39% yield, 100% purity) as a white solid. MS (ESI) m/z 528.2 [M+H]+.
[000275] Isomer 1 : 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.60 (s, 1H), 9.38 (br d, J= 8.2 Hz, 1H), 8.67 - 8.58 (m, 2H), 7.88 (t, J= 2.1 Hz, 1H), 4.38 - 4.33 (m, 2H), 3.85 (dd, J= 5.3, 10.3 Hz, 1H), 3.70 (d, J= 10.5 Hz, 1H), 1.89 (s, 3H), 1.61 (dd, J = 5.5, 7.2 Hz, 1H), 1.36 (d, J = 7.7 Hz, 1H), 1.05 (s, 3H), 0.93 - 0.88 (m, 12H).
[000276] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.43 (s, 1H), 9.35 (br d, J= 7.0 Hz, 1H), 8.63 (dd, J= 2.2, 3.7 Hz, 2H), 7.93 (t, J= 2.2 Hz, 1H), 4.39 - 4.31 (m, 2H), 3.82 (dd, J = 5.5, 10.4 Hz, 1H), 3.65 (d, J= 10.5 Hz, 1H), 1.89 (s, 3H), 1.51 (dd, J = 5.3, 7.5 Hz, 1H), 1.29 (d, J= 7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.86 (s, 3H).
Example 17: Synthesis of (lR,2S,5S)-N-[l-cyano-l-(3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 117)
Figure imgf000191_0001
190
SUBSTITUTE SHEET RULE 26 Step 1: 2-amino-2-(3-pyridyl)propanenitrile
[000277] To a solution of l-(3-pyridyl)ethanone (2 g, 16.51 mmol, 1.82 mL, 1 eq) in NHa/MeOH (10 mL) was added NH4CI (2.65 g, 49.53 mmol, 3 eq), and the resluting mixture was stirred at 25 °C for 1 h. TMSCN (4.91 g, 49.53 mmol, 6.20 mL, 3 eq) was added at 0 °C. The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with water (30 mL) and extracted with DCM (15 mL * 6). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to and was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 2: 1 to 1 : 1 to 0: 1) to give the 2-amino-2- (3-pyridyl)propanenitrile (1.1 g, 6.28 mmol, 38.03% yield, 84% purity) as yellow oil. MS (ESI) m/z 148.2 [M+H]+
Step 2: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(3-pyridyl)ethyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000278] To a solution of 2-amino-2-(3-pyridyl)propanenitrile (200 mg, 1.36 mmol, 1 eq) in ACN (2 mL) was added (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (500.71 mg, 1.36 mmol, 1 eq), and then 1 -methylimidazole (390.50 mg, 4.76 mmol, 379.13 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (762.56 mg, 2.72 mmol, 2 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was quenched by addition H2O (15 mL) and extracted with DCM (10 mL * 6). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 2:1 to 1 : 1) to give the product tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(3-pyridyl)ethyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate (460 mg, 924.39 umol, 68.02% yield) as white solid. MS (ESI) m/z 498.3 [M+H]+
Step 3: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[l-cyano-l-(3-pyridyl)ethyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[000279] To a solution of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[l-cyano-l-(3- pyridyl)ethyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (460 mg, 924.39 umol, 1 eq) in DCM (10 mL) was added TFA (7.70 g, 67.53
191
SUBSTITUTE SHEET RULE 26 mmol, 5 mL, 73.06 eq). The mixture was stirred at 0 °C for 1.5 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2-amino-3,3- dimethyl-butanoyl]-N-[l-cyano-l-(3-pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (350 mg, crude) as yellow oil. MS (ESI) m/z 398.3 [M+H]+
Step 4: (lR,2S,5S)-N-[l-cyano-l-(3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000280] To a solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[l-cyano-l- (3-pyridyl)ethyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (350 mg, 880.48 umol, 1 eq) in DCM (15 mL) was added DIEA (398.27 mg, 3.08 mmol, 536.76 uL, 3.5 eq) and TFAA (295.88 mg, 1.41 mmol, 195.95 uL, 1.6 eq). The mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by addition NaHCCh (30 mL) and extracted with DCM (8 mL * 6) and blow-dried with N2 and purifited by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40mm * lOum; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 8 min) to give the product (lR,2S,5S)-N-[l-cyano-l-(3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (110 mg, 218.43 umol, 24.81% yield, 98% purity) as white solid. MS (ESI) m/z 494.3 [M+H]+
Step 5: (lR,2S,5S)-N-[l-cyano-l-(3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000281] The (lR,2S,5S)-N-[l-cyano-l-(3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (0.11 g, 222.89 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 36%-36%, 10 min) to afford (1R,2S,5S)-N-[1- cyano-l-(3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 1, 13 mg, 25.55 umol, 11.46% yield, 97% purity) as a white solid. MS (ESI) m/z 494.1 [M+H]+ and (lR,2S,5S)-N-[l-cyano-l- (3-pyridyl)ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 2, 65 mg, 117.86 umol, 40.62% yield, 100% purity) as white solid. MS (ESI) m/z 494.1 [M+H]+
[000282] Isomer 1 : 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.53 (s, 1H), 9.35 (br d, J = 6.7 Hz, 1H), 8.69 (d, J= 2.4 Hz, 1H), 8.51 (d, J= 3.9 Hz, 1H), 7.84 (br d, J= 8.1 Hz, 1H), 7.30 (dd, J =
192
SUBSTITUTE SHEET RULE 26 4.8, 8.1 Hz, 1H), 4.40 (s, 1H), 4.34 (br d, J= 6.6 Hz, 1H), 3.85 (dd, 7 = 5.4, 10.3 Hz, 1H), 3.71 (br d, J= 10.4 Hz, 1H), 1.85 (s, 3H), 1.63 - 1.57 (m, 1H), 1.36 (d, J= 7.6 Hz, 1H), 1.05 (s, 3H), 0.91 (s, 9H), 0.90 (br s, 3H).
[000283] Isomer 2: 'H NMR (400 MHz, DMSO-t76) 5 = 9.38 (s, 1H), 9.32 (d, J= 8.2 Hz, 1H), 8.66 (d, J= 2.4 Hz, 1H), 8.55 (dd, J= 1.4, 4.7 Hz, 1H), 7.88 - 7.80 (m, 1H), 7.46 (dd, J = 4.8, 8.1 Hz, 1H), 4.38 - 4.31 (m, 2H), 3.84 - 3.76 (m, 1H), 3.66 (d, J= 10.5 Hz, 1H), 1.86 (s, 3H), 1.50 (dd, J= 5.4, 7.5 Hz, 1H), 1.30 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 0.98 (s, 9H), 0.87 (s, 3H).
Example 18: Synthesis of (lR,2S,5S)-N-[cyano(lH-pyrrolo[2,3-c]pyridin-4-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 136)
Figure imgf000194_0001
Step 1: 2-amino-2-(lH-pyrrolo[2,3-c]pyridin-4-yl)acetonitrile
[000284] To a solution of lH-pyrrolo[2,3-c]pyridine-4-carbaldehyde (400 mg, 2.74 mmol, 1 eq) in NHLMeOH (20 mL) (7M) was added NH4CI (292.81 mg, 5.47 mmol, 2 eq). After 2 hours, TMSCN (543.05 mg, 5.47 mmol, 684.81 uL, 2 eq) was added drop-wise, and the mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NH MeOH. The residue was diluted with H2O 30 mL and extracted with EA (30 mL * 6). The combined organic layers were dried over Na2SO4, filtered and
193
SUBSTITUTE SHEET RULE 26 concentrated under reduced pressure to give 2-amino-2-(lH-pyrrolo[2,3-c]pyridin-4- yl)acetonitrile (400 mg, crude) as a yellow solid. MS (ESI) m/z 173.1 [M+H]+.
Step 2: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(lH-pyrrolo[2,3-c]pyridin-4- yl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate
[000285] A solution of 2-amino-2-(lH-pyrrolo[2,3-c]pyridin-4-yl)acetonitrile (400 mg, 2.09 mmol, 90% purity, 1 eq) in ACN (20 mL) was cooled to 0 °C, and then 1 -methylimidazole (686.64 mg, 8.36 mmol, 666.64 uL, 4 eq) was added. (lR,2S,5S)-3-[(2S)-2-(tert- butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (770.37 mg, 2.09 mmol, 1 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (703.95 mg, 2.51 mmol, 1.2 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 4 h. Upon completion, the reaction mixture was quenched by addition H2O 100 mL at 0 °C, and then extracted with EA (50 mL * 3). The combined organic layers were washed with brine 60 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, ethyl acetate: petroleum ether = 1:0 to 0: 1) to afford tert-butyl N-[(lS)-l-[(lR,2S,5S)-2- [[cyano(lH-pyrrolo[2,3-c]pyridin-4-yl)methyl]carbamoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate (600 mg, 1.03 mmol, 49.42% yield, 90% purity) as a yellow solid. MS (ESI) m/z 523.3 [M+H]+.
Step 3: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(lH-pyrrolo[2,3- c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000286] A solution of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(lH-pyrrolo[2,3-c]pyridin- 4-yl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (420 mg, 803.62 umol, 1 eq) in DCM (9 mL) was cooled to 0 °C, and then TFA (4.85 g, 42.54 mmol, 3.15 mL, 52.94 eq) was added drop-wise. The mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by addition NaHCCh 40 mL at 0 °C, and then extracted with EA (20 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(lH-pyrrolo[2,3-c]pyridin-4-
194
SUBSTITUTE SHEET RULE 26 yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (280 mg, crude) as a yellow solid. MS (ESI) m/z 423.3 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano(lH-pyrrolo[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000287] To a solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(lH- pyrrolo[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (280 mg, 662.69 umol, 1 eq) in MeOH (8 mL) was added drop-wise Et3N (201.17 mg, 1.99 mmol, 276.72 uL, 3 eq), and then methyl 2,2,2-trifluoroacetate (848.57 mg, 6.63 mmol, 668.17 uL, 10 eq) was added drop- wise. The resluting mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75 * 30 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 8 mm) to afford (lR,2S,5S)-N-[cyano(lH-pyrrolo[2,3- c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (79.24 mg, 152.82 umol, 23.06% yield, 100% purity) as a yellow solid. MS (ESI) m/z 519.2 [M+H]+.
[000288] 'H NMR (400 MHz, DMSO-t/r,) 5 = 11.87 (br d, J= 9.5 Hz, 1H), 9.47 (t, J= 7.3 Hz, 1H), 9.38 (br s, 1H), 8.80 (s, 1H), 8.24 (d, J= 8.3 Hz, 1H), 7.76 - 7.59 (m, 1H), 6.70 - 6.58 (m, 1H), 6.48 (d, J= 7.8 Hz, 1H), 4.52 - 4.38 (m, 1H), 4.30 (d, J= 5.5 Hz, 1H), 3.91 (br d, J = 5.4 Hz, 1H), 3.78 - 3.64 (m, 1H), 1.60 - 1.47 (m, 1H), 1.36 - 1.10 (m, 1H), 1.03 - 0.87 (m, 12H), 0.82 (d, J = 5.5 Hz, 3H).
195
SUBSTITUTE SHEET RULE 26 Example 19: Synthesis of (1R, 2S, 5S)-N-[cyano(isoxazol-3-yl)methyl]-3-[(2S)-3,3-dimethyl-
2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 141)
Figure imgf000197_0001
Step 1: 2-amino-2-isoxazol-3-yl-acetonitrile
[000289] A solution of isoxazole-3-carbaldehyde (500 mg, 5.15 mmol, 1 eq) in NHa/MeOH (7 M, 5 mL, 6.80 eq) was added NH4CI (551.05 mg, 10.30 mmol, 2 eq) at 25 °C. After the addition, the mixture was stirred at 25 °C for 2 h, and then TMSCN (1.02 g, 10.30 mmol, 1.29 mL, 2 eq) was added. The resulting mixture was stirred at 25 °C for 12 h. Upon completion, the combined concentrated under reduced pressure to give a residue. The residue was diluted with water (15 mL) and extracted with ethyl acetate (10 mL * 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 10:1 to 0:1) to afford 2- amino-2-isoxazol-3-yl-acetonitrile (108 mg, 877.25 umol, 17.03% yield) as a yellow oil.
Step 2: tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(isoxazol-3-yl)methyl]carbamoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl-propyl]carbamate
[000290] A solution of 2-amino-2-isoxazol-3-yl-acetonitrile (108 mg, 877.25 umol, 1.2 eq) in ACN (3 mL) was added (lR,2S,5S)-3-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (269.36 mg, 731.04 umol, 1 eq) followed by 1 -methylimidazole (210.07 mg, 2.56 mmol, 203.96 uL, 3.5 eq).
196
SUBSTITUTE SHEET RULE 26 [Chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (246.14 mg, 877.25 umol, 1.2 eq) was added, and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (5 mL * 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 0:1) to give tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(isoxazol-3- yl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (115 mg, 242.84 umol, 33.22% yield) as a yellow oil.
Step 3: (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N-[cyano(isoxazol-3-yl)methyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000291] A solution of tert-butyl N-[(lS)-l-[(lR,2S,5S)-2-[[cyano(isoxazol-3- yl)methyl]carbamoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-3-carbonyl]-2,2-dimethyl- propyl] carbamate (90 mg, 190.05 umol, 1 eq) in DCM (1 mL) was added TFA (770.00 mg, 6.75 mmol, 0.5 mL, 35.53 eq). The mixture was stirred at 25 °C for 0.5 h. Upon completion, the reaction mixture is blow-dried with nitrogen to give (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl- butanoyl]-N-[cyano(isoxazol-3-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (71 mg, crude, TFA) as a yellow oil.
Step 4: (lR,2S,5S)-N-[cyano(isoxazol-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000292] A solution of (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-N- [cyano(isoxazol-3-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (70 mg, 187.44 umol, 1 eq) in DCM (1 mL) was added DIEA (48.45 mg, 374.88 umol, 65.30 uL, 2 eq) and then TFAA (31.49 mg, 149.95 umol, 20.86 uL, 0.8 eq) was added drop-wise in DCM (0.5 mL). The mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (column: Phenomenex C18 100 * 30 mm * 10 um; mobile phase: [water(NH4HCO3)- ACN]; B%: 35%-65%, 10 min to give (lR,2S,5S)-N-[cyano(isoxazol-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (11.73 mg, 24.99 umol, 14.33% yield) as yellow oil. MS (ESI) m/z 470.1 [M+H]+
197
SUBSTITUTE SHEET RULE 26 [000293] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (t, J= 8.1 Hz, 1H), 9.45 - 9.21 (m, 1H), 9.01 (dd, J= 1.6, 7.6 Hz, 1H), 6.61 (dd, J= 1.7, 18.8 Hz, 1H), 6.51 - 6.39 (m, 1H), 4.40 (d, J = 2.2 Hz, 1H), 4.26 (d, J= 17.4 Hz, 1H), 3.96 - 3.85 (m, 1H), 3.70 (br d, J= 10.3 Hz, 1H), 1.61 - 1.52 (m, 1H), 1.36 (dd, J= 7.6, 19.9 Hz, 1H), 1.05 - 0.98 (m, 12H), 0.85 (d, J= 3.1 Hz, 3H)
Example 20: Synthesis of (lR,2S,5S)-N-[cyano(lH-pyrazol-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 138)
Figure imgf000199_0001
Step 1: 2-amino-2-(lH-pyrazol-4-yl)acetonitrile
[000294] To a solution of lH-pyrazole-4-carbaldehyde (400 mg, 4.16 mmol, 1 eq) in NHa/MeOH (7M, 5 mL) was added NH4CI (445.36 mg, 8.33 mmol, 2 eq), and the mixture was stirred at 25 °C for 1.5 h. TMSCN (825.97 mg, 8.33 mmol, 1.04 mL, 2 eq) was added, and the mixture was stirred at 25 °C for 1.5 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL) at 0 °C, and then extracted with EA (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(lH-pyrazol-4-yl)acetonitrile (340 mg, crude) as a white solid. MS (ESI) m/z 123.2 [M+H]+
Step 2: (lR,2S,5S)-N-[cyano(lH-pyrazol-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000295] A solution of 2-amino-2-(lH-pyrazol-4-yl)acetonitrile (200 mg, 1.60 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (584.75 mg, 1.60 mmol, 1 eq) in ACN (8 mL) was added with 1 -methylimidazole (527.04 mg, 6.42 mmol, 511.69 uL, 4 eq), and then [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (540.35 mg, 1.93 mmol, 1.2 eq) was added at 0 °C. The mixture was stirred at 25 °C for 2 h. Upon completion, the
198
SUBSTITUTE SHEET RULE 26 reaction mixture was quenched by addition H2O (20 mL) at 0 °C, and extracted with EA (15 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (column: Phenomenex Luna Cl 8 75 * 30 mm * 3 um; mobile phase: [water(LA)-ACN]; B%: 35%-65%, 8 min) to give (lR,2S,5S)-N-[cyano(lH-pyrazol-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Isomer 1, 24.70 mg, 52.72 umol, 3.29% yield, 100% purity) as a white solid, MS (ESI) m/z 469.2 [M+H]+, and (lR,2S,5S)-N-[cyano(lH-pyrazol-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Isomer 2, 30.02 mg, 64.08 umol, 3.99% yield, 100% purity) as a white solid. MS (ESI) m/z 469.2 [M+H]+.
[000296] Isomer 1 : 'H NMR (400 MHz, DMSO-t/r,) 5 = 13.60 - 12.61 (m, 1H), 9.37 (br d, J = 8.2 Hz, 1H), 9.26 - 9.18 (m, 1H), 7.73 (br d, J= 8.2 Hz, 2H), 6.09 - 5.96 (m, 1H), 4.40 (d, J = 8.2 Hz, 1H), 4.27 (s, 1H), 3.90 (dd, J= 5.3, 10.3 Hz, 1H), 3.69 (d, J= 10.5 Hz, 1H), 1.52 (dd, J =
5.4, 7.5 Hz, 1H), 1.35 - 1.25 (m, 1H), 1.02 - 0.98 (m, 12H), 0.86 - 0.83 (m, 3H).
[000297] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 13.95 - 12.34 (m, 1H), 9.36 (br d, J = 8.1 Hz, 1H), 9.24 (d, J= 7.6 Hz, 1H), 7.72 (br s, 2H), 6.07 (d, J= 7.5 Hz, 1H), 4.41 (d, J= 8.3 Hz, 1H), 4.26 (s, 1H), 3.91 (dd, J= 5.4, 10.3 Hz, 1H), 3.71 (br d, J= 10.4 Hz, 1H), 1.57 (dd, J =
5.5, 7.5 Hz, 1H), 1.40 - 1.31 (m, 1H), 1.05 - 0.97 (m, 12H), 0.87 - 0.83 (m, 3H).
Example 21: Synthesis of (lR,2S,5S)-N-(cyano(thiazol-5-yl)methyl)-3-((S)-3,3-dimethyl-2- (2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (Compound 139)
Figure imgf000200_0001
199
SUBSTITUTE SHEET RULE 26 Step 1: 2-amino-2-(thiazol-5-yl)acetonitrile
[000298] A solution of thiazole-5-carbaldehyde (500 mg, 4.42 mmol, 1 eq) in DCM (10 mL) was added Ti(i-PrO)4 (1.88 g, 6.63 mmol, 1.96 mL, 1.5 eq) and NHVMeOH (7 M, 2.53 mL, 4 eq), and the resulting mixture was stirred at 25 °C for 1 h. TMSCN (1.32 g, 13.26 mmol, 1.66 mL, 3 eq) was added at 0 °C, and then the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was quenched by addition H2O 40 mL, and then extracted with DCM (30 mL * 5). The combined organic layers were washed with brine (30 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(thiazol-5-yl) acetonitrile (380 mg, crude) as a brown solid.
Step 2 : (lR,2S,5S)-N-(cyano(thiazol-5-yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000299] To a solution of 2-amino-2-(thiazol-5-yl)acetonitrile (150 mg, 646.65 umol, 60% purity, 1 eq) in DCM (4 mL) was added (lR,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxylic acid (235.61 mg, 646.65 umol, 1 eq), DMAP (237.00 mg, 1.94 mmol, 3 eq) and EDCI (247.93 mg, 1.29 mmol, 2 eq), and then the mixture was stirred at 25 °C for 2 h. The resulting mixture was stirred for another 14 h at 25 °C. Upon completion, the reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC column: Cl 8-1 150 * 30 mm * 5 um; mobile phase: [water (FA)-ACN]; B%: 35%-65%, 10 min, which was further separated by column: DAI CEL CHIRALPAK AD(250 mm * 30 mm, 10 um); mobile phase: [0.1%NH3H2O EtOH]; B%: 10%-10%, 15 min to give (lR,2S,5S)-N-(cyano(thiazol-5- yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 1, 21.23 mg, 43.73 umol, 6.76% yield, 100% purity) as a white solid (MS (ESI) m/z 486.1 [M+H]+) and (lR,2S,5S)-N-(cyano(thiazol-5- yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide ((Isomer 2, 16.10 mg, 33.16 umol, 5.13% yield, 100% purity) as a white solid (MS (ESI) m/z 486.1 [M+H]+).
[000300] Isomer 1 : 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.70 - 9.60 (m, 1H), 9.42 - 9.32 (m, 1H), 9.14 (dd, J= 0.6, 2.6 Hz, 1H), 7.99 (d, J= 3.4 Hz, 1H), 6.62 - 6.53 (m, 1H), 4.39 (d, J= 8.1
200
SUBSTITUTE SHEET RULE 26 Hz, 1H), 4.24 (d, J= 2.2 Hz, 1H), 3.96 - 3.87 (m, 1H), 3.73 - 3.59 (m, 1H), 1.61 - 1.52 (m, 1H), 1.36 - 1.24 (m, 1H), 1.04 - 0.98 (m, 12H), 0.89 - 0.84 (m, 3H).
[000301] Isomer 2: 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.63 (dd, J= 3.4, 7.6 Hz, 1H), 9.47 - 9.31 (m, 1H), 9.21 - 9.07 (m, 1H), 7.99 (d, J= 3.4 Hz, 1H), 6.58 (dd, J= 8.1, 10.0 Hz, 1H), 4.39 (br d, J= 7.6 Hz, 1H), 4.25 (d, J= 2.2 Hz, 1H), 4.00 - 3.86 (m, 1H), 3.77 - 3.65 (m, 1H), 1.63 - 1.51 (m, 1H), 1.25 (br d, J= 7.7 Hz, 1H), 1.04 - 0.95 (m, 12H), 0.85 (d, J= 3.5 Hz, 3H)
Example 22: Synthesis of (1R, 2S, 5S)-N-[cyano(l,7-naphthyridin-5-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 148)
Figure imgf000202_0001
Step 1: 5-vinyl-l,7-naphthyridine
[000302] A mixture of trifluoro-potassio-vinyl-boron(l-) (1.28 g, 9.56 mmol, 2.00 eq) and 5- bromo-l,7-naphthyridine (1 g, 4.78 mmol, 1 eq) in H2O (10 mb) and dioxane (40 mL) was added CS2CO3 (4.68 g, 14.36 mmol, 3.00 eq), and then cyclopentyl(diphenyl)phosphane; dichloromethane; dichloropalladium; iron (391 mg, 478.79 umol, 0.1 eq) were added. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O 30 mL, and then extracted with EA (15 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, Petroleum
201
SUBSTITUTE SHEET RULE 26 ether:Ethyl acetate = 1:0 to 44:56) to give 5-vinyl-l,7-naphthyridine (385 mg, 2.39 mmol, 50.02% yield, 97% purity) as a yellow oil. MS (ESI) m/z 157.1 [M+H]+
Step 2: l,7-naphthyridine-5-carbaldehyde
[000303] A mixture of 5-vinyl-l,7-naphthyridine (385 mg, 2.47 mmol, 1 eq) in H2O (5 mL) and dioxane (25 mL) was added K2OSO4.2H2O (181.65 mg, 493.01 umol, 0.2 eq) and 2,6- dimethyl(l 15N)pyridine (533.17 mg, 4.93 mmol, 2 eq). The resluting mixture was stirred at 25 °C for 30 min, and then NalCh (2.64 g, 12.33 mmol, 682.97 uL, 5 eq) was added to the mixture at 0 °C, and stirred at 25 °C for 15 hr. Upon completion, the reaction mixture was quenched by addition 20 mL (H2O: EA = 1:1), and then stirred at 25 °C for 10 min. The mixture was extracted with ethyl acetate (15 mL * 3), washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:0 to 42:58) to give a 1,7- naphthyridine-5-carbaldehyde (280 mg, 1.58 mmol, 63.92% yield, 89% purity) as a white solid. MS (ESI) m/z 159.0 [M+H]+.
Step 3: 2-amino-2-(l,7-naphthyridin-5-yl)acetonitrile
[000304] A solution of l,7-naphthyridine-5-carbaldehyde (200 mg, 1.26 mmol, 1 eq) in DCM (5 mL) was added NEh/MeOH (7 M, 3.63 mL, 20.09 eq) and Ti(i-PrO)4 (539.11 mg, 1.90 mmol, 559.83 uL, 1.5 eq) was stirred at 25 °C for 1 hr, and then TMSCN (376.36 mg, 3.79 mmol, 474.60 uL, 3 eq) was added to the mixture at 0 °C. The resulting mixture was stirred at 25 °C for 14 hr. Upon completion, the reaction mixture was quenched by addition H2O 30 mL, and then extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine 20 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue to give a 2-amino-2-(l,7-naphthyridin-5-yl)acetonitrile (483 mg, crude) as a yellow solid. MS (ESI) m/z 185.1 [M+H]+
Step 4: (lR,2S,5S)-N-[cyano(l,7-naphthyridin-5-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000305] A solution of 2-amino-2-(l,7-naphthyridin-5-yl)acetonitrile (483 mg, 2.62 mmol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (955.42 mg, 2.62 mmol, 1 eq) in ACN (10 mL) was
202
SUBSTITUTE SHEET RULE 26 added 1 -methylimidazole (753.52 mg, 9.18 mmol, 731.57 uL, 3.5 eq) [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (1.47 g, 5.24 mmol, 2 eq), and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 15 mL, and then extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine 20 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80 * 40 mm * 3 um; mobile phase: [water( NH4HCO3)- ACN]; B%: 25%-45%, 8 min) to give (lR,2S,5S)-N-[cyano(l,7-naphthyridin-5-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (36 mg, 66.97 umol, 2.55% yield, 98.7% purity) as a white solid.
[000306] 'H NMR (400 MHz, DMSO- e) 5 = 9.67 - 9.50 (m, 1H), 9.49 - 9.47 (m, 1H), 9.41 - 9.30 (m, 1H), 9.19 - 9.11 (m, 1H), 8.87 - 8.78 (m, 1H), 8.50 - 8.35 (m, 1H), 7.96 - 7.74 (m, 1H), 7.07 - 6.90 (m, 1H), 4.42 - 4.32 (m, 1H), 4.23 - 4.14 (m, 1H), 3.97 - 3.87 (m, 1H), 3.74 - 3.63 (m, 1H), 1.64 - 1.52 (m, 1H), 1.37 - 1.14 (m, 1H), 1.03 - 0.87 (m, 12H), 0.84 - 0.78 (m, 3H).
Example 23: Synthesis of (lR,2S,5S)-N-[cyano(lH-indazol-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 162)
Figure imgf000204_0001
Step 1: 2-amino-2-(lH-indazol-4-yl)acetonitrile
[000307] To lH-indazole-4-carbaldehyde (500 mg, 3.42 mmol, 1 eq) and TMSCN (339.41 mg, 3.42 mmol, 428.01 uL, 1 eq) was added NH4CI (549.02 mg, 10.26 mmol, 3 eq) and NH MeOH (7 M, 5 mL, 10.23 eq). The mixture was stirred at 25 °C for 4 h. Upon completion, the reaction mixture was partitioned between H2O 20 mL and EtOAc 10 mL. The water phase was separated, washed with EtOAc 50 mL (10 mL * 5), dried over Na2SO4, filtered and
203
SUBSTITUTE SHEET RULE 26 concentrated under reduced pressure to give 2-amino-2-(lH-indazol-4-yl)acetonitrile (600 mg, crude) as a gray solid. MS (ESI) m/z 173.0 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano(lH-indazol-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000308] To a solution of 2-amino-2-(lH-indazol-4-yl)acetonitrile (50 mg, 290.38 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (105.80 mg, 290.38 umol, 1 eq) in DCM (5 m ) was added HATU (220.82 mg, 580.77 umol, 2 eq) and DIEA (112.59 mg, 871.15 umol, 151.74 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction solution was dried over N2 to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75 * 30 mm * 3 um; mobile phase: [water(FA)-ACN];B%: 30%-60%, 8 min) to give desired compound (60 mg, purity 92%) as a white solid, which was further separated by SFC (column: DAICEL CHIRALPAK AD(250 mm * 30 mm, 10 um); mobile phase: [0.1%NH3H2O ETOH]; B%: 20%-20%, 9 min). Then the seperated two peaks were combined and lyophilized to give (lR,2S,5S)-N-[cyano(lH-indazol-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (28 mg, 54.00 umol, 18.60% yield, 100% purity) as a white solid. MS (ESI) m/z 519.3 [M+H]+.
[000309] 1H NMR (400MHz, DMSO-t76) 5 = 13.37 - 13.22 (m, 1H), 9.54 (dd, J= 7.9, 14.4 Hz, 1H), 9.40 (br t, J = 7.2 Hz, 1H), 8.22 - 8.06 (m, 1H), 7.61 (d, J= 8.3 Hz, 1H), 7.47 - 7.36 (m, 1H), 7.34 - 7.25 (m, 1H), 6.60 (dd, J= 7.8, 18.9 Hz, 1H), 4.46 - 4.38 (m, 1H), 4.31 (d, J= 7.5 Hz, 1H), 3.92 (dt, J= 5.4, 11.0 Hz, 1H), 3.77 - 3.64 (m, 1H), 1.64 - 1.51 (m, 1H), 1.34 (d, J= 7.6 Hz, 1H), 1.06 - 0.95 (m, 12H), 0.83 (d, J= 5.5 Hz, 3H).
204
SUBSTITUTE SHEET RULE 26 Example 24: Synthesis of (lR,2S,5S)-N-[cyano(tetrahydropyran-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 164)
Figure imgf000206_0001
Step 1: 2-amino-2-tetrahydropyran-3-yl-acetonitrile
[000310] A solution of tetrahydropyran-3-carbaldehyde (600 mg, 5.26 mmol, 1 eq) in NHVMeOH (10 mL) was added NH4CI (843.55 mg, 15.77 mmol, 3 eq) and the mixture was stirred at 25 °C for 1 h, then the mixture was added TMSCN (1.56 g, 15.77 mmol, 1.97 mL, 3 eq) and stirred at 25 °C for 15 h. Upon completion, the mixture was concentrated in the vacuum, quenched by H2O (50 mL) and extracted with DCM (30 mL * 3). The combined organic phase was washed with brine (100 mL), dried with anhydrous Na2SO4, filtered, concentrated in vacuum and purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:4 to 0: 1) to give 2-amino-2-tetrahydropyran-3-yl-acetonitrile (380 mg, 2.71 mmol, 51.57% yield) as yellow oil. MS (ESI) m/z 141.1 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano(tetrahydropyran-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000311] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (486.05 mg, 1.33 mmol, 1.1 eq) in DCM (5 mL) was added HATU (691.66 mg, 1.82 mmol, 1.5 eq), and then 2-amino-2-tetrahydropyran-3-yl-acetonitrile (170 mg, 1.21 mmol, 1 eq) followed by DIEA (470.20 mg, 3.64 mmol, 633.69 uL, 3 eq). The resulting mixture was stirred at 25 °C for 2 h. Upon completion, the reaction was quenched by H2O (20 mL), and was extracted with DCM (20 mL * 3). The combined organic phase was washed with brine (20 mL), dried with anhydrous Na2SO4, filtered, concentrated in vacuum and purified by prep-HPLC (column: Phenomenex C18 80*40mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-
205
SUBSTITUTE SHEET RULE 26 50%,8min) to give (lR,2S,5S)-N-[cyano(tetrahydropyran-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 369.97 umol, 30.51% yield, 100% purity) as white solid. MS (ESI) m/z 487.2 [M+H]+.
[000312] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.38 (br s, 1H), 9.04 - 8.83 (m, 1H), 4.95 - 4.63 (m, 1H), 4.45 - 4.35 (m, 1H), 4.26 - 4.17 (m, 1H), 3.94 - 3.61 (m, 4H), 3.42 - 3.33 (m, 1H), 3.30 - 3.02 (m, 1H), 2.05 - 1.78 (m, 2H), 1.64 - 1.24 (m, 5H), 1.06 - 0.94 (m, 12H), 0.91 - 0.81 (m, 3H)
[000313] 'H NMR (400 MHz, DMSO+D2O-t76) 5 = 4.91 - 4.61 (m, 1H), 4.39 - 4.34 (m, 1H), 4.21 - 4.17 (m, 1H), 3.92 - 3.61 (m, 4H), 3.40 - 3.04 (m, 2H), 2.03 - 1.76 (m, 2H), 1.59 - 1.20 (m, 5H), 1.03 - 0.92 (m, 12H), 0.90 - 0.80 (m, 3H)
Example 25: Synthesis of (lR,2S,5S)-N-[cyano(tetrahydrofuran-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 165)
Figure imgf000207_0001
Step 1: 2-amino-2-tetrahydrofuran-3-yl-acetonitrile
[000314] A solution of tetrahydrofuran-3-carbaldehyde (1 g, 9.99 mmol, 900.90 uL, 1 eq) in NH MeOH (7 M, 3 mL) was added NH4CI (1.60 g, 29.97 mmol, 3 eq). The mixture was stirred at 25 °C for 1 h. Then TMSCN (1.98 g, 19.98 mmol, 2.50 mL, 2 eq) was added at 25 °C. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 3: 1 to 0: 1) to give the product 2-amino-2-tetrahydrofuran- 3-yl-acetonitrile (800 mg, 6.34 mmol, 63.49% yield) as a yellow oil. MS (ESI) m/z 127.3 [M+H]+
Step 2: (lR,2S,5S)-N-[cyano(tetrahydrofuran-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
206
SUBSTITUTE SHEET RULE 26 [000315] To a solution of 2-amino-2-tetrahydrofuran-3-yl-acetonitrile (20 mg, 158.53 umol, 1 eq) in DCM (2 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (115.53 mg, 158.53 umol, 50% purity, 1 eq), then added HATU (90.42 mg, 237.80 umol, 1.5 eq) and DIEA (61.47 mg, 475.60 umol, 82.84 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with DCM (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S 1O2, Petroleum ether:Ethyl acetate = 10: 1 to 0: 1) to give the product (lR,2S,5S)-N-[cyano(tetrahydrofuran-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (2.71 mg, 183.85 umol, 11.60% yield, 97.60% purity) as a white solid. MS (ESI) m/z 473.3 [M+H]+
[000316] 'H NMR (400 MHz, MeOD- /4) 5 = 4.84 - 4.68 (m, 1H), 4.62 - 4.49 (m, 1H), 4.40 - 4.25 (m, 1H), 4.07 - 3.60 (m, 6H), 2.87 - 2.71 (m, 1H), 2.27 - 2.05 (m, 1H), 1.93 - 1.75 (m, 1H), 1.68 - 1.58 (m, 1H), 1.44 - 1.35 (m, 1H), 1.11 - 0.92 (m, 15H)
Example 26: Synthesis of (lR,2S,5S)-N-[cyano-[4-(3-pyridyl)-3-pyridyl]methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 167)
Figure imgf000208_0001
207
SUBSTITUTE SHEET RULE 26 Step 1: 4-bromopyridine-3-carbaldehyde
[000317] To a solution of 4-bromopyridine (1 g, 6.33 mmol, 1 eq) in THF (15 mb) was added LDA (2 M, 4.75 mL, 1.5 eq) at -78 °C. After stirring at -78 °C for 1 h under N2, DMF (1.85 g, 25.32 mmol, 1.95 mL, 4 eq) was added, and the solution was stirred at -78 °C for 1 h under N2. Upon completion, the reaction mixture was diluted with sat. NH4CI (40 mL) and extracted with ethyl acetate (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:0 to 20:1) to give 4-bromopyridine-3- carbaldehyde (0.45 g, 2.18 mmol, 34.40% yield, 90% purity) as a yellow solid.
Step 2: 4-(3-pyridyl)pyridine-3-carbaldehyde
[000318] To a solution of 4-bromopyridine-3-carbaldehyde (0.4 g, 2.15 mmol, 1 eq) and 3- pyridylboronic acid (475.79 mg, 3.87 mmol, 1.8 eq) in dioxane (5 mL) and H2O (1.5 mL) was added CS2CO3 (2.10 g, 6.45 mmol, 3 eq). Then, Pd(dppf)Ch.CH2C12 (175.62 mg, 215.05 umol, 0.1 eq) was added under N2, and the solution was stirred at 100 °C for 2 h under N2. Upon completion, the reaction mixture was diluted with water (20 mL) and filtered and the filtrate was extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 0: 1) to give 4-(3- pyridyl)pyridine-3-carbaldehyde (0.2 g, 977.23 umol, 45.44% yield, 90% purity) as a yellow oil.
Step 3: 2-amino-2-[4-(3-pyridyl)-3 -pyridyl] acetonitrile
[000319] To a solution of 4-(3-pyridyl)pyridine-3-carbaldehyde (0.2 g, 1.09 mmol, 1 eq) in NH3/MeOH (7 M, 4 mL, 25.79 eq) was added NH4C1 (174.24 mg, 3.26 mmol, 3 eq) and TMSCN (323.17 mg, 3.26 mmol, 407.53 uL, 3 eq) at 0 °C. The resulting solution was stirred at 25 °C for 12 h under N2. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with EA (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- TLC (SiCh, petroleum etherethyl acetate = 0:1) to give 2-amino-2-[4-(3-pyridyl)-3- pyridyl] acetonitrile (0.05 g, 190.26 umol, 17.52% yield, 80% purity) as a yellow oil.
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SUBSTITUTE SHEET RULE 26 Step 4: (lR,2S,5S)-N-[cyano-[4-(3-pyridyl)-3-pyridyl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000320] To a solution of 2-amino-2-[4-(3-pyridyl)-3-pyridyl]acetonitrile (0.04 g, 190.26 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (69.32 mg, 190.26 umol, 1 eq) in DCM (2 mL) was added DIEA (73.77 mg, 570.79 umol, 99.42 uL, 3 eq), and then HATU (217.03 mg, 570.79 umol, 3 eq). The solution was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with DCM (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 25%-55%,8min) to give (lR,2S,5S)-N-[cyano-[4-(3-pyridyl)-3-pyridyl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (17.66 mg, 29.79 umol, 15.66% yield, 93.9% purity) as a white solid. MS (ESI) m/z 557.2 [M+H]+.
[000321] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.58 - 9.19 (m, 2H), 8.93 (d, J= 6.1 Hz, 1H), 8.75 (d, J= 4.9 Hz, 1H), 8.70 - 8.57 (m, 2H), 8.02 - 7.74 (m, 1H), 7.56 - 7.37 (m, 2H), 6.21 - 5.80 (m, 1H), 4.44 - 4.30 (m, 1H), 4.24 - 4.06 (m, 1H), 3.79 (br d, J= 4.9 Hz, 1H), 3.64 (dd, J = 10.5, 18.6 Hz, 1H), 1.61 - 1.37 (m, 1H), 1.27 - 1.13 (m, 1H), 1.05 - 0.89 (m, 12H), 0.85 - 0.73 (m, 3H).
209
SUBSTITUTE SHEET RULE 26 Example 27: Synthesis of N-[l-[ [cyano(4-isoquinolyl)methyl] carbamoyl] cyclobutyl] -5- cyclopropyl-lH-pyrrole-2-carboxamide (Compound 152)
Figure imgf000211_0001
Step 1: 5-cyclopropyl-lH-pyrrole-2-carboxylic acid
[000322] A solution of methyl 5-cyclopropyl-lH-pyrrole-2-carboxylate (400 mg, 2.42 mmol, 1 eq) in THF (3 mL):MeOH (3 mL):H20 (3 mL) = 1 :1 :1 was added L1OH.H2O (304.84 mg, 7.26 mmol, 3 eq) then was stirred at 60 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O 15 mL, the aqueous phase was added HC1 (IM) to pH = 3 and extracted with DCM (15 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give 5-cyclopropyl-lH- pyrrole-2-carboxylic acid (188 mg, crude) as a brown solid. MS (ESI) m/z 152.1 [M+H]+.
Step 2: 1 -(tert- butoxy carbonylamino)cy cl obutanecarboxy lie acid
[000323] A solution of 1 -aminocyclobutanecarboxy lie acid (1 g, 8.69 mmol, 1 eq) in dioxane
(10 mL) and H2O (10 mL) was added NaHCCh (2.48 g, 29.53 mmol, 1.15 mL, 3.4 eq), BOC2O (2.27 g, 10.42 mmol, 2.39 mL, 1.2 eq) at 0 °C. The mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O 20 mL, the aqueous phase was added HC1 (IM) to pH = 3 and extracted with EA (25 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced
210
SUBSTITUTE SHEET RULE 26 pressure to give 1 -(tert- butoxy carbonylamino)cy cl obutanecarboxylic acid (1 g, crude) as a white solid. MS (ESI) m/z 216.1 [M+H]+.
Step 3: tert-butyl N-[l-[[cyano(4-isoquinolyl)methyl]carbamoyl]cyclobutyl]carbamate
[000324] A solution of l-(tert-butoxycarbonylamino)cy cl obutanecarboxylic acid (700 mg, 3.25 mmol, 1 eq), 2-amino-2-(4-isoquinolyl)acetonitrile (595.81 mg, 3.25 mmol, 1 eq) in DCM (25 m ) was added DMAP (794.60 mg, 6.50 mmol, 2 eq) and EDCI (935.15 mg, 4.88 mmol, 1.5 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 30 mL, and then extracted with DCM (25 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 1:0 to 0:1) to give tert-butyl N-[l-[[cyano(4- isoquinolyl)methyl] carbamoyl] cyclobutyl] carbamate (530 mg, 1.18 mmol, 36.41% yield, 85% purity) as a white solid. MS (ESI) m/z 381.2 [M+H]+.
Step 4: 1 -amino-N-[cyano(4-isoquinolyl)methyl]cyclobutanecarboxamide
[000325] A solution of tert-butyl N-[l-[[cyano(4-isoquinolyl)methyl]carbamoyl] cyclobutyl] carbamate (110 mg, 289.14 umol, 1 eq) in TFA (2 mL) and DCM (4 mL) was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition NaHCCh 30 mL, and then extracted with DCM (25 mL * 3). The combined organic layers were washed with brine 40 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give 1- amino-N-[cyano(4-isoquinolyl)methyl] cyclobutanecarboxamide (98 mg, crude) as a white solid. MS (ESI) m/z 281.1 [M+H]+.
Step 5: N-[l-[[cyano(4-isoquinolyl)methyl]carbamoyl]cyclobutyl]-5-cyclopropyl-lH-pyrrole-2- carboxamide
[000326] A solution of l-amino-N-[cyano(4-isoquinolyl)methyl]cyclobutanecarboxamide (91 mg, 324.62 umol, 1 eq) and 5-cyclopropyl-lH-pyrrole-2-carboxylic acid (49.07 mg, 324.62 umol, 1 eq) in DCM (5 mL) was added HATU (246.86 mg, 649.25 umol, 2 eq) and DIPEA (125.87 mg, 973.87 umol, 169.63 uL, 3 eq). The resulting solution was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 10 mL, and then extracted with DCM (10 mL * 3). The combined organic layers were washed with brine 20 mL, dried over
211
SUBSTITUTE SHEET RULE 26 Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30 mm * 10 um; mobile phase: [water(NH4HCO3)-ACN]; B%: 35%-65%, 8 min) to give N-[l-[[cyano(4- isoquinolyl)methyl] carbamoyl] cyclobutyl] -5-cyclopropyl-lH-pyrrole-2-carboxamide (18 mg, 41.36 umol, 12.74% yield, 95% purity) as a white solid. MS (ESI) m/z 414.2 [M+H]+.
[000327] 1H NMR (400 MHz, DMSO-t76) 5 = 11.23 (br s, 1H), 9.35 (s, 1H), 8.98 (d, J = 1.9 Hz, 1H), 8.68 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.85 (dt, J = 1.2, 7.7 Hz, 1H), 7.77 - 7.71 (m, 1H), 6.80 (d, J = 7.9 Hz, 1H), 6.71 - 6.67 (m, 1H), 5.70 - 5.64 (m, 1H), 2.64 - 2.57 (m, 1H), 2.43 - 2.38 (m, 1H), 2.21 - 2.10 (m, 2H), 1.93 - 1.83 (m, 3H), 0.86 - 0.79 (m, 2H), 0.63 - 0.56 (m, 2H).
Example 28: Synthesis of N-[l-[ [cyano(4-isoquinolyl)methyl] carbamoyl] cyclohexyl] -5- cyclopropyl-lH-pyrrole-2-carboxamide (Compound 154)
Figure imgf000213_0001
Step 1: 5-cyclopropyl-lH-pyrrole-2-carboxylic acid
[000328] To a solution of methyl 5-cyclopropyl-lH-pyrrole-2-carboxylate (250 mg, 1.51 mmol, 1 eq) in MeOH (3 mL), THF (3 mL) and H2O (3 mL) was added LiOH.H2O (190.53 mg, 4.54 mmol, 3 eq). The mixture was stirred at 60 °C for 16 h. Upon completion, The mixture was adjusted pH = 3 with 1 M HCI, and was extracted with EA (2.5 mL*2), dried with Na2SO4, filtered and concentrated in vacuum to give 5-cyclopropyl-lH-pyrrole-2-carboxylic acid (230 mg, crude) as a brown solid.
Step 2: methyl l-[(5-cyclopropyl-lH-pyrrole-2-carbonyl)amino]cyclohexanecarboxylate
212
SUBSTITUTE SHEET RULE 26 [000329] A solution of 5-cyclopropyl-lH-pyrrole-2-carboxylic acid (229 mg, 1.51 mmol, 1 eq) in DCM (1 mL) was added methyl 1 -aminocyclohexanecarboxylate (285.79 mg, 1.82 mmol, 1.2 eq) and DMAP (462.69 mg, 3.79 mmol, 2.5 eq). And then was added EDCI (580.83 mg, 3.03 mmol, 2 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the combined was diluted with water 10 mL and extracted with ethyl acetate (5mL * 2). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 2:1) to give methyl l-[(5-cyclopropyl-lH-pyrrole-2-carbonyl)amino]cyclohexanecarboxylate (280 mg, 915.15 umol, 60.41% yield, 94.9% purity) as a yellow oil.
Step 3: l-[(5-cyclopropyl-lH-pyrrole-2-carbonyl)amino]cyclohexanecarboxylic acid
[000330] A solution of methyl 1 -[(5 -cyclopropyl- lH-pyrrole-2- carbonyl)amino]cyclohexanecarboxylate (280 mg, 964.33 umol, 1 eq) in THF (3 mL) and lLO (3 mL) was added LiOH.TLO (121.40 mg, 2.89 mmol, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the mixture was concentrated in vacuum and was adjusted pH = 3 with 1 mol/L HCI, and was extracted with ethyl acetate (4 mL*2), dried with Na2SO4, filtered and concentrated in vacuum to give 1 -[(5 -cyclopropyl- lH-pyrrole-2- carbonyl)amino]cyclohexanecarboxylic acid (230 mg, crude) as a yellow oil.
Step 4: N- [ 1 - [ [cyano(4-isoquinolyl)methyl] carbamoyl] cyclohexyl] -5 -cyclopropyl- 1 H-pyrrole-2- carboxamide
[000331] To a solution of l-[(5-cyclopropyl-lH-pyrrole-2- carbonyl)amino]cyclohexanecarboxylic acid (230 mg, 832.34 umol, 1 eq) in ACN (10 mL) was added 2-amino-2-(4-isoquinolyl)acetonitrile (182.99 mg, 998.81 umol, 1.2 eq) and 1- methylimidazole (205.01 mg, 2.50 mmol, 199.04 uL, 3 eq). [Chloro(dimethylamino)methylene]- dimethyl-ammonium;hexafluorophosphate (467.07 mg, 1.66 mmol, 2 eq) was added, and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The crude product was purified by prep-HPLC (neutral condition) to give desired compound (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water( NH4HCO3)-ACN];B%: 35%-65%,8min) to give N-[l-[[cyano(4- isoquinolyl)methyl]carbamoyl]cyclohexyl]-5-cyclopropyl-lH-pyrrole-2-carboxamide (44 mg, 97.46 umol, 11.71% yield, 97.8% purity) as an off-white solid. MS (ESI) m/z 442.1 [M+H]+
213
SUBSTITUTE SHEET RULE 26 [000332] 'H NMR (400 MHz, DMSO-de) 5 = 11.30 (br s, 1H), 9.36 (s, 1H), 8.98 (d, J= 7.5 Hz, 1H), 8.70 (s, 1H), 8.19 (d, J= 8.0 Hz, 1H), 7.99 (d, J= 8.4 Hz, 1H), 7.85 (dt, J= 1.2, 7.7 Hz, 1H), 7.78 - 7.70 (m, 1H), 7.14 (s, 1H), 6.73 - 6.65 (m, 2H), 5.69 - 5.63 (m, 1H), 2.16 - 2.05 (m, 1H), 1.98 - 1.85 (m, 2H), 1.80 - 1.68 (m, 2H), 1.54 - 1.42 (m, 5H), 1.30 - 1.17 (m, 1H), 0.87 - 0.80 (m, 2H), 0.63 - 0.57 (m, 2H)
Example 29: Synthesis of (lR,2S,5S)-N-[cyano-[4-(3-pyridyl)-3-pyridyl]methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 175)
Figure imgf000215_0001
Step 1: 7-chloroisoquinoline-4-carbaldehyde
[000333] To a solution of n-BuLi (2.5 M, 907.22 uL, 1.1 eq) in THF (3 mL) was added 4- bromo-7-chloro-isoquinoline (0.5 g, 2.06 mmol, 1 eq) in THF (1 mL) drop-wise at -60 °C. After stirring at -60 °C for 1 h, DMF (376.75 mg, 5.15 mmol, 396.58 uL, 2.5 eq) in THF (1 mL) was added drop-wise. The solution was stirred at -60 °C for 1 h under N2. Upon completion, aq. HC1 (1 M, 5 mL) was added to the reaction drop-wise at -60 °C, then reaction mixture was diluted with water (10 mL) and extracted with EA (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum etherethyl acetate = 2:1) to give 7-chloroisoquinoline-4- carbaldehyde (0.15 g, 547.98 umol, 26.58% yield, 70% purity) as a yellow solid.
214
SUBSTITUTE SHEET RULE 26 Step 2: 2-amino-2-(7-chloro-4-isoquinolyl)acetonitrile
[000334] To a solution of 7-chloroisoquinoline-4-carbaldehyde (0.15 g, 782.83 umol, 1 eq) in NH3/MeOH (3 mL) was added TMSCN (155.33 mg, 1.57 mmol, 195.87 uL, 2 eq) and NH4C1 (83.75 mg, 1.57 mmol, 2 eq). The solution was stirred at 40 °C for 12 h. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO-i, filtered and concentrated under reduced pressure to give 2-amino-2-(7-chloro-4-isoquinolyl)acetonitrile (0.17 g, crude) as a yellow oil.
Step 3: (lR,2S,5S)-N-[(7-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000335] To a solution of 2-amino-2-(7-chloro-4-isoquinolyl)acetonitrile (170.78 mg, 784.62 umol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (285.88 mg, 784.62 umol, 1 eq), and 1 -methylimidazole (128.83 mg, 1.57 mmol, 125.08 uL, 2 eq), and then [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (440.29 mg, 1.57 mmol, 2 eq) was added. The solution was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-70%,8min) and re-purified by prep-TLC (Petroleum ether:Ethyl acetate = 0: 1) to give (lR,2S,5S)-N-[(7-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (7 mg, 11.79 umol, 1.50% yield, 95% purity) as a white solid. MS (ESI) m/z 564.1 [M+H]+.
[000336] 'H NMR (400 MHz, DMSO-t76) 5 = 9.86 - 9.52 (m, 2H), 9.47 - 9.18 (m, 1H), 8.67 (dd, J= 5.7, 7.8 Hz, 1H), 8.17 (dd, J= 5.6, 8.8 Hz, 1H), 8.00 (dd, J= 5.7, 8.6 Hz, 1H), 7.93 (dd, J= 7.3, 8.8 Hz, 1H), 7.02 (t, J= 6.9 Hz, 1H), 4.46 - 4.27 (m, 2H), 3.86 (dt, J= 5.5, 11.1 Hz, 1H), 3.69 (d, J= 10.4 Hz, 1H), 1.61 - 1.23 (m, 2H), 1.07 - 1.01 (m, 6H), 0.96 - 0.79 (m, 9H).
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SUBSTITUTE SHEET RULE 26 Example 30: Synthesis of (lR,2S,5S)-N-[cyano-(8-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 177)
Figure imgf000217_0001
Step 1: 4-bromo-8-fluoro-isoquinoline
[000337] To a solution of 8-fluoroisoquinoline (2.5 g, 16.99 mmol, 1 eq) in CCh (60 mL) was added NBS (6.05 g, 33.98 mmol, 2 eq) and AIBN (2.79 g, 16.99 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was concentrated in a vacuum to dryness to afford a residue. The residue was purified by column (SiCh, petroleum etherethyl acetate = 20:1 to 1: 1) to afford 4-bromo-8-fluoro-isoquinoline (1 g, 3.40 mmol, 20.01% yield, 76.847% purity) as yellow solid.
Step 2: 8-fluoro-4-vinyl-isoquinoline
[000338] To a solution of 4-bromo-8-fluoro-isoquinoline (600 mg, 2.65 mmol, 1 eq) in toluene (4 mL) and H2O (1 mL) was added potassium trifluoro(vinyl)boranuide (533.32 mg, 3.98 mmol, 1.5 eq). Followed by the addition of ditert- butyl(cyclopentyl)phosphane;dichloropalladium;iron (173.00 mg, 265.43 umol, 0.1 eq) and CS2CO3 (2.59 g, 7.96 mmol, 3 eq), the mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was quenched by 2 mL water (aq.). The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuum to give a residue. The residue was purified by
216
SUBSTITUTE SHEET RULE 26 prep-TLC (SiCh, petroleum ether: ethyl acetate = 3:1) to afford 8-fluoro-4-vinyl-isoquinoline (400 mg, 2.31 mmol, 87.01% yield) as yellow solid. MS (ESI) m/z 173.9 [M+H]+.
Step 3: 8-fluoroisoquinoline-4-carbaldehyde
[000339] To a mixture of 8-fhioro-4-vinyl-isoquinoline (200 mg, 1.15 mmol, 1 eq) in dioxane (5 mL) and H2O (1 mL) was added K2OSO4.2H2O (85.10 mg, 230.97 umol, 0.2 eq) and 2,6- dimethyl(15N)pyridine (249.78 mg, 2.31 mmol, 2 eq). The mixture was stirred at 25 °C for 30 min, and then was added NalCh (1.24 g, 5.77 mmol, 319.96 uL, 5 eq) at 0 °C. The resulting mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was filtered and the mother liquer was diluted with H2O 2 mL and extracted with ethyl acetate (13 mL * 3). The combined organic layers were washed with brine 5 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum etherethyl acetate = 0: 1) to afford 8-fluoroisoquinoline-4-carbaldehyde (70 mg, 399.64 umol, 34.61% yield) as black solid.
Step 4: 2-amino-2-(8-fluoro-4-isoquinolyl)acetonitrile
[000340] A solution of 8-fluoroisoquinoline-4-carbaldehyde (70 mg, 399.64 umol, 1 eq) in NH MeOH (5 mL) was added NH4CI (42.75 mg, 799.27 umol, 2 eq) stirred at 25 °C for 1 h, and then was added TMSCN (79.29 mg, 799.27 umol, 99.99 uL, 2 eq) at 0 °C. The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NEb/MeOH. The reaction mixture was quenched by addition H2O 10 mL, and then extracted with DCM (15 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 0: 1) to afford 2-amino-2-(8-fluoro- 4-isoquinolyl)acetonitrile (50 mg, 248.51 umol, 62.18% yield) as yellow solid. MS (ESI) m/z 202.1 [M+H]+
Step 5: (lR,2S,5S)-N-[cyano-(8-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000341] A mixture of 2-amino-2-(8-fluoro-4-isoquinolyl)acetonitrile (40 mg, 198.81 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (72.44 mg, 198.81 umol, 1 eq) in DCM
217
SUBSTITUTE SHEET RULE 26 (10 mL) was added DMAP (72.86 mg, 596.43 umol, 3 eq) and EDCI (76.22 mg, 397.62 umol, 2 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the recationg mixture was quenched by water (0.5ml), blowed by N2 to dryness and diluted by DMF (1ml) to afford a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-80%,8min) to afford (lR,2S,5S)-N-[cyano-(8-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (20 mg, 36.53 umol, 18.37% yield, 100% purity) as white solid. MS (ESI) m/z 548.2 [M+H]+
[000342] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.70 - 9.50 (m, 2H), 9.37 (br dd, J= 8.4, 13.1 Hz, 1H), 8.83 (d, J= 6.8 Hz, 1H), 7.99 - 7.74 (m, 2H), 7.61 (dd, J= 7.6, 10.1 Hz, 1H), 6.90 (dd, J= 7.8, 15.8 Hz, 1H), 4.43 - 4.33 (m, 1H), 4.22 (d, J= 13.4 Hz, 1H), 3.93 (td, J= 5.0, 9.9 Hz, 1H), 3.79 - 3.61 (m, 1H), 1.66 - 1.50 (m, 1H), 1.36 - 1.12 (m, 1H), 1.03 (s, 3H), 1.00 - 0.91 (m, 9H), 0.81 (d, J = 7.0 Hz, 3H).
Example 31: Synthesis of (lR,2S,5S)-N-[cyano-[7-(trifluoromethyl)-4-isoquinolyl]methyl]- 3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 176)
Figure imgf000219_0001
Step 1: [7-(trifluoromethyl)-4-isoquinolyl]methanol
[000343] To a solution of 7-(trifluoromethyl)isoquinoline-4-carboxylic acid (300 mg, 1.24 mmol, 1 eq) in THF(3 mL) was added LAH(118.02 mg, 3.11 mmol, 2.5 eq) at 0 °C, and the
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SUBSTITUTE SHEET RULE 26 resulting mixture was stirred at 20 °C for 6 h. Upon completion, the reaction mixture was cooled to 0 °C, added 0.11 mL H2O and 0.11 mL 15% NaOH (aq), dried over NA2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiC>2, EA:PE = 1:3) to give [7-(trifluoromethyl)-4-isoquinolyl] methanol (70 mg, 308.12 umol, 24.77% yield) as a yellow solid. MS (ESI) m/z 228.0 [M+H]+.
Step 2: 7-(trifhioromethyl)isoquinoline-4-carbaldehyde
[000344] A solution of [7-(trifhioromethyl)-4-isoquinolyl]methanol (70 mg, 308.12 umol, 1 eq) in DCM (1 mL) was added with MnCh (535.74 mg, 6.16 mmol, 20 eq), and the resulting mixture was stirred at 20 °C for 14 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give 7-(trifluoromethyl)isoquinoline-4-carbaldehyde (60 mg, crude) as a red solid. MS (ESI) m/z 225.9 [M+H]+.
Step 3: 2-amino-2-[7-(trifluoromethyl)-4-isoquinolyl]acetonitrile
[000345] A solution of 7-(trifluoromethyl)isoquinoline-4-carbaldehyde (50 mg, 222.06 umol, 1 eq) in NEh/MeOH (1 mL) was added with NH4CI (35.63 mg, 666.17 umol, 3 eq) and the resulting solution was stirred at 20 °C for 1 h. Then the mixture was added with TMSCN (66.09 mg, 666.17 umol, 83.34 uL, 3 eq) at 0 °C, and stirred at 20 °C for 3 h. Upon completion, the solution was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, EA) to afford 2-amino-2-[7-(trifluoromethyl)-4- isoquinolyl]acetonitrile (20 mg, 79.62 umol, 35.85% yield) as yellow solid. MS (ESI) m/z 252.0 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-[7-(trifluoromethyl)-4-isoquinolyl]methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000346] To a mixture of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (29.01 mg, 79.62 umol, 1 eq) in ACN (1 mL) was added 2-amino-2-[7-(trifhioromethyl)-4- isoquinolyl]acetonitrile (20 mg, 79.62 umol, 1 eq), [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (33.51 mg, 119.42 umol, 1.5 eq) and 1 -methylimidazole (19.61
219
SUBSTITUTE SHEET RULE 26 mg, 238.85 umol, 19.04 uL, 3 eq) at 0 °C, and the resulting mixture was stirred at 0 °C for 2 h. Upon completion, the reaction mixture was quenched by water (1 mL), and then extracted with EA (2 mL * 3). The combined organic layers weredried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC(column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-70%,8min) to give (lR,2S,5S)-N-[cyano-[7-(trifluoromethyl)-4-isoquinolyl]methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (2.38 mg, 3.96 umol, 4.97% yield, 99.4% purity) as a white solid. MS (ESI) m/z 598.2 [M+H]+.
[000347] 'H NMR (400 MHz, DMSO-de) 5 = 9.69 - 9.43 (m, 2H), 9.36 (br dd, J = 7.2, 17.3 Hz, 1H), 8.87 (d, J = 7.0 Hz, 1H), 8.78 (s, 1H), 8.25 - 7.96 (m, 2H), 7.04 - 6.91 (m, 1H), 4.36 (br dd, J = 7.4, 16.1 Hz, 1H), 4.19 (d, J = 19.6 Hz, 1H), 3.96 - 3.89 (m, 1H), 3.74 - 3.61 (m, 1H), 1.64 - 1.52 (m, 1H), 1.38 - 1.15 (m, 1H), 1.04 - 0.85 (m, 12H), 0.80 (d, J = 7.5 Hz, 3H)
Example 32: Synthesis of (lR,2S,5S)-N-[cyano-(8-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 177)
Figure imgf000221_0001
Step 1: 4-bromo-8-fluoro-isoquinoline
[000348] To a solution of 8-fluoroisoquinoline (2.5 g, 16.99 mmol, 1 eq) in CCI4 (60 mL) was added NBS (6.05 g, 33.98 mmol, 2 eq) and AIBN (2.79 g, 16.99 mmol, 1 eq). The mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was concentrated in a vacuum to dryness to afford a residue. The residue was purified by column (SiCh, petroleum
220
SUBSTITUTE SHEET RULE 26 etherethyl acetate = 20:1 to 1: 1) to afford 4-bromo-8-fluoro-isoquinoline (1 g, 3.40 mmol, 20.01% yield, 76.847% purity) as yellow solid.
Step 2: 8-fluoro-4-vinyl-isoquinoline
[000349] To a solution of 4-bromo-8-fluoro-isoquinoline (600 mg, 2.65 mmol, 1 eq) in Tol. (4 mb) and H2O (1 mL) was added potassium;trifluoro(vinyl)boranuide (533.32 mg, 3.98 mmol, 1.5 eq) followed by ditert-butyl(cyclopentyl)phosphane;dichloropalladium;iron (173.00 mg, 265.43 umol, 0.1 eq) and CS2CO3 (2.59 g, 7.96 mmol, 3 eq). After addition, the mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was quenched by 2 mL water (aq.). The organic layer was separated, dried over Na2SO4, filtered, and concentrated in vacuum to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 3: 1) to afford 8-fhioro-4-vinyl-isoquinoline (400 mg, 2.31 mmol, 87.01% yield) as yellow solid. MS (ESI) m/z 173.9 [M+H]+.
Step 3: 8-fluoroisoquinoline-4-carbaldehyde
[000350] To a mixture of 8-fluoro-4-vinyl-isoquinoline (200 mg, 1.15 mmol, 1 eq) in dioxane (5 mL) and H2O (1 mL) was added K2OSO4.2H2O (85.10 mg, 230.97 umol, 0.2 eq) and 2,6- dimethyl(l15N)pyridine (249.78 mg, 2.31 mmol, 2 eq). The mixture was stirred at 25 °C for 30 min, and then was added NalCh (1.24 g, 5.77 mmol, 319.96 uL, 5 eq) at 0 °C. The resulting mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was filtered and the mother liquer was diluted with H2O 2 mL and extracted with ethyl acetate (13 mL * 3). The combined organic layers were washed with brine 5 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum etherethyl acetate = 0: 1) to afford 8-fluoroisoquinoline-4-carbaldehyde (70 mg, 399.64 umol, 34.61% yield) as black solid.
Step 4: 2-amino-2-(8-fluoro-4-isoquinolyl)acetonitrile
[000351] A solution of 8-fluoroisoquinoline-4-carbaldehyde (70 mg, 399.64 umol, 1 eq) in NHVMeOH (5 mL) was added NH4CI (42.75 mg, 799.27 umol, 2 eq), and the resulting solution was stirred at 25 °C for 1 h. TMSCN (79.29 mg, 799.27 umol, 99.99 uL, 2 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was
221
SUBSTITUTE SHEET RULE 26 concentrated under reduced pressure to remove NH MeOH. The reaction mixture was quenched by addition H2O 10 mL, and then extracted with DCM (15 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 0: 1) to afford 2-amino-2-(8-fluoro-4-isoquinolyl)acetonitrile (50 mg, 248.51 umol, 62.18% yield) as yellow solid. MS (ESI) m/z 202.1 [M+H]+
Step 5: (lR,2S,5S)-N-[cyano-(8-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trif luoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000352] A mixture of 2-amino-2-(8-fluoro-4-isoquinolyl)acetonitrile (40 mg, 198.81 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (72.44 mg, 198.81 umol, 1 eq) in DCM (10 mL) was added DMAP (72.86 mg, 596.43 umol, 3 eq) and EDCI (76.22 mg, 397.62 umol, 2 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the recationg mixture was quenched by water (0.5ml), blowed by N2 to dryness and diluted by DMF (1ml) to afford a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-80%,8min) to afford (lR,2S,5S)-N-[cyano-(8-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (20 mg, 36.53 umol, 18.37% yield, 100% purity) as white solid. MS (ESI) m/z 548.2 [M+H]+
[000353] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.70 - 9.50 (m, 2H), 9.37 (br dd, J= 8.4, 13.1 Hz, 1H), 8.83 (d, J= 6.8 Hz, 1H), 7.99 - 7.74 (m, 2H), 7.61 (dd, J= 7.6, 10.1 Hz, 1H), 6.90 (dd, J= 7.8, 15.8 Hz, 1H), 4.43 - 4.33 (m, 1H), 4.22 (d, J= 13.4 Hz, 1H), 3.93 (td, J= 5.0, 9.9 Hz, 1H), 3.79 - 3.61 (m, 1H), 1.66 - 1.50 (m, 1H), 1.36 - 1.12 (m, 1H), 1.03 (s, 3H), 1.00 - 0.91 (m, 9H), 0.81 (d, J = 7.0 Hz, 3H).
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SUBSTITUTE SHEET RULE 26 Example 33: Synthesis of (lR,2S,5S)-N-[cyano-[8-(trifluoromethyl)-4-isoquinolyl]methyl]- 3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 179)
Figure imgf000224_0001
Step 1: [8-(trifluoromethyl)-4-isoquinolyl]methanol
[000354] To a mixture of 8-(trifluoromethyl)isoquinoline-4-carboxylic acid (500 mg, 2.07 mmol, 1 eq) in THF (9 mL) was added TEA (230.77 mg, 2.28 mmol, 317.43 uL, 1.1 eq) at 0 °C, and then isobutyl carbonochloridate (283.16 mg, 2.07 mmol, 272.27 uL, 1 eq) was added and stirred at 25 °C for 30 min. The resulting mixture was filtered, added with H2O (1 mL), and then NaBH4 (167.07 mg, 4.42 mmol, 2.13 eq) was added at 0 °C in portions. The resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was extracted with DCM (15 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, petroleum ether: ethyl acetate = 1:0 to 3: 1) to afford [8-(trifluoromethyl)-4-isoquinolyl]methanol (350 mg, 1.14 mmol, 54.99% yield, 74% purity) as yellow oil.
Step 2: 8-(trifhioromethyl)isoquinoline-4-carbaldehyde
[000355] To a solution of [8-(trifluoromethyl)-4-isoquinolyl]methanol (350 mg, 1.54 mmol, 1 eq) in DCM (5 mL) was added MnCh (2.68 g, 30.81 mmol, 20 eq). The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was filtered and concentrated under
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SUBSTITUTE SHEET RULE 26 reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 1: 1) to afford 8-(trifluoromethyl)isoquinoline-4-carbaldehyde (280 mg, 1.13 mmol, 73.45% yield, 91% purity) as light yellow solid.
Step 3: 2-amino-2-[8-(trifhioromethyl)-4-isoquinolyl]acetonitrile
[000356] A solution of 8-(trifluoromethyl)isoquinoline-4-carbaldehyde (280 mg, 1.24 mmol, 1 eq) in DCM (2 mL) and NH3/MeOH (7 M, 710.59 uL, 4 eq) was added Ti(i-PrO)4 (530.14 mg, 1.87 mmol, 550.51 uL, 1.5 eq), and the resulting solution was stirred at 25 °C for 1 h. TMSCN (370.10 mg, 3.73 mmol, 50.76 uL, 3 eq) was added at 0 °C, and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (2 mL) at 20 °C, and then extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine 10 mL, dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 0:1) to afford 2-amino-2-[8-(trifluoromethyl)-4-isoquinolyl]acetonitrile (200 mg, 732.46 umol, 58.90% yield, 92% purity) as white solid.
Step 4: (lR,2S,5S)-N-[cyano-[8-(trifluoromethyl)-4-isoquinolyl]methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3. L0]hexane-2- carboxamide
[000357] To a mixture of 2-amino-2-[8-(trifluoromethyl)-4-isoquinolyl]acetonitrile (150 mg, 597.12 umol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (217.57 mg, 597.12 umol, 1 eq), [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (251.31 mg, 895.68 umol, 1.5 eq) and 1 -methylimidazole (147.08 mg, 1.79 mmol, 142.79 uL, 3 eq) at 0 °C. The resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by water (0.1 mL). The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75*30mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 50%-70%,8min) to afford (lR,2S,5S)-N-[cyano-[8- (trifluoromethyl)-4-isoquinolyl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (20 mg, 33.47 umol, 5.61% yield, 100% purity) as white solid. MS (ESI) m/z 589.1 [M+H]
224
SUBSTITUTE SHEET RULE 26 [000358] 'H NMR (400 MHz, DMSO-de) 5 = 9.77 - 9.46 (m, 2H), 9.44 - 9.26 (m, 1H), 8.91 (d, J = 5.7 Hz, 1H), 8.39 - 8.19 (m, 2H), 8.10 - 7.91 (m, 1H), 7.07 - 6.95 (m, 1H), 4.36 (br d, J = 18.6 Hz, 1H), 4.19 (d, J = 14.7 Hz, 1H), 3.93 (td, J = 5.1, 10.1 Hz, 1H), 3.76 - 3.61 (m, 1H), 1.65 - 1.52 (m, 1H), 1.38 - 1.16 (m, 1H), 1.08 - 0.89 (m, 9H), 0.85 (s, 3H), 0.81 (d, J = 8.0 Hz, 3H).
Example 34: Synthesis of (lR,2S,5S)-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 180)
Figure imgf000226_0001
[000359] To a solution of 2-amino-2-(7,8-difluoro-4-isoquinolyl)acetonitrile (100 mg, 456.23 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (199.48 mg, 547.47 umol, 1.2 eq), 1- methylimidazole (112.37 mg, 1.37 mmol, 109.10 uL, 3 eq) in ACN (5 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (256.01 mg, 912.45 umol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O 35 mL at 20 °C, and then extracted with EtOAc (40 mL *3). The combined organic layers were washed with brine 35 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)- ACN];B%: 45%-70%,8min) to give (lR,2S,5S)-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (181.7 mg, 321.29 umol, 70.42% yield, 100% purity) as a white solid. MS (ESI) m/z 566.3 [M+H]+
225
SUBSTITUTE SHEET RULE 26 [000360] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.72 - 9.51 (m, 2H), 9.43 - 9.30 (m, 1H), 8.82 (d, J= 8.6 Hz, 1H), 8.17 - 7.86 (m, 2H), 6.95 (dd, J= 7.9, 12.3 Hz, 1H), 4.37 (dd, J= 8.6, 10.5 Hz, 1H), 4.20 (d, J= 14.4 Hz, 1H), 3.98 - 3.84 (m, 1H), 3.68 (dd, J= 10.5, 17.2 Hz, 1H), 1.64 - 1.51 (m, 1H), 1.37 - 1.12 (m, 1H), 1.00 (m, 12H), 0.81 (d, J= 7.1 Hz, 3H).
Example 35: Synthesis of (lR,2S,5S)-N-[cyano-(7,8-dichloro-4-isoquinolyl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 181)
Figure imgf000227_0001
[000361] A mixture of 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile (60 mg, 238.00 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (95.39 mg, 261.80 umol, 1.1 eq) in ACN (3 mL) was added 1 -methylimidazole (58.62 mg, 714.00 umol, 56.91 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (133.56 mg, 476.00 umol, 2 eq), and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Phenomenex Cl 8 75*30mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 45%-75%,8min) to give (lR,2S,5S)-N-[cyano-(7,8- dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (73 mg, 120.76 umol, 50.74% yield, 99% purity) as a white solid. MS (ESI) m/z 598.2 [M+l]+
[000362] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.77 - 9.47 (m, 2H), 9.34 (br d, J = 2.1 Hz, 1H), 8.87 (d, J = 7.6 Hz, 1H), 8.15 - 7.88 (m, 2H), 7.08 - 6.88 (m, 1H), 4.36 (br d, J = 18.4 Hz, 1H),
226
SUBSTITUTE SHEET RULE 26 4.18 (d, J = 18.1 Hz, 1H), 3.97 - 3.86 (m, 1H), 3.75 - 3.59 (m, 1H), 1.65 - 1.51 (m, 1H), 1.40 -
1.15 (m, 1H), 1.05 - 0.91 (m, 9H), 0.86 - 0.79 (m, 6H)
Example 36: Synthesis of (lR,2S,5S)-N-[cyano-(4-phenyl-3-pyridyl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 182)
Figure imgf000228_0001
Step 1: 4-bromopyridine-3-carbaldehyde
[000363] To a solution of 4-bromopyridine (2 g, 12.66 mmol, 1 eq) in THF (30 mb) was added LDA (2 M, 9.49 mb, 1.5 eq) at -78 °C. After stirring at -78 °C for 1 h under N2, DMF (3.70 g, 50.63 mmol, 3.90 mL, 4 eq) was added slowly, and the mixture was stirred at -78 °C for 1 h under N2. Upon completion, the reaction mixture was diluted with NH4CI (30 mL) and extracted with ethyl acetate (10 mL * 3), the combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 20 : 1) to give 4-bromopyridine-3-carbaldehyde (700 mg, 3.76 mmol, 29.73% yield) as a yellow solid. MS (ESI) m/z 185.9 [M+H]+
Step 2: 4-phenylpyridine-3-carbaldehyde
227
SUBSTITUTE SHEET RULE 26 [000364] To a mixture of 4-bromopyridine-3-carbaldehyde (700 mg, 3.39 mmol, 90% purity, 1 eq), phenylboronic acid (495.57 mg, 4.06 mmol, 1.2 eq) and K2CO3 (936.20 mg, 6.77 mmol, 2 eq) in DME (20 mL) and H2O (4 mL), was added Pd(PPh3)4 (391.39 mg, 338.70 umol, 0.1 eq) was added under N2. The resulting mixture was stirred at 80 °C for 3 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL * 3), the combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 5 : 1) to give 4-phenylpyridine-3- carbaldehyde (450 mg, 2.21 mmol, 65.27% yield, 90% purity) as a red oil. MS (ESI) m/z 184.1 [M+H]+
Step 3: 2-amino-2-(4-phenyl-3-pyridyl)acetonitrile
[000365] To a solution of 4-phenylpyridine-3-carbaldehyde (400 mg, 2.18 mmol, 1 eq) in NH3/MeOH (7 M, 8.00 mL, 25.65 eq) was added NH4C1 (233.58 mg, 4.37 mmol, 2 eq) and TMSCN (433.20 mg, 4.37 mmol, 546.28 uL, 2 eq). The mixture was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-phenyl-3- pyridyl)acetonitrile (420 mg, crude) as a white solid, and used directly next step. MS (ESI) m/z 210.1 [M+H]+
Step 4: (lR,2S,5S)-N-[cyano-(4-phenyl-3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000366] To a solution of 2-amino-2-(4-phenyl-3-pyridyl)acetonitrile (420 mg, 2.01 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (731.34 mg, 2.01 mmol, 1 eq) in DCM (10 mL) was added DIEA (778.25 mg, 6.02 mmol, 1.05 mL, 3 eq) and HATU (763.20 mg, 2.01 mmol, 1 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with DCM (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Cl 8
228
SUBSTITUTE SHEET RULE 26 (250*50 mm*10 um); mobile phase: [water (NH4HCCh)-ACN]; B%: 40%-60%, lOmin) to give (lR,2S,5S)-N-[cyano-(4-phenyl-3-pyridyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (225.6 mg, 386.56 umol, 19.26% yield, 95.2% purity) as a white solid. MS (ESI) m/z 556.2 [M+H]+
[000367] 'H NMR (400 MHz, DMSO-de) 5 = 9.60 - 9.27 (m, 2H), 8.93 - 8.90 (d, J = 10.4 Hz, 1H), 8.71 (dd, J = 1.3, 4.7 Hz, 1H), 7.50 - 7.38 (m, 6H), 6.09 - 5.78 (m, 1H), 4.43 - 4.33 (m, 1H), 4.27 - 4.14 (m, 1H), 3.79 - 3.70 (m, 1H), 3.70 - 3.60 (m, 1H), 1.59 - 1.28 (m, 2H), 1.04 - 0.95 (m, 12H), 0.84 - 0.75 (m, 3H)
Example 37: Synthesis of (lR,2S,5S)-N-(cinnolin-5-yl(cyano)methyl)-3-((S)-3,3-dimethyl-2- (2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (Compound 184)
Figure imgf000230_0001
229
SUBSTITUTE SHEET RULE 26 Step 1: l-(2-amino-6-bromophenyl)ethanone
[000368] A suspension of 2-amino-6-bromo-benzonitrile (40 g, 203.01 mmol, 1 eq) in THF (600 mL) was added slowly to MeMgBr (3 M, 473.7 mb, 7 eq) at 25 °C, and the mixture was heated to 45 °C for 16 h. The mixture was cooled to 0 °C, and HC1 (6 M, 473.7 mL, 14 eq) was added drop- wise. After the addition, the mixutre was stirred at 100 °C for 2 h. Upon completion, the reaction mixture was cooled to 25 °C and the mixture was adjusted to pH of about 6-7 with Na2CO3. The mixture was extracted with EtOAc (1000 mL * 2). The organic layer was dried over Na2SO4, concentratred to afford a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 10:1 to 0: 1). The residue was triturated with PE:EtOAc (5:1) at 25 °C for 30 min and fitlered. The filter cake was dried to give l-(2- amino-6-bromo-phenyl)ethanone (26 g, 121.46 mmol, 29.91% yield) as a yellow solid. MS (ESI) m/z 214.1 [M+H]+.
Step 2: 5-bromocinnolin-4-ol
[000369] To a solution of l-(2-amino-6-bromo-phenyl)ethanone (2 g, 9.34 mmol, 1 eq) in H2O (12 mL) was added HC1 (12 M, 12.00 mL, 15.41 eq) at 0°C. The resulting mixture was stirred for 20 min. Then a solution of NaNCh (670.42 mg, 9.72 mmol, 1.04 eq) in H2O (5.00 g, 277.54 mmol, 5.00 mL, 29.71 eq) was added at 0 °C. The mixture was stirred at 0°C for 2 h. Then the mixture was warmed to 65 °C, and the mixture was stirred at 65 °C for 2 h. Five batchs were set up in parallel. Upon completion, the reaction mixture was diluted with ethyl acetate (100 mL) and the solution was filtered and the filter cake was concentrated under reduced pressure to give 5-bromocinnolin-4-ol (1.4 g, 5.55 mmol, 11.89% yield, 89.27% purity) as a yellow solid. MS (ESI) m/z 225.1 [M+H]+.
Step 3: 5-bromo-4-chlorocinnoline
[000370] To a solution of 5-bromocinnolin-4-ol (1.4 g, 6.22 mmol, 1 eq) in THF (28 mL) was added POCI3 (1.43 g, 9.33 mmol, 867.17 uL, 1.5 eq), and the resulting mixture was stirred at 80 °C for 1 h. Upon completion, the reaction mixture was quenched by water (10 mL), and then adjust pH = 7~8 by aq.NaHCCh and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated
230
SUBSTITUTE SHEET RULE 26 under reduced pressure to give 5-bromo-4-chloro-cinnoline (1.4 g, 5.75 mmol, 92.42% yield) as a yellow solid. MS (ESI) m/z 245.0 [M+H]+.
Step 4: N'-(5-bromocinnolin-4-yl)-4-methylbenzenesulfonohydrazide
[000371] To a stirred solution of 5-bromo-4-chloro-cinnoline (1.4 g, 5.75 mmol, 1 eq) in DCE (1 mL), under nitrogen atmosphere, was added 4-methylbenzenesulfonohydrazide (1.07 g, 5.75 mmol, 1 eq) drop wise at 15°C and the mixture heated at 70 °C for 3 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with DCM (5 mL) at 15 °C for 30 min to give N'-(5-bromocinnolin-4-yl)-
4-methyl-benzenesulfonohydrazide (2 g, 5.09 mmol, 88.45% yield) as a brown solid. MS (ESI) m/z 393.1 [M+H]+.
Step 5: 5-bromocinnoline
[000372] To a stirred solution of N'-(5-bromocinnolin-4-yl)-4-methyl- benzenesulfonohydrazide (2 g, 5.09 mmol, 1 eq) in H2O (50 mL) was added a solution of Na2CO3 (1.46 g, 13.78 mmol, 2.71 eq) in H2O (10 mL) drop-wise at 15 °C. The resluting mixture was heated at 100 °C for 2 h under nitrogen atmosphere. Upon completion, the reaction mixture was extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give
5-bromocinnoline (0.917 g, 4.39 mmol, 86.25% yield) as a brown solid. MS (ESI) m/z 211.0 [M+H]+.
Step 6: 5-vinylcinnoline
[000373] A mixture of 5-bromocinnoline (1.06 g, 5.07 mmol, 1 eq), potassium;trifhroro(vinyl)boranuide (815.07 mg, 6.08 mmol, 1.2 eq), CS2CO3 (4.96 g, 15.21 mmol, 3 eq) and Pd(dppf)Ch (371.03 mg, 507.07 umol, 0.1 eq) in dioxane (40 mL) and H2O (8 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 70 °C for 18 h under N2 atmosphere. Upon completion, the reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (30 mL *3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl
231
SUBSTITUTE SHEET RULE 26 acetate = 10:1 to 0: 1) to give 5-vinylcinnoline (615 mg, 3.94 mmol, 77.65% yield) as a red oil. MS (ESI) m/z 157.2 [M+H]+.
Step 7: cinnoline-5-carbaldehyde
[000374] To a solution of 5-vinylcinnoline (615 mg, 3.94 mmol, 1 eq) in dioxane (10 mL) and H2O (2 mL) was added 2,6-dimethyl(l 15N)pyridine (851.69 mg, 7.88 mmol, 2 eq) and K2OSO4.2H2O (290.17 mg, 787.53 umol, 0.2 eq). The mixture was stirred at 15 °C for 0.5 h. The solution was cooled to 0°C, and then NalCh (4.21 g, 19.69 mmol, 1.09 mL, 5 eq) was added at 0 °C. The mixture was stirred at 15 °C for 17.5 h. Upon completion, the reaction mixture was diluted with water (20 mL), filtered, and the mother liquer was extracted with ethyl acetate (8 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 10: 1 to 0:1) to give cinnoline-5- carbaldehyde (470 mg, 2.97 mmol, 75.47% yield) as a yellow solid. MS (ESI) m/z 274.8 [M+H]+.
Step 8: 2-amino-2-(cinnolin-5-yl)acetonitrile
[000375] To a solution of cinnoline-5-carbaldehyde (100 mg, 632.29 umol, 1 eq) in DCM (1 mL) was added Ti(i-PrO)4 (269.56 mg, 948.43 umol, 279.91 uL, 1.5 eq) and NIU/MeOH (7 M, 361.31 uL, 4 eq). The mixture was stirred at 25 °C for 0.5 h. Then TMSCN (188.18 mg, 1.90 mmol, 237.30 uL, 3 eq) was added at 25 °C. The mixture was stirred at 25 °C for 17.5 h. Upon completion, the reaction mixture was diluted with water (2 mL) and filtered, and the mother liquer was extracted with DCM:MeOH = 4:1 (1 mL * 10). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-cinnolin- 5-yl-acetonitrile (0.11 g, 597.19 umol, 94.45% yield) as a brown solid. MS (ESI) m/z 185.1 [M+H]+.
Step 9: (lR,2S,5S)-N-(cinnolin-5-yl(cyano)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000376] To a solution of 2-amino-2-cinnolin-5-yl-acetonitrile (110 mg, 597.19 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-
232
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxylic acid (217.59 mg, 597.19 umol, 1 eq) in ACN (3 mL) was added 1 -methylimidazole (147.09 mg, 1.79 mmol, 142.81 uL, 3 eq) [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (718.98 mg, 2.56 mmol, 2 eq). The mixture was stirred at 15 °C for 1 h. Upon completion, the residue was purified by prep-HPLC (column: C18 (250 * 50 mm * 10 um); mobile phase: [water(NH4HCO3)-ACN]; B%: 30%-50%, 10 min) to give lR,2S,5S)-N-[cinnohn-5-yl(cyano)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (250 mg, 466.03 umol, 78.04% yield, 98.9% purity) as a white solid. MS (ESI) m/z
531.3 [M+H]+.
Step 10: (lR,2S,5S)-N-(cinnolin-5-yl(cyano)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000377] The residue was purified by SFC (column: DAICEL CHIRALCEL OD (250 mm * 30 mm, 10 um);mobile phase: [Neu-IPA]; B%: 17%-17%, 14 min) to give (1R,2S,5S)-N- [cinnolin-5-yl(cyano)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (78.18 mg, 147.36 umol, 31.27% yield) as a white solid. 'H NMR (400 MHz, DMSO-de) 5 = 9.68 - 9.27 (m, 3H), 8.56 (br d, J= 8.6 Hz, 1H), 8.22 - 7.99 (m, 3H), 7.10 - 6.94 (m, 1H), 4.46 - 4.31 (m, 1H), 4.25 - 4.09 (m, 1H), 3.93 (dt, J = 5.6, 9.7 Hz, 1H), 3.77 - 3.61 (m, 1H), 1.66 - 1.51 (m, 1H), 1.37 - 1.13 (m, 1H), 1.07 - 0.91 (m, 9H), 0.86 (s, 3H), 0.84 - 0.77 (m, 3H)
[000378] (lR,2S,5S)-N-[cinnolin-5-yl(cyano)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 2 (64.13 mg, 120.88 umol, 25.65% yield) was obtained as a white solid. 'H NMR (400 MHz, DMSO-de) 5 = 9.53 - 9.47 (m, 2H), 9.38 (d, J= 8.3 Hz, 1H), 8.55 (d, J= 8.5 Hz, 1H), 8.15 (dd, J = 6.4, 13.3 Hz, 2H), 8.04 (dd, J= 7.3, 8.4 Hz, 1H), 6.98 (d, J= 8.0 Hz, 1H), 4.34 (d, J= 8.4 Hz, 1H), 4.21 (s, 1H), 3.92 (dd, J= 5.5, 10.3 Hz, 1H), 3.64 (d, J= 10.5 Hz, 1H), 1.55 (dd, J= 5.6,
7.3 Hz, 1H), 1.16 (d, J= 7.6 Hz, 1H), 0.94 (s, 3H), 0.86 (s, 9H), 0.79 (s, 3H).
233
SUBSTITUTE SHEET RULE 26 Example 38: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 186)
Figure imgf000235_0001
Step 1: methyl (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino) butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate
[000379] To a mixture of methyl (lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylate (1 g, 4.86 mmol, 1 eq, HC1) and (2S,3R)-3-tert-butoxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)butanoic acid (1.93 g, 4.86 mmol, 1 eq) in DMF (10 mL) was added DIEA (1.89 g, 14.59 mmol, 2.54 mL, 3 eq), and then HATU (3.70 g, 9.72 mmol, 2 eq) was added. The mixture was stirred at 25 °C for 30 min. Upon completion, the reaction mixture was diluted with H2O 40 mL and extracted with ethyl acetate 80 mL (40 mL * 2). The combined organic layers were washed with brine 80 mL (80 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 15: 1 to 8: 1). Methyl (lR,2S,5S)-3- [(2S,3R)-3-tert-butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (2.35 g, 4.11 mmol, 84.57% yield, 96% purity) was obtained as a colourless oil. MS (ESI) m/z 493.4 [M+H-56]+.
Step 2: (lR,2S,5S)-3-[(2S,3R)-2-amino-3-tert-butoxy-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxylic acid
234
SUBSTITUTE SHEET RULE 26 [000380] To a solution of methyl (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate (2.3 g, 4.19 mmol, 1 eq) in THF (15 mL) and H2O (5 mL) was added LiOH.HzO (615.63 mg, 14.67 mmol, 3.5 eq). The mixture was stirred at 40 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S,3R)-2-amino-3-tert-butoxy- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (6.8 g, crude) as a white solid. MS (ESI) m/z 311.3 [M-H]’.
Step 3: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000381] To a mixture of (lR,2S,5S)-3-[(2S,3R)-2-amino-3-tert-butoxy-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.3 g, 4.16 mmol, 1 eq) and TEA (1.26 g, 12.48 mmol, 1.74 mL, 3 eq) was added methyl 2,2,2-trifluoroacetate (5.33 g, 41.61 mmol, 4.20 mL, 10 eq). After the addition, the resulting mixture was stirred at 25 °C for 4 h. Upon completion, the reaction mixture was concentrated in a vacuum to remove most of methyl 2,2,2- trifluoroacetate to afford a residue. The combined residue was diluted with EtOAc (20 mL) and washed with 1 M aqueous HC1 (20 mL). The organic layer was seprated and the aqueous layer was extracted with EtOAc (20 mL * 2). The combined orangic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was triturated with petroleum ether: ethyl acetate = 20: 1 (20 mL) at 25 °C for 15 min, and then filtered to give a crude product. The crude product was washed with petroleum ether: ethyl acetate (20: 1) (200 mL) to give the desired product. (lR,2S,5S)-3-[(2S,3R)-3-Tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (1.08 g, 2.59 mmol, 62.15% yield, 97.8% purity) was obtained as a white solid. MS (ESI) m/z 407.2 [M-H]’.
Step 4: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000382] To a mixture of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (200 mg, 489.70 umol, 1 eq) in DMF (5 mL) was added DIEA (189.87 mg, 1.47 mmol, 255.89 uL, 3
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SUBSTITUTE SHEET RULE 26 eq), then 2-amino-2-(4-isoquinolyl)acetonitrile (89.72 mg, 489.70 umol, 1 eq) and HATU (372.40 mg, 979.40 umol, 2 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with ethyl acetate 40 mL (20 mL * 2). The combined organic layers were washed with brine 40 mL (40 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by neutral prep-HPLC (column: Waters Xbridge BEH Cl 8 100 * 30mm * lOum; mobile phase: [water( NH4HCO3)-ACN]; B%:40%-70%, 10 mm). (lR,2S,5S)-3-[(2S,3R)-3-Tert-butoxy- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (125 mg, 211.82 umol, 43.25% yield, 97.2% purity) was obtained as a white solid. MS (ESI) m/z 574.2 [M+H]+.
[000383] 'H NMR (400 MHz, DMSO-d6) 5 = 9.71 - 9.56 (m, 1H), 9.47 - 9.34 (m, 2H), 8.77 - 8.67 (m, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.09 - 7.72 (m, 3H), 6.87 (dd, J = 7.8, 13.1 Hz, 1H), 4.44 - 4.32 (m, 1H), 4.22 (d, J = 2.8 Hz, 1H), 4.07 - 3.95 (m, 1H), 3.93 - 3.82 (m, 1H), 3.73 - 3.59 (m, 1H), 1.64 - 1.53 (m, 1H), 1.37 (d, J = 7.5 Hz, 1H), 1.19 - 1.08 (m, 9H), 1.05 - 0.88 (m, 6H), 0.80 (d, J = 8.9 Hz, 3H).
Example 39: Synthesis of (lR,2S,5S)-N-[cyano(pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 187)
Figure imgf000237_0001
Step 1: 2-amino-2-pyrazolo[l,5-a]pyridin-3-yl-acetonitrile
[000384] A solution of pyrazolo[l,5-a]pyridine-3-carbaldehyde (200 mg, 1.37 mmol, 1 eq) in NHLMeOH (2 mL) was added NH4CI (219.60 mg, 4.11 mmol, 3 eq) first and then stirred at 25 °C for 1 h. The resluting mixture was added with TMSCN (407.31 mg, 4.11 mmol, 513.63 uL, 3 eq) at 0 °C and stirred at 25 °C for 15 h. Upon completion, the reaction mixture was
236
SUBSTITUTE SHEET RULE 26 filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, ethyl acetate) to give 2-amino-2-pyrazolo[l,5-a]pyridin-3-yl-acetonitrile (100 mg, 580.77 umol, 42.44% yield) as a yellow solid.MS (ESI) m/z 173.0 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano(pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000385] A solution of 2-amino-2-pyrazolo[l,5-a]pyridin-3-yl-acetonitrile (100 mg, 580.77 umol, 1 eq) in ACN (2 mL) was added with (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (211.61 mg, 580.77 umol, 1 eq), [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (244.43 mg, 871.15 umol, 1.5 eq) and 1 -methylimidazole (143.04 mg, 1.74 mmol, 138.88 uL, 3 eq) at 0 °C, and then the resulting mixture was stirred at 0 °C for 2 h. Upon completion, the reaction mixture was quenched by water (0.5 mL), and then extracted with ethyl acetate (2 mL * 3). The combined organic layers weredried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep- HPLC(column: Phenomenex Cl 8 75*30mm*3um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-60%,8min), then further purified by SLC(column: DAICEL CHIRALPAK AD(250mm*30mm,10um);mobile phase: [0.1%NH3H2O IPA];B%: 20%-20%,8min) to afford (lR,2S,5S)-N-[cyano(pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (27.62 mg, 53.27 umol, 9.17% yield, 100% purity) as off-white solid. MS (ESI) m/z 519.2 [M+H]+.
[000386] 'H NMR (400 MHz, DMSO-de) 5 = 9.40 - 9.27 (m, 2H), 8.73 (d, J = 7.0 Hz, 1H), 8.18 - 8.05 (m, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.36 (dd, J = 7.2, 8.4 Hz, 1H), 6.99 (t, J = 0.8, 6.8 Hz, 1H), 6.40 (d, J = 7.4 Hz, 1H), 4.39 (s, 1H), 4.29 - 4.20 (m, 1H), 3.90 (dd, J = 5.5, 10.3 Hz, 1H), 3.74 - 3.65 (m, 1H), 1.60 - 1.48 (m, 1H), 1.38 - 1.16 (m, 1H), 1.04 - 0.93 (m, 12H), 0.85 - 0.78 (m, 3H)
237
SUBSTITUTE SHEET RULE 26 Example 40: Synthesis of (lR,2S,5S)-N-[cyano(imidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 188)
Figure imgf000239_0001
Step 1: 2-amino-2-imidazo[l,2-a]pyridin-3-yl-acetonitrile
[000387] A solution of imidazo[l,2-a]pyridine-3-carbaldehyde (300 mg, 2.05 mmol, 1 eq) in NHa/MeOH (15 mL) was added NH4CI (329.41 mg, 6.16 mmol, 3 eq). The mixture was stirred at 25 °C for 1 h. TMSCN (407.29 mg, 4.11 mmol, 513.61 uL, 2 eq) was then added at 0 °C. The mixture was stirred at 25 °C for 16 h. Upon completion, the mixture was concentrated under the reduced pressure to give 2-amino-2-imidazo[l,2-a]pyridin-3-yl-acetonitrile (353 mg, crude) as a yellow oil. MS (ESI) m/z 173.2 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano(imidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000388] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (634.82 mg, 1.74 mmol, 1 eq) in DCM (5 mL) was added HATU (993.71 mg, 2.61 mmol, 1.5 eq) and DIEA (675.54 mg, 5.23 mmol, 910.43 uL, 3 eq). The mixture was stirred at 25 °C for 30 min. Then, 2-amino-2-imidazo[l,2-a]pyridin-3-yl-acetonitrile (300 mg, 1.74 mmol, 1 eq) was added at 25 °C. The mixture was stirred at 25 °C for 1 h. Upon completion, the residue was quenched by H2O (5 mL) and was extracted with DCM (5 mL * 3). The combined organic phase was washed with brine (5 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex C 18 75 * 30 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%- 65%, 8 min) to give the product (lR,2S,5S)-N-[cyano(imidazo[l,2-a]pyridin-3-yl)methyl]-3-
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SUBSTITUTE SHEET RULE 26 [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (160 mg, 308.56 umol, 61.54% yield) as a white solid. MS (ESI) m/z 519.2 [M+H]+
[000389] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.60 - 9.30 (m, 2H), 8.24 - 8.05 (m, 1H), 7.79 - 7.64 (m, 2H), 7.43 - 7.32 (m, 1H), 7.11 - 6.75 (m, 2H), 4.46 - 4.34 (m, 1H), 4.21 (d, J= 19.4 Hz, 1H), 3.99 - 3.88 (m, 1H), 3.69 (dd, J= 10.4, 18.8 Hz, 1H), 1.65 - 1.53 (m, 1H), 1.41 - 1.21 (m, 1H), 1.08 - 0.88 (m, 12H), 0.82 (d, J= 3.8 Hz, 3H)
Example 41: Synthesis of (lR,2S,5S)-N-[cyano(imidazo[l,5-a]pyridin-5-yl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 189)
Figure imgf000240_0001
Step 1: 2-amino-2-imidazo[l,5-a]pyridin-5-yl-acetonitrile
[000390] To a mixture of imidazo[l,5-a]pyridine-5-carbaldehyde (300 mg, 2.05 mmol, 1 eq) in ammonia (7 M, 8 mL, 14.73 eq) was added NH4CI (109.81 mg, 2.05 mmol, 1 eq) in one portion at 20°C. The mixture was stirred at 20°C for 30 min, and then TMSCN (407.29 mg, 4.10 mmol, 513.61 uL, 2 eq) was added at 20°C and stirred for 16 h. Upon completion, The reaction mixture was concentrated under reduced pressure to afford 2-amino-2-imidazo[l,5-a]pyridin-5- yl-acetonitrile (264 mg, crude) as a black brown solid. MS (ESI) m/z 173.4 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano(imidazo[l,5-a]pyridin-5-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000391] To a mixture of 2-amino-2-imidazo[l,5-a]pyridin-5-yl-acetonitrile (200 mg, 1.16 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (338.57 mg, 929.23 umol, 0.8 eq) in DCM (3 mL) was added HATU (883.30 mg, 2.32 mmol, 2 eq) and DIEA (300.24 mg, 2.32
239
SUBSTITUTE SHEET RULE 26 mmol, 404.64 uL, 2 eq) in one portion at 25°C. The mixture was stirred at 25°C for 1 h. Upon completion, the reaction mixture was quenched by addition water 5 mL, and then extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC(column: Waters Xbridge BEH Cl 8 100*30mm*10um;mobile phase: [water (NH3H2O+NH4HCO3) - ACN]; B%: 30%-50%, 10mm) to give a (1R,2S,5S)-N- [cyano(imidazo[l,5-a]pyridin-5-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (26.02 mg, 50.03 umol, 28.82% yield, 99.7% purity) as a yellow solid. MS (ESI) m/z 519.3 [M+H]+.
[000392] 1H NMR (400 MHz, DMSO-t76) 5 = 9.76 - 9.59 (m, 1H), 9.39 (br s, 1H), 8.19 - 8.09 (m, 1H), 7.71 (d, J= 9.1 Hz, 1H), 7.54 (d, J= 7.2 Hz, 1H), 7.03 - 6.95 (m, 1H), 6.89 (td, J= 6.0, 9.0 Hz, 1H), 6.81 (br d, J= 7.7 Hz, 1H), 4.45 - 4.34 (m, 1H), 4.24 (d, J= 12.3 Hz, 1H), 3.99 - 3.88 (m, 1H), 3.76 - 3.65 (m, 1H), 1.65 - 1.55 (m, 1H), 1.35 - 1.16 (m, 1H), 1.05 - 0.94 (m, 12H), 0.83 (d, J= 5.6 Hz, 3H)
Example 42: Synthesis of (lR,2S,5S)-N-[cyano(imidazo[l,2-a]pyridin-5-yl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 190)
Figure imgf000241_0001
[000393] To a solution of imidazo[l,2-a]pyridine-5-carbaldehyde (300 mg, 2.05 mmol, 1 eq) in DCM (5 mL) was added NHa/MeOH (7 M, 1.17 mL, 4 eq) and tetraisopropoxytitanium (875.13 mg, 3.08 mmol, 908.76 uL, 1.5 eq) at 25 °C. After addition, the mixture was stirred at 25 °C for 1 h. And then, TMSCN (610.94 mg, 6.16 mmol, 770.41 uL, 3 eq) was added, and the resulting mixture was stirred at 25 °C for 15 h. Upon completion, the combined reaction mixture was diluted with water (10 mL) and extracted with Ethyl acetate (8 mL), filtered to give a mother liquor. The mother liquor was extracted with ethyl acetate (5 mL * 2). The combined organic
240
SUBSTITUTE SHEET RULE 26 layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give z-amino- 2-imidazo[l,2-a]pyridin-5-yl-acetonitrile (320 mg, crude) as a yellow solid.
Step 2: (lR,2S,5S)-N-[cyano(imidazo[l,2-a]pyridin-5-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000394] To a solution of 2-amino-2-imidazo[l,2-a]pyridin-5-yl-acetonitrile (320 mg, 1.86 mmol, 1.1 eq) in DCM (10 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (615.5 mg, 1.69 mmol, 1 eq) and DIEA (545.89 mg, 4.22 mmol, 735.70 uL, 2.5 eq). HATU (963.60 mg, 2.53 mmol, 1.5 eq) was added, and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by prep-HPLC (neutral condition) (column: C18 (250*50mm*10 um);mobile phase: [water(NH4HCO3)-ACN];B%: 45%-65%,10min) to give (lR,2S,5S)-N-[cyano(imidazo[l,2- a]pyridin-5-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (250 mg, 476.36 umol, 28.20% yield, 99.6% purity) as a white solid. MS (ESI) m/z 519.3 [M+H]+
[000395] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.77 - 9.60 (m, 1H), 9.39 (dd, J= 3.4, 8.2 Hz, 1H), 7.78 - 7.56 (m, 3H), 7.36 (ddd, J= 2.4, 6.9, 9.1 Hz, 1H), 7.26 - 7.17 (m, 1H), 6.80 (dd, J = 3.5, 8.0 Hz, 1H), 4.43 - 4.36 (m, 1H), 4.24 (d, J= 11.3 Hz, 1H), 3.98 - 3.91 (m, 1H), 3.75 - 3.67 (m, 1H), 1.64 - 1.56 (m, 1H), 1.37 - 1.19 (m, 1H), 1.05 - 0.95 (m, 12H), 0.89 - 0.81 (m, 3H)
241
SUBSTITUTE SHEET RULE 26 Example 43: Synthesis of (lR,2S,5S)-N-[cyano-[4-(4-fluorophenyl)-l,2,4-triazol-3- yl] methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 191)
Figure imgf000243_0001
Step 1: 4-(4-fluorophenyl)-3-(methoxymethyl)-l,2,4-triazole
[000396] To a solution of 2-methoxyacetohydrazide (5.15 g, 49.50 mmol, 1.1 eq) in MeCN (45 mL) was added dropwise DMF/DMA (6.60 g, 55.35 mmol, 7.35 mL, 1.23 eq) at 25 °C. After the addition, the mixture was stirred at 50 °C for 0.5 h, and then a solution of 4-fluoroaniline (5 g, 45.00 mmol, 4.31 mL, 1 eq) in MeCN (20 mL) and AcOH (28.37 g, 472.48 mmol, 27.02 mL, 10.5 eq) were added at 50 °C. The resulting mixture was stirred at 120 °C for 3.5 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10:1 to 1:1) to afford 4-(4-fluorophenyl)-3-(methoxymethyl)-l,2,4-triazole (3.7 g, 17.86 mmol, 39.68% yield) as a white solid. MS (ESI) m/z 208.2 [M+H]+.
Step 2: [4-(4-fluorophenyl)-l,2,4-triazol-3-yl]methanol
[000397] To a solution of 4-(4-fluorophenyl)-3-(methoxymethyl)-l,2,4-triazole (3.7 g, 17.86 mmol, 1 eq) in DCM (40 mL) was added BBn (5.37 g, 21.43 mmol, 2.06 mL, 1.2 eq) at -30 °C. The mixture was stirred at 25 °C for 0.5 h, and then BBn (5.37 g, 21.43 mmol, 2.06 mL, 1.2 eq) was added at -30 °C. The mixture was stirred at 25 °C for 0.5 h. Upon completion, the reaction
242
SUBSTITUTE SHEET RULE 26 mixture was quenched by addition methanol 20 mL at 0 °C. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 5: 1 to Ethyl acetate/Methanol = 20:1) to give [4-(4-fluorophenyl)-l,2,4-triazol-3-yl]methanol (1.9 g, 8.20 mmol, 45.94% yield, 83.4% purity) as a brown solid. MS (ESI) m/z 194.2 [M+H]+
Step 3: 4-(4-fluorophenyl)-l,2,4-triazole-3-carbaldehyde
[000398] To a solution of [4-(4-fluorophenyl)-l,2,4-triazol-3-yl]methanol (1.9 g, 9.84 mmol, 1 eq) in CHCh (30 mL) was added MnCh (17.10 g, 196.71 mmol, 20 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction solution was filtered with diatomite to remove the manganese dioxide and then the mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to Ethyl acetate: Methanol = 20: 1) to afford 4-(4-fluorophenyl)-l,2,4-triazole-3- carbaldehyde (838 mg, 4.38 mmol, 44.57% yield) as a white solid. MS (ESI) m/z 192.2 [M+H]+.
Step 4: 2-amino-2-[4-(4-fluorophenyl)-l ,2,4-triazol-3-yl]acetonitrile
[000399] To a solution of 4-(4-fluorophenyl)-l,2,4-triazole-3-carbaldehyde (838 mg, 4.38 mmol, 1 eq) in NEh/MeOH (7 M, 40 mL, 63.87 eq) was added NH4CI (468.98 mg, 8.77 mmol, 2 eq) at 25 °C. After the addition, the mixture was stirred at 25 °C for 1 h. TMSCN (869.79 mg, 8.77 mmol, 1.10 mL, 2 eq) was added, and the resulting mixture was stirred at 25 °C for 15 h. Upon completion, the mixture was concentrated under reduced pressure to give a residue. The residue was diluted with water (10 mL) and extracted with ethyl acetate (8 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, DCM:MeOH = 20: 1) to give 2-amino-2-[4-(4-fluorophenyl)-l, 2, 4-triazol-3-yl] acetonitrile (80 mg, 368.32 umol, 8.40% yield) as a yellow solid. MS (ESI) m/z 218.2 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-[4-(4-fhiorophenyl)-l,2,4-triazol-3-yl]methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000400] To a solution of 2-amino-2-[4-(4-fluorophenyl)-l,2,4-triazol-3-yl]acetonitrile (78.69 mg, 362.28 umol, 1.2 eq) in DCM (3 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-
243
SUBSTITUTE SHEET RULE 26 [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxylic acid (110 mg, 301.90 umol, 1 eq) and DIEA (97.55 mg, 754.75 umol, 131.46 uL, 2.5 eq). HATU (172.19 mg, 452.85 umol, 1.5 eq) was added, and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 100 * 30mm * lOum; mobile phase: [water ( NH4HCO3) - ACN]; B%: 35%-65%, 10 min) to give (lR,2S,5S)-N-[cyano-[4-(4- fluorophenyl)-l,2,4-triazol-3-yl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxamide (45.6 mg, 79.95 umol, 26.48% yield, 98.8% purity) as a yellow solid. MS (ESI) m/z 564.3 [M+H]+.
Figure imgf000245_0001
NMR (400 MHz, DMSO-de) 5 = 9.54 - 9.34 (m, 1H), 8.91 (d, J= 2.8 Hz, 1H), 7.63 - 7.38 (m, 5H), , 6.68 - 6.27 (m, 1H), 4.36 (d, J= 19.5 Hz, 1H), 4.15 - 3.95 (m, 1H), 3.88 - 3.70 (m, 1H), 3.58 (dd, J= 10.5, 16.5 Hz, 1H), 1.53 - 1.27 (m, 1H), 0.91 (s, 12H), 0.88 (br d, J= 7.9 Hz, 0.5H), 0.82 - 0.70 (m, 3H), 0.14 (d, J = 7.6 Hz, 0.5H)
Example 44: Synthesis of (lR,2S,5S)-N-[[4-(3-chlorophenyl)-l,2,4-triazol-3-yl]-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 192)
Figure imgf000245_0002
Step 1: 4-(3-chlorophenyl)-3-(methoxymethyl)-l,2,4-triazole
[000401] A solution of 2-methoxyacetohydrazide (11 mmol, 1.1 eq) in ACN (30 mL) was added DMFDMA (4.67 g, 39.19 mmol, 5.21 mL, 1 eq) and the mixture was stirred at 50 °C for 0.5 h. The mixture was added with 3 -chloroaniline (5 g, 39.19 mmol, 4.17 mL, 1 eq) in ACN (15
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SUBSTITUTE SHEET RULE 26 mL), and then CH3COOH (24.71 g, 411.53 mmol, 23.54 mL, 10.5 eq) was added and the mixture was stirred at 120 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O 50 mL at 25 °C, and extracted with ethyl acetate 60 mL (20 mL * 3). The combined organic layers were dried over Na2SO4, concentrated in vacuum. The residue was purified by chromatography (SiCh, ethyl acetate: petroleum ether = 5: 1) to afford 4-(3-chlorophenyl)-3- (methoxymethyl)-l,2,4-triazole (2.3 g, 8.33 mmol, 21.25% yield, 81% purity) as a white solid. MS (ESI) m/z 224.1 [M+H]+
Step 2: [4-(3-chlorophenyl)-l,2,4-triazol-3-yl]methanol
[000402] A solution of 4-(3-chlorophenyl)-3-(methoxymethyl)-l,2,4-triazole (2.0 g, 7.24 mmol, 81% purity, 1 eq) in DCM (50 mL) was added with tribromoborane (2.18 g, 8.69 mmol, 837.50 uL, 1.2 eq) dropwise at -60 °C in N2 atmosphere. The mixture was stirred at 25 °C for 2.5 h. Upon completion, the reaction mixture was quenched by addition methanol (absolute) 50 mL at 0 °C, triturated with DCM at 25 °C for 0.5 h, and then the mixture was filtered and concentrated under reduced pressure to get the residue. The residue was purified by chromatography (SiCh, di chloromethane: methanol = 10:1) to afford [4-(3 -chlorophenyl)- 1,2,4- triazol-3-yl]methanol (360 mg, 1.51 mmol, 20.86% yield, 88% purity) as a white solid. MS (ESI) m/z 210.1 [M+H]+
Step 3: 4-(3-chlorophenyl)-l,2,4-triazole-3-carbaldehyde
[000403] A solution of [4-(3-chlorophenyl)-l,2,4-triazol-3-yl]methanol (300 mg, 1.26 mmol, 88% purity, 1 eq) in CHCh (15 mL) was added MnCh (2.19 g, 25.19 mmol, 20 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the solution was filtered with diatomite to remove the manganese dioxide and then the mixture was concentrated under reduced pressure. The residue was purified by prep-TLC (SiCh, dichloromethane:methanol = 10:1) to afford 4-(3- chlorophenyl)-l,2,4-triazole-3-carbaldehyde (87 mg, 238.85 umol, 18.97% yield, 57% purity) as a white solid. MS (ESI) m/z 208.0 [M+H]+
Step 4: 2-amino-2- [4-(3 -chlorophenyl)- 1 ,2,4-triazol-3 -y 1] acetonitrile
[000404] A mixture of 4-(3-chlorophenyl)-l,2,4-triazole-3-carbaldehyde (87 mg, 419.04 umol, 1 eq), NEb/MeOH (7 M, 239.45 uL, 4 eq) and tetraisopropoxytitanium (178.65 mg, 628.56
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SUBSTITUTE SHEET RULE 26 umol, 185.51 uL, 1.5 eq) in DCM (2 mL) was stirred at 25 °C for 1 h, then TMSCN (124.72 mg, 1.26 mmol, 157.27 uL, 3 eq) was added at 0 °C. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep-TLC (SiCh, dichloromethane:methanol = 10:1) to afford 2-amino-2-[4-(3- chlorophenyl)-l,2,4-triazol-3-yl]acetonitrile (45 mg, 177.18 umol, 42.28% yield, 92% purity) as a white solid. MS (ESI) m/z 234.0 [M+H]+
Step 5: (lR,2S,5S)-N-[[4-(3-chlorophenyl)-l,2,4-triazol-3-yl]-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000405] A mixture of 2-amino-2-[4-(3-chlorophenyl)-l,2,4-triazol-3-yl]acetonitrile (40 mg, 171.19 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifhroroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (68.61 mg, 188.31 umol, 1.1 eq) in ACN (1 mL) was added with 1 -methylimidazole (42.17 mg, 513.57 umol, 40.94 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphate (96.07 mg, 342.38 umol, 2 eq). The resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure and then the residue was purified by prep-HPLC (neutral condition, column: Waters Xbridge BEH C18 100*30mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 35%-60%,10min.) to give (lR,2S,5S)-N-[[4-(3- chlorophenyl)- 1 ,2,4-triazol-3 -yl] -cyano-methyl] -3 - [(2S)-3 , 3 -dimethy 1-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (43 mg, 71.17 umol, 41.57% yield, 96% purity) as a white solid. MS (ESI) m/z 580.2 [M+H]+.
[000406] 1 H NMR (400 MHz, DMSO-de) 5 = 9.62 - 9.14 (m, 2H), 9.05 - 8.87 (m, 1 H), 7.73 -
7.43 (m, 4H), 6.82 - 6.43 (m, 1H), 4.34 (br s, 1H), 4.09 - 3.96 (m, 1H), 3.91 - 3.70 (m, 1H), 3.65 - 3.45 (m, 1H), 1.48 - 1.24 (m, 1H), 1.01 - 0.86 (m, 12H), 0.76 - 0.67 (m, 3H), 0.13 (d, J = 7.6 Hz, 1H).
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SUBSTITUTE SHEET RULE 26 Example 45: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 193)
Figure imgf000248_0001
[000407] To a solution of 2-amino-2-(7,8-difluoro-4-isoquinolyl)acetonitrile (100 mg, 456.23 umol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (279.49 mg, 684.34 umol, 1.5 eq), 1- methylimidazole (112.37 mg, 1.37 mmol, 109.10 uL, 3 eq) in ACN (5 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (256.01 mg, 912.45 umol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O 30 mL at 20 °C, and then extracted with EtOAc (35 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)- ACN];B%: 45%-70%,8min) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (117.7 mg, 192.31 umol, 42.15% yield, 99.6% purity) as a white solid. MS (ESI) m/z 610.2 [M+H]+.
[000408] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.90 - 9.35 (m, 3H), 8.89 - 8.73 (m, 1H), 8.16 - 7.86 (m, 2H), 7.01 - 6.70 (m, 1H), 4.36 (d, J= 7.1 Hz, 1H), 4.18 (d, J= 5.1 Hz, 1H), 3.98 (dd, J = 5.4, 10.4 Hz, 1H), 3.85 (ddd, J= 6.4, 7.6, 13.9 Hz, 1H), 3.62 (t, J= 11.2 Hz, 1H), 1.62 - 1.45 (m, 1H), 1.38 - 1.16 (m, 1H), 1.14 - 0.79 (m, 18H).
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SUBSTITUTE SHEET RULE 26 Example 46: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-dichloro-4-isoquinolyl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 194)
Figure imgf000249_0001
Step 1: 4-bromo-7,8-dichloro-isoquinoline
[000409] To a solution of 7,8-dichloroisoquinoline (2 g, 10.10 mmol, 1 eq) in AcOH (20 mL) was added NBS (2.16 g, 12.12 mmol, 1.2 eq), and the mixture was stirred at 110 °C for 2 h. Upon completion, the reaction mixture was removed with solvent, poured into H2O 50 mL at 20 °C, and then NaHCOi was added to adjust pH to 8. The resulting mixture was extracted with EtOAc (50 mL * 3), and the combined organic layers were washed with brine 45 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give 4-bromo-7,8-dichloro- isoquinoline (2.6 g, crude) as a yellow solid. MS (ESI) m/z 278.0 [M+H]+.
Step 2: 7,8-dichloro-4-vinyl-isoquinoline
[000410] To a solution of 4-bromo-7,8-dichloro-isoquinoline (2.4 g, 8.67 mmol, 1 eq) in toluene (24 mL) was added PPhi (227.30 mg, 866.60 umol, 0.1 eq) and Pd(dba)2 (498.30 mg, 866.60 umol, 0.1 eq). The resulting mixture was stirred for 30 min at 20 °C, and then tributyl(vinyl)stannane (4.12 g, 13.00 mmol, 3.78 mL, 1.5 eq) in toluene (3 mL) was added. The mixture was stirred at 110 °C for 15.5 h. Upon completion, the mixture was quenched by
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SUBSTITUTE SHEET RULE 26 addition of H2O 60 mL, extracted with ethyl acetate (60 mL * 3), and then the combined organic layers were washed with brine (60 mL) and dried over Na2SO4. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 90:1 to 30: 1) to give 7,8-dichloro-4- vinyl-isoquinoline (1.4 g, 5.94 mmol, 68.49% yield, 95% purity) as a yellow solid. MS (ESI) m/z 224.1 [M+H]+.
Step 3: 7,8-dichloroisoquinoline-4-carbaldehyde
[000411] To a solution of 7,8-dichloro-4-vinyl-isoquinoline (1.4 g, 6.25 mmol, 1 eq) in THF (15 mL) and H2O (3 mL) was added K2OSO4.2H2O (230.20 mg, 624.76 umol, 0.1 eq), and the resulting mixture was stirred for 30 min. NaICU (4.01 g, 18.74 mmol, 1.04 mL, 3 eq was added, and the mixture was stirred at 20 °C for 5.5 h. Upon completion, the reaction mixture was poured into H2O 45 mL at 20 °C, and then extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 90:1 to 20: 1) to give 7,8-dichloroisoquinoline-4-carbaldehyde (1.2 g, 5.04 mmol, 80.72% yield, 95% purity) as a yellow solid. MS (ESI) m/z 226.0 [M+H]+.
Step 4: 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile
[000412] A solution of 7,8-dichloroisoquinoline-4-carbaldehyde (1.2 g, 5.31 mmol, 1 eq), Ti(i-PrO)4 (2.26 g, 7.96 mmol, 2.35 mL, 1.5 eq), NH3/MeOH (7 M, 3.03 mL, 4 eq) in DCM (12 mL) was stirred 1 h at 20 °C, and then TMSCN (1.58 g, 15.93 mmol, 1.99 mL, 3 eq) was added. The mixture was stirred at 20 °C for 5 h. Upon completion, the reaction mixture was poured into H2O 50 mL at 20°C, and then extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 100:0 to 95:5) to give 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile (1.2 g, 4.43 mmol, 83.39% yield, 93% purity) as a yellow solid. MS (ESI) m/z 252.1 [M+H]+.
Step 5: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(7,8-dichloro-4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
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SUBSTITUTE SHEET RULE 26 [000413] To a solution of 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile (90 mg, 357.00 umol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (189.55 mg, 464.10 umol, 1.3 eq), 1- methylimidazole (87.93 mg, 1.07 mmol, 85.37 uL, 3 eq in ACN (5 mL), was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (200.33 mg, 714.00 umol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O 25 mL at 20 °C, and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine 25 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)- ACN];B%: 50%-80%,8min) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-dichloro-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (120 mg, 186.21 umol, 52.16% yield, 99.7% purity) as a white solid. MS (ESI) m/z 642.1 [M+H]+.
[000414] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.85 - 9.31 (m, 3H), 9.05 - 8.82 (m, 1H), 8.20 - 7.92 (m, 2H), 6.98 (dd, J= 4.3, 7.9 Hz, 1H), 4.34 (d, J= 19.1 Hz, 1H), 4.16 (d, J= 9.9 Hz, 1H), 3.98 (dd, J= 5.0, 10.1 Hz, 1H), 3.90 - 3.71 (m, 1H), 3.68 - 3.56 (m, 1H), 1.62 - 1.45 (m, 1H), 1.40 - 1.19 (m, 1H), 1.11 - 0.74 (m, 18H).
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SUBSTITUTE SHEET RULE 26 Example 47: Synthesis of N-((lS,2R)-2-((cyano(7,8-difluoroisoquinolin-4- yl)methyl)carbamoyl)-[l,l'-bi(cyclopropan)]-2-yl)-6,7-difluoro-lH-indole-2-carboxamide (Compound 196)
Figure imgf000252_0001
Step 1: ethyl l-(tert-butoxycarbonylamino)-2-cyclopropyl-cyclopropanecarboxylate
[000415] To a solution of ZnEt2 (1 M, 13.04 mL, 4.16 eq) in DCM (80 mL) was added TFA (1.17 g, 10.28 mmol, 760.95 uL, 3.28 eq) in DCM (15 mL) at 0 °C under N2, and then the mixture was stirred at 0 °C for 0.5 h. CH2I2 (3.47 g, 12.94 mmol, 1.04 mL, 4.13 eq) was added drop- wise, and then the mixture was stirred at 0 °C under N2 for 0.5 h. Ethyl l-(tert- butoxycarbonylamino)-2-vinyl-cyclopropanecarboxylate (800 mg, 3.13 mmol, 1 eq) in DCM (15 mL) was added at 0 °C, and then the mixture was stirred at 30 °C for 20 h. Upon completion, the mixture was quenched by sat. NH4CI (100 mL) and was extracted with DCM (30 mL * 3), and
251
SUBSTITUTE SHEET RULE 26 then the organic phase was concentrated in vacuum to obtain etnyl 1 -( tert- butoxycarbonylamino)-2-cyclopropyl-cyclopropanecarboxylate (800 mg, crude) as a yellow oil.
Step 2: ethyl l-(tert-butoxycarbonylamino)-2-cyclopropyl-cyclopropanecarboxylate
[000416] A solution of ethyl 1 -(tert- butoxy carbonylamino)-2-cy cl opropyl- cyclopropanecarboxylate (800 mg, 2.97 mmol, 1 eq) in HCl/dioxane (4 M, 8 mL) was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum to afford ethyl 1- amino-2-cyclopropyl-cyclopropanecarboxylate (600 mg, crude, HC1) as a yellow oil.
Step 3: l-amino-2-cyclopropyl-cyclopropanecarboxylic acid
[000417] To a solution of ethyl l-amino-2-cyclopropyl-cyclopropanecarboxylate (250.00 mg,
1.48 mmol, 1 eq) in THF (3 mL) and H2O (1.5 mL) was added LiOH.TLO (371.94 mg, 8.86 mmol, 6 eq), and the mixture was stirred at 60 °C for 16 h. Upon completion, the mixture was used to next step and was obtained l-amino-2-cyclopropyl-cyclopropanecarboxylic acid (210 mg, crude) as a yellow liquid.
Step 4: 1 -(tert- butoxy carbonylamino)-2-cyclopropyl-cy cl opropanecarboxylic acid
[000418] To a solution of 1 -amino-2-cyclopropyl-cy cl opropanecarboxylic acid (210.00 mg,
1.49 mmol, 1 eq) and DMAP (181.74 mg, 1.49 mmol, 1 eq) in THF (4 mL) and H2O (0.5 mL) was added BOC2O (973.99 mg, 4.46 mmol, 1.03 mL, 3 eq), the mixture was stirred at 20 °C for 3 h. Upon completion, the mixture was adjusted to pH~l with IN HC1 (10 mL) and was extracted with ethyl acetate (4 mL * 3). The organic phase was concentrated in vacuum to afford l-(tert- butoxycarbonylamino)-2-cyclopropyl-cyclopropanecarboxylic acid (250 mg, crude) as a yellow gum.
Step 5: tert-butyl N-[l-[[cyano-(7,8-difluoro-4-isoquinolyl)methyl]carbamoyl]-2-cyclopropyl- cyclopropy 1] carbamate
[000419] To a solution of 1 -(tert-butoxycarbonylamino)-2-cyclopropyl- cyclopropanecarboxylic acid (150.00 mg, 621.68 umol, 1.5 eq) and 2,3-diamino-3-(7,8-difluoro- 4-isoquinolyl) propanenitrile (102.88 mg, 414.45 umol, 1 eq) in ACN (10 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (232.57 mg, 828.90 umol, 2 eq) and 1 -methylimidazole (102.08 mg, 1.24 mmol, 99.11 uL, 3 eq), and the
252
SUBSTITUTE SHEET RULE 26 mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum and was extracted with DCM (5 mL * 3), then the organic phase was concerntrated in vacuum. The crude product was purified by column (SiCh, PE:EA = 1 :0 to 2: 1) to afford tert-butyl N-[l- [ [cyano-(7, 8-difhioro-4-isoquinoly l)methyl] carbamoyl] -2-cyclopropyl-cyclopropy 1] carbamate (180 mg, 345.80 umol, 83.43% yield, 85% purity) as a white solid. MS (ESI) m/z 443.2 [M+H]+
Step 6: l-amino-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-2-cyclopropyl- cyclopropanecarboxamide
[000420] A solution of tert-butyl N-[l-[[cyano-(7,8-difluoro-4- isoquinolyl)methyl]carbamoyl]-2-cyclopropyl-cyclopropyl]carbamate (180 mg, 345.80 umol, 85% purity, 1 eq) in HCl/dioxane (4 M, 3 mL) was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum to obtained l-amino-N-[cyano-(7,8-difluoro-4- isoquinolyl)methyl]-2-cyclopropyl-cyclopropanecarboxamide (150 mg, crude, HC1) as a red gum.
Step 7: N-[l-[[(lS)-2-amino-l-[[(3R)-5,5-dimethyl-2-oxo-pyrrolidin-3-yl]methyl]-2-oxo- ethyl]carbamoyl]-2-cyclopropyl-cyclopropyl]-6,7-difluoro-lH-indole-2-carboxamide
[000421] To a solution of l-amino-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-2- cyclopropyl-cyclopropanecarboxamide (150.00 mg, 395.98 umol, 1 eq, HC1) and 6,7-difluoro- lH-indole-2-carboxylic acid (78.06 mg, 395.98 umol, 1 eq) in DCM (5 mL) was added DIEA (153.53 mg, 1.19 mmol, 206.92 uL, 3 eq) and HATU (150.57 mg, 395.98 umol, 1 eq), and then the resulting mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was quenched by water (15 mL) and was extracted with DCM (5 mL * 3), then the organic phase was concentrated in vacuum and was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75 * 30mm * 3um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35 %-75 %, 8min) and re-purified by prep-HPLC (column: Phenomenex Luna Cl 8 75 * 30mm * 3um; mobile phase: [water (FA)- ACN]; B%: 35%-75%, 8min) to afford N-[l-[[cyano-(7,8-difluoro-4- isoquinolyl)methyl]carbamoyl]-2-cyclopropyl-cyclopropyl]-6,7-difluoro-lH-indole-2- carboxamide (8 mg, 14.53 umol, 3.67% yield, 94.7% purity) as a red solid. MS (ESI) m/z 522.2 [M+H]+
253
SUBSTITUTE SHEET RULE 26 [000422] 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.35 - 12.16 (m, 1H), 9.63 - 9.24 (m, 2H), 8.99 - 8.46 (m, 2H), 8.67 - 7.38 (m, 3H), 7.26 - 7.17 (m, 1H), 7.13 - 7.03 (m, 2H), 1.74 - 1.64 (m, 1H), 1.49 - 1.28 (m, 1H), 1.15 - 1.08 (m, 1H), 0.90 - 0.66 (m, 1H), 0.56 - 0.36 (m, 2H), 0.32 - 0.10 (m, 2H).
Example 48: Synthesis of (lR,2S,5S)-N-[cyano-(2-oxo-lH-quinolin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 197)
Figure imgf000255_0001
Step 1: 4-(hydroxymethyl)-lH-quinolin-2-one
[000423] A mixture of 4-(bromomethyl)-lH-quinolin-2-one (5.00 g, 21.00 mmol, 1 eq) in DMSO (20.00 g, 255.98 mmol, 20 mL, 12.19 eq) was stirred for 0.5 h at 70 °C and aq. Na2CCh (44.52 g, 21.00 mmol, 2 mL, 5% purity, 1 eq) was added dropwise at 20 °C. The mixture was stirred at 110 °C for 16 h. Upon completion, the reaction mixture was cooled, diluted with ice cooled water and filtered, and then the filter cake was dried under reduced pressure to give 4- (hydroxymethyl)-lH-quinolin-2-one (1.6 g, crude) as a yellow solid. MS (ESI) m/z 176.2 [M+H]+.
Step 2: 2-oxo- lH-quinoline-4-carbaldehy de
[000424] To a solution of 4-(hydroxymethyl)-lH-quinolin-2-one (1.3 g, 7.42 mmol, 1 eq) in DMSO (10 mL) was added Dess-Martin (3.78 g, 8.90 mmol, 2.76 mL, 1.2 eq). The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was poured into H2O (30 mL) at
254
SUBSTITUTE SHEET RULE 26 20 °C, and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10:1 to 5: 1) to give 2-oxo-lH-quinoline-4-carbaldehyde (1.29 g, 7.45 mmol, 100.00% yield) as a yellow solid. MS (ESI) m/z 174.2 [M+H]+.
Step 3: 2-oxo- lH-quinoline-4-carbaldehy de
[000425] To a solution of 2-oxo-lH-quinoline-4-carbaldehyde (1.29 g, 7.45 mmol, 1 eq) in DCM (10 mL) was added Ti(i-PrO)4 (3.18 g, 11.17 mmol, 3.30 mL, 1.5 eq) and NH3/MeOH (7 M, 4.26 mL, 4 eq). The resulting mixture was stirred for 1 h at 20 °C before TMSCN (2.22 g, 22.35 mmol, 2.80 mL, 3 eq) was added at 20 °C. The mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was poured into H2O (30 mL) at 20 °C, and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM:MeOH = 100:0 to 90:10) to give 2-amino-2-(2- oxo-lH-quinolin-4-yl)acetonitrile (150 mg, 669.40 umol, 8.99% yield, 88.9% purity) as a yellow solid. MS (ESI) m/z 200.2 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(2-oxo-lH-quinohn-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000426] To a solution of 2-amino-2-(2-oxo-lH-quinolin-4-yl)acetonitrile (140 mg, 702.78 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (281.67 mg, 773.06 umol, 1.1 eq and 1- methylimidazole (173.10 mg, 2.11 mmol, 168.06 uL, 3 eq) in ACN (10 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (394.37 mg, 1.41 mmol, 2 eq at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O 30 mL at 25 °C, and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 100 * 30mm * lOum; mobile phase: [water( NELHCCh)- ACN]; B%: 40%-60%, 8min) to give (lR,2S,5S)-N-[cyano-(2-oxo-lH-quinolin-4-yl)methyl]-3-
255
SUBSTITUTE SHEET RULE 26 [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (130 mg, 229.95 umol, 32.72% yield, 96.5% purity) as a white solid. MS (ESI) m/z 546.3 [M+H]+.
[000427] 'H NMR (400 MHz, DMSO-t/r,) 5 = 12.14 - 11.84 (m, 1H), 9.68 - 9.27 (m, 2H), 7.72 - 7.49 (m, 2H), 7.37 (t, J= 7.2 Hz, 1H), 7.27 - 7.07 (m, 1H), 6.84 - 6.59 (m, 2H), 4.39 (s, 1H), 4.23 (d, J= 8.9 Hz, 1H), 3.99 - 3.87 (m, 1H), 3.77 - 3.63 (m, 1H), 1.66 - 1.48 (m, 1H), 1.33 - 1.15 (m, 1H), 1.10 - 0.90 (m, 12H), 0.82 (d, J= 6.1 Hz, 3H).
Example 49: Synthesis of (lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-N-((5-chloroisoquinolin-4-yl)(cyano)methyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 198)
Figure imgf000257_0001
[000428] To a solution of 2-amino-2-(5-chloroisoquinolin-4-yl)acetonitrile (31.98 mg, 146.91 umol, 1 eq) in ACN (3 mL) was added (lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (60 mg, 146.91 umol, 1 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (82.44 mg, 293.82 umol, 2 eq). Then, 1 -methylimidazole (36.19 mg, 440.73 umol, 35.13 uL, 3 eq) was added, and then the mixture was stirred for 1 hour at 25 °C. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with aqueous NaCl (10 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE:EA = 0: 1), which was further separated by SEC column: DAICEL CHIRALPAK AD(250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 20%-20%, 8 min to give (lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2-
256
SUBSTITUTE SHEET RULE 26 trifluoroacetamido)butanoyl)-N-((5-chloroisoquinolin-4-yl)(cyano)methyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (18.01 mg, 27.74 umol, 18.89% yield, 93.67% purity) as a white solid. MS (ESI) m/z 608.2 [M+H]+. 'H NMR (400 MHz, DMSO-de) 5 = 9.68 - 9.57 (m, 1H), 9.56 - 9.46 (m, 1H), 9.37 (br d, J = 7.0 Hz, 1H), 8.87 (s, 1H), 8.29 (br d, J = 7.9 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.83 - 7.74 (m, 1H), 7.34 (br s, 1H), 4.39 (br d, J = 7.8 Hz, 1H), 4.24 (s, 1H), 4.00 - 3.85 (m, 2H), 3.64 (br d, J = 10.6 Hz, 1H), 1.60 - 1.51 (m, 1H), 1.30 (br d, J = 7.5 Hz, 1H), 1.16 - 1.09 (m, 9H), 1.02 (br d, J = 6.0 Hz, 3H), 0.99 - 0.95 (m, 3H), 0.80 (s, 3H).
[000429] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (31.49 mg, 50.98 umol, 34.70% yield, 98.44% purity) was obtained as a white solid. MS (ESI) m/z 608.1 [M+H]+. 'H NMR (400 MHz, DMSO-de) 5 = 9.74 - 9.58 (m, 1H), 9.50 (br s, 2H), 8.92 - 8.83 (m, 1H), 8.35 - 8.20 (m, 1H), 8.13 - 8.00 (m, 1H), 7.81 - 7.67 (m, 1H), 7.42 - 7.20 (m, 1H), 4.42 - 4.35 (m, 1H), 4.32 - 4.24 (m, 1H), 4.00 - 3.85 (m, 2H), 3.78 - 3.57 (m, 1H),
1.62 - 1.54 (m, 1H), 1.40 (br d, J = 7.3 Hz, 1H), 1.06 (s, 12H), 1.04 - 1.01 (m, 3H), 0.83 (s, 3H)
Example 50: Synthesis of (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-
3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 199)
Figure imgf000258_0001
257
SUBSTITUTE SHEET RULE 26 Step 1: 5-bromoisoquinoline-4-carbaldehyde
[000430] A mixture of COI1C.H2SO4 (50 mL) and isoquinoline-4-carbaldehyde (5 g, 31.81 mmol, 1 eq) at 0 °C was added NBS (7.02 g, 39.45 mmol, 1.24 eq) slowly and the reaction mixture was allowed to warm to 25 °C and stirred for 16 h. Upon completion, the mixture was adjust to pH=7~9 by sat. NaOH (100 mL), and extracted with ethyl acetate (40 mL * 2). The combined organic layers were washed with brine (40 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 3: 1 to 0: 1) to give the product 5- bromoisoquinoline-4-carbaldehyde (2 g, 8.47 mmol, 26.63% yield) as a yellow solid. MS (ESI) m/z 236.0 [M+H]+
Step 2: 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile
[000431] A solution of 5-bromoisoquinoline-4-carbaldehyde (200 mg, 847.23 umol, 1 eq) in NHVMeOH (7 M, 5 mL) was added with NH4CI (135.96 mg, 2.54 mmol, 3 eq) and stirred at 25 °C for 1 h. TMSCN (252.15 mg, 2.54 mmol, 317.97 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHVMeOH. The reaction mixture was quenched by addition H2O (20 mL), and then extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE:EA = 0: 1) to give the product 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (120 mg, 457.83 umol, 54.04% yield) as a yellow oil. MS (ESI) m/z 262.0 [M+H]+
Step 3: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000432] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (80 mg, 195.88 umol, 1 eq) in ACN (3 mL) was added 2-amino-2-(5-bromo-4- isoquinolyl)acetonitrile (51.34 mg, 195.88 umol, 1 eq), and then 1 -methylimidazole (56.29 mg, 685.58 umol, 54.65 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphate (65.95 mg, 235.06 umol, 1.2 eq) were added. The mixture was stirred at
258
SUBSTITUTE SHEET RULE 26 25 °C for 1 h. Upon completion, the residue was quenched by H2O (10 mL) and was extracted with EA (10 mL * 3). The combined organic phase was washed with brine (10 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 100 * 30 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 8 min) to afford (lR,2S,5S)-N-[(5- bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (62 mg, 95.02 umol, 48.51% yield) as a white solid. MS (ESI) m/z 654.1 [M+H]+
[000433] 'H NMR (400 MHz, DMSO-de) 5 = 9.75 - 9.17 (m, 3H), 8.94 - 8.82 (m, 1H), 8.37 - 8.21 (m, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.49 (br t, J = 5.8 Hz, 1H), 4.54 - 4.15 (m, 2H), 4.07 - 3.78 (m, 2H), 3.65 (br dd, J = 6.3, 10.3 Hz, 1H), 1.63 - 1.52 (m, 1H), 1.46 - 1.32 (m, 1H), 1.12 - 0.96 (m, 15H), 0.82 (d, J = 13.6 Hz, 3H)
Step 4: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000434] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (40 mg, 61.30 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-Ol(250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 45%-45%, 10 min) to afford (lR,2S,5S)-N-[(5- bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 1 (17.46 mg, 26.76 umol, 43.65% yield) as a white solid. MS (ESI) m/z 654.1 [M+H]+ 'H NMR (400 MHz, DMSO-de) 5 = 9.70 - 9.31 (m, 3H), 8.88 (s, 1H), 8.31 (dd, J = 7.8, 19.0 Hz, 2H), 7.68
(t, J = 7.8 Hz, 1H), 7.50 (d, J = 6.8 Hz, 1H), 4.44 - 4.34 (m, 1H), 4.24 (s, 1H), 4.03 - 3.86 (m, 2H), 3.64 (d, J = 10.5 Hz, 1H), 1.59 - 1.53 (m, 1H), 1.35 (d, J = 7.7 Hz, 1H), 1.07 (br d, J = 6.1 Hz, 12H), 0.99 - 0.96 (m, 3H), 0.80 (s, 3H)
[000435] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 2) (18.89 mg, 28.95 umol, 47.23% yield) was obtained as a white solid. MS (ESI) m/z 654.1 [M+H]+ 'H NMR (400 MHz, DMSO-de) 5 = 9.66 (br d, J = 8.1 Hz, 1H), 9.55 (br d, J = 6.8 Hz, 1H), 9.50 (s, 1H), 8.88 (s, 1H), 8.37 - 8.22 (m, 2H), 7.68 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 6.6
259
SUBSTITUTE SHEET RULE 26 Hz, 1H), 4.38 (t, J = 7.9 Hz, 1H), 4.31 (s, 1H), 3.97 (dd, J = 5.4, 10.4 Hz, 1H), 3.92 - 3.81 (m, 1H), 3.66 (d, J = 10.5 Hz, 1H), 1.62 - 1.55 (m, 1H), 1.43 (d, J = 7.5 Hz, 1H), 1.10 - 1.02 (m, 15H), 0.83 (s, 3H)
Example 51: Synthesis of (1R, 2S, 5S)-N-[cyano-(5-methyl-4-isoquinolyl) methyl] -3- [(2S)-3, 3-dimethyl-2-[(2, 2, 2-trifluoroacetyl) amino] butanoyl]-6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (Compound 201)
Figure imgf000261_0001
Step 1: 5-methylisoquinoline-4-carbaldehyde
[000436] To a solution of 5-bromoisoquinoline-4-carbaldehyde (200 mg, 847.23 umol, 1 eq), methylboronic acid (152.14 mg, 2.54 mmol, 3 eq) in dioxane (8 mL) was added K2CO3 (292.73 mg, 2.12 mmol, 2.5 eq) and Pd(PPh3)4 (97.90 mg, 84.72 umol, 0.1 eq) under N2, and the resulting mixture was stirred at 100 °C for 14 h. Upon completion, the mixture was concentrated under reduce pressure to remove dioxane, then diluted with H2O (20 mL) and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:0 to 71:29) to give 5-methylisoquinoline-4-carbaldehyde (124 mg, 507.02 umol, 59.85% yield, 70% purity) as a yellow solid. MS (ESI) m/z 172.1 [M+H]+.
Step 2: 2-amino-2-(5-chloro-3-pyridyl) propanenitrile
260
SUBSTITUTE SHEET RULE 26 [000437] A solution of 5-methylisoquinoline-4-carbaldehyde (114 mg, 665.91 umol, 1 eq) in NHa/MeOH (5 mL) (7 M) was added with NH4CI (106.86 mg, 2.00 mmol, 3 eq), and the mixture was stirred at 20 °C for 1 h. TMSCN (198.19 mg, 2.00 mmol, 249.92 uL, 3 eq) was then added to the mixture at 0 °C and stirred at 20 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O (5 mL), and then extracted with ethyl acetate (5mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to afford 2-amino-2-(5-methyl-4-isoquinolyl) acetonitrile (95 mg, crude) as a yellow oil. MS (ESI) m/z 198.1 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano-(5-methyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000438] To a sloution of 2-amino-2-(5-methyl-4-isoquinolyl)acetonitrile (90 mg, 456.31 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3azabicyclo[3.1.0]hexane-2-carboxylic acid (166.26 mg, 456.31 umol, 1 eq) in ACN (6 mL) was added 1 -methylimidazole (131.13 mg, 1.60 mmol, 127.31 uL, 3.5 eq), [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (256.06 mg, 912.62 umol, 2 eq), and the mixure was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and then extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC(column: Waters Xbridge BEHC18 100 * 30 mm * 10 um; mobile phase:
[water( NH4HCO3)-ACN]; B%: 45%-65%, 8 min) to afford (lR,2S,5S)-N-[cyano-(5-methyl-4- isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide(45 mg, 81.96 umol, 17.96% yield, 99% purity) as a white solid. The residue was further separated by SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; %: 5%-25%, 16 min) to give (lR,2S,5S)-N-[cyano-(5-methyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 1 as a white solid. MS (ESI) m/z 544.3 [M+H]+.
[000439] 'H NMR (400 MHz, DMSO-t/e) 5 = 9.47 - 9.30 (m, 3H), 8.83 - 8.77 (m, 1H), 8.09 (dd, J= 2.0, 7.4 Hz, 1H), 7.67 - 7.62 (m, 2H), 6.91 - 6.85 (m, 1H), 4.40 (br s, 1H), 4.21 (s, 1H),
261
SUBSTITUTE SHEET RULE 26 3.95 (dd, J = 5.5, 10.4 Hz, 1H), 3.78 - 3.68 (m, 1H), 2.83 - 2.71 (m, 3H), 1.66 - 1.59 (m, 1H),
1.31 (d, J = 7.6 Hz, 1H), 1.03 (s, 11H), 0.92 (s, 1H), 0.80 (s, 3H)
[000440] (lR,2S,5S)-N-[cyano-(5-methyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide Isomer 2 was obtained as a white solid. MS (ESI) m/z 544.3 [M+H]+.
[000441] 'H NMR (400 MHz, DMSO-t/e) 5 = 9.42 - 9.22 (m, 3H), 8.83 - 8.78 (m, 1H), 8.17 - 8.02 (m, 1H), 7.75 - 7.56 (m, 2H), 6.97 - 6.83 (m, 1H), 4.39 - 4.31 (m, 1H), 4.28 - 4.19 (m, 1H), 3.99 - 3.90 (m, 1H), 3.69 - 3.60 (m, 1H), 2.82 - 2.75 (m, 3H), 1.62 - 1.51 (m, 1H), 1.25 - 1.13 (m, 1H), 1.02 - 0.86 (m, 12H), 0.85 - 0.73 (m, 3H)
Example 52: Synthesis of (lR,2S,5S)-N-[cyano-(5-cyclopropyl-4-isoquinolyl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 203)
Figure imgf000263_0001
Step 1: 5-cyclopropylisoquinoline-4-carbalde
[000442] A solution of 5-bromoisoquinoline-4-carbaldehyde (50 mg, 211.81 umol, 1 eq), cyclopropylboronic acid (36.39 mg, 423.61 umol, 2 eq) and K2CO3 (73.18 mg, 529.52 umol, 2.5 eq) in dioxane (2 mL) and H2O (0.5 mL) was added with Pd(PPhs)4 (48.95 mg, 42.36 umol, 0.2 eq) under N2. The mixture was stirred at 90 °C for 15 h. Upon completion, the reaction mixture was diluted with H2O (10 Ml) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (20 mL). The combined organic layers were dried over
262
SUBSTITUTE SHEET RULE 26 Na2SO4, concentrated in vacuum. The residue was purified by chromatography (S1O2, Petroleum ether:Ethyl acetate = 1:2 ) to afford 5-cyclopropylisoquinoline-4-carbaldehyde (25 mg, 126.75 umol, 19.95% yield) was obtained as a white solid. MS (ESI) m/z 198.1 [M+H]+.
Step 2: 2-amino-2-(5-cyclopropyl-4-isoquinolyl)acetonitrile
[000443] A mixture of 5-cyclopropylisoquinoline-4-carbaldehyde (25 mg, 126.75 umol, 1 eq) in NIE/MeOH (1 mL) was added NH4CI (13.56 mg, 253.51 umol, 2 eq) and stirred at 25 °C for 1 h. Then, TMSCN (25.15 mg, 253.51 umol, 31.71 uL, 2 eq) was added the former mixture at 0 °C, and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give 2-amino-2-(5-cyclopropyl-4- isoquinolyl)acetonitrile(26 mg, crude) and used directly without further purification. MS (ESI) m/z 224.1 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano-(5-cyclopropyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000444] A mixture of 2-amino-2-(5-cyclopropyl-4-isoquinolyl)acetonitrile (20 mg, 89.58 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (32.64 mg, 89.58 umol, 1 eq) in ACN (2 mL) was added 1 -methylimidazole (22.06 mg, 268.73 umol, 21.42 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (50.27 mg, 179.15 umol, 2 eq). The resulting mixture was stirred at 25 °C for 1 h. Upon completion, the mixture was concentrated under reduced pressure to give residue. The residue was purified by pre-TLC (SiCh, dichloromethane: methanol= 10: 1) to afford (lR,2S,5S)-N-[cyano-(5- cy clopropyl-4-isoquinolyl)methy 1] -3-[(2S)-3, 3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (15 mg, 17.55% yield, 96% purity) as white solid. MS (ESI) m/z 570.3 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(5-cyclopropyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 and Isomer 2
263
SUBSTITUTE SHEET RULE 26 [000445] (lR,2S,5S)-N-[cyano-(5-cyclopropyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (15 mg, 96% purity) was separated by SFC (column: (s,s) WHELK-01 (250mm * 30mm, 5um); mobile phase: [Neu-IPA]; B%: 10%-35%,15min) to afford (lR,2S,5S)-N-[cyano- (5-cyclopropyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl) amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Isomer 1) (4.9 mg, 8.56 umol, 9.56% yield, 99.5% purity) as a white solid. MS (ESI) m/z 570.2 [M+H]+. 'H NMR (400 MHz, DMSO-t/e) 5 = 9.45 - 9.27 (m, 3H), 8.86 (s, 1H), 8.11 (br d, J= 7.8 Hz, 1H), 7.75 (br d, J= Hz, 1H), 7.71 - 7.62 (m, 2H), 4.35 (br d, J= 6.5 Hz, 1H), 4.25 (s, 1H), 3.92 (br dd, J = 5.4, 10.3 Hz, 1H), 3.64 (br d, J= 10.3 Hz, 1H), 1.57 - 1.51 (m, 1H), 1.14 - 1.05 (m, 3H), 0.96 (br s, 3H), 0.90 (s, 9H), 0.80 (s, 6H).
[000446] (lR,2S,5S)-N-[cyano-(5-cyclopropyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2carboxamide (Isomer 2) (7.8 mg, 13.58 umol, 15.16% yield, 99.2% purity) was obtained as a white solid. MS (ESI) m/z 570.2 [M+H]+. 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.51 (br d, J = 5.7 Hz, 1H), 9.45 - 9.29 (m, 2H), 8.86 (s, 1H), 8.12 (br d, J= 7.7 Hz, 1H), 7.77 - 7.62 (m, 2H), 7.58 (br d, J= 5.7 Hz, 1H), 4.40 (br d, J= 6.4 Hz, 1H), 4.18 (s, 1H), 3.92 (br dd, J= 5.2, 9.7 Hz, 1H), 3.71 (br d, J = 10.4 Hz, 1H), 1.61 (br t, J= 6.1 Hz, 1H), 1.34 (br d, .7= 7,5 Hz, 1H), 1.24 - 1.15 (m, 1H), 1.06 - 0.97 (m, 15H), 0.82 - 0.68 (m, 4H).
Example 53: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(5- cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 205)
Figure imgf000265_0001
Step 1: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(5-cyclopropyl-lH-pyrrole-2- carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
264
SUBSTITUTE SHEET RULE 26 [000447] To a solution of (lR,2S,5S)-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxylic acid (111.28 mg, 385.93 umol, 1.2 eq) and 2-amino-2- (5-chloro-4-isoquinolyl)acetonitrile (70 mg, 321.61 umol, 1 eq) in ACN (4 mL) was added 1- methylimidazole (79.22 mg, 964.83 umol, 76.91 uL, 3 eq) and TCFH (180.47 mg, 643.22 umol, 2 eq), and the resulting mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (6 mL) and then was extracted with ethyl acetate (2 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40% - 70%, 8 min) to give the product (lR,2S,5S)-N-[(5- chloro-4-isoquinolyl)-cyano-methyl]-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 200.83 umol, 62.44% yield, 98% purity) as yellow solid. MS (ESI) m/z 488.1 [M+H]+
Step 2: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(5-cyclopropyl-lH-pyrrole-2- carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 and 2
[000448] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(5-cyclopropyl-lH- pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 204.93 umol, 1 eq) was separated by SFC (column: (s,s) WHELK-01 (250 mm * 30 mm, 5 um); mobile phase: [0.1% NH3H2O MeOH]; B%: 50%-50%, 18 mm) to give (lR,2S,5S)-N-[(5-chloro-4- isoquinolyl)-cyano-methyl]-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (35.74 mg, 71.63 umol, 34.95% yield, 97.802% purity) as white solid. MS (ESI) m/z 488.1 [M+H]+ 'H NMR (400 MHz, DMSO-t/r,) 5 ppm 11.30 - 11.19 (m, 1H), 9.74 - 9.57 (m, 1H), 9.49 (s, 1H), 8.91 - 8.83 (m, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.07 - 7.98 (m, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.49 - 7.32 (m, 1H), 6.57 - 6.12 (m, 1H), 5.76 - 5.42 (m, 1H), 4.58 - 4.50 (m, 1H), 3.98 (dd, J= 5.4, 10.4 Hz, 1H), 3.87 - 3.67 (m, 1H), 1.96 - 1.85 (m, 1H), 1.66 - 1.52 (m, 1H), 1.38 (d, J= 7.6 Hz, 1H), 1.05 (s, 3H), 0.89 - 0.77 (m, 5H), 0.67 - 0.51 (m, 2H).
[000449] ( 1 R,2S, 5 S)-N- [(5 -chloro-4-isoquinolyl)-cyano-methyl] -3 -(5 -cy clopropyl- 1 H- pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (23.4 mg, 47.73 umol, 23.29% yield, 99.537% purity) was obtained as white solid. MS (ESI) m/z 488.1 [M+H]+ 'H NMR (400 MHz, DMSO-ti,) 5 ppm 11.38 - 11.20 (m, 1H), 9.69 - 9.53 (m, 1H), 9.52
265
SUBSTITUTE SHEET RULE 26 - 9.47 (m, 1H), 8.91 - 8.82 (m, 1H), 8.33 - 8.25 (m, 1H), 8.11 - 8.01 (m, 1H), 7.83 - 7.72 (m, 1H), 7.45 - 7.27 (m, 1H), 6.61 - 6.18 (m, 1H), 5.80 - 5.51 (m, 1H), 4.58 - 4.41 (m, 1H), 3.98 (dd, J = 5.4, 10.2 Hz, 1H), 3.87 - 3.67 (m, 1H), 2.04 - 1.86 (m, 1H), 1.63 (dd, J= 5.4, 7.4 Hz, 1H), 1.30 - 1.22 (m, 1H), 1.06 - 0.95 (m, 3H), 0.87 - 0.79 (m, 5H), 0.69 - 0.55 (m, 2H).
Example 54: Synthesis of (lR,2S,5S)-N-[(lS)-l-cyano-2-[(6R)-5-oxo-4-azaspiro[2.4]heptan- 6-yl] ethyl] -3- [(2S)-2- [(3,3-difluoropyrrolidine- l-carbonyl)amino] -3,3-dimethyl-butanoyl] - 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 206)
Figure imgf000267_0001
Step 1: 5-cyclopropyl-lH-pyrrole-2-carboxylic acid
[000450] To a solution of methyl 5-cyclopropyl-lH-pyrrole-2-carboxylate (0.2 g, 1.21 mmol, 1 eq) in THF (1 mL) and H2O (1 mL) was added LiOH.H2O (152.41 mg, 3.63 mmol, 3 eq). The mixture was stirred at 60 °C for 16 h. Upon completion, the mixture was adjust to pH = 1 with 1 M HC1 (8 mL) and was extracted with ethyl acetate (15 mL * 6), then was concentrated under reduced pressure to get crude product 5 -cyclopropyl- lH-pyrrole-2-carboxylic acid (180 mg, crude) as yellow solid. MS (ESI) m/z 150.2 [M+H]+
Step 2: (2S)-2-(tert-butoxycarbonylamino)-3-(l-fluorocyclopropyl)propanoic acid
[000451] To a solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(l- fluorocyclopropyl)propanoate (0.2 g, 765.44 umol, 1 eq) in THF (1 mL) and H2O (1 mL) was
266
SUBSTITUTE SHEET RULE 26 added LiOH.IfcO (96.35 mg, 2.30 mmol, 3 eq). The mixture was stirred at 20 °C for 3 h. Upon completion, the mixture was adjusted to pH = 1 with 1 M HC1 (8 mL) and extracted with ethyl acetate (15 mL * 6), and then was concentrated under reduced pressure to get crude product (2S)-2-(tert-butoxycarbonylamino)-3-(l-fluorocyclopropyl)propanoic acid (180 mg, crude) as yellow solid. MS (ESI) m/z 246.2 [M+H]+
Step 3: tert-butyl N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]carbamate
[000452] To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-(l- fluorocyclopropyl)propanoic acid (113.60 mg, 459.44 umol, 1 eq) in ACN (4 mL) was added 2- amino-2-(5-chloro-4-isoquinolyl)acetonitrile (0.1 g, 459.44 umol, 1 eq), and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (257.82 mg, 918.89 umol, 2 eq), and then 1 -methylimidazole (113.17 mg, 1.38 mmol, 109.87 uL, 3 eq) was added at 20 °C. The mixture was stirred at 20 °C for 1.5 h. Upon completion, the reaction mixture was quenched by addition water (25 mL) and extracted with DCM (20 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 0: 1 ) to afford tertbutyl N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl] carbamate (129 mg, 248.24 umol, 54.03% yield, 86% purity) as white solid. MS (ESI) m/z 447.1 [M+H]+
Step 4: (2S)-2-amino-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(l- fluorocyclopropyl)propanamide
[000453] A solution of tert-butyl N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano-methyl]amino]- l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]carbamate (85 mg, 190.20 umol, 1 eq) in DCM (2 mL) and TEA (1 mL) was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was quenched by addition NaHCCh (60 mL) and extracted with DCM (10 mL * 8) and blow-dried with N2 to give (2S)-2-amino-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(l- fluorocyclopropyl)propanamide (70 mg, crude) as yellow oil. MS (ESI) m/z 347.1 [M+H]+
Step 5: N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-5-cyclopropyl-lH-pyrrole-2-carboxamide
267
SUBSTITUTE SHEET RULE 26 [000454] To a solution of (2S)-2-amino-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-(l- fluorocyclopropyl)propanamide (70 mg, 201.85 umol, 1 eq) in DCM (3 mL) was added 5- cyclopropyl-lH-pyrrole-2-carboxylic acid (30.51 mg, 201.85 umol, 1 eq), HATU (153.50 mg, 403.71 umol, 2 eq) and DIEA (78.26 mg, 605.56 umol, 105.48 uL, 3 eq). The mixture was stirred at 20 °C for 13 h. Upon completion, the reaction mixture was quenched by addition water (25 mL) and extracted with DCM (20 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purifited by prep-HPLC (column: Waters Xbridge BEH Cl 8 100 * 30 mm * 10 um; mobile phase: [water (NH4HCO3) - ACN]; B%: 35% - 55%, 8 min) to give N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano- methyl]amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-5-cyclopropyl-lH-pyrrole-2- carboxamide (30 mg, 62.51 umol, 30.97% yield) as white solid. MS (ESI) m/z 480.1 [M+H]+
Step 6: N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-5-cyclopropyl-lH-pyrrole-2-carboxamide
[000455] The N- [( 1 S)-2- [ [(5 -chloro-4-isoquinolyl)-cyano-methyl] amino] - 1 - [( 1 - fluorocyclopropyl)methyl]-2-oxo-ethyl]-5-cyclopropyl-lH-pyrrole-2-carboxamide (30 mg, 62.51 umol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu - IP A]; B%: 50% - 50%, 15 min)to give the product N-[(lS)-2-[[(5- chloro-4-isoquinolyl)-cyano-methy l]amino] - 1 - [( 1 -fluorocy clopropy l)methyl] -2-oxo-ethyl] -5- cyclopropyl-lH-pyrrole-2-carboxamide Isomer 1 (8 mg, 16.67 umol, 26.67% yield, 100% purity) as white solid. MS (ESI) m/z 480.1 [M+H]+.
[000456] 1 H NMR (400 MHz, DMSO-de) 5 = 11.28 (br s, 1H), 9.64 (br d, J = 6.6 Hz, 1H),
9.50 (s, 1H), 8.86 (s, 1H), 8.29 (dd, J = 1.2, 8.2 Hz, 1H), 8.05 (dd, J = 1.2, 7.6 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.37 (br d, J = 3.8 Hz, 1H), 6.72 (dd, J = 2.8, 3.4 Hz, 1H), 5.69 (dd, J = 2.6, 3.3 Hz, 1H), 4.79 (ddd, J = 4.2, 8.4, 10.3 Hz, 1H), 2.40 - 2.31 (m, 1H), 2.08 - 1.93 (m, 1H), 1.93 - 1.84 (m, 1H), 0.87 - 0.79 (m, 5H), 0.66 - 0.58 (m, 2H), 0.53 - 0.41 (m, 1H).
[000457] N-[(lS)-2-[[(5-chloro-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-5-cyclopropyl-lH-pyrrole-2-carboxamide Isomer 2 (8 mg, 16.50 umol, 26.40% yield, 99% purity) was obtained as a white solid. MS (ESI) m/z 480.1 [M+H]+. 'H NMR (400 MHz, DMSO-de) 5 = 11.25 (br s, 1H), 9.61 (br d, J = 7.0 Hz, 1H), 9.49 (s, 1H), 8.89 (s, 1H), 8.27 (d, J = 8.2 Hz, 1H), 8.05 - 7.99 (m, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.75
268
SUBSTITUTE SHEET RULE 26 (t, J = 7.8 Hz, 1H), 7.36 (br d, J = 4.6 Hz, 1H), 6.73 - 6.66 (m, 1H), 5.71 - 5.64 (m, 1H), 4.81 (dt, J = 5.1, 8.8 Hz, 1H), 2.39 - 2.32 (m, 1H), 2.08 - 1.96 (m, 1H), 1.92 - 1.81 (m, 1H), 0.92 - 0.79 (m, 5H), 0.63 - 0.59 (m, 2H), 0.58 - 0.51 (m, 1H).
Example 55: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(thiazol-2- ylamino)butanoyl]-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 208)
Figure imgf000270_0001
[000458] To a solution of 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (50.00 mg, 229.72 umol, 1 eq) and (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(thiazol-2-ylamino)butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (92.45 mg, 229.72 umol, 1 eq, Li) in ACN (3 mL) was added with [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (128.91 mg, 459.44 umol, 2 eq) and 1 -methylimidazole (66.01 mg, 804.02 umol, 64.09 uL, 3.5 eq). The resulting mixture was stirred at 25 °C for 2 hours. Upon completion, the reaction mixture was diluted with H2O (10 mL) and then extracted with ethyl acetate (10 mL * 2). The combined organic layers were washed with 15 % citric acid (10 mL * 2), NaHCCh (10 mL) and brine (10 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (S 1O2 petroleum ether: ethyl acetate = 0: 1 ), Then the the residue purified by SFC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu- IPA]; B%: 33%-33%, 16 min) to afford (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(thiazol-2- ylamino)butanoyl]-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (2.23 mg, 3.65 umol, 1.59% yield, 97.3% purity) as a white solid. MS (ESI) m/z 595.3 [M+H]+. 'H NMR (400 MHz, DMSO-t/e) 5 = 9.55 - 9.40 (m, 1H), 9.01 - 8.69 (m, 2H), 8.28 (br d, J= 8.1 Hz, 1H), 8.09 - 7.94 (m, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.65 - 7.46 (m, 1H), 7.42 - 7.25 (m, 1H), 6.94 - 6.70 (m, 1H), 6.63 - 6.40 (m, 1H),
269
SUBSTITUTE SHEET RULE 26 4.43 (br dd, J= 6.2, 8.7 Hz, 1H), 4.33 - 4.21 (m, 1H), 4.01 - 3.89 (m, 2H), 3.85 - 3.72 (m, 1H),
1.58 (br d, J= 7.2 Hz, 1H), 1.39 - 1.33 (m, 1H), 1.10 - 0.70 (m, 18H).
[000459] (1R, 2S, 5S)-3-[(2S, 3R)-3-tert-butoxy-2-(thiazol-2-ylamino)butanoyl]-N-[(5- chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (10.75 mg, 18.06 umol, 7.86% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 595.3 [M+H]+. 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.50 (s, 1H), 8.98 - 8.80 (m, 2H), 8.34 - 8.22 (m, 1H), 8.08 - 7.92 (m, 1H), 7.76 (t, J= 7.8 Hz, 1H), 7.60 (d, J= 8.7 Hz, 1H), 7.44 - 7.27 (m, 1H), 6.95 - 6.72 (m, 1H), 6.64 - 6.40 (m, 1H), 4.49 - 4.34 (m, 1H), 4.28 (s, 1H), 4.01 - 3.88 (m, 2H), 3.83 (quin, J= 6.0 Hz, 1H), 1.62 - 1.54 (m, 1H), 1.38 (d, J= 7.6 Hz, 1H), 1.09 (d, J= 6.2 Hz, 4H), 1.03 - 1.03 (m, 4H), 0.94 (s, 10H).
Example 56: Synthesis of (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 209)
Figure imgf000271_0001
Step 1: 8-vinyl-l,6-naphthyridine
[000460] To a solution of 8-bromo-l,6-naphthyridine (500 mg, 2.39 mmol, 1 eq) in dioxane (10 mL) and H2O (2.5 mL) was added CS2CO3 (2.34 g, 7.18 mmol, 3 eq) and ditert- butyl(cyclopentyl)phosphane;dichloropalladium;iron (155.89 mg, 239.19 umol, 0.1 eq), and then was added potassium;trifluoro(vinyl)boranuide (320.39 mg, 2.39 mmol, 1 eq) under N2 atmosphere. The mixture was stirred at 80 °C for 2 h. Upon completion, the reaction mixture was
270
SUBSTITUTE SHEET RULE 26 filtered and the mother liquer was diluted with water (3 mL) and extracted with ethyl acetate (2 mL * 3). The combined organic layers were washed with brine (1.5 mL), dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10: 1 to 0:1) to give 8-vinyl-l,6- naphthyridine (230 mg, 1.47 mmol, 61.57% yield) as a yellow solid. MS (ESI) m/z 157.2 [M+H]+.
Step 2: l,6-naphthyridine-8-carbaldehyde
[000461] To a mixture of 8-vinyl-l,6-naphthyridine (230 mg, 1.47 mmol, 1 eq) in dioxane (2.5 mL) and H2O (0.5 mL) was added K2OSO4.2H2O (108.52 mg, 294.53 umol, 0.2 eq) and 2,6- dimethyl(l15N)pyridine (318.52 mg, 2.95 mmol, 2 eq). The mixture was stirred at 25 °C for 30 min, and then NalCh (1.57 g, 7.36 mmol, 408.01 uL, 5 eq) was added at 0 °C. The mixture was stirred at 25 °C for 15.5 h. Upon completion, the reaction mixture was diluted with water (3 mL) and filtered, the mother liquer was extracted with ethyl acetate (2 mL * 3). The combined organic layers were washed with brine (3 mL), dried over Na2SO4 filtered and concentrated under reduced pressure to give l,6-naphthyridine-8-carbaldehyde (230 mg, crude) as a yellow solid. MS (ESI) m/z 159.1 [M+H]+.
Step 3: 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile
[000462] To a solution of l,6-naphthyridine-8-carbaldehyde (230 mg, 1.45 mmol, 1 eq) in DCM (4 mL) was added Ti(i-PrO)4 (619.98 mg, 2.18 mmol, 643.80 uL, 1.5 eq) and NEh/MeOH (7 M, 831.00 uL, 4 eq) at 25 °C. After the addition, the mixture was stirred at 25 °C for 1 h. TMSCN (432.81 mg, 4.36 mmol, 545.79 uL, 3 eq) was added, and the resulting mixture was stirred at 25 °C for 15 h. Upon completion, the combined was diluted with water (4 mL) and extracted with ethyl acetate (3 mL, and then filtered to give a solution which was extracted with ethyl acetate (3 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, DCM:MeOH = 20: 1) to give 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile (65 mg, 352.88 umol, 24.27% yield) as a yellow solid. MS (ESI) m/z 185.2 [M+H]+.
Step 4: 2-amino-2-[4-(4-fluorophenyl)-l ,2,4-triazol-3-yl]acetonitrile
271
SUBSTITUTE SHEET RULE 26 [000463] To a solution of 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile (60 mg, 325.74 umol, 1.2 eq) in ACN (3 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (98.90 mg, 271.45 umol, 1 eq) and 1 -methylimidazole (66.86 mg, 814.35 umol, 64.91 uL, 3 eq). Then, [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (152.33 mg, 542.90 umol, 2 eq) was added and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by (column: Waters Xbridge BEH Cl 8 100 * 30mm * lOum; mobile phase: [water( NH4HCO3)-ACN]; B%: 45%-75%, 8min) to give (lR,2S,5S)-N-[cyano(l,6- naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (30 mg, 56.55 umol, 20.83% yield) as a white solid. MS (ESI) m/z 531.4 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000464] The (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide was separated by SFC (column: DAICEL CHIRALPAK AD(250mm * 30mm, lOum); mobile phase: [Neu-ETOH]; B%: 5%-30%, 16min) to give Isomer 1 (10 mg, 18.85 umol, 40.00% yield) as a white solid, which chiral purity is not good and further separated again by SFC (column: DAICEL CHIRALPAK AD(250mm * 30mm, lOum); mobile phase: [Neu-ETOH]; B%: 20%- 20%,16min) to give (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (4.5 mg, 8.48 umol, 18.00% yield) as a white solid. MS (ESI) m/z 531.1 [M+H]+.
[000465] 'H NMR (400 MHz, METHANOL-^) 5 = 9.45 - 9.41 (m, 1H), 9.26 - 9.23 (m, 1H), 8.99 - 8.94 (m, 1H), 8.68 - 8.60 (m, 1H), 7.85 - 7.76 (m, 1H), 6.90 (s, 1H), 4.56 - 4.49 (m, 1H), 4.42 (s, 1H), 4.02 - 3.93 (m, 1H), 3.88 - 3.81 (m, 1H), 1.65 - 1.58 (m, 1H), 1.49 - 1.31 (m, 1H), 1.14 - 1.04 (m, 3H), 1.03 - 0.99 (m, 3H), 0.91 - 0.88 (m, 9H).
[000466] (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1. 0] hexane-2-carboxamide
272
SUBSTITUTE SHEET RULE 26 Isomer 2 (4.5 mg, 8.48 umol, 18.00% yield) was obtained as a white solid. MS (ESI) m/z 531.3 [M+H]+
[000467] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.53 - 9.50 (m, 1H), 9.46 (d, J= 7.6 Hz, 1H), 9.41 (br d, J= 6.0 Hz, 1H), 9.28 - 9.25 (m, 1H), 8.96 - 8.92 (m, 1H), 8.72 - 8.69 (m, 1H), 7.85 (dd, J= 4.2, 8.3 Hz, 1H), 6.88 - 6.80 (m, 1H), 4.44 - 4.31 (m, 2H), 3.90 (dd, J= 5.4, 10.3 Hz, 1H), 3.69 (br d, J= 10.4 Hz, 1H), 1.55 - 1.50 (m, 1H), 1.25 - 1.22 (m, 1H), 0.99 (s, 8H), 0.95 (s, 3H), 0.86 - 0.80 (m, 3H).
Example 57: Synthesis of (lR,2S,5S)-N-(cyano(2,7-naphthyridin-4-yl)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 210)
Figure imgf000274_0001
Step 1: 4-vinyl-2,7-naphthyridine
[000468] A solution of 4-bromo-2,7-naphthyridine (300 mg, 1.44 mmol, 1 eq) and potassium;trifluoro(vinyl)boranuide (230.68 mg, 1.72 mmol, 1.2 eq) in H2O (1 mL) and toluene (4 mL) was added CS2CO3 (1.40 g, 4.31 mmol, 3 eq), and then ditert- butyl(cyclopentyl)phosphane; dichloropalladium; iron (93.53 mg, 143.51 umol, 0.1 eq) was added under N2 atmosphere. The mixture was stirred at 80 °C for 2 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 4: 1 to
273
SUBSTITUTE SHEET RULE 26 1 : 1) to give 4- vinyl-2, 7-naphthyridine (205 mg, 1.31 mmol, 91.46% yield) as a yellow solid. MS (ESI) m/z 157.2 [M+H]+.
Step 2: 2,7-naphthyridine-4-carbaldehyde
[000469] A solution of 4- vinyl-2, 7-naphthyridine (205 mg, 1.31 mmol, 1 eq) in H2O (2.5 mL) and THF (5 mL) was added dipotassium;dioxido(dioxo)osmium;dihydrate (48.36 mg, 131.26 umol, 0.1 eq) and stirred for 30 min, then sodium;periodate (1.12 g, 5.25 mmol, 290.93 uL, 4 eq) was added. The mixture was stirred at 20 °C for 15.5 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (10 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroluem ether: ethyl acetate = 0: 1) to give 2,7-naphthyridine-4-carbaldehyde (140 mg, 885.20 umol, 67.44% yield) as a white solid.
Step 3: 2-amino-2-(2,7-naphthyridin-4-yl)acetonitrile
[000470] A solution of 2,7-naphthyridine-4-carbaldehyde (140 mg, 885.20 umol, 1 eq), Ti(i- PrO)4 (377.38 mg, 1.33 mmol, 391.88 uL, 1.5 eq) and NH3/MeOH (7 M, 505.83 uL, 4 eq) in DCM (3 mL) was stirred for 1 h, and then TMSCN (263.45 mg, 2.66 mmol, 332.22 uL, 3 eq) was added. The mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NH3/MeOH. The reaction mixture was quenched by addition H2O (3 mL), and then extracted with ethyl acetate (3 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(2,7-naphthyridin-4-yl)acetonitrile (130 mg, crude) as a yellow solid. MS (ESI) m/z 185.2 [M+H]+.
Step 4: (lR,2S,5S)-N-(cyano(2,7-naphthyridin-4-yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000471] A solution of 2-amino-2-(2,7-naphthyridin-4-yl)acetonitrile (130 mg, 705.77 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (308.58 mg, 846.92 umol, 1.2 eq) in ACN (3 mL) was added 1 -methylimidazole (231.77 mg, 2.82 mmol, 225.02 uL, 4 eq). Then, TCEH
274
SUBSTITUTE SHEET RULE 26 (237.63 mg, 846.92 umol, 1.2 eq) was added to the mixture and tne mixture was sitrred at ZU
Figure imgf000276_0001
for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (5 mL) at 0°C, and extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate=l :0 to 3: 1) then purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-55%, 8 min) to give (1R,2S,5S)-N- [cyano(2,7-naphthyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide (85.36MG, 160.41 umol, 22.73% yield, 99.7% purity) as a white solid. MS (ESI) m/z 531.2 [M+H]+.
[000472] 'H NMR (400 MHz, DMSO-t76) 5 = 9.69 - 9.49 (m, 3H), 9.44 - 9.23 (m, 1H), 8.92 (d, J = 6.0 Hz, 1H), 8.90 - 8.74 (m, 1H), 7.87 (dd, J = 6.0, 14.8 Hz, 1H), 6.96 - 6.85 (m, 1H), 4.37 (br d, J = 12.8 Hz, 1H), 4.24 - 4.16 (m, 1H), 3.97 - 3.88 (m, 1H), 3.74 - 3.63 (m, 1H), 1.63 - 1.53 (m, 1H), 1.16 (d, J = 7.6 Hz, 1H), 1.04 - 0.98 (m, 6H), 0.95 - 0.86 (m, 6H), 0.84 - 0.78 (m, 3H).
Example 58: Synthesis of (lR,2S,5S)-N-[(5-chloro-7,8-difhioro-4-isoquinolyl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 211)
Figure imgf000276_0002
275
SUBSTITUTE SHEET RULE 26 Step 1 : (Z)-l-(5-chloro-2,3-difluoro-phenyl)-N-(2,2-dimethoxyethyl)methammine
[000473] To a mixture of 5-chloro-2,3-difluoro-benzaldehyde (3 g, 16.99 mmol, 1 eq) in toluene (30 mL) was added 2,2-dimethoxyethanamine (1.79 g, 16.99 mmol, 1.85 mL, 1 eq) at 25 °C. The mixture was stirred at 150 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent to afford (Z)-l-(5-chloro-2,3-difluoro- phenyl)-N-(2,2-dimethoxyethyl)methanimine (4.5 g, crude) as a yellow solid.
Step 2: 5-chloro-7,8-difluoro-isoquinoline
[000474] (Z)-l-(5-chloro-2,3-difluoro-phenyl)-N-(2,2-dimethoxyethyl)methanimine (4.5 g, 17.07 mmol, 1 eq) was added with H2SO4 (45 mL) portion- wise at 0 °C. The mixture was stirred at 140 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (5 L) and then adjust pH to 8 with aq. NaOH, extracted with DCM (5 L * 3). The combined organic layers were washed with brine (5000 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column (petroleum ether: ethyl acetate = 98:2 to 97:3) to give 5-chloro-7,8-difluoro-isoquinoline (600 mg, crude) as a yellow solid.
Step 3: 4-bromo-5-chloro-7,8-difluoro-isoquinoline
[000475] To a solution of 5-chloro-7,8-difluoro-isoquinoline (600 mg, 3.01 mmol, 1 eq) in AcOH (12 mL) was added NBS (802.58 mg, 4.51 mmol, 1.5 eq). The mixture was stirred at 100 °C for 2 h. Upon completion, the reaction mixture was remove solvent, then poured into H2O (10 mL) at 20 °C, NaHCCh was added, adjust pH to 8, then extracted with EtOAc (10 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column (petroleum ether: ethyl acetate = 95:5) to give 4-bromo-5-chloro-7,8-difluoro- isoquinoline (400 mg, 1.44 mmol, 47.78% yield) as a light yellow solid.
Step 4: 5-chloro-7,8-difluoro-isoquinoline-4-carbaldehyde
[000476] A solution of 4-bromo-5-chloro-7,8-difluoro-isoquinoline (400 mg, 1.44 mmol, 1 eq) and morpholine-4-carbaldehyde (496.11 mg, 4.31 mmol, 431.40 uL, 3 eq) in THF (4 mL) was cooled to -70 °C, and then was added n-BuLi (2.5 M, 2.30 mL, 4 eq). The reaction was
276
SUBSTITUTE SHEET RULE 26 stirred at -70 °C for 2 h. Upon completion, the reaction mixture was quenched with sat.NEhCl (20 mL) and extracted with ethyl acetate (10 mL *2). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 95:5) to afford 5-chloro-7,8-difluoro-isoquinoline-4-carbaldehyde (135 mg, 533.84 umol, 37.17% yield, 90% purity) as a yellow solid.
Step 5: 2-amino-2-(5-chloro-7,8-difluoro-4-isoquinolyl)acetonitrile
[000477] A mixture of 5-chloro-7,8-difluoro-isoquinoline-4-carbaldehyde (135 mg, 593.16 umol, 1 eq) in DCM (3 mL) was added NHa/MeOH (7 M, 338.95 uL, 4 eq) and Ti(i-PrO)4 (252.88 mg, 889.74 umol, 262.59 uL, 1.5 eq), and the resulting mixture was stirred for 1 h. TMSCN (176.54 mg, 1.78 mmol, 222.62 uL, 3 eq) was added and the mixture was stirred at 25 °C for 12 h. Upon completion, the reaction was poured into H2O (10 mL), filtered and extracted with ethyl acetate (30 mL * 2). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuum to give 2-amino-2- (5-chloro-7,8-difluoro-4-isoquinolyl)acetonitrile (200 mg, crude) as a off-white solid.
Step 6 : (lR,2S,5S)-N-[(5-chloro-7,8-difhioro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000478] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (268.63 mg, 737.28 umol, 1.1 eq) and 2-amino-2-(5-chloro-7,8-difluoro-4- isoquinolyl)acetonitrile (170 mg, 670.25 umol, 1 eq) in ACN (5 mL) was added 1- methylimidazole (192.60 mg, 2.35 mmol, 186.99 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (206.87 mg, 737.28 umol, 1.1 eq), and then the mixture was stirred at 25 °C for 2 h. Upon completion, the residue was poured into water (20 mL), the aqueous phase was extracted with ethyl acetate (20 mL * 2). The combined organic phase was washed with brine (10 mL * 2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC(column: Waters Xbridge BEH C18 100 * 30 mm * 10 um; mobile phase: [water( NH4HCO3)- ACN] ; B%:
277
SUBSTITUTE SHEET RULE 26 48%-78%, 8 min), then was purified by SFC (column: DAICEL CHIRALCEL OD(250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 17%-17%, 8 min) to give (Rt=2.033 min) (lR,2S,5S)-N-[(5-chloro-7,8-difluoro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (62.98 mg, 104.97 umol, 15.66% yield, 100% purity) as a white solid. MS (ESI) m/z 600.2[M+H]+.
[000479] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.73 - 9.58 (m, 2H), 9.38 (br d, J= 7.5 Hz, 1H), 9.04 - 8.93 (m, 1H), 8.52 - 8.29 (m, 1H), 7.27 (br d, J= 3.9 Hz, 1H), 4.39 (br d, J= 7.6 Hz, 1H), 4.30 (s, 1H), 3.91 (dd, J= 5.5, 10.3 Hz, 1H), 3.72 (d, J= 10.4 Hz, 1H), 1.65 - 1.55 (m, 1H), 1.38 (d, J= 7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.84 (s, 3H).
[000480] (lR,2S,5S)-N-[(5-chloro-7,8-difluoro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (73.20 mg, 122.00 umol, 18.20% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 600.3 [M+H]+.
[000481] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.76 - 9.60 (m, 1H), 9.50 (br d, J= 6.7 Hz, 1H), 9.46 - 9.35 (m, 1H), 9.01 - 8.91 (m, 1H), 8.49 - 8.37 (m, 1H), 7.37 - 7.21 (m, 1H), 4.47 - 4.31 (m, 1H), 4.25 (s, 1H), 3.97 - 3.83 (m, 1H), 3.68 (br d, J= 10.1 Hz, 1H), 1.67 - 1.50 (m, 1H), 1.33 - 1.18 (m, 1H), 1.07 - 0.92 (m, 12H), 0.85 - 0.78 (m, 3H).
Example 59: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Compound 213)
Figure imgf000279_0001
SUBSTITUTE SHEET RULE 26 Step 1: 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[000482] To a solution of 5-bromoisoquinoline-4-carbaldehyde (600 mg, 2.54 mmol, 1 eq) in DMSO (10 mb) was added i-PnNH (771.58 mg, 7.63 mmol, 1.08 mL, 3 eq), and then was added Cui (193.63 mg, 1.02 mmol, 0.4 eq) and Pd(PPhs)4 (587.41 mg, 508.34 umol, 0.2 eq), and ethynyl(trimethyl)silane (499.28 mg, 5.08 mmol, 704.20 uL, 2 eq) slowly under N2. The resulting mixture was stirred at 25 °C for 16 h. Upon completion, the reaction was quenched by addition H2O (30 mL), and filtered through celite then extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 2: 1) to afford 5-(2- trimethylsilylethynyl)isoquinoline-4-carbaldehyde (460 mg, 1.69 mmol, 66.43% yield, 93% purity) as yellow solid. MS (ESI) m/z 254.1 [M+H]+.
Step 2: 5-ethynylisoquinoline-4-carbaldehyde
[000483] To a solution of 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (460 mg, 1.82 mmol, 1 eq) in MeOH (7 mL) was added K2CO3 (627.31 mg, 4.54 mmol, 2.5 eq), and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was filtered to give the 5-ethynylisoquinoline-4-carbaldehyde (330 mg, crude) as yellow liquid (solution in MeOH). MS (ESI) m/z 182.0 [M+H]+
Step 3: 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile
[000484] To a solution of 5-ethynylisoquinoline-4-carbaldehyde (330 mg, 1.82 mmol, 1 eq) in DCM (5 mL) was added Ti(i-PrO)4 (776.45 mg, 2.73 mmol, 806.28 uL, 1.5 eq) and NHVMeOH (7 M, 15 mL). The resulting mixture was stirred at 25 °C for 1 h, and then TMSCN (542.07 mg, 5.46 mmol, 683.57 uL, 3 eq) was added at 0 °C. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was diluted with H2O (15 mL) and then was extracted with DCM (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum to give 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (300 mg, crude) as black brown solid. MS (ESI) m/z 208.1 [M+H]+
Step 4: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
279
SUBSTITUTE SHEET RULE 26 [000485] To a solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (300 mg, 1.45 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (632.96 mg, 1.74 mmol, 1.2 eq) in ACN (8 mb) was added 1 -methylimidazole (356.56 mg, 4.34 mmol, 346.17 uL, 3 eq) and TCFH (963.14 mg, 2.90 mmol, 2 eq), and then the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was quenched by addition H2O (20 mL), and filtered through celite then extracted with ethyl acetate (7 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3) - ACN]; B%: 35% - 65%, 8 min) to give the product (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (220 mg, 290.11 umol, 20.04% yield, 73% purity) as white solid. MS (ESI) m/z 554.4 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000486] The (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (220 mg, 397.42 umol, 1 eq) was separated by SFC (column: REGIS (S, S) WHELK - 01 (250 mm * 25 mm, 10 um); mobile phase: [Neu - ETOH]; B%: 27% - 27%, 6 min) to get product (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (66.6 mg, 112.66 umol, 28.35% yield, 93.639% purity) as white solid. MS (ESI) m/z 554.2 [M+H]+.
[000487] 'H NMR (400 MHz, MeOD-d4) 5 ppm 9.39 (s, 1H), 8.91 (s, 1H), 8.30 - 8.24 (m, 1H), 8.16 (d, J = 7.2 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.65 (s, 1H), 4.56 (s, 1H), 4.35 (s, 1H), 4.10 - 3.91 (m, 2H), 3.88 (d, J = 10.4 Hz, 1H), 1.61 (dd, J = 5.4, 7.6 Hz, 1H), 1.52 - 1.47 (m, 1H), 1.07 (s, 9H), 1.03 - 1.01 (m, 3H), 0.88 (s, 3H).
[000488] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
280
SUBSTITUTE SHEET RULE 26 Isomer 2 (63.25 mg, 110.19 umol, 27.73% yield, 96.441% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+.
[000489] 'H NMR (400 MHz, MeOD-d4) 5 ppm 9.35 (s, 1H), 8.91 (s, 1H), 8.25 (dd, J = 1.0, 8.2 Hz, 1H), 8.18 - 8.09 (m, 1H), 7.78 (d, J = 6.8 Hz, 1H), 7.77 - 7.69 (m, 1H), 4.63 - 4.52 (m, 1H), 4.44 (s, 1H), 4.11 - 3.91 (m, 2H), 3.87 (d, J = 10.4 Hz, 1H), 1.69 - 1.57 (m, 1H), 1.50 (d, J = 7.6 Hz, 1H), 1.11 - 1.07 (m, 3H), 0.99 (s, 9H), 0.91 (s, 3H).
Example 60: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino] butanoyl] -N-(3-cyano-6-azatricyclo [6.3.1.04,12] dodeca- l(ll),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 214)
Figure imgf000282_0001
Step 1: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]
-N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[000490] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (300 mg, 734.55 umol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (180.93 mg, 2.20 mmol, 175.66 uL, 3 eq), TCFH (412.20 mg, 1.47 mmol, 2 eq), Then 3-amino-6- azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaene-3-carbonitrile (143.40 mg, 734.55 umol, 1 eq) was added, and the mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was diluted by addition H2O (30 mL), and then extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purifed by pre-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40mm * lOum; mobile phase:
281
SUBSTITUTE SHEET RULE 26 [water( NH4HCO3)-ACN]; B%: 45%-75%, 8 min) to give the product (lR,2S,5S)-3-[(2S,3R)-3- tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-(3-cyano-6- azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 306.45 umol, 41.72% yield, 99.7% purity) as a brown solid. MS (ESI) m/z 586.2 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-(3- cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 and Isomer 2
[000491] The (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l 1), 4(12), 5,7, 9-pentaen-3-yl)-6, 6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 307.37 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK IG (250mm * 30mm, 10 um); mobile phase: [Neu-ETOH];
B%: 12%-12%, 15 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (53.33 mg, 90.52 umol, 29.45% yield, 99.4% purity) as a white solid. MS (ESI) m/z 586.2 [M+H]+.
[000492] 'H NMR (400 MHz, DMSO-de) 5 = 9.57 (br d, J = 8.3 Hz, 1H), 9.48 - 9.39 (m, 2H), 8.96 - 8.87 (m, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.83 - 7.69 (m, 2H), 4.40 (t, J = 7.9 Hz, 1H), 4.28 (d, J = 18.2 Hz, 1H), 4.21 (s, 1H), 4.01 (dd, J = 5.3, 10.4 Hz, 1H), 3.92 (dd, J = 6.3, 7.3 Hz, 1H), 3.78 (d, J = 18.4 Hz, 1H), 3.65 (br d, J = 10.4 Hz, 1H), 1.60 (dd, J = 5.4, 7.5 Hz, 1H), 1.37 (d, J = 7.5 Hz, 1H), 1.17 - 1.09 (m, 11H), 1.03 - 0.98 (m, 4H), 0.81 (s, 3H).
[000493] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- (3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (55.86 mg, 85.66 umol, 27.87% yield, 89.8% purity) was obtained as a white solid. MS (ESI) m/z 586.2 [M+H]+.
[000494] 'H NMR (400 MHz, DMSO-de) 5 = 9.57 (br d, J = 8.2 Hz, 1H), 9.45 (s, 1H), 9.38 (s, 1H), 8.82 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.82 - 7.69 (m, 2H), 4.39 (t, J = 7.9 Hz, 1H), 4.31 - 4.20 (m, 2H), 4.04 - 3.86 (m, 3H), 3.66 (d, J = 10.5 Hz, 1H), 1.58 (dd, J = 5.4, 7.4 Hz, 1H), 1.42 (d, J = 7.6 Hz, 1H), 1.16 - 1.11 (m, 12H), 1.01 - 0.98 (m, 3H), 0.81 (s, 3H).
282
SUBSTITUTE SHEET RULE 26 Example 61: Synthesis of (lR,2S,5S)-N-[(8-chlorophthalazin-l-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 223)
N,N,N,N, -tetramethyl
Figure imgf000284_0001
Figure imgf000284_0002
Figure imgf000284_0003
Figure imgf000284_0004
Step 1: N-tert-butyl-2-chloro-benzamide
[000495] A solution of 2-chlorobenzoyl chloride (10 g, 57.14 mmol, 8.40 mL, 1 eq) in DCM (95 mL) was cooled to 0 °C, and then a solution of 2-methylpropan-2-amine (4.18 g, 57.14 mmol, 6.00 mL, 1 eq) and TEA (14.45 g, 142.85 mmol, 19.88 mL, 2.5 eq) in DCM (47.5 mL) was added dropwise. The mixture was stirred at 20 °C for 3 h. Upon completion, the reaction mixture was diluted with NaHCCh (200 mL) and extracted with DCM (100 mL * 3). The combined organic layers were washed with aqueous citric acid solution (100 mL), then the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:0 to 1: 1) to give N-tert-butyl-2-chloro-benzamide (12 g, 51.02 mmol, 89.29% yield, 90% purity) as a white solid.
Step 2: 2-tert-butyl-7-chloro-3-hydroxy-isoindolin-l-one
283
SUBSTITUTE SHEET RULE 26 [000496] To a solution of N-tert-butyl-2-chloro-benzamide (10 g, 47.24 mmol, 1 eq) in THF (1016 mb) was added N,N,N',N' -tetramethylethane- 1,2-diamine (17.51 g, 150.69 mmol, 22.74 mL, 3.19 eq). s-BuLi (1.3, 115.92 mb, 3.19 eq) was added dropwise at -65 °C for 0.5 h under argon atmosphere, followed by stirring at the same temperature for 2.5 h, then the mixture was added with DMF (8 mL) and warmed from -65 °C to 20 °C for 2 h. Upon completion, the reaction mixture was quenched by aq.NLLiCl (1500 mL) at 0 °C, and then extracted with ethyl acetate (300 mL * 3). The combined organic layers were washed with brine (200 mL * 2) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Diehl oromethane: Methanol = 10:1 to 1: 1) to give 2- tert-butyl-7-chloro-3-hydroxy-isoindolin-l-one (8 g, 30.04 mmol, 63.59% yield, 90% purity) as a white solid.
Step 3: 8-chloro-2H-phthalazin- 1 -one
[000497] 2-Tert-butyl-7-chloro-3-hydroxy-isoindolin-l-one (5 g, 20.86 mmol, 1 eq) was suspended in AcOH (20 mL) under nitrogen. The resulting thick slurry was heated to 90 °C. At approximately 90 °C, a homogeneous solution was obtained. NH2NH2.H2O (3.13 g, 62.58 mmol, 3.04 mL, 3 eq) was added drop wise (exotherm), keeping the internal temperature at 90 °C over approximately 4 h. Water (40 mL) preheated to 80 °C was added, maintaining the mixture at 90 °C followed by ramping down to 20 °C over approximately 1 h. Upon completion, the resulting suspension was transferred onto a filter. The filter cake was rinsed with water (10 mL * 3). The filter cake was evaporated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10: 1 to 0:1) to give 8- chloro-2H-phthalazin-l-one (2.35 g, 12.75 mmol, 61.14% yield, 98% purity) as a yellow solid.
Step 4: 1 -bromo- 8-chloro-phthalazine
[000498] A mixture of 8-chloro-2H-phthalazin-l-one (1 g, 5.54 mmol, 1 eq) and POBn (7.94 g, 27.69 mmol, 2.81 mL, 5 eq) was stirred at 130 °C for 0.5 h. Upon completion, the residue was diluted with THF (20 mL), and then the filter cake was evaporated under reduced pressure to to give l-bromo-8-chloro-phthalazine (1.5 g, crude) as a brown solid.
Step 5: 2-(benzhydrylideneamino)-2-(8-chlorophthalazin- 1 -yl)acetonitrile
284
SUBSTITUTE SHEET RULE 26 [000499] A mixture of 2-(benzhydrylideneamino)acetonitrile (1.36 g, 6.16 mmol, 1 eq) in THF (40 mL) was added LiHMDS (1 M, 15.40 mL, 2.5 eq) at -65 °C under N2, then the mixture was stirred at -65 °C for 1 h, then was added l-bromo-8-chloro-phthalazine (1.5 g, 6.16 mmol, 1 eq) in THF (40 mL) dropwised to the mixture. The resulting mixture was stirred at -65 °C for 0.5 h, the mixture was warmed to 20 °C, and the mixture was stirred at 20 °C for 3.5 h. Upon completion, the reaction mixture was diluted with NH4CI (500 mL) and extracted with DCM (200 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 100: 1 to petroleum ether:DCM = 0: 1) to give 2- (benzhydrylideneamino)-2-(8-chlorophthalazin-l-yl)acetonitrile (1.1 g, 2.59 mmol, 41.98% yield, 90% purity) as a red solid.
Step 6: 2-amino-2-(8-chlorophthalazin-l-yl)acetonitrile
[000500] A mixture of 2-(benzhydrylideneamino)-2-(8-chlorophthalazin-l-yl)acetonitrile (300 mg, 783.61 umol, 1 eq) in aq. HC1 (3 M, 10 mL, 38.28 eq) and DCM (10 mL) was stirred at 25 °C for 36 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with DCM (10 mL) at 25 °C for 0.5 h, the filter cake was evaporated under reduced pressure to give 2-amino-2-(8-chlorophthalazin-l- yl)acetonitrile (150 mg, 548.84 umol, 70.04% yield, 80% purity) as a brown solid.
Step 7: (lR,2S,5S)-N-[(8-chlorophthalazin-l-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000501] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (249.97 mg, 686.05 umol, 1 eq) in ACN (15 mL) was added 1 -methylimidazole (112.65 mg, 1.37 mmol, 109.37 uL, 2 eq) and TCFH (384.99 mg, 1.37 mmol, 2 eq) at 0 °C, and then 2-amino-2- (8-chlorophthalazin-l-yl)acetonitrile (150 mg, 686.05 umol, 1 eq) was added. The resulting mixture was stirred at 0 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (30 mL) and extracted with DCM (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30 mm *10 um; mobile phase:
285
SUBSTITUTE SHEET RULE 26 [water( NH4HCCh)-ACN]; B%: 38% - 68%, 8 min) to give (lR,2S,5S)-N-[(8-chlorophthalazin- l-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (60 mg, 106.20 umol, 42.86% yield, 100% purity) as a white solid. MS (ESI) m/z 564.2 [M+H]+.
[000502] 1H NMR (400 MHz, DMSO-t76) 5 = 9.84 (s, 1H), 9.54 (dd, J= 7.2, 11.2 Hz, 1H), 9.45 - 9.24 (m, 1H), 8.29 (t, J= 7.1 Hz, 1H), 8.25 - 8.12 (m, 1H), 8.12 - 8.01 (m, 1H), 7.36 (t, J = 6.7 Hz, 1H), 4.46 - 4.35 (m, 1H), 4.29 (d, J= 10.5 Hz, 1H), 3.99 - 3.88 (m, 1H), 3.78 - 3.66 (m, 1H), 1.64 - 1.52 (m, 1H), 1.33 - 1.23 (m, 1H), 1.06 - 0.93 (m, 12H), 0.79 (d, J= 3.8 Hz, 3H).
Example 62: Synthesis of (Compound 224)
Figure imgf000287_0001
Step 1: 3,7-dibromo-3H-isobenzofuran-l-one
[000503] To a solution of 7-bromo-3H-isobenzofuran-l-one (2 g, 9.39 mmol, 1 eq) in CH3CI (10 mL) was added AIBN (76.92 mg, 468.42 umol, 4.99e-2 eq) and NBS (1.84 g, 10.33 mmol, 1.1 eq). The mixture was stirred at 70 °C for 2 h under N2 atmosphere. Upon completion, the reaction mixture was diluted with H2O (40 mL) and extracted with DCM (40 mL * 2). The combined organic layers were washed with brine (40 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column
286
SUBSTITUTE SHEET RULE 26 chromatography (SiCh, petroleum etherethyl acetate = 20: 1 to 10:1). 3,7-Dibromo-3H- isobenzofuran-l-one (2.59 g, 8.43 mmol, 89.78% yield, 95% purity) was obtained as a white solid. MS (ESI) m/z 292.8 [M+H]+.
Step 2: 8-bromo-2H-phthalazin-l-one
[000504] 3,7 -Dibromo-3H-isobenzofuran-l-one (2.5 g, 8.56 mmol, 1 eq) was suspended in
AcOH (10 mL) under nitrogen. The resulting thick slurry was heated to 90 °C. NH2NH2.H2O (1.29 g, 25.69 mmol, 1.25 mL, 3 eq) was added dropwise, keeping the internal temperature at 90 °C over approximately 2 h. Upon completion, water (20 mL) preheated to 80 °C was added, maintaining the mixture at 80-90 °C followed by ramping down to 20 °C over approximately 3 h. The resulting suspension was transferred onto a filter and give the crude product, the crude product was washed with water (10 mL * 3) to give 8-bromo-2H-phthalazin-l-one (1.3 g, 5.37 mmol, 62.73% yield, 93% purity) as a yellow solid. MS (ESI) m/z 225.0 [M+H]+.
Step 3: 1,8-dibromophthalazine
[000505] A mixture of 8-bromo-2H-phthalazin-l-one (450 mg, 2.00 mmol, 1 eq) and POBn (2.87 g, 10.00 mmol, 1.02 mL, 5 eq) was stirred at 130 °C for 0.5 h. Upon completion, the residue was diluted with THE (10 mL), then filtered to give the crude product. The crude product was washed with THF (100 mL) to give Compound 1,8-dibromophthalazine (400 mg, 1.03 mmol, 51.41% yield, 74% purity) as a yellow solid. MS (ESI) m/z 288.9 [M+H]+.
Step 4: 2-(benzhydrylideneamino)-2-(8-bromophthalazin-l-yl)acetonitrile
[000506] A mixture of 2-(benzhydrylideneamino)acetonitrile (305.99 mg, 1.39 mmol, 1 eq) in THF (8 mL) was added LiHMDS (1 M, 3.47 mL, 2.5 eq) at -65 °C under N2, and then the mixture was stirred at -65 °C for 0.5 h. 1,8-Dibromophthalazine (400 mg, 1.39 mmol, 1 eq) in THF (8 mL) dropwised to the mixture, and then the mixture was stirred at -65 °C for 0.5 h. The mixture was warmed to 25 °C, and the mixture was stirred at 25 °C for 4 h. Upon completion, the reaction mixture was diluted with water 50 mL and extracted with DCM ( 50 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 100:1 to petroleum ether:DCM = 0:1). 2-(Benzhydrylideneamino)-2-(8-
287
SUBSTITUTE SHEET RULE 26 bromophthalazin- 1 -yl)acetonitrile (360 mg, 676.53 umol, 48.70% yield, 80.3% purity) was obtained as a yellow solid. MS (ESI) m/z 427.0 [M+H]+.
Step 5: 2-amino-2-(8-bromophthalazin-l-yl)acetonitrile
[000507] A mixture of 2-(benzhydrylideneamino)-2-(8-bromophthalazin-l-yl)acetonitrile (300 mg, 702.09 umol, 1 eq) in HC1 (3 M, 6.00 mL, 25.64 eq) and THF (6 mL) was stirred at 25 °C for 24 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water(HCl)-ACN]; B%: 5%-20%, 8 min). Compound 2-amino-2-(8- bromophthalazin-l-yl)acetonitrile (80 mg, 267.06 umol, 38.04% yield, HC1) was obtained as a white solid. MS (ESI) m/z 263.1 [M+H]+.
Step 6: (lR,2S,5S)-N-[(8-bromophthalazin-l-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000508] To a mixture of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (97.31 mg, 267.06 umol, 1 eq) in ACN (8 mL) was added 1 -methylimidazole (65.78 mg, 801.19 umol, 63.86 uL, 3 eq) and TCFH (149.87 mg, 534.13 umol, 2 eq). Then 2-amino-2-(8- bromophthalazin-l-yl)acetonitrile (80 mg, 267.06 umol, 1 eq, HC1) was added and the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL) at 25 °C, and then extracted with DCM (20 mL * 2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75 * 30 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-70%, 8 min).
Compound (lR,2S,5S)-N-[(8-bromophthalazin-l-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (30 mg, 238.01 umol, 17.82% yield, 98.3% purity) was obtained as a white solid.
[000509] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.84 (s, 1H), 9.56 (dd, J= 6.9, 19.8 Hz, 1H), 9.36 (br dd, J= 8.3, 17.7 Hz, 1H), 8.43 (ddd, J= 1.0, 7.7, 15.3 Hz, 1H), 8.33 (ddd, J= 0.8, 4.2, 8.0 Hz, 1H), 8.00 - 7.91 (m, 1H), 7.53 - 7.42 (m, 1H), 4.41 (t, J= 8.2 Hz, 1H), 4.31 (d, J= 5.2
288
SUBSTITUTE SHEET RULE 26 Hz, 1H), 3.97 - 3.87 (m, 1H), 3.72 (dd, J= 10.4, 17.2 Hz, 1H), 1.62 - 1.52 (m, 1H), 1.34 (dd, J = 4.5, 7.6 Hz, 1H), 1.07 - 0.95 (m, 12H), 0.82 - 0.79 (m, 3H).
Example 63: Synthesis of (S)-2-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanamido)- 3-(l-fluorocyclopropyl)propanoic acid
Figure imgf000290_0001
Step 1: (S)-methyl 2-amino-3-(l-fluorocyclopropyl)propanoate hydrochloride
[000510] A solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(l- fluorocyclopropyl)propanoate (150 mg, 574.08 umol, 1 eq) in HCl/MeOH (4 M, 5 mb, 34.84 eq) was stirred at 20 °C for 1 h. Upon completion the reaction mixture was concentrated under reduced pressure to give methyl (2S)-2-amino-3-(l-fluorocyclopropyl)propanoate (135 mg, crude, HCI) as white solid. MS (ESI) m/z 162.1 [M+H]+.
Step 2: (S)-methyl 2-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)-3-(l- fluorocyclopropyl)propanoate
[000511] A solution of methyl (2S)-2-amino-3-(l-fluorocyclopropyl)propanoate (135 mg, 683.08 umol, 1 eq, HCI) in DCM (5 mL) was added DMAP (250.35 mg, 2.05 mmol, 3 eq), and added (2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoic acid (189.59 mg, 819.69 umol, 1.2 eq) was added at 0 °C under N2 atmosphere. Then, EDCI (261.90 mg, 1.37 mmol, 2 eq) was added to the mixture and the mixture was sitrred at 25 °C for 2 h. Upon completion the reaction mixture was quenched by addition H2O 5 mL at 0 °C, and extracted with DCM 15 mL (5 mL * 3). The combined organic layers were washed with brine 5 mL (5 mL * 1), dried over Na2SO4,
289
SUBSTITUTE SHEET RULE 26 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 1/0 to 3/1) to give methyl (2S)-2- [[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]amino]-3-(l- fluorocyclopropyl)propanoate (200 mg, 432.64 umol, 63.34% yield, 81% purity) as a white solid. MS (ESI) m/z 375.3 [M+H]+.
Step 3: (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanamido)-3-(l- fluorocyclopropyl)propanoic acid
[000512] To a solution of methyl (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]amino]-3-(l-fluorocyclopropyl)propanoate (200 mg, 534.12 umol, 1 eq) in THF (5 mL) and H2O (0.5 mL) was added LiOH H2O (44.83 mg, 1.07 mmol, 2 eq) and the resulting mixture was sitrred at 25 °C for 2 h. Upon completion the reaction mixture, the mixture was diluted with 5 mL H2O, adjusted pH = 3, and then was extracted with ethyl acetate (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]amino]-3-(l-fluorocyclopropyl)propanoic acid (135 mg, crude) as a white solid. MS (ESI) m/z 361.2 [M+H]+.
Step 4: (S)-2-((S)-2-amino-3,3-dimethylbutanamido)-3-(l-fluorocyclopropyl)propanoic acid hydrochloride
[000513] A solution of (2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]amino]-3-(l-fluorocyclopropyl)propanoic acid (135 mg, 374.56 umol, 1 eq) in HCl/dioxane (5 mL) was stirred at 20 °C for 1 h. Upon completion the reaction mixture was concentrated under reduced pressure to give (2S)-2-[[(2S)-2-amino-3,3-dimethyl- butanoyl]amino]-3-(l-fluorocyclopropyl)propanoic acid (90 mg, crude, HC1) as a white solid. MS (ESI) m/z 261.1 [M+H]+.
Step 5: (S)-2-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanamido)-3-(l- fluorocyclopropyl)propanoic acid
[000514] A solution of (2S)-2-[[(2S)-2-amino-3,3-dimethyl-butanoyl]amino]-3-(l- fluorocyclopropyl)propanoic acid (110 mg, 422.58 umol, 1 eq) in methyl 2,2,2-trifluoroacetate
290
SUBSTITUTE SHEET RULE 26 (13.97 g, 109.09 mmol, 11.00 mL, 258.15 eq) was added TEA (128.28 mg, 1.27 mmol, 176.46 uL, 3 eq) and stirred at 20 °C for 3 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (10 Ml) and adjusted to pH = 3 and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (10 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with petroleum ether/ethyl acetate = 30/1 (15 mL) at 20 °C for 1 h to give (2S)-2-[[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]amino]-3-(l-fluorocyclopropyl)propanoic acid (130 mg, 353.90 umol, 83.75% yield, 97% purity) as a white solid. Then 30 mg of the product was furthur purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water(NHiHCO3)-ACN]; B%: 10%-40%, 8 min) to give (2S)-2-[[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]amino]-3-(l-fluorocyclopropyl)propanoic acid (17.6 mg, 49.39 umol, 58.67% yield, 100% purity) as a white solid. MS (ESI) m/z 357.2 [M+H]+.
[000515] 'H NMR (400 MHz, METHANOL-d4) 5 = 4.53 - 4.62 (dd, J= 4.3, 8.3 Hz, 1H), 4.46 (s, 1H), 2.36 - 2.51 (ddd, J= 4.3, 15.1, 19.5 Hz, 1H), 2.08 - 1.92 (m, 1H), 1.04 (s, 9H), 0.92 - 0.80 (m, 2H), 0.69 - 0.48 (m, 2H)
Example 64: Synthesis of (S)-N-((S)-l-(((5-bromoisoquinolin-4-yl)(cyano)methyl)amino)-3- (l-fhiorocyclopropyl)-l-oxopropan-2-yl)-3,3-dimethyl-2-(2,2,2- trifluoroacetamidojbutanamide (Compound 227)
Figure imgf000292_0001
Step 1: 2-amino-2-(5-bromoisoquinolin-4-yl)acetonitrile
291
SUBSTITUTE SHEET RULE 26 [000516] A solution of 5-bromoisoquinoline-4-carbaldehyde (290 mg, 1.23 mmol, 1 eq) in NFh/MeOH (5 mL) was added NH4CI (197.14 mg, 3.69 mmol, 3 eq) under N2 atmosphere. Then TMSCN (365.62 mg, 3.69 mmol, 461.06 uL, 3 eq) was added to the mixture and the mixture was sitrred at 20 °C for 16 h. Upon completion the reaction mixture was concentrated under reduced pressure to remove NHs/MeOH. The reaction mixture was quenched by addition H2O (5 mL), and then extracted with ethyl acetate (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 0:1) to give 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (170 mg, 603.19 umol, 49.10% yield, 93% purity) as a yellow solid. MS (ESI) m/z 262.1 [M+H]+.
Step 2: (2S)-N-((2S)-l-(((5-bromoisoquinolin-4-yl)(cyano)methyl)amino)-3-(l- fluorocyclopropyl)-l-oxopropan-2-yl)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanamide
[000517] A solution of 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (160 mg, 610.44 umol, 1 eq) and (2S)-2-[[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]amino]-3-(l- fluorocyclopropyl)propanoic acid (326.26 mg, 915.66 umol, 1.5 eq) in ACN (5 mL) was added with TCFH (205.53 mg, 732.53 umol, 1.2 eq) and 1 -methylimidazole (200.47 mg, 2.44 mmol, 194.63 uL, 4 eq). Then the mixture was sitrred at 25 °C for 2 h. Upon completion the reaction mixture was quenched by addition H2O 5 mL at 0°C, and extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate=l/O to 3/1) to get (270 mg, 77% purity), a residue which was further separated by SFC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 8 min) to give (2S)-N- [2- [ [(5-bromo-4-isoquinoly l)-cyano-methyl] amino] - 1 - [( 1 -fluorocy clopropy l)methy 1] -2-oxo- ethyl]-3, 3-dimethyl-2-[(2, 2, 2-trifluoroacetyl)amino]butanamide Isomer 1 (29.35 mg, 48.88 umol, 8.01% yield, 100% purity) as a white solid. MS (ESI) m/z 602.0 [M+H]+.
[000518] 1H NMR (400 MHz, DMSO-t/6) 5 = 9.71 (br d, J= 6.1 Hz, 1H), 9.50 (s, 1H), 9.18 (br d, J= 10.0 Hz, 1H), 8.89 (s, 1H), 8.47 (br d, J= 7.9 Hz, 1H), 8.33 (d, J= 7.9 Hz, 1H), 8.25 (d, J= 7.5 Hz, 1H), 7.67 (t, J= 7.7 Hz, 1H), 7.48 (br d, J= 5.7 Hz, 1H), 4.71 (br d, J= 7.2 Hz,
292
SUBSTITUTE SHEET RULE 26 1H), 4.41 (br d, J= 8.4 Hz, 1H), 2.19 (br s, 1H), 2.03 (br d, J= 7.5 Hz, 1H), 0.89 (s, 9H), 0.83 - 0.65 (m, 3H), 0.61 - 0.55 (m, 1H)
[000519] (2S)-N-[2-[[(5-bromo-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide Isomer 2 (68.30 mg, 113.76 umol, 18.64% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 602.0 [M+H]+.
[000520] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.57 (br d, J= 6.7 Hz, 1H), 9.50 (s, 1H), 9.20 (br d, .7= 9,3 Hz, 1H), 8.89 (s, 1H), 8.44 (d, J= 8.2 Hz, 1H), 8.33 (d, J= 8.1 Hz, 1H), 8.27 (d, J = 7.5 Hz, 1H), 7.68 (t, J= 7.8 Hz, 1H), 7.52 (br d, J= 6.7 Hz, 1H), 4.77 - 4.68 (m, 1H), 4.40 (d, J= 9.3 Hz, 1H), 2.29 - 2.22 (m, 1H), 1.98 - 1.84 (m, 1H), 0.92 (s, 9H), 0.80 - 0.70 (m, 3H), 0.50 - 0.42 (m, 1H)
[000521] (2S)-N-[2-[[(5-bromo-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide Isomer 3 (70.55 mg, 116.21 umol, 19.04% yield, 98.9% purity) was obtained as a white solid. MS (ESI) m/z 602.0 [M+H]+.
[000522] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.64 (br d, J= 6.2 Hz, 1H), 9.50 (s, 1H), 9.28 (br d, .7= 7.9 Hz, 1H), 8.87 (s, 1H), 8.59 (br d, J= 7.8 Hz, 1H), 8.33 (d, J= 8.1 Hz, 1H), 8.27 (d, J= 7.5 Hz, 1H), 7.68 (t, J= 7.8 Hz, 1H), 7.53 (br d, J= 5.5 Hz, 1H), 4.69 - 4.60 (m, 1H), 4.48 - 4.40 (m, 1H), 2.33 - 2.24 (m, 1H), 1.89 (br s, 1H), 0.95 (s, 9H), 0.87 - 0.75 (m, 3H), 0.47 (br d, J = 7.6 Hz, 1H)
[000523] (2S)-N-[2-[[(5-bromo-4-isoquinolyl)-cyano-methyl]amino]-l-[(l- fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide Isomer 4 (69.44 mg, 113.57 umol, 18.61% yield, 98.2% purity) was obtained as a white solid. MS (ESI) m/z 602.0 [M+H]+.
[000524] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.60 (br d, J= 6.4 Hz, 1H), 9.50 (s, 1H), 9.15 (br d, .7= 9.5 Hz, 1H), 8.88 (s, 1H), 8.63 (d, J= 8.2 Hz, 1H), 8.32 (d, J = 8.1 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 7.68 (t, J= 7.8 Hz, 1H), 7.49 - 7.39 (m, 1H), 4.68 (br d, J= 6.5 Hz, 1H), 4.41 (d, J= 9.3 Hz, 1H), 2.31 - 2.20 (m, 1H), 2.00 (s, 1H), 0.92 (s, 9H), 0.90 - 0.74 (m, 1H), 0.64 - 0.55 (m, 1H).
293
SUBSTITUTE SHEET RULE 26 Example 65: Synthesis ofN-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- l(ll),4(12),5,7,9-pentaen-3-yl)amino]-l-[(l-flnorocyclopropyl)methyl]-2-oxo-ethyl]-3,3- dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanamide (Compound 228)
Figure imgf000295_0001
Step 1: (2S)-2-amino-3,3-dimethyl-butanoic acid
[000525] A solution of (2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoic acid (1 g, 4.32 mmol, 1 eq) in HCl/Dioxane (4 M, 10 mL, 9.25 eq) was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated in vacuum to give (2S)-2-amino-3,3- dimethyl-butanoic acid (750 mg, crude, HC1) as a white solid.
Step 2: (2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoic acid
[000526] To a solution of (2S)-2-amino-3,3-dimethyl-butanoic acid (650 mg, 4.96 mmol, 1 eq) in methyl 2,2,2-trifluoroacetate (1.90 g, 14.87 mmol, 1.50 mL, 3 eq) was added TEA (1.50 g, 14.87 mmol, 2.07 mL, 3 eq), and then the mixture was stirred at 25 °C for 12 h. Upon completion, the residue was concentrated and adjusted to pH~4 with 2 N HC1, and then extracted with ethyl acetate (20 mL * 2). The combined organic phase was washed with brine (20 mL),
294
SUBSTITUTE SHEET RULE 26 dried over Na2SO4. and then concentrated to give (2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoic acid (985 mg, 4.34 mmol, 87.50% yield) as a yellow oil.
Step 3: (2S)-2-(tert-butoxycarbonylamino)-3-(l-fluorocyclopropyl)propanoic acid
[000527] To a solution of methyl (2S)-2-(tert-butoxycarbonylamino)-3-(l -fluorocyclopropyl) propanoate (200 mg, 765.44 umol, 1 eq) and LiOH.FLO (112.41 mg, 2.68 mmol, 3.5 eq) in THF (3 mb) and H2O (3 mL) was stirred at 25 °C for 1 h. Upon completion, the residue was concentrated and adjusted to pH~4 with 2 N HC1, and was extracted with ethyl acetate (10 mL * 2), the combined organic phase was washed with brine (10 mL), dried over Na2SO4 and concentrated, to give (2S)-2-(tert-butoxycarbonylamino)-3-(l-fluorocyclopropyl) propanoic acid (200 mg, crude) as a white solid.
Step 4: tert-butyl N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]carbamate
[000528] To a solution of 3-amino-6-azatricyclo[6.3.1.04, 12] dodeca- 1(11), 4(12), 5,7,9- pentaene-3-carbonitrile (165.80 mg, 849.30 umol, 1 eq) and (2S)-2-(tert-butoxycarbonylamino)- 3-(l-fluorocyclopropyl)propanoic acid (420 mg, 1.70 mmol, 2 eq) in ACN (5 mL) was added TCFH (476.59 mg, 1.70 mmol, 2 eq) and 1 -methylimidazole (244.05 mg, 2.97 mmol, 236.94 uL, 3.5 eq). The resulting mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and then extracted with ethyl acetate (10 mL * 2). The combined organic layers were washed with 15% citric acid (10 mL * 2), NaHCCh (10 mL) and brine (10 * 2 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate=2: l to 1 :1) to give tert-butyl N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- 1(11), 4(12), 5 ,7,9-pentaen-3 -yl)amino] - 1 - [( 1 -fluorocyclopropy l)methy 1] -2-oxo-ethyl] carbamate (360 mg, 602.17 umol, 70.90% yield, 71% purity) as a yellow solid.
Step 5: (2S)-2-amino-N-(3-cyano-6-azatricyclo[6.3.1.04, 12]dodeca-l (11), 4(12), 5,7, 9-pentaen-3- y 1) - 3 -( 1 -fluorocyclopropy l)propanamide
[000529] A solution of tert-butyl N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- 1(11), 4(12), 5 ,7,9-pentaen-3 -yl)amino] - 1 - [( 1 -fluorocyclopropy l)methy 1] -2-oxo-ethyl] carbamate
295
SUBSTITUTE SHEET RULE 26 (330 mg, 777.45 umol, 1 eq) in DCM (4 mL) and HCl/dioxane (2 mL) was stirred at 20 °C for 1 h. Upon completion, the residue was concentrated in vacuum to give (2S)-2-amino-N-(3-cyano- 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-3-(l- fluorocyclopropyl)propanamide (240 mg, crude) as a white solid.
Step 6: N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide
[000530] To a solution of (2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoic acid (493.79 mg, 2.17 mmol, 3 eq) and (2S)-2-amino-N-(3 -cyano-6-azatricyclo[6.3.1.04, 12] dodecaIQ 1), 4(12), 5,7, 9-pentaen-3-yl)-3-(l-fluorocyclopropyl)propanamide (235 mg, 724.52 umol, 1 eq) in DCM (5 mL) was added with DMAP (265.54 mg, 2.17 mmol, 3 eq) and EDCI (277.78 mg, 1.45 mmol, 2 eq), and then the mixture was stirred at 25 °C for 3 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and then extracted with ethyl acetate (10 mL * 2). The combined organic layers were washed with 15% citric acid (10 mL * 2), NaHCCh (10 mL) and brine (20 mL), then dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue, the residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30mm * 10 urn; mobile phase: [water (NH4HCO3) -ACN]; B%: 25%-55%, 8 mins) to give N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04, 12]dodeca-l(l l), 4(12), 5,7, 9-pentaen- 3 -y l)amino] - 1 - [( 1 -fluorocyclopropyl)methyl] -2-oxo-ethy 1] -3 ,3 -dimethyl-2- [(2,2,2- trifluoroacetyl)amino]butanamide (130 mg) as a white solid.
Step 7: N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide
[000531] N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide was further separated by SFC (column: DAICEL CHIRALPAK IG (250mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 10%-35%, 14min) to give (Rt= 1.486 min) N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2-
296
SUBSTITUTE SHEET RULE 26 trifluoroacetyl)amino]butanamide (Isomer 1) (21.19 mg, 39.32 umol, 5.43% yield, 99.0% purity) as a white solid. MS (ESI) m/z 534.2 [M+H]+.
[000532] 1H NMR (400 MHz, DMSO-t/6) 5 = 9.55 (s, 1H), 9.40 (s, 1H), 9.15 (br d, J = 8.2 Hz, 1H), 8.83 (s, 1H), 8.48 (d, J = 7.0 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.80 - 7.75 (m, 1H),
7.72 - 7.68 (m, 1H), 4.58 (q, J = 7.2 Hz, 1H), 4.40 (br d, J = 7.8 Hz, 1H), 4.25 (d, J = 18.2 Hz, 1H), 3.86 (d, J = 18.1 Hz, 1H), 2.18 - 2.11 (m, 1H), 2.10 - 2.04 (m, 1H), 0.93 (s, 9H), 0.86 (br s, 1H), 0.85 - 0.75 (m, 2H), 0.75 - 0.75 (m, 1H), 0.73 - 0.64 (m, 1H), 0.57 - 0.46 (m, 1H).
[000533] N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide (Isomer 1) (10.66 mg, 19.78 umol, 21.11% yield, 99.0% purity) as a white soild. MS (ESI) m/z 534.2 [M+H]+.
[000534] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.53 (s, 1H), 9.39 (s, 1H), 9.29 - 9.07 (m, 1H), 8.84 (s, 1H), 8.64 - 8.62 (m, 1H), 8.60 (br d, J= 7.1 Hz, 1H), 8.03 (d, J= 8.1 Hz, 1H), 7.82 -
7.73 (m, 1H), 7.70 (d, J= 6.9 Hz, 1H), 4.62 - 4.54 (m, 1H), 4.43 (s, 1H), 4.26 (br d, J= 18.1 Hz, 1H), 3.86 (br d, J= 18.3 Hz, 1H), 2.30 - 2.12 (m, 1H), 0.92 (s, 9H), 0.87 - 0.73 (m, 3H), 0.55 - 0.47 (m, 1H).
[000535] N-[(lS)-2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanamide Isomer 2 (23.49 mg, 43.63 umol, 46.56% yield, 99.1% purity) was obtained as a white soild. MS (ESI) m/z 534.2 [M+H]+.
[000536] XH NMR (400 MHz, DMSO-t/e) 5 = 9.52 (s, 1H), 9.36 (s, 1H), 9.11 (br d, J= 7.1 Hz, 1H), 8.85 (s, 1H), 8.49 (br d, J= 7.6 Hz, 1H), 8.02 (d, J= 8.0 Hz, 1H), 7.80 - 7.75 (m, 1H),
7.74 - 7.70 (m, 1H), 4.63 (q, J= 7.3 Hz, 1H), 4.39 (br d, J= 6.5 Hz, 1H), 4.23 (br d, J= 18.1 Hz, 1H), 3.96 (br d, J= 18.3 Hz, 1H), 2.29 - 2.16 (m, 1H), 2.11 - 1.89 (m, 1H), 0.86 (s, 11H), 0.76 - 0.67 (m, 1H), 0.64 - 0.54 (m, 1H).
[000537] (Rt= 2.093 min) N-[2-[(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-
1(1 l),4(12),5,7,9-pentaen-3-yl)amino]-l-[(l-fluorocyclopropyl)methyl]-2-oxo-ethyl]-3,3- dimethyl-2-[(2, 2, 2-trifluoroacetyl)amino]butanamide Isomer 3 (13.69 mg, 25.33 umol, 3.50% yield, 98.7% purity) as a white solid. MS (ESI) m/z 534.2 [M+H]+.
297
SUBSTITUTE SHEET RULE 26 [000538] 'H NMR (400 MHz, DMSO- e) 5 = 9.49 (s, 1H), 9.37 (s, 1H), 9.21 (br d, J= 9.3 Hz, 1H), 8.84 (s, 1H), 8.58 (br d, J= 7.6 Hz, 1H), 8.02 (d, J= 8.1 Hz, 1H), 7.80 - 7.75 (m, 1H), 7.74 - 7.67 (m, 1H), 4.67 - 4.58 (m, 1H), 4.41 (br d, J= 9.0 Hz, 1H), 4.24 (br d, J= 18.1 Hz, 1H), 3.92 (br d, J= 18.3 Hz, 1H), 2.28 - 2.15 (m, 1H), 2.03 - 1.82 (m, 1H), 0.90 (s, 9H), 0.87 - 0.71 (m, 3H), 0.62 - 0.55 (m, 1H).
Example 66: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 229)
Figure imgf000299_0001
Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[5-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000539] A solution of 2-chloro-5-(trifluoromethyl)pyrazine (500 mg, 2.74 mmol, 1.2 eq) and tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (740.64 mg, 2.28 mmol, 1 eq) in dioxane (10 mL) was added BINAP (1.42 g, 2.28 mmol, 1 eq) and CS2CO3 (1.49 g, 4.57 mmol, 2 eq), and then Pd(OAc)2 (51.25 mg, 228.27 umol, 0.1 eq) was added under N2 atmosphere. The mixture was stirred at 100 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 20/1 to 8/1) to give tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[5-
(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
298
SUBSTITUTE SHEET RULE 26 carboxylate (425 mg, 614.20 umol, 26.91% yield, 68% purity) as a yellow oil. MS (ESI) m/z 471.2 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[5-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid
[000540] A solution of tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[5- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylate (425 mg, 903.24 umol, 1 eq) in HCl/dioxane (8 mL) was stirred at 40 °C for 2 hr. Upon completion, the mixture was concentrated under reduced pressure to give (lR,2S,5S)-3- [(2S)-3,3-dimethyl-2-[[5-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (305 mg, crude) as a yellow oil. MS (ESI) m/z 415.2 [M+H]+.
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[5- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000541] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[5-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (305 mg, 735.97 umol, 1 eq) in ACN (10 mL) was added with TCFH (247.80 mg, 883.16 umol, 1.2 eq) and 1- methylimidazole (241.70 mg, 2.94 mmol, 234.66 uL, 4 eq). The mixture was stirred at 25°C for 5 min. Then, 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (160.19 mg, 735.97 umol, 1 eq) was added to the mixture and stirred at 25 °C for 55 min. Upon completion, the reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, petroleum ether/ethyl acetate = 20/1 to 1/1) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)- cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[5-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (400 mg, 514.61 umol, 69.92% yield, 79% purity) as a yellow oil.
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SUBSTITUTE SHEET RULE 26 Step 4: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[5- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000542] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[5- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide was separated by SFC (condition: column: DAICEL CHIRALPAK IC(250 mm * 30 mm, 10 um); mobile phase: [Neu-ETOH]; B%: 35%-35%,10 min ). Then Isomer 1 was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 100 * 30mm * lOum; mobile phase: [water( NH4HCO3)-ACN]; B%: 50%-80%, 8 min). Then the mixture was separated by SFC (column: DAICEL CHIRALPAK IC(250 mm * 30 mm, 10 um); mobile phase: [Neu-ETOH]; B%: 30%-30%, 8 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[[5-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (52.67 mg, 84.83 umol, 32.56% yield, 98.9% purity) as a white solid. MS (ESI) m/z 614.2 [M+H]+.
[000543] 1H NMR (400 MHz, DMSO ) 5 = 9.59 (br d, J = 6.5 Hz, 1H), 9.49 (s, 1H), 8.88 (s, 1H), 8.28 - 8.24 (m, 3H), 8.04 - 7.99 (m, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.29 (br s, 1H), 4.44 (d, J = 8.3 Hz, 1H), 4.28 (s, 1H), 4.05 - 3.99 (m, 1H), 3.97 - 3.92 (m, 1H), 1.62 (dd, J = 5.4, 7.5 Hz, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.06 - 1.00 (m, 12H), 0.85 (s, 3H).
[000544] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[5- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (52.44 mg, 83.01 umol, 31.86% yield, 97.2% purity) as a white solid. MS (ESI) m/z 614.2 [M+H]+.
[000545] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.50 (s, 1H), 9.42 (br d, J= 7.2 Hz, 1H), 8.87 (s, 1H), 8.32 - 8.23 (m, 3H), 8.05 (dd, J= 1.1, 7.6 Hz, 1H), 7.93 (d, J= 8.2 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.30 (br d, J= 2.9 Hz, 1H), 4.44 (d, J= 8.3 Hz, 1H), 4.24 (s, 1H), 4.04 - 3.98 (m, 1H), 3.97 - 3.91 (m, 1H), 1.63 - 1.51 (m, 1H), 1.25 (d, J= 7.7 Hz, 1H), 1.05 (s, 9H), 0.95 (br s, 3H), 0.85 - 0.80 (m, 3H)
300
SUBSTITUTE SHEET RULE 26 Example 67: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo [3.1.0] hexane-2-carboxamide
Figure imgf000302_0001
Step 1: 7-bromo-l-methyl-pyrrolo[3,2-c]pyridine
[000546] A solution of 7-bromo-lH-pyrrolo[3,2-c]pyridine (2.5 g, 12.69 mmol, 1 eq) in DMF (25 mL) was added NaH (761.23 mg, 19.03 mmol, 60% purity, 1.5 eq) at 0 °C for 0.5 h. Then, CH3I (2.70 g, 19.03 mmol, 1.18 mL, 1.5 eq) was added dropwise under N2. The mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by H2O (20 mL) and extracted with EA (20 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 100: 1 to 20:1) to give 7-bromo-l-methyl-pyrrolo[3,2-c]pyridine (1.1 g, 5.16 mmol, 40.67% yield, 99% purity) as a white solid. MS (ESI) m/z 213.0 [M+H]+.
Step 2: l-methyl-7-vinyl-pyrrolo[3,2-c]pyridine
[000547] A solution of 7-bromo-l-methyl-pyrrolo[3,2-c]pyridine (1.1 g, 5.21 mmol, 1 eq) in THF (10 mL) and H2O (2.5 mL) was added potassium trifluoro(vinyl)boranuide (837.75 mg, 6.25 mmol, 1.2 eq) and K3PO4 (2.21 g, 10.42 mmol, 2 eq). The mixture was added Xphos-Pd-G2 (410.07 mg, 521.18 umol, 0.1 eq) under N2 and stirred at 80 °C for 16 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced
301
SUBSTITUTE SHEET RULE 26 pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 100: 1 to 10:1) to give l-methyl-7-vinyl-pyrrolo[3,2-c]pyridine (300 mg, 1.90 mmol, 36.39% yield) as a white solid. MS (ESI) m/z 159.1 [M+H]+.
Step 3: l-methylpyrrolo[3,2-c]pyridine-7-carbaldehyde
[000548] A solution of l-methyl-7-vinyl-pyrrolo[3,2-c]pyridine (300 mg, 1.90 mmol, 1 eq) in THF (6 mL) and H2O (3 mL) was added K2OSO4.2H2O (69.87 mg, 189.63 umol, 0.1 eq) and stirred at 20 °C for 30 min, then NalCh (1.22 g, 5.69 mmol, 315.24 uL, 3 eq) was added. The mixture was stirred at 20 °C for 5.5 h. Upon completion, the reaction mixture was poured into H2O (5 mL) and extracted with ethyl acetate (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (S1O2, Ethyl acetate) to give 1- methylpyrrolo[3,2-c]pyridine-7-carbaldehyde (100 mg, 624.33 umol, 32.92% yield) as a yellow solid. MS (ESI) m/z 161.1 [M+H]+.
Step 4: 2-amino-2-(l-methylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile
[000549] A solution of l-methylpyrrolo[3,2-c]pyridine-7-carbaldehyde (100 mg, 624.33 umol, 1 eq), Ti(i-PrO)4 (266.16 mg, 936.49 umol, 276.39 uL, 1.5 eq), and NH3/MeOH (7 M, 356.76 uL, 4 eq) in DCM (1 mL) was stirred at 20 °C for 1 h, and then TMSCN (185.81 mg, 1.87 mmol, 234.32 uL, 3 eq) was added. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (1 mL) and extracted with DCM (1 mL * 3). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, DCM:MeOH = 10: 1) to give 2-amino-2-(l-methylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile (35 mg, 182.32 umol, 29.20% yield, 97% purity) as a yellow solid. MS (ESI) m/z 187.2 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000550] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
302
SUBSTITUTE SHEET RULE 26 (67.04 mg, 183.99 umol, 1 eq), 2-amino-2-thiazolo[4,5-c]pyridin-7-yl-acetonitrile (35 mg, 183.99 umol, 1 eq), 1 -methylimidazole (45.32 mg, 551.98 umol, 44.00 uL, 3 eq) in ACN (1 mL) was added TCFH (103.25 mg, 367.99 umol, 2 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the residue was diluted with H2O (1 mL) and extracted with ethyl acetate (1 ml * 3). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, EA) to give (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]- 3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (30 mg, 56.33 umol, 30.62% yield) as a white solid. MS (ESI) m/z 533.2 [M+H]+.
Step 6: (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3. L0]hexane-2- carboxamide
[000551] The (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide (30 mg, 56.33 umol, 30.62% yield) was separated by SFC (column: DAICEL CHIRALPAK AD(250mm * 30mm, lOum); mobile phase: [Neu-IPA]; B%: 10%-35%, lOmin) to give (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3. L0]hexane-2- carboxamide Isomer 1 (8.43 mg, 15.83 umol, 8.60% yield) as a white solid. MS (ESI) m/z 533.2 [M+H]+.
[000552] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.53 (d, J= 7.0 Hz, 1H), 9.33 (d, J= 8.0 Hz, 1H), 8.90 (s, 1H), 8.39 (s, 1H), 7.46 (d, J= 3.1 Hz, 1H), 6.88 (d, J= 6.9 Hz, 1H), 6.67 (d, J= 3.1 Hz, 1H), 4.40 (d, J= 8.1 Hz, 1H), 4.23 (s, 1H), 3.95 (dd, J = 5.5, 10.4 Hz, 1H), 3.91 (s, 3H), 3.74 (d, J= 10.5 Hz, 1H), 1.63 (dd, J= 5.5, 7.4 Hz, 1H), 1.29 (d, J= 7.6 Hz, 1H), 1.03 (s, 12H), 0.81 (s, 3H)
[000553] (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide Isomer 2 (8.35 mg, 15.68 umol, 8.52% yield) was obtained as a white solid. MS (ESI) m/z 533.2 [M+H]+.
303
SUBSTITUTE SHEET RULE 26 [000554] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.40 (br d, J= 7.5 Hz, 1H), 9.26 (br d, J= 7.5 Hz, 1H), 8.87 (s, 1H), 8.40 (s, 1H), 7.42 (br s, 1H), 6.78 (br d, J= 7.3 Hz, 1H), 6.66 (br s, 1H), 4.35 (br d, J= 8.0 Hz, 1H), 4.23 (s, 1H), 3.93 (m, 4H), 3.62 (br d, J= 10.4 Hz, 1H), 1.56 (br t, J = 6.1 Hz, 1H), 1.24 (br d, J= 6.8 Hz, 1H), 0.98 (s, 3H), 0.88 (s, 9H), 0.80 (s, 3H).
Example 68: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 231)
Figure imgf000305_0001
Step 1 : (4-bromopyrazolo [1,5 -a]pyridin-3 -yl)-imidazol- 1 -yl-methanone
[000555] To a solution of 4-bromopyrazolo[l,5-a]pyridine-3-carboxylic acid (350 mg, 1.45 mmol, 1 eq) in THF (4 mL) was added CDI (353.17 mg, 2.18 mmol, 1.5 eq), the mixture was stirred at 25 °C for 18 h. Upon completion, the reaction mixture was used to next step without any worked up and to give the product (4-bromopyrazolo[l,5-a]pyridin-3-yl)-imidazol-l-yl- methanone (400 mg, crude) as colorless liquid (solution in THF). MS (ESI) m/z 293.0 [M+H]+.
Step 2: (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol
[000556] To a solution of (4-bromopyrazolo[l,5-a]pyridin-3-yl)-imidazol-l-yl-methanone (400 mg, 1.37 mmol, 1 eq) in THF (2 mL) and H2O (2 mL) at 0 °C, was added with NaBFh (259.91 mg, 6.87 mmol, 5 eq) at 0 °C under N2, and the resulting mixture was stirred at 25 °C for 30 min. Upon completion, the reaction mixture was diluted with sat. NH4CI (20 mL) and then extracted with ethyl acetate (7 mL * 3). The combined organic layers were dried over Na2SO4,
304
SUBSTITUTE SHEET RULE 26 filtered and concentrated in vacuum and was purified by prep-TLC (S1O2, Petroleum ether:Ethyl acetate = 2: 1) to give the product (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol (170 mg, 741.22 umol, 53.94% yield, 99% purity) as white solid. MS (ESI) m/z 227.0 [M+H]+.
Step 3: 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde
[000557] To a solution of (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol (170 mg, 748.71 umol, 1 eq) in DCM (5 mL) was added MnCh (976.39 mg, 11.23 mmol, 15 e ), the mixture was stirred at 25 °C for 96 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (160 mg, crude) as white solid. MS (ESI) m/z 225.1 [M+H]+.
Step 4: 2-amino-2-(4-bromopyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
[000558] To a solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (160 mg, 710.98 umol, 1 eq) in DCM (4 mL) was added Ti(i-PrO)4 (303.10 mg, 1.07 mmol, 314.75 uL, 1.5 eq) and NEh/MeOH (7 M, 4 mL), and the mixture was stirred at 25 °C for 1 h. TMSCN (211.61 mg, 2.13 mmol, 266.85 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was diluted with H2O (8 mL) and then was extracted with DCM (3 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum to give 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (150 mg, crude) as gray solid. MS (ESI) m/z 251.0 [M+H]+.
Step 5: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000559] To a solution of 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (150 mg, 597.41 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (217.67 mg, 597.41 umol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (147.14 mg, 1.79 mmol, 142.86 uL, 3 eq) and TCEH (335.24 mg, 1.19 mmol, 2 eq), and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was quenched by addition H2O (10 mL), and then extracted with EA (3 mL * 3). The combined organic layers were dried over Na2SO4, filtered and
305
SUBSTITUTE SHEET RULE 26 concentrated in vacuum and was purified by prep-HPLC (column: Waters Xbndge Prep OBD C18 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3) - ACN]; B%: 40%-70%, 8 min) to give the product (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (180 mg, 298.28 umol, 49.93% yield, 99% purity) as white solid. MS (ESI) m/z 597.1 [M+H]+.
Step 6: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 and Isomer 2
[000560] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (180 mg, 301.29 umol, 1 eq) was separated by SFC (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu - MeOH]; B%: 20% - 20%, 4 min) to get (lR,2S,5S)-N-[(4- bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (52.78 mg, 88.35 umol, 29.32% yield, 100% purity) as white solid. MS (ESI) m/z 597.1 [M+H]+.
[000561] 'H NMR (400 MHz, DMSO-t/r,) 5 ppm 9.42 (d, J= 6.4 Hz, IH), 9.35 (br s, IH), 8.83 (d, J= 6.8 Hz, IH), 8.31 (s, IH), 7.68 (d, J= 7.4 Hz, IH), 6.94 (t, J = 7.2 Hz, IH), 6.57 (d, J= 6.4 Hz, IH), 4.51 - 4.39 (m, IH), 4.32 (s, IH), 3.90 (br dd, J= 5.4, 10.4 Hz, IH), 3.72 (br d, J= 10.4 Hz, IH), 1.60 - 1.55 (m, IH), 1.36 (d, J = 7.6 Hz, IH), 1.03 (s, 3H), 0.97 (s, 9H), 0.86 - 0.82 (m, 3H).
[000562] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (52.78 mg, 82.91 umol, 27.52% yield, 93.842% purity) as white solid. MS (ESI) m/z 597.1 [M+H]+.
[000563] 'H NMR (400 MHz, DMSO-t/r,) 5 ppm 9.35 (br d, J= 7.0 Hz, 2H), 8.83 (d, J= 7.0 Hz, IH), 8.35 - 8.24 (m, IH), 7.69 (d, J= 7.2 Hz, IH), 6.94 (t, J= Hz, IH), 6.58 (d, J= 6.8
306
SUBSTITUTE SHEET RULE 26 Hz, 1H), 4.40 (br s, 1H), 4.27 (s, 1H), 3.91 (br dd, J= 5.4, 10.2 Hz, 1H), 3.73 - 3.66 (m, 1H), 1.54 (br t, J= 6.2 Hz, 1H), 1.32 (d, J = 7.6 Hz, 1H), 1.01 (s, 9H), 0.98 (s, 3H), 0.83 (s, 3H).
Example 69: Synthesis of (lR,2S,5S)-N-[cyano-(4-methylpyrazolo[l,5-a]pyridin-3- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 232)
Figure imgf000308_0001
Step 1: (4-methylpyrazolo[l,5-a]pyridin-3-yl)methanol
[000564] To a solution of 4-methylpyrazolo[l,5-a]pyridine-3-carboxylic acid (4 g, 22.71 mmol, 1 eq) in THF (50 mL) was added LiAlHt (1.72 g, 45.41 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was cooled to 0 °C, quenched by addition of 1.72 mL of H2O, followed by 1.72 mL of 15% aqueous NaOH, then 10 mL of H2O was added. After being stirred at room temperature for 0.5 h, the mixture was filtered through celite pad to remove by-product. The filtrate was concentrated to dryness to give (4- methylpyrazolo[l,5-a]pyridin-3-yl)methanol (3.6 g, crude) as a yellow oil. MS (ESI) m/z 163.2 [M+H]+.
Step 2: 4-methylpyrazolo[l,5-a]pyridine-3-carbaldehyde
[000565] To a solution of (4-methylpyrazolo[l,5-a]pyridin-3-yl)methanol (3.6 g, 22.20 mmol, 1 eq) in DCM (20 mL) was added PCC (9.57 g, 44.39 mmol, 2 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (30 mL) at 25 °C, and then extracted with DCM (30 mL * 3). The combined organic layers were washed 307
SUBSTITUTE SHEET RULE 26 with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to 3: 1) to give 4-methylpyrazolo[l,5-a]pyridine-3-carbaldehyde (430 mg, 1.88 mmol, 8.47% yield, 70% purity) as a yellow solid. MS (ESI) m/z 161.2 [M+H]+.
Step 3: 2-oxo- lH-quinoline-4-carbaldehy de
[000566] To a solution of 4-methylpyrazolo[l,5-a]pyridine-3-carbaldehyde (390 mg, 2.43 mmol, 1 eq) in DCM (15 mL) was added Ti(i-PrO)4 (1.04 g, 3.65 mmol, 1.08 mL, 1.5 eq) and ammonia (7 M, 1.39 mL, 4 eq) stirred 1 h at 25 °C before TMSCN (724.67 mg, 7.30 mmol, 913.83 uL, 3 eq) was added at 25 °C. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was poured into H2O (30 mL) at 25 °C, and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 4: 1 to 1 : 1) to give 2- amino-2-(4-methylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile (230 mg, 629.92 umol, 25.87% yield, 51% purity) as a yellow oil. MS (ESI) m/z 187.2 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(4-methylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000567] A mixture of 2-amino-2-(4-methylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile (230 mg, 1.24 mmol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (450.04 mg, 1.24 mmol, 1 eq), 1- methylimidazole (304.23 mg, 3.71 mmol, 295.37 uL, 3 eq) in ACN (10 mL) was added with [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (693.11 mg, 2.47 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL) at 20 °C, and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: C18 (250 * 50 mm * 10 um); mobile phase: [water( NH4HCO3)-ACN]; B%: 45%-65%, 10 min) to give (lR,2S,5S)-N-[cyano-(4-methylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-
308
SUBSTITUTE SHEET RULE 26 dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (250 mg, 468.49 umol, 37.93% yield, 99.8% purity) as a white solid. MS (ESI) m/z 533.4 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-(4-methylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000568] ( 1 R,2S , 5 S)-N - [cyano-(4-methy lpyrazolo[ 1 , 5 -a]pyri din-3 -yl)methyl] -3 - [(2S)-3 , 3 - dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (250 mg) was separated by SFC (column: DAICEL CHIRALPAK IC(250mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 15%- 15%, 9 min) to give (lR,2S,5S)-N-[cyano-(4- methylpyrazolo[ 1 , 5-a]pyridin-3 -yl)methy 1] -3 - [(2S)-3 , 3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 1 (117.6 mg, 217.95 umol, 46.43% yield, 98.7% purity) as a white solid. MS (ESI) m/z 533.4 [M+H]+.
[000569] 'H NMR (400 MHz, MeOD-t74) 5 = 8.42 (d, J= Hz, 1H), 8.19 (s, 1H), 7.07 (d, J = 6.9 Hz, 1H), 6.87 (t, J= Hz, 1H), 6.51 (s, 1H), 4.58 - 4.51 (m, 1H), 4.32 (s, 1H), 4.04 (dd, J = 5.5, 10.3 Hz, 1H), 3.88 (d, J= 10.4 Hz, 1H), 2.60 (s, 3H), 1.66 (dd, J= 5.4, 7.5 Hz, 1H), 1.42 (d, J= 7.6 Hz, 1H), 1.08 (s, 3H), 1.04 (s, 9H), 0.91 (s, 3H).
[000570] (lR,2S,5S)-N-[cyano-(4-methylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (107.1 mg, 197.49 umol, 42.07% yield, 98.2% purity) was obtained as a white solid. MS (ESI) m/z 533.4 [M+H]+.
[000571] 'H NMR (400 MHz, MeOD-t74) 5 = 8.43 (d, J= 6.9 Hz, 1H), 8.18 (s, 1H), 7.09 (d, J = 6.9 Hz, 1H), 6.88 (t, J= 6.9 Hz, 1H), 6.47 (s, 1H), 4.52 (s, 1H), 4.33 (s, 1H), 4.02 (dd, J= 5.5, 10.3 Hz, 1H), 3.84 (d, J= 10.4 Hz, 1H), 2.62 (s, 3H), 1.64 - 1.60 (m, 1H), 1.32 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 0.96 (s, 9H), 0.89 (s, 3H).
Example 70: Synthesis of (lR,2S,5S)-N-[cyano-(5-methylimidazo[l,2-a]pyridin-3- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 234)
309
SUBSTITUTE SHEET RULE 26
Figure imgf000311_0001
Step 1 : 2-amino-2-( 5 -methy limidazo [ 1 , 2-a] py ridin- 3 -y l)acetonitrile
[000572] A mixture of 5-methylimidazo[l,2-a]pyridine-3-carbaldehyde (600 mg, 3.75 mmol, 1 eq), Ti(i-PrO)4 (1.60 g, 5.62 mmol, 1.66 mL, 1.5 eq) and ammonia (7 M, 2.14 mL, 4 eq) was added in DCM (6 ml) was stirred at 20 °C for 30 min, and then TMSCN (1.11 g, 11.24 mmol, 1.41 mL, 3 eq) was added at 25 °C and stirred for 16 h. Upon completion, the residue was poured into water (5 mL). The aqueous phase was extracted with DCM (10 mL*3).The combined organic phase was washed with brine (10 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by prep-TLC (ethyl acetate : methanol = 5 : 1, Rf = 0.52) to afford 2-amino-2-(5-methylimidazo[l,2-a]pyridin-3-yl)acetonitrile (600 mg, 3.06 mmol, 81.71% yield, 95% purity) as a yellow solid. MS (ESI) m/z 187.2 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano-(5-methylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000573] To a mixture of 2-amino-2-(5-methylimidazo[l,2-a]pyridin-3-yl)acetonitrile (600 mg, 3.22 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.06 g, 2.90 mmol, 0.9 eq ) in DCM (10 mL) was added HATU (1.84 g, 4.83 mmol, 1.5 eq) and DIEA (832.87 mg, 6.44 mmol, 1.12 mL, 2 eq) in one portion at 25°C. The mixture was stirred at 25°C for 1 h. Upon completion, the reaction mixture was quenched by addition water 10 mL, and then extracted with DCM (10 mL * 3). The combined organic layers were washed with brine (10 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether/ethyl acetate=50/l, 3:1) to give a (lR,2S,5S)-N-[cyano- (5-methylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)
310
SUBSTITUTE SHEET RULE 26 amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (1.2 g, 2.03 mmol, 62.94% yield, 90% purity) as a red oil. MS (ESI) m/z 533.3 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano-(5-methylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 and Isomer 2
[000574] ( 1 R,2S , 5 S)-N - [cyano-(5 -methy limidazo [ 1 ,2-a] pyridin-3 -yl)methyl] -3 - [(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (1.2 g, 2.03 mmol, 1 eq ) was separated by SFC. The product was separated by SFC(column: DAICEL CHIRALPAK AD(250mm * 50mm, lOum); mobile phase: [Neu-IPA]; B%: 15%-l 5%, 4.5min) to give (lR,2S,5S)-N-[cyano-(5-methylimidazo[l,2-a]pyridin-3- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (68.82 mg, 127.69 umol, 5.67% yield, 100% purity) as white solid. MS (ESI) m/z 533.2 [M+H]+.
[000575] 'H NMR (400 MHz, DMSO-de) 5 = 9.56 (d, J = 6.6 Hz, 1H), 9.43 - 9.25 (m, 1H), 7.82 (s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.24 (dd, J = 6.9, 9.0 Hz, 1H), 6.84 (d, J = 6.6 Hz, 1H), 6.76 (d, J = 6.7 Hz, 1H), 4.40 (s, 1H), 4.21 (s, 1H), 3.94 (dd, J = 5.3, 10.2 Hz, 1H), 3.73 (d, J = 10.5 Hz, 1H), 2.73 (s, 3H), 1.62 (dd, J = 5.5, 7.5 Hz, 1H), 1.27 (d, J = 7.6 Hz, 1H), 1.03 (s, 12H), 0.81 (s, 3H)
[000576] (lR,2S,5S)-N-[cyano-(5-methylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (82.02 mg, 150.89 umol, 6.70% yield, 98% purity) was obtained as white solid. MS (ESI) m/z 533.2 [M+H]+.
[000577] 'H NMR (400 MHz, DMSO-de) 5 = 9.41 (br d, J = 7.2 Hz, 1H), 9.27 (d, J = 7.5 Hz, 1H), 7.83 (s, 1H), 7.50 (d, J = 8.9 Hz, 1H), 7.24 (dd, J = 7.0, 8.9 Hz, 1H), 6.78 (dd, J = 7.2, 13.6 Hz, 2H), 4.35 (br d, J = 7.3 Hz, 1H), 4.21 (s, 1H), 3.96 (dd, J = 5.6, 10.2 Hz, 1H), 3.62 (br d, J = 10.4 Hz, 1H), 2.73 (s, 3H), 1.62 - 1.50 (m, 1H), 1.24 (d, J = 7.6 Hz, 1H), 0.99 (s, 3H), 0.89 (s, 9H), 0.80 (s, 3H).
311
SUBSTITUTE SHEET RULE 26 Example 71: Synthesis of (lR,2S,5S)-N-((3-bromothieno[2,3-c]pyridin-4-yl)(cyano)methyl)- 3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo [3.1.0] hexane-2-carboxamide
Figure imgf000313_0001
Step 1: ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate
[000578] Ethyl 2-sulfanylacetate (2.27 g, 18.87 mmol, 2.06 mL, 1 eq) and CS2CO3 (7.99 g, 24.54 mmol, 1.3 eq) was added to a solution of 3,5-dibromopyridine-4-carbaldehyde (5 g, 18.87 mmol, 1 eq) in THF (100 mL). The mixture was stirred at 60 °C for 3 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give ethyl 4- bromothieno[2,3-c]pyridine-2-carboxylate (4.5 g, crude) as yellow solid.
Step 2: 4-bromothieno[2,3-c]pyridine-2-carboxylic acid
[000579] To a solution of ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate (4 g, 13.98 mmol, 1 eq) in THF (50 mL) and MeOH (20 mL) was added NaOH (7 M, 8.00 mL, 4.01 eq). The mixture was stirred at 20 °C for 6 h. Upon completion, the reaction mixture was quenched by addition THF and MeOH, H2O (60 mL) was added to the residue, IM HC1 was added to pH = 3, solid was precipitated and filtered out. The solid was washed with H2O (20 mL), dired under
312
SUBSTITUTE SHEET RULE 26 vacuum to give 4-bromothieno[2,3-c]pyridine-2-carboxylic acid (3.5 g, crude) as white solid. MS (ESI) m/z 255.9 [M-H]+.
Step 3: 4-bromothieno[2,3-c]pyridine
[000580] A solution of 4-bromothieno[2,3-c]pyridine-2-carboxylic acid (3.7 g, 14.34 mmol, 1 eq), 1 , 10-phenanthroline (2.07 g, 11.47 mmol, 0.8 eq) and Q12O (2.05 g, 14.34 mmol, 1.47 mL, 1 eq) in NMP (50 mL) and H2O (l mL), and quinoline (18.80 g, 145.58 mmol, 17.25 mL, 10.15 eq) was stirred at 160 °C for 4 h. Upon completion, the reaction was poured into H2O (300 mL) and then extracted with ethyl acetate (150 mL * 3). The combined organic phase was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (neutral condition; column: Agela DuraShell Cl 8 250 * 70mm * lOum; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%) to give 4- bromothieno[2,3-c]pyridine (1.2 g, 5.61 mmol, 39.10% yield, N/A purity) as white solid. MS (ESI) m/z 213.9 [M+H]+.
Step 4: 4-vinylthieno[2,3-c]pyridine
[000581] To a solution of 4-bromothieno[2,3-c]pyridine (1.1 g, 5.14 mmol, 1 eq) in toluene (12 mL) and H2O (3 mL) was added K3PO4 (2.18 g, 10.28 mmol, 2 eq) and potassium trifluoro(vinyl)boranuide (825.91 mg, 6.17 mmol, 1.2 eq). The mixture was added ditert- butyl(cyclopentyl)phosphane; dichloropalladium; iron (334.88 mg, 513.82 umol, 0.1 eq) under N2. The mixture was stirred at 80 °C for 16 h under N2. Upon completion, the reaction was pour into H2O (70 mL) and then extracted with ethyl acetate (30 mL * 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 20/1 to 4/1) to give 4-vinylthieno[2,3-c]pyridine (0.8 g, 4.37 mmol, 84.98% yield, 88% purity) as black brown oil. MS (ESI) m/z 162.1 [M+H]+.
Step 5: thieno[2,3-c]pyridine-4-carbaldehyde
[000582] To a solution of 4-vinylthieno[2,3-c]pyridine (0.8 g, 4.96 mmol, 1 eq) in THE (8 mL) and H2O (1.6 mL) was added dipotassium dioxido(dioxo)osmium dihydrate (182.83 mg, 496.21 umol, 0.1 eq). The mixture was stirred for 0.5 h, then NalCh (3.18 g, 14.89 mmol, 824.88
313
SUBSTITUTE SHEET RULE 26 uL, 3 eq) was added. The mixture was stirred at 20 °C for 12 h. Upon completion, the reaction was pour into H2O (30 mL) and then extracted with ethyl acetate (20 mL * 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 1/0 to 4/1) to give thieno[2,3-c]pyridine-4-carbaldehyde (400 mg, 2.45 mmol, 49.40% yield, N/A purity) as yellow solid. MS (ESI) m/z 164.0 [M+H]+.
Step 6: 3-bromothieno[2,3-c]pyridine-4-carbaldehyde
[000583] To a solution of thieno[2,3-c]pyridine-4-carbaldehyde (30 mg, 183.83 umol, 1 eq) in H2SO4 (0.5 mL) was added l,3-dibromo-5,5-dimethyl-imidazolidine-2, 4-dione (63.07 mg, 220.59 umol, 1.2 eq) at 0 °C. The mixture was stirred at 25 °C for 14 h. Upon completion, the reaction solution is then poured onto crushed ice. Then was adjusted to pH~8 With NaHCCh aq, then was extrated with ethyl acetate (50 mL * 5), and the combined organic phase was washed with brine (150 mL), dried over Na2SO4 and concentrated in vacuum to give 3-bromothieno[2,3- c]pyridine-4-carbaldehyde (30 mg, crude) as yellow solid. MS (ESI) m/z 241.9 [M+H]+.
Step 7 : 2-amino-2-(3 -bromothieno [2,3 -c] pyridin-4-yl)acetonitrile
[000584] A mixture of 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (300 mg, 1.24 mmol, 1 eq) and NHVMeOH (7 M, 2 mL, 11.30 e ), Ti(i-PrO)4 (528.30 mg, 1.86 mmol, 548.60 uL, 1.5 eq) in DCM (4 mL) was stirred at 25 °C for 1 h. Then was added TMSCN (368.81 mg, 3.72 mmol, 465.08 uL, 3 eq) at 0 °C. The mixture was stirred at 25 °C for 14 h. Upon completion, the reaction was pour into H2O (100 mL) and then extracted with DCM (30 mL * 5). The combined organic phase was washed with brine (70 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give 2-amino-2-(3-bromothieno[2,3-c]pyridin-4-yl)acetonitrile (300 mg, crude) as black brown oil. MS (ESI) m/z 267.9 [M+H]+.
Step 8: (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000585] To a solution of 2-amino-2-(3-bromothieno[2,3-c]pyridin-4-yl)acetonitrile (300 mg, 727.25 umol, 65% purity, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-
314
SUBSTITUTE SHEET RULE 26 trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (264.98 mg, 727.25 umol, 1 eq) in ACN (8 mL) was added TCFH (265.27 mg, 945.43 umol, 1.3 eq) and 1 -methylimidazole (119.42 mg, 1.45 mmol, 115.94 uL, 2 eq). The mixture was stirred at 25 °C for 3 h. Upon completion, the reaction was pour into H2O (50 mL) and then extracted with EA (20 mL * 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 20/1 to 1/1) to give desired compound (350 mg, purity 92%) as a white solid, which was further separated by SFC (condition: column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 urn); mobile phase: [Neu-MeOH]; B%: 33%- 33%, 6 min) to give (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (110 mg, 165.95 umol, 22.82% yield, 92.7% purity) as a white solid and (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 159.32 umol, 21.91% yield, 97.9% purity) as a white solid. MS (ESI) m/z 614.2 [M+H]+.
[000586] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.64 - 9.40 (m, 2H), 9.36 (br s, 1H), 8.75 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 9.9 Hz, 1H), 7.10 (br d, J = 6.1 Hz, 1H), 4.45 - 4.23 (m, 2H), 3.91 (br dd, J = 5.2, 9.9 Hz, 1H), 3.71 (t, J = 10.4 Hz, 1H), 1.57 (ddd, J = 5.6, 7.1, 12.5 Hz, 1H), 1.44 - 1.27 (m, 1H), 1.04 - 0.95 (m, 12H), 0.87 - 0.81 (m, 3H).
Step 9: (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000587] (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (110 mg, 165.95 umol, 92.7% purity) was purified by prep-HPLC (neutral condition;column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water( NH4HCCh)-ACN]; B%: 35%-75%, 8 min) to give (lR,2S,5S)-N-[(3-bromothieno[2,3- c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (90 mg, 146.47 umol, 88.26% yield).
315
SUBSTITUTE SHEET RULE 26 And (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (90 mg, 97.9% purity), which was further separated by SFC (condition: column: DAICEL CHIRALPAK IC(250 mm * 30 mm, 10 urn); mobile phase: [Neu-MeOH];B%: 5%-25%, 15 min ) to give (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (0.09 g, 146.47 umol, 50.00% yield, 100% purity) as white solid. MS (ESI) m/z 614.2 [M+H]+.
[000588] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (br d, J = 5.9 Hz, 1H), 9.45 (s, 1H), 9.36 (br s, 1H), 8.75 (s, 1H), 8.46 (d, J = 0.6 Hz, 1H), 7.09 (br d, J = 4.3 Hz, 1H), 4.39 (br s, 1H), 4.33 (s, 1H), 3.91 (br dd, J = 5.4, 10.2 Hz, 1H), 3.72 (br d, J = 10.5 Hz, 1H), 1.61 - 1.55 (m, 1H), 1.39 (br d, J = 7.6 Hz, 1H), 1.03 - 0.95 (m, 11H), 0.83 (s, 3H).
[000589] (lR,2S,5S)-N-[(3-bromothieno[2,3-c]pyridin-4-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (0.035 g, 56.16 umol, 19.17% yield, 98.59% purity) was obtained as a white solid. MS (ESI) m/z 614.2 [M+H]+.
[000590] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.50 - 9.40 (m, 2H), 9.37 (br s, 1H), 8.75 (s, 1H), 8.49 (s, 1H), 7.09 (br d, J = 6.7 Hz, 1H), 4.39 (br s, 1H), 4.26 (s, 1H), 3.91 (br dd, J = 5.6, 10.5 Hz, 1H), 3.69 (br d, J = 10.4 Hz, 1H), 1.60 - 1.50 (m, 1H), 1.34 (d, J = 7.6 Hz, 1H), 1.03 - 0.96 (m, 11H), 0.84 - 0.80 (m, 3H).
316
SUBSTITUTE SHEET RULE 26 Example 72: Synthesis of (lR,2S,5S)-N-((3-chlorothieno[2,3-c]pyridin-4-yl)(cyano)methyl)- 3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 236)
Figure imgf000318_0001
Step 1: ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate
[000591] Ethyl 2-mercaptoacetate (2.50 g, 20.76 mmol, 2.27 mL, 1 eq) and CS2CO3 (8.79 g, 26.99 mmol, 1.3 eq) were added to a solution of 3,5-dibromoisonicotinaldehyde (5.5 g, 20.76 mmol, 1 eq) in THF (100 mL). The mixture was stirred at 60 °C for 3 h. Upon completion, the mixture was filtered and concentrated under reduced pressure to give a residue ethyl 4- bromothieno [2,3 -c]pyridine-2-carboxy late (12.36 g, crude) as a yellow solid.
Step 2 : 4-bromothieno[2,3-c]pyridine-2-carboxylic acid
[000592] To a solution of ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate (12.36 g, 43.19 mmol, 1 eq) in THF (100 mL) and MeOH (40 mL) was added NaOH (7 M, 24.74 mL, 4.01 eq). The mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was quenched by addition THF and MeOH, H2O (100 mL) was added to the residue, IM HC1 was added to pH=3, solid was precipitated and filtered out. The solid was washed with H2O (200 mL), dired
317
SUBSTITUTE SHEET RULE 26 under vacuum to give 4-bromothieno[2,3-c]pyridine-2-carboxylic acid (11 g, crude) as a white solid.
Step 3 : 4-bromothieno[2,3-c]pyridine
[000593] A solution of 4-bromothieno[2,3-c]pyridine-2-carboxylic acid (11 g, 42.62 mmol, 1 eq), 1 , 10-phenanthroline (6.14 g, 34.10 mmol, 0.8 eq) and cupriooxycopper (6.10 g, 42.62 mmol, 4.36 mL, 1 eq) in NMP (150 mL) and H2O (3 mL), quinoline (55.87 g, 432.60 mmol, 51.26 mL, 10.15 eq) was stirred at 160 °C for 4 h. Upon completion, the reaction was pour into H2O (300 mL) and then extracted with ethyl acetate (150 mL*3). The combined organic phase was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-HPLC column: XtimateC18 lOu 250mm*80mm; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 24 min to give 4-bromothieno [2,3-c]pyridine (4 g, 18.68 mmol, 43.84% yield) as a yellow solid.
Step 4 : 4-bromo-3-chlorothieno[2,3-c]pyridine
[000594] To a mixture of concentrated H2SO4 (15 mL) and 4-bromothieno[2,3-c]pyridine (1.5 g, 7.01 mmol, 1 eq) at 0 0 C was added with NCS (1.87 g, 14.01 mmol, 2 eq) slowly and the reaction mixture was allowed to warm up to 25 °C was stirred for 16 h. Upon completion, the mixture was adjust pH to 7 by NaOH (100 mL), and extracted with ethyl acetate (50 mL * 2). The combined organic layers were washed with brine (50 mL*2), dried over Na2SO4, filtered and concentratedunder reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 10:1 to 3: 1) to give 4-bromo-3- chlorothieno[2,3-c]pyridine (750 mg, 3.02 mmol, 43.07% yield) as a light yellow solid.
Step 5 : 3-chloro-4-vinylthieno[2,3-c]pyridine
[000595] To a solution of 4-bromo-3-chlorothieno [2,3 -c]pyri dine (750 mg, 3.02 mmol, 1 eq) in Tol. (8 mL) and H2O (2 mL) was added K3PO4 (1.28 g, 6.04 mmol, 2 eq) and potassium trifluoro (vinyl) borate (485.08 mg, 3.62 mmol, 1.2 eq). The mixture was added ditert-butyl (cyclopentyl) phosphane dichloropalladium; iron (196.68 mg, 301.78 umol, 0.1 eq) under N2. The mixture was stirred at 80 °C for 16 h under N2. Upon completion, the resulting solution was poured into H2O (20 mL), and then extracted with ethyl acetate (30 mL * 2), the combined
318
SUBSTITUTE SHEET RULE 26 organic phase was washed brine (30 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 3:1 to 1: 1) to give 3-chloro-4-vinylthieno[2,3-c]pyridine (340 mg, 1.74 mmol, 57.58% yield) as a yellow solid.
Step 6 : 3-chlorothieno[2,3-c]pyridine-4-carbaldehyde
[000596] To a solution of 3-chloro-4-vinylthieno[2,3-c]pyridine (330 mg, 1.69 mmol, 1 eq) in THF (5 mL) and H2O (1 mL) was added dipotassium dioxido(dioxo)osmium dihydrate (62.14 mg, 168.65 umol, 0.1 eq), and the resulting solution was stirred for 0.5 h. Sodium periodate (1.08 g, 5.06 mmol, 280.36 uL, 3 eq) was added, and the mixture was stirred at 25 °C for 12 h. Upon completion, the reaction was pour into H2O (10 mL) and then extracted with ethyl acetate (10 mL*7). The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 3: 1 to 0:1) to give 3-chlorothieno[2,3-c]pyridine-4- carbaldehyde (150 mg, 758.95 umol, 45.00% yield) as a white solid.
Step 7 : 2-amino-2-(3-chlorothieno[2,3-c]pyridin-4-yl)acetonitrile
[000597] A solution of 3-chlorothieno[2,3-c]pyridine-4-carbaldehyde (140 mg, 708.35 umol, 1 eq) in NFU/MeOH (3 mL) was added with NH4CI ( 113.67 mg, 2.13 mmol, 3 eq), and then the mixture was stirred at 25 °C for 1 h. TMSCN (210.83 mg, 2.13 mmol, 265.86 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 7 h. Upon completion, the reaction mixture was quenched by addition H2O (10 Ml), and then extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (30 mL*2), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(3-chlorothieno[2,3-c]pyridin-4- yl)acetonitrile (130 mg, crude) as a yellow solid.
Step 8 : (lR,2S,5S)-N-((3-chlorothieno[2,3-c]pyridin-4-yl)(cyano)methyl)-3-((S)-3,3-dimethyl- 2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000598] To a solution of 2-amino-2-(3-chlorothieno[2,3-c]pyridin-4-yl)acetonitrile (130 mg, 581.18 umol, 1 eq) in ACN (3 mL) was added (lR,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxylic acid (211.76
319
SUBSTITUTE SHEET RULE 26 mg, 581.18 umol, 1 eq), [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (195.68 mg, 697.42 umol, 1.2 eq) and 1 -methylimidazole (167.00 mg, 2.03 mmol, 162.14 uL, 3.5 eq), and then the mixture was stirred at 25 °C for 0.5 h. Upon completion, the resulting solution was concentrated in vacuum. The residue was purified by prep-HPLC column: Phenomenex Cl 8 80 * 40 mm * 3 um; mobile phase: [water (NH-iHCCT)-ACN]; B%: 40%-60%, 8 min, which was further separated by SFC column: DAICEL CHIRALPAK IC (250mm * 30mm, 10 um);mobile phase: [Neu-MeOH];B%: 33%- 33%, 10 min to give (lR,2S,5S)-N-((3-chlorothieno[2,3-c]pyridin-4-yl)(cyano)methyl)-3-((S)- 3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (35.41 mg, 61.64 umol, 10.61% yield, 99.22% purity) as a white solid. MS (ESI) m/z 570.1 [M+H]+.
[000599] 'H NMR (400 MHz, DMSO-t76) 5 = 9.58 - 9.49 (m, 1H), 9.48 - 9.40 (m, 1H), 9.39 - 9.26 (m, 1H), 8.79 - 8.70 (m, 1H), 8.35 (s, 1H), 7.01 - 6.93 (m, 1H), 4.40 (s, 1H), 4.31 (s, 1H), 3.95 - 3.87 (m, 1H), 3.73 (br d, J = 10.4 Hz, 1H), 1.63 - 1.57 (m, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.06 - 1.01 (m, 3H), 0.98 (s, 9H), 0.85 - 0.80 (m, 3H)
[000600] (lR,2S,5S)-N-((3-chlorothieno[2,3-c]pyridin-4-yl)(cyano)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (46.93 mg, 77.06 umol, 13.26% yield, 93.60% purity) was obtained as a white solid. MS (ESI) m/z 570.1 [M+H]+.
[000601] 1H NMR (400 MHz, DMSO-t76) 5 = 9.45 - 9.42 (m, 1H), 9.42 - 9.38 (m, 1H), 9.37 - 9.26 (m, 1H), 8.76 - 8.70 (m, 1H), 8.40 - 8.33 (m, 1H), 6.96 (br d, J = 7.2 Hz, 1H), 4.42 - 4.35 (m, 1H), 4.29 - 4.22 (m, 1H), 3.94 - 3.87 (m, 1H), 3.70 - 3.65 (m, 1H), 1.58 - 1.54 (m, 1H), 1.27 - 1.25 (m, 1H), 0.99 - 0.96 (m, 12H), 0.82 - 0.80 (m, 3H)
320
SUBSTITUTE SHEET RULE 26 Example 73: Synthesis of (lR,2S,5S)-N-[cyano(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 238)
Figure imgf000322_0001
Step 1: 4,5,6,7-tetrahydropyrazolo[l ,5-a]pyridine-3-carbaldehyde
[000602] To a mixture of pyrazolo[l,5-a]pyridine-3-carbaldehyde (1 g, 6.84 mmol, 1 eq) in EtOH (20 mL) was added Pd/C (10 mg, 10% purity) at 25 °C. The mixture was stirred at 60 °C for 16 h under H2 (15 PSI). Upon completion, the reaction mixture filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether/ethyl acetate = 50/1-0/1) to give a 4,5,6,7-tetrahydropyrazolo[l,5-a]pyridine-3-carbaldehyde (320 mg, 532.70 umol, 8.00% yield, 25% purity) as a colorless oil. MS (ESI) m/z 151.2 [M+H]+.
Step 2: 2-amino-2-(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[000603] To a mixture of 4,5,6,7-tetrahydropyrazolo[l,5-a]pyridine-3-carbaldehyde (320 mg, 2.13 mmol, 1 eq) in NHa/MeOH (4 mL) was added NH4CI (341.94 mg, 6.39 mmol, 3 eq) in one portion at 20 °C. The mixture was stirred at 20 °C for 30 min, and then TMSCN (634.17 mg, 6.39 mmol, 799.71 uL, 3 eq) at 20 °C was added and the mixture was stirred for 16 h. Upon completion, the residue was poured into water (5 mL). The aqueous phase was extracted with DCM (5 mL * 3). The combined organic phase was washed with brine (5 mL * 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give a 2-amino-2-(4, 5,6,7-
321
SUBSTITUTE SHEET RULE 26 tetrahydropyrazolo[l,5-a]pyridin-3-yl)acetonitrile (300 mg, crude) as a white solid. MS (ESI) m/z 177.2 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000604] To a mixture of 2-amino-2-(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3- yl)acetonitrile (200 mg, 567.48 umol, 50% purity, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (206.77 mg, 567.48 umol, 1 eq) in DCM (2 mL) was added with HATU (323.66 mg, 851.22 umol, 1.5 eq) and DIEA (146.69 mg, 1.13 mmol, 197.69 uL, 2 eq) in one portion at 25 °C. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition water 5 mL, and then extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (column: Waters Xbridge BEH C18 100 * 30 mm * 10 um; mobile phase: [water (NH4HCO3)- ACN] ; B%: 30%-60%, 10 min) to give a (lR,2S,5S)-N-[cyano(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (150 mg, 279.01 umol, 49.17% yield, 97.2% purity) as a white solid. MS (ESI) m/z 523.3 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000605] (lR,2S,5S)-N-[cyano(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (150 mg, 287.05 umol, 1 eq) was separated by SFC. The product was separated by SFC(column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 5%-25%, 17 min) to give (lR,2S,5S)-N-[cyano(4,5,6,7- tetrahydropyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-
322
SUBSTITUTE SHEET RULE 26 trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (42.14 mg, 77.50 umol, 27.00% yield, 96.1% purity) as white solid. MS (ESI) m/z 523.3 [M+H]+
[000606] 'H NMR (400 MHz, DMSO-de) 5 = 9.40 - 9.19 (m, 1H), 9.15 (d, J = 7.5 Hz, 1H), 7.58 - 7.46 (m, 1H), 6.01 (d, J = 7.5 Hz, 1H), 4.48 - 4.36 (m, 1H), 4.24 (s, 1H), 4.09 - 3.99 (m, 2H), 3.90 (dd, J = 5.4, 10.2 Hz, 1H), 3.72 (br d, J = 10.4 Hz, 1H), 2.76 - 2.65 (m, 2H), 2.01 - 1.86 (m, 2H), 1.75 (quin, J = 5.9 Hz, 2H), 1.59 - 1.54 (m, 1H), 1.33 - 1.28 (m, 1H), 1.06 - 0.93 (m, 12H), 0.88 - 0.82 (m, 3H).
[000607] (lR,2S,5S)-N-[cyano(4,5,6,7-tetrahydropyrazolo[l,5-a]pyridin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (56.03 mg, 105.94 umol, 36.91% yield, 98.8% purity) was obtained as white solid. MS (ESI) m/z 523.2 [M+H]+
[000608] 'H NMR (400 MHz, DMSO-de) 5 = 9.37 (br s, 1H), 9.07 (d, J = 7.4 Hz, 1H), 7.46 (s, 1H), 5.93 - 5.87 (m, 1H), 4.39 (br d, J = 5.0 Hz, 1H), 4.25 (s, 1H), 4.10 - 4.00 (m, 2H), 3.90 (dd, J = 5.4, 10.3 Hz, 1H), 3.68 (d, J = 10.5 Hz, 1H), 2.86 - 2.75 (m, 1H), 2.73 - 2.61 (m, 1H), 2.00 - 1.87 (m, 2H), 1.84 - 1.73 (m, 2H), 1.53 (dd, J = 5.4, 7.5 Hz, 1H), 1.22 (d, J = 7.6 Hz, 1H), 0.99 (s, 12H), 0.83 (s, 3H).
Example 74: Synthesis of (lR,2S,5S)-N-((5-bromoisoquinolin-4-yl)(cyano)methyl)-3-((S)- 3,3-dimethyl-2-(thiazol-2-ylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 240)
Figure imgf000324_0001
Step 1 : 2-amino-2-(5-bromoisoquinolin-4-yl)acetonitrile
[000609] To a solution of 5-bromoisoquinoline-4-carbaldehyde (300 mg, 1.27 mmol, 1 eq) in NHa/MeOH (10 mL) was added NH4CI (203.94 mg, 3.81 mmol, 3 eq), and the resulting mixture was stirred at 25 °C for 1 h. TMSCN (378.23 mg, 3.81 mmol, 476.96 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was
323
SUBSTITUTE SHEET RULE 26 concentrated under reduced pressure to remove NH MeOH. The reaction mixture was quenched by addition H2O (30 mL), and then extracted with ethyl acetate (30 mL * 5). The combined organic layers were washed with brine (30 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE: EA = 0: 1) to give 2-amino-2-(5-bromoisoquinolin-4-yl)acetonitrile (210 mg, 801.21 umol, 63.05% yield) as yellow oil.
Step 2 : (lR,2S,5S)-N-((5-bromoisoquinolin-4-yl)(cyano)methyl)-3-((S)-3,3-dimethyl-2-(thiazol-
2-ylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000610] To a solution of 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (100 mg, 381.53 umol, 1.2 eq) in ACN (3 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-(thiazol-2- ylamino)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (113.95 mg, 317.94 umol, 1 eq, Li) and [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (178.41 mg, 635.88 umol, 2 eq). Then, 1 -methylimidazole (78.31 mg, 953.82 umol, 76.03 uL, 3 eq) was added, and the mixture was stirred for 1 h at 25 °C. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with aqueous NaCl (10 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80 * 40mm * 3um; mobile phase: [water( NH4HCO3)-ACN]; B%: 40%-70%, 8 min) to give (lR,2S,5S)-N-[(5-bromo-4- isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-(thiazol-2-ylamino)butanoyl]-6,6-dimethyl-
3-azabicyclo[3.1.0]hexane-2-carboxamide (26.30 mg, 42.49 umol, 13.36% yield, 96.21% purity) as a gray solid. MS (ESI) m/z 597.1 [M+H]+.
[000611] 'H NMR (400 MHz, DMSO-t76) 5 = 9.66 - 9.58 (m, 1H), 9.49 (d, J = 8.6 Hz, 1H), 9.44 - 9.38 (m, 1H), 9.72 - 9.31 (m, 1H), 8.90 (d, J = 8.3 Hz, 1H), 8.39 - 8.19 (m, 2H), 7.73 - 7.64 (m, 1H), 7.57 (dd, J = 4.9, 8.8 Hz, 1H), 7.45 (br dd, J = 6.5, 13.4 Hz, 1H), 6.98 - 6.79 (m, 1H), 6.66 - 6.46 (m, 1H), 4.23 (s, 1H), 4.24 - 4.20 (m, 1H), 4.13 (br dd, J = 3.1, 10.1 Hz, 1H), 3.95 - 3.78 (m, 1H), 1.68 - 1.49 (m, 1H), 1.38 - 1.24 (m, 1H), 1.03 - 1.00 (m, 4H), 0.97 (s, 4H), 1.07 - 0.92 (m, 1H), 0.89 - 0.85 (m, 1H), 0.84 (d, J = 13.0 Hz, 2H).
324
SUBSTITUTE SHEET RULE 26 Example 75: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2- [(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 242)
Figure imgf000326_0001
Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-
6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate
[000612] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (0.5 g, 1.54 mmol, 1 eq), 2-bromo-5-chloro- pyrazine (357.70 mg, 1.85 mmol, 1.2 eq) in dioxane (10 mL) was added t-BuONa (296.20 mg, 3.08 mmol, 2 eq) and [2-(2-aminophenyl)phenyl] -methylsulfonyloxy-palladium; ditert-butyl- [2- (2,4,6-triisopropylphenyl)phenyl]phosphane (122.42 mg, 154.10 umol, 0.1 eq) under N2. The mixture was stirred at 110 °C for 16 h. Four batches were set up in parallel. Upon completion, the reaction mixture was poured into H2O (120 mL), and then extracted with EtOAc (120 mL * 3). The combined organic layers were washed with brine (120 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 10:1 to 5: 1) to give tert-butyl (1R,2S,5S)- 3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (300 mg, 686.52 umol, 11.14% yield) as a yellow oil. MS (ESI) m/z 437.3 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
325
SUBSTITUTE SHEET RULE 26 [000613] A mixture of tert-butyl (lR,2S,5S)-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3- dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (430 mg, 984.04 umol, 1 eq) in HCl/dioxane (4 M, 30 mL, 121.95 eq) was stirred at 40 °C for 2 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (366 mg, crude) as a yellow solid.
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(5-chloropyrazin-2- yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000614] A mixture of (lR,2S,5S)-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (366 mg, 960.96 umol, 1 eq), 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (209.16 mg, 960.96 umol, 1 eq) and 1- methylimidazole (236.70 mg, 2.88 mmol, 229.80 uL, 3 eq) in ACN (5 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (539.25 mg, 1.92 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC(column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 65%-85%, 8 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)- cyano-methyl]-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (290 mg, 499.56 umol, 51.99% yield, 100% purity) as a yellow solid. MS (ESI) m/z 580.3 [M+H]+.
[000615] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.63 - 9.38 (m, 2H), 8.92 - 8.83 (m, 1H), 8.32 - 8.22 (m, 1H), 8.08 - 7.97 (m, 2H), 7.91 (d, J= 1.1 Hz, 1H), 7.76 (dt, J= 4.6, 7.8 Hz, 1H), 7.31 (d, J= 8.5 Hz, 2H), 4.38 - 4.29 (m, 1H), 4.28 - 4.21 (m, 1H), 4.02 - 3.86 (m, 2H), 1.64 - 1.54 (m, 1H), 1.37 - 1.22 (m, 1H), 1.06 - 0.96 (m, 12H), 0.87 - 0.78 (m, 3H).
Step 4: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(5-chloropyrazin-2- yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide [000616] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(5- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
326
SUBSTITUTE SHEET RULE 26 carboxamide (239 mg) was separated by SFC (column: ChiralPak IH, 250 * 30mm, lOum; mobile phase: [Neu-MeOH]; B%: 30%-30%, 7 min) to give (lR,2S,5S)-N-[(5-chloro-4- isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(5-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (72.29 mg, 124.53 umol, 30.25% yield, 100% purity) as a white solid. MS (ESI) m/z 580.3 [M+H]+.
[000617] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.58 (d, J= 6.5 Hz, IH), 9.49 (s, IH), 8.89 (s, IH), 8.34 - 8.22 (m, IH), 8.06 - 7.97 (m, 2H), 7.92 (d, J= 1.1 Hz, IH), 7.76 (t, J= 7.9 Hz, IH), 7.31 (d, J= 8.5 Hz, 2H), 4.32 (d, J= 8.6 Hz, IH), 4.27 (s, IH), 4.02 - 3.90 (m, 2H), 1.66 - 1.57 (m, IH), 1.35 (d, J= 7.6 Hz, IH), 1.07 - 0.98 (m, 12H), 0.85 (s, 3H).
[000618] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(5- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (67.79 mg, 116.78 umol, 28.36% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 580.3 [M+H]+.
[000619] XH NMR (400 MHz, DMSO-t/e) 5 = 9.50 (s, IH), 9.40 (d, J= 7.1 Hz, IH), 8.87 (s, IH), 8.34 - 8.22 (m, IH), 8.10 - 7.98 (m, 2H), 7.91 (d, J= 1.1 Hz, IH), 7.77 (t, J = 7.9 Hz, IH), 7.31 (d, J= 8.5 Hz, 2H), 4.32 (d, J= 8.6 Hz, IH), 4.22 (s, IH), 4.04 - 3.85 (m, 2H), 1.62 - 1.52 (m, IH), 1.24 (d, J= 7.6 Hz, IH), 1.08 - 0.95 (m, 12H), 0.81 (s, 3H).
Example 76: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2- [(6-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 243)
Figure imgf000328_0001
327
SUBSTITUTE SHEET RULE 26 Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoylJ- 6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate
[000620] To a solution of 2,6-dichloropyrazine (275.50 mg, 1.85 mmol, 2 eq), tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylate (300 mg, 924.62 umol, 1 eq) in dioxane (10 mL) was added Pd(OAc)2 (20.76 mg, 92.46 umol, 0.1 eq), BINAP (57.57 mg, 92.46 umol, 0.1 eq), and Cs2CO3 (753.15 mg, 2.31 mmol, 2.5 eq). The mixture was stirred at 110 °C for 6 h. Upon comptetion, the reaction mixture was poured into H2O (30 mL) at 20 °C, and then extracted with EtOAc (40 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 99:1 to 1:1) to give tert-butyl (lR,2S,5S)-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (285 mg, 586.99 umol, 63.48% yield, 90% purity) as a yellow solid. MS (ESI) m/z 437.3 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000621] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3- dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (285 mg, 652.21 umol, 1 eq) in HCl/dioxane (4 M, 5 mL, 30.66 eq). The mixture was stirred at 50 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent to give (lR,2S,5S)-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (248 mg, crude) as a yellow solid.
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(6-chloropyrazin-2- yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000622] To a solution of (lR,2S,5S)-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (248 mg, 651.14 umol, 1 eq), 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (212.59 mg, 976.72 umol, 1.5 eq), 1- methylimidazole (160.38 mg, 1.95 mmol, 155.71 uL, 3 eq) in ACN (6 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (365.39 mg, 1.30
328
SUBSTITUTE SHEET RULE 26 mmol, 2 eq). The mixture was stirred at 20 °C for 2 h. Upon comptetion, the reaction mixture was poured into H2O (35 mL) at 20 °C, and then extracted with EtOAc (35 mL *3). The combined organic layers were washed with brine (35 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C 18 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%-75%, 8 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)- cyano-methyl]-3-[(2S)-2-[(6-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (110 mg, 189.49 umol, 29.10% yield, 100% purity) as a white solid. MS (ESI) m/z 580.1 [M+H]+.
Step 4: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(6-chloropyrazin-2- yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000623] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(6- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (110 mg, 189.49 umol, 1 eq) was separated by (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu-MeOH]; B%: 25%-25%, 15 min) to give (lR,2S,5S)-N-[(5- chloro-4-isoquinolyl)-cyano-methy 1] -3 - [(2S)-2- [(6-chloropyrazin-2-yl)amino] -3 ,3 -dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (63 mg, 107.22 umol, 56.59% yield, 98.8% purity) as a white solid. MS (ESI) m/z 580.1 [M+H]+.
[000624] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.59 (d, J= 5.8 Hz, IH), 9.49 (s, IH), 8.89 (s, IH), 8.27 (dd, J= 0.8, 8.1 Hz, IH), 8.11 (s, IH), 8.01 (d, J= 6.9 Hz, IH), 7.75 (t, .7 = 7,9 Hz, IH), 7.70 (s, IH), 7.60 (d, J= 9.4 Hz, IH), 7.30 (s, IH), 4.42 (d, J= 9.4 Hz, IH), 4.28 (s, IH), 4.09 - 4.03 (m, IH), 4.02 - 3.95 (m, IH), 1.63 (dd, J= 5.4, 7.4 Hz, IH), 1.36 (d, J= 7.6 Hz, IH), 1.06 - 0.98 (m, 12H), 0.80 (s, 3H).
[000625] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(6- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (24.6 mg, 41.36 umol, 21.83% yield, 97.6% purity) was obtained as a white solid. MS (ESI) m/z 580.1 [M+H]+.
[000626] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.50 (d, J= 6.1 Hz, IH), 9.42 (d, J= 7.3 Hz, IH), 8.88 (d, J= 4.3 Hz, IH), 8.32 - 8.24 (m, IH), 8.12 (d, J= 7.6 Hz, IH), 8.03 (dd, J= 1.0,
329
SUBSTITUTE SHEET RULE 26 7.6, 15.8 Hz, 1H), 7.76 (dt, J = 3.6, 7.9 Hz, 1H), 7.70 (s, 1H), 7.60 (dd, J= 4.1, 9.3 Hz, 1H), 7.30 (s, 1H), 4.42 (d, J = 9.3 Hz, 1H), 4.26 (d, J = 17.3 Hz, 1H), 4.09 - 4.02 (m, 1H), 4.02 - 3.94 (m, 1H), 1.61 (dd, J = 5.3, 7.3, 12.9 Hz, 1H), 1.37 - 1.24 (m, 1H), 1.05 - 0.95 (m, 12H), 0.78 (d, J = 11.2 Hz, 3H).
Example 77: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2- [(3-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 244)
Figure imgf000331_0001
Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-
6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate
[000627] To a mixture of tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (2 g, 5.54 mmol, 1 eq, HC1) and 2,3- di chloropyrazine (908.11 mg, 6.10 mmol, 1.1 eq) in dioxane (20 mL) was added BrettPhos Pd G3 (502.33 mg, 554.14 umol, 0.1 eq) and NaOtBu (2 M, 5.54 mL, 2 eq) in one portion at 25 °C under N2. The mixture was stirred at 110 °C for 48 h. Upon completion, the reaction mixture was quenched by addition water 20 mL, and then extracted with Ethyl acetate (20mL * 3). The combined organic layers were washed with brine (20 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 200 * 40 mm * 10 um; mobile phase: [water(FA)-ACN]; B%: 40%-95%, 8 min) to give tert-butyl (lR,2S,5S)-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]-3,3-
330
SUBSTITUTE SHEET RULE 26 dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (200 mg, 457.69 umol, 8.26% yield) as a red oil. MS (ESI) m/z 437.3 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000628] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]-3,3- dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (120 mg, 274.62 umol, 1 eq) was added DCM (1 mL) and TFA (1 mL) in one portion. The mixture was stirred at 40 °C for 2 h. Upon completion, the reaction was concentrated to give (lR,2S,5S)-3-[(2S)-2-[(3- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (120 mg, crude) as a red oil. MS (ESI) m/z 381.3 [M+H]+.
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(3-chloropyrazin-2- yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000629] To a mixture of (lR,2S,5S)-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (104 mg, 273.06 umol, 1 eq) and 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (71.32 mg, 327.67 umol, 1.2 eq) in ACN (1 mL) was added TCFH (153.23 mg, 546.12 umol, 2 eq) and 1 -methylimidazole (78.47 mg, 955.71 umol, 76.18 uL, 3.5 eq) in one portion at 25°C. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition water (5 mL), and then extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (Petroleum ether : ethyl acetate=0: 1, Rf=0.58) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]- 3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (150 mg, 220.93 umol, 80.91% yield, 85.5% purity) as a white oil. MS (ESI) m/z 580.3 [M+H]+.
Step 4: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(3-chloropyrazin-2- yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 & Isomer 2
331
SUBSTITUTE SHEET RULE 26 [000630] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(3- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (150 mg, 258.39 umol, 1 eq ) was separated by SFC (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu-MeOH]; B%: 25%-25%, 5 min) to give (lR,2S,5S)-N-[(5- chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(3-chloropyrazin-2-yl)amino]-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (60 mg, 103.15 umol, 39.92% yield, 99.8% purity) as white solid. MS (ESI) m/z 580.3 [M+H]+
[000631] 'H NMR (400 MHz, DMSO-de) 5 = 9.61 (br d, J = 5.2 Hz, IH), 9.49 (s, IH), 8.87 (s, IH), 8.27 (d, J = 8.0 Hz, IH), 8.08 - 7.97 (m, 2H), 7.76 (t, J = 7.7 Hz, IH), 7.68 (d, J = 2.6 Hz, IH), 7.37 - 7.23 (m, IH), 5.64 (d, J = 8.9 Hz, IH), 4.50 (d, J = 9.1 Hz, IH), 4.27 (s, IH), 4.03 - 3.86 (m, 2H), 1.67 - 1.57 (m, IH), 1.37 (d, J = 7.6 Hz, IH), 1.04 (s, 12H), 0.79 (s, 3H).
[000632] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-2-[(3- chloropyrazin-2-yl)amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (70 mg, 120.58 umol, 46.67% yield, 100.0% purity) as white solid. MS (ESI) m/z 580.3 [M+H]+
[000633] 'H NMR (400 MHz, DMSO-de) 5 = 9.51 (s, IH), 9.43 (br d, J = 7.0 Hz, IH), 8.86 (s, IH), 8.29 (d, J = 8.0 Hz, IH), 8.06 (d, J = 7.5 Hz, IH), 8.01 (d, J = 2.6 Hz, IH), 7.77 (t, J = 7.8 Hz, IH), 7.69 (d, J = 2.7 Hz, IH), 7.29 (br d, J = 6.1 Hz, IH), 5.66 (d, J = 9.1 Hz, IH), 4.51 (d, J = 9.2 Hz, IH), 4.21 (s, IH), 4.01 - 3.90 (m, 2H), 1.64 - 1.56 (m, IH), 1.29 - 1.21 (m, IH), 1.06 (s, 8H), 0.99 - 0.93 (m, 3H), 0.77 (s, 3H).
Example 78: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[[6-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo [3.1.0] hexane-2-carboxamide
332
SUBSTITUTE SHEET RULE 26
Figure imgf000334_0001
Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000634] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (500 mg, 1.54 mmol, 1 eq) in dioxane (10 mL) was added 2-chloro-6-(trifluoromethyl)pyrazine (309.42 mg, 1.70 mmol, 1.1 eq) and CS2CO3 (1.00 g, 3.08 mmol, 2 eq). Xantphos Pd G4 (148.31 mg, 154.10 umol, 0.1 eq) was added, and the mixture was stirred at 110 °C for 16 h. Upon completion, the reaction mixture was diluted with water (3 mL) and filtered, the mother liquer was extracted with ethyl acetate (2 mL * 3). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 10:1 to 3: 1) to give tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (220 mg, 467.56 umol, 30.34% yield) as a white solid. MS (ESI) m/z 471.3 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid
[000635] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-
(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
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SUBSTITUTE SHEET RULE 26 carboxylate (210 mg, 446.31 umol, 1 eq) in DCM (2.1 mL) was added TFA (3.23 g, 28.36 mmol, 2.1 mL, 63.55 eq). The mixture was stirred at 40 °C for 2 h. Upon completion, the reaction mixture was was blow-dried with N2 to give (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (180 mg, crude) as a yellow oil. MS (ESI) m/z 415.2 [M+H]+
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000636] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (180.00 mg, 434.34 umol, 1 eq) in ACN (6 mL) was added 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (94.54 mg, 434.34 umol, 1 eq) and 1 -methylimidazole (106.98 mg, 1.30 mmol, 103.87 uL, 3 eq). Then, [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (243.73 mg, 868.68 umol, 2 eq) was added at 25 °C and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water(HCl)-ACN]; B%: 50%-80%, 8 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)- cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 162.85 umol, 37.49% yield) as a white solid. MS (ESI) m/z 614.3 [M+H]+.
Step 4: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6 (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000637] The (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [[6-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide was separated by SEC (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu-MeOH]; B%: 25%-25%, 6 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[[6-(trifhioromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (27 mg, 43.97 umol, 10.12% yield) as a
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SUBSTITUTE SHEET RULE 26 white solid, which chiral purity is not good then further separated again by SFC (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu-MeOH]; B%: 25%-25%, 10 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (17.8 mg, 28.99 umol, 6.67% yield) as a white solid. MS (ESI) m/z 614.3 [M+H]+.
[000638] 'H NMR (400 MHz, DMSO-de) 5 = 9.64 - 9.38 (m, 2H), 8.88 (d, J = 3.9 Hz, IH), 8.44 (d, J= 8.0 Hz, IH), 8.28 (ddd, J= 1.0, 5.3, 8.1 Hz, IH), 8.11 - 7.98 (m, 2H), 7.88 - 7.73 (m, 2H), 7.30 (br s, IH), 4.65 (d, J= 9.8 Hz, IH), 4.26 (d, J= 16.8 Hz, IH), 4.04 - 3.93 (m, 2H), 1.65 - 1.53 (m, IH), 1.37 - 1.24 (m, IH), 1.07 - 0.91 (m, 12H), 0.64 (d, J= 11.9 Hz, 3H).
[000639] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (24 mg, 39.08 umol, 9.00% yield) was obtained as a white solid. MS (ESI) m/z 614.3 [M+H]+.
[000640] 'H NMR (400 MHz, DMSO-de) 5 = 9.51 (s, IH), 9.43 (br d, J= Hz, IH), 8.88 (s, IH), 8.45 (s, IH), 8.29 (d, J= 8.0 Hz, IH), 8.11 - 8.02 (m, 2H), 7.87 - 7.73 (m, 2H), 7.35 - 7.27 (m, IH), 4.65 (d, J= 9.8 Hz, IH), 4.24 (s, IH), 4.02 - 3.93 (m, 2H), 1.60 - 1.55 (m, IH), 1.26 (br d, J= 7.6 Hz, IH), 1.02 (s, 9H), 0.93 (s, 3H), 0.62 (s, 3H).
Example 79: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[[3-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 246)
335
SUBSTITUTE SHEET RULE 26
Figure imgf000337_0001
Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000641] To a solution of 2-chloro-3-(trifluoromethyl)pyrazine (300 mg, 1.64 mmol, 1 eq) and tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (533.26 mg, 1.64 mmol, 1 eq) in DMSO (12 mL) was added DIEA (637.25 mg, 4.93 mmol, 858.83 uL, 3 eq). The mixture was stirred at 120 °C for 16 h. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and then extracted with Ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate=99: l to 94:6) to give tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (300 mg, 561.07 umol, 34.14% yield, 88% purity) as a yellow oil.
Step 2: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)pyrazin-2-yl]amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid
[000642] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylate (300 mg, 637.58 umol, 1 eq) in TEA (11.55 g, 101.30 mmol, 7.5 mL, 158.87 eq) and DCM (7.5 mL). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture
336
SUBSTITUTE SHEET RULE 26 was concentrated by N2 to remove solvent. The residue was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL * 2). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, concentrated by N2 to give (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (260 mg, crude) as a yellow oil.
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000643] To a solution of 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (136.55 mg, 627.38 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)pyrazin-2- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (260 mg, 627.38 umol, 1 eq) in ACN (10 mL) was added [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (211.24 mg, 752.86 umol, 1.2 eq) and 1 -methylimidazole (206.03 mg, 2.51 mmol, 200.03 uL, 4 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by H2O (10 mL), and then extracted with ethyl acetate (20 mL * 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by by prep-HPLC (column: Welch Xtimate Cl 8 250 * 70 mm # 10 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 50%-80%, 20 min), then further separated by SFC (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu-IPA]; B%: 20%-20%, 7.5 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (104 mg, 168.99 umol, 26.94% yield, 99.78% purity) as a white solid. MS (ESI) m/z 614.1 [M+H]+
[000644] 'H NMR (400 MHz, DMSO-de) 5 = 9.64 (br d, J = 5.7 Hz, IH), 9.49 (s, IH), 8.87 (s, IH), 8.36 (d, J = 2.2 Hz, IH), 8.29 - 8.26 (m, IH), 8.07 - 7.99 (m, 2H), 7.76 (t, J = 7.8 Hz, IH), 7.36 - 7.26 (m, IH), 5.50 (br d, J = 8.8 Hz, IH), 4.62 (d, J = 8.8 Hz, IH), 4.30 (s, IH), 3.97 (d, J = 2.6 Hz, 2H), 1.67 - 1.60 (m, IH), 1.38 (d, J = 7.6 Hz, IH), 1.17 (d, J = 6.5 Hz, IH), 1.06 - 0.92 (m, 13H), 0.80 (s, 3H)
337
SUBSTITUTE SHEET RULE 26 [000645] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (106 mg, 171.95 umol, 27.41% yield, 99.61% purity) was obtained as a light yellow solid. MS (ESI) m/z 614.1 [M+H]+
[000646] 'H NMR (400 MHz, DMSO-de) 5 = 9.52 (s, 1H), 9.47 (br d, J = 7.1 Hz, 1H), 8.87 (s, 1H), 8.36 (d, J = 2.1 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.36 - 7.26 (m, 1H), 5.54 (br d, J = 8.6 Hz, 1H), 4.64 (d, J = 8.7 Hz, 1H), 4.23 (s, 1H), 4.04 - 3.90 (m, 2H), 1.65 - 1.58 (m, 1H), 1.28 (d, J = 7.6 Hz, 1H), 1.18 (d, J = 6.5 Hz, 1H), 1.06 (s, 9H), 1.00 - 0.95 (m, 3H), 0.78 (s, 3H).
Example 80: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2- [ [6-(trifhioromethyl)pyridazin-3-yl] amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 247)
Figure imgf000339_0001
Step 1: tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyridazin-3- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000647] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (600 mg, 1.85 mmol, 1 eq) in dioxane (12 mL) was added 3-chloro-6-(trifluoromethyl)pyridazine (371.30 mg, 2.03 mmol, 1.1 eq) and NaOtBu (355.44 mg, 3.70 mmol, 2 eq). tBuXPhos Pd-G3 (146.90 mg, 184.92 umol, 0.1 eq) was added, and then the mixture was stirred at 110 °C for 16 h. Upon completion, the reaction mixture was diluted with water (15 mL) and filtered. The mother liquer was extracted with ethyl
338
SUBSTITUTE SHEET RULE 26 acetate (12 mL * 3). The combined organic layers were washed with brine (12 mL), dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, [etroleum ether: ethyl acetate = 20:1 to 5: 1) to give tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyridazin-3- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (280 mg, 595.08 umol, 32.18% yield) as a white solid. MS (ESI) m/z 471.3 [M+H]+
Step 2: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyridazin-3-yl]amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid
[000648] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylate (210.00 mg, 446.31 umol, 1 eq) in DCM (2.1 mL) was added TFA (3.23 g, 28.36 mmol, 2.1 mL, 63.55 eq). The mixture was stirred at 40 °C for 2 h. Upon completion, the reaction mixture was was blow-dried with N2 to give (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyridazin-3-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (237 mg, crude) as a yellow oil. MS (ESI) m/z 415.2 [M+H]+
Step 3: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyrazin-2-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000649] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[6-(trifluoromethyl)pyridazin- 3-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (237 mg, 571.88 umol, 1 eq) in ACN (4 mL) was added 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (124.47 mg, 571.88 umol, 1 eq) and 1 -methylimidazole (140.86 mg, 1.72 mmol, 136.76 uL, 3 eq). Then, [chloro(dimethylamino)methylene] -dimethyl-ammonium;hexafluorophosphate (320.92 mg, 1.14 mmol, 2 eq) was added and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30 mm * 10 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 35%-65%, 8 min) to give (lR,2S,5S)-N-[(5-chloro-4- isoquinolyl)-cyano-methyl] -3 - [(2S)-3 ,3 -dimethyl-2- [ [6-(trifluoromethyl)pyridazin-3 -
339
SUBSTITUTE SHEET RULE 26 yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (150 mg, 244.28 umol, 42.71% yield) as a white solid. MS (ESI) m/z 614.3 [M+H]+
Step 4: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyridazin-3-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000650] The (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [[6-(trifluoromethyl)pyridazin-3-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide was separated by SFC (column: DAICEL CHIRALPAK AD (250mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 50%-50%, 13.5 min) to give (lR,2S,5S)-N-[(5-chloro-4- isoquinolyl)-cyano-methyl] -3 - [(2S)-3 , 3 -dimethyl-2- [ [6-(trifluoromethyl)pyridazin-3 - yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (30.68 mg, 49.96 umol, 8.74% yield) as a white solid. MS (ESI) m/z 614.3 [M+H]+.
[000651] 'H NMR (400 MHz, DMSO-de) 5 = 9.50 (s, 1H), 9.40 (br d, J= 7.2 Hz, 1H), 8.87 (s, 1H), 8.29 (d, J= 8.0 Hz, 1H), 8.06 (d, J= 7.5 Hz, 1H), 7.77 (t, J= 7.8 Hz, 1H), 7.64 (d, J = 9.4 Hz, 1H), 7.51 (d, J= 8.6 Hz, 1H), 7.37 - 7.20 (m, 2H), 4.72 (d, J= 8.6 Hz, 1H), 4.27 - 4.12 (m, 2H), 3.96 (dd, J= 5.5, 10.0 Hz, 1H), 1.62 - 1.54 (m, 1H), 1.24 (d, J= 7.6 Hz, 1H), 1.05 (s, 9H), 0.96 (s, 3H), 0.75 (s, 3H).
[000652] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[6- (trifluoromethyl)pyridazin-3-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (45.68 mg, 74.39 umol, 13.01% yield) was obtained as a white solid. MS (ESI) m/z 614.3 [M+H]+.
[000653] 'H NMR (400 MHz, DMSO-de) 5 = 9.57 (br d, J= 5.2 Hz, 1H), 9.49 (s, 1H), 8.88 (s, 1H), 8.27 (d, J= 8.0 Hz, 1H), 8.01 (d, J= 7.5 Hz, 1H), 7.75 (t, J= 7.9 Hz, 1H), 7.64 (d, J = 9.4 Hz, 1H), 7.51 (br d, .7= 8.6 Hz, 1H), 7.34 - 7.18 (m, 2H), 4.72 (d, J= 8.6 Hz, 1H), 4.28 (s, 1H), 4.15 (br d, J= 10.3 Hz, 1H), 3.96 (br dd, J = 5.5, 9.9 Hz, 1H), 1.64 - 1.58 (m, 1H), 1.34 (d, J = 7.5 Hz, 1H), 1.08 - 0.98 (m, 12H), 0.78 (s, 3H).
Example 81: Synthesis of (lR,2S,5S)-N-[(5-chloroimidazo[l,2-a]pyridin-3-yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 248)
340
SUBSTITUTE SHEET RULE 26
Figure imgf000342_0001
Step 1: 5-chloroimidazo[l,2-a]pyridine-3-carbaldehyde
[000654] 5-Chloroimidazo[l,2-a]pyridine (4 g, 26.22 mmol, 1 eq) and HMTA (7.35 g, 52.43 mmol, 2 eq) in AcOH (40 mL) was stirred at 85 °C for 16 hr. Upon completion, the mixture was cooled to room temperature, water (lOOmL) and ethyl acetate (lOOmL) was added, and the mixture was adjusted to pH 9 with 2 M NaOH solution. The aqueous layer was separated, and the aqueous layer was extracted with ethyl acetate (100mL*3), and the combined organic layers was washed with brine (200mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography with (SiCh, petroleum ether/ethyl acetate = 2:3) and concentrated to give 5-chloroimidazo[l,2-a]pyridine-3-carbaldehyde (1.0 g, 4.43 mmol, 16.90% yield, 80% purity) as a light yellow solid. MS (ESI) m/z 180.9 [M+H]+.
[000655] 'H NMR (400 MHz, CHLOROFORM-d) 5 = 10.75 (s, 1H), 8.54 (s, 1H), 7.79 (dd, J = 0.9, 8.9 Hz, 1H), 7.44 (dd, J = 7.5, 8.8 Hz, 1H), 7.21 (dd, J = 0.7, 7.5 Hz, 1H).
Step 2: 2-amino-2-(5-chloroimidazo[l,2-a]pyridin-3-yl)acetonitrile
[000656] To a mixture of 5-chloroimidazo[l,2-a]pyridine-3-carbaldehyde(500 mg, 2.77 mmol, 1 eq) in DCM (20 mL) was added Ti(i-PrO)4 (1.18 g, 4.15 mmol, 1.23 mL, 1.5 eq) and NH MeOH (7 M, 1.54 mL, 3.89 eq), the mixture was stirred at 20 °C for 1 h, TMSCN (824.02 mg, 8.31 mmol, 1.04 mL, 3 eq) was added, and the mixture was stirred at 20 °C for 15 h. Upon completion, the mixture was quenched with water (100 mL) and ethyl acetate (100 mL), filtered
341
SUBSTITUTE SHEET RULE 26 with a silica gel, the organic phase was separated, washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(5- chloroimidazo[l,2-a]pyridin-3-yl)acetonitrile (500 mg, crude) as brown oil which was used for next step without further purification. MS (ESI) m/z 207.0 [M+H]+.
Step 3: (lR,2S,5S)-N-[(5-chloroimidazo[l,2-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide & (lR,2S,5S)-N-[(5-chloroimidazo[l,2-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000657] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (600 mg, 1.65 mmol, 1 eq), 2-amino-2-(5-chloroimidazo[l,2-a]pyridin-3-yl)acetonitrile (408.32 mg, 1.98 mmol, 1.2 eq), and TCFH (924.08 mg, 3.29 mmol, 2 eq) in ACN (25 mL) was added 1- methylimidazole (405.61 mg, 4.94 mmol, 393.79 uL, 3 eq), the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (50 mL) at 20 °C, and then diluted with ethyl acetate (50 mL) and extracted with ethyl acetate (30 mL*2). The combined organic layers were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was further purified by prep- HPLC (column: Welch Xtimate Cl 8 250 * 70mm # lOum; mobile phase: [water( NH4HCO3)- ACN]; B%: 36%-66%, 20 min) to give (lR,2S,5S)-N-[(5-chloroimidazo[l,2-a]pyridin-3-yl)- cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (450 mg, 786.92 umol, 47.79% yield, 96.7% purity) as white solid, then the product was further purified by SFC (column: DAICEL CHIRALPAK IG (250mm * 30mm, lOum); mobile phase: [Neu-MeOH]; B%: 15%-l 5%, 11 min) to give (lR,2S,5S)-N-[(5-chloroimidazo[l,2-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (100 mg, 180.84 umol, 10.98% yield, 100% purity) as a white solid. MS (ESI) m/z 553.1 [M+H]+.
[000658] 'H NMR (400 MHz, DMSO-d6) 5 = 9.59 (d, J = 6.2 Hz, 1H), 9.37 (br d, J = 4.2 Hz, 1H), 7.91 (s, 1H), 7.69 (dd, J = 0.9, 9.0 Hz, 1H), 7.36 (dd, J = 7.3, 8.9 Hz, 1H), 7.17 (dd, J = 0.6,
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SUBSTITUTE SHEET RULE 26 7.2 Hz, 1H), 6.90 (d, J = 6.1 Hz, 1H), 4.41 (br d, J = 5.0 Hz, 1H), 4.28 (s, 1H), 3.92 (dd, J = 5.4, 10.3 Hz, 1H), 3.73 (d, J = 10.4 Hz, 1H), 1.59 (dd, J = 5.4, 7.4 Hz, 1H), 1.32 (d, J = 7.6 Hz, 1H), 1.06 - 0.98 (m, 12H), 0.82 (s, 3H).
[000659] (lR,2S,5S)-N-[(5-chloroimidazo[l,2-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (90 mg, 155.35 umol, 9.43% yield, 95.45% purity) was obtained as a white solid. MS (ESI) m/z 553.1 [M+H]+.
[000660] 'H NMR (400 MHz, DMSO-d6) 5 = 9.44 (d, J = 7.1 Hz, 1H), 9.37 (br d, J = 6.5 Hz, 1H), 7.91 (s, 1H), 7.75 - 7.65 (m, 1H), 7.41 - 7.32 (m, 1H), 7.21 (d, J = 6.7 Hz, 1H), 6.89 (br d, J = 7.1 Hz, 1H), 4.38 (br d, J = 7.0 Hz, 1H), 4.24 (s, 1H), 3.92 (br dd, J = 5.4, 10.3 Hz, 1H), 3.69 (br d, J = 10.4 Hz, 1H), 1.57 (dd, J = 5.6, 7.4 Hz, 1H), 1.28 (d, J = 7.6 Hz, 1H), 1.00 - 0.95 (m, 12H), 0.82 (s, 3H).
Example 82: Synthesis of (lR,2S,5S)-N-[(4-chloropyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 249)
Figure imgf000344_0001
Step 1: (4-chloropyrazolo[l,5-a]pyridin-3-yl)methanol
[000661] A solution of methyl 4-chloropyrazolo[l,5-a]pyridine-3-carboxylate (1.3 g, 6.17 mmol, 1 eq) in THF (15 mL) was added with LiBH4 (672.17 mg, 30.86 mmol, 5 eq) at 0 °C, and then the mixture was stirred at 45 °C for 16 h. Upon completion, the reaction mixture was
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SUBSTITUTE SHEET RULE 26 quenched by H2O (20 mL) and extracted with ethyl acetate (50 mL* 2). The organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, PE:EA = 99: 1 to 0: 1) to give (4-chloropyrazolo[l,5-a]pyridin-3-yl)methanol (800 mg, 4.38 mmol, 70.98% yield) as a yellow solid. MS (ESI) m/z 183.1 [M+H]+.
Step 2: 4-chloropyrazolo[l,5-a]pyridine-3-carbaldehyde
[000662] A solution of 4-chloropyrazolo[l,5-a]pyridin-3-yl)methanol (800 mg, 3.72 mmol, 85% purity, 1 eq) in DCM (8 mL) was added MnCh (6.48 g, 74.48 mmol, 20 eq) and stirred at 45 °C for 14 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give 4-chloropyrazolo[l,5-a]pyridine-3-carbaldehyde (750 mg, crude) as a yellow solid. MS (ESI) m/z 181.0 [M+H]+.
Step 3: 2-amino-2-(4-chloropyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[000663] A solution of 4-chloropyrazolo[l,5-a]pyridine-3-carbaldehyde (700 mg, 3.88 mmol, 1 eq) in DCM (10 mL) was added Ti(i-PrO)4 (1.10 g, 3.88 mmol, 1.14 mL, 1 eq) and NEh/MeOH (7 M, 1.66 mL, 3 eq) then stirred at 20 °C for 1 h, and then added TMSCN (1.15 g, 11.63 mmol, 1.45 mL, 3 eq) and stirred at 20 °C for 15 h. Upon completion, the mixture was concentrated under reduced pressure to remove NEE/MeOH. The reaction mixture was quenched by water (10 mL), and then extracted with EA (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, concentrated in vacuum. The residue was purified by was purified by prep-TLC (SiCh, EA) to afford 2-amino-2-(4-chloropyrazolo[l,5- a]pyri din-3 -yl)acetonitrile (500 mg, 2.42 mmol, 62.43% yield) as yellow solid. MS (ESI) m/z 207.1 [M+H]+.
Step 4: (lR,2S,5S)-N-[(4-chloropyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000664] A mixture of 2-amino-2-(4-chloropyrazolo[l,5-a]pyridin-3-yl)acetonitrile (500 mg, 2.42 mmol, 1 eq) in ACN (6 mL) was added with (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
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SUBSTITUTE SHEET RULE 26 (881.66 mg, 2.42 mmol, 1 eq), [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (1.02 g, 3.63 mmol, 1.5 eq) and 1 -methylimidazole (595.99 mg, 7.26 mmol, 578.63 uL, 3 eq) and stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by water (6 mL), and then extracted with ethyl acetate (6 mL * 3). The combined organic layers were washed with brine (6 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the residue. The residue was purified by prep- HPLC(column: Agela DuraShell Cl 8 250 * 70 mm * 10 um; mobile phase: [water(NH4HCO3)- ACN]; B%: 45%-70%, 20 min) (lR,2S,5S)-N-[(4-chloropyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (800 mg, 1.43 mmol, 59.01% yield, 98.7% purity) as white solid. MS (ESI) m/z 553.3 [M+H]+.
Step 5: tert-butyl 2-[[(2S)-2-(benzyloxycarbonylamino)-3,3-dimethyl-butanoyl]amino]-3- trimethylsilyl-propanoate
[000665] (lR,2S,5S)-N-[(4-chloropyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (800 mg, 1.45 mmol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK AD(250 mm * 30 mm, 10 um); mobile phase: [Neu-ETOH]; B%: 16%-16%, 5 min) to give (lR,2S,5S)-N-[(4-chloropyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (360.12 mg, 651.24 umol, 45.02% yield, 100% purity) as a white solid. MS (ESI) m/z 553.3 [M+H]+.
[000666] 'H NMR (400 MHz, DMSO-de) 5 = 9.42 - 9.33 (m, 2H), 8.79 (d, J = 7.0 Hz, 1H), 8.29 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.51 (d, J = 6.6 Hz, 1H), 4.40 (br d, J = 7.2 Hz, 1H), 4.30 (s, 1H), 3.90 (dd, J = 5.4, 10.2 Hz, 1H), 3.72 (d, J = 10.3 Hz, 1H), 1.58 (dd, J = 5.4, 7.3 Hz, 1H), 1.33 (d, J = 7.5 Hz, 1H), 1.07 - 0.92 (m, 12H), 0.86 - 0.81 (m, 3H).
[000667] (lR,2S,5S)-N-[(4-chloropyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (305.46 mg, 551.84 umol, 38.14% yield, 99.9% purity) was obtained as a white solid. MS (ESI) m/z 553.3 [M+H]+.
345
SUBSTITUTE SHEET RULE 26 [000668] 'H NMR (400 MHz, DMSO-de) 5 = 9.87 - 9.30 (m, 2H), 8.80 (d, J = 6.8 Hz, 1H), 8.29 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.50 (d, J = 7.0 Hz, 1H), 4.39 (br d, J = 7.5 Hz, 1H), 4.26 (s, 1H), 3.91 (dd, J = 5.5, 10.3 Hz, 1H), 3.73 - 3.66 (m, 1H), 1.55 (dd, J = 5.5, 7.5 Hz, 1H), 1.26 (d, J = 7.7 Hz, 1H), 1.03 - 0.92 (m, 12H), 0.82 (s, 3H).
Example 83: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)- 3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 250)
Figure imgf000347_0001
Step 1: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide [000669] To a solution of 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (230 mg, 1.06 mmol, 1 eq) and (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (388.31 mg, 1.06 mmol, 1 eq) in ACN (10 mL) and then TCFH (594.83 mg, 2.12 mmol, 2 eq and 1 -methylimidazole (304.59 mg, 3.71 mmol, 295.72 uL, 3.5 eq was added. The resulting mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (20 mL) and then extracted with ethyl acetate (20 mL * 2). The combined organic layers were washed with 15 % citric acid (20 mL * 2), NaHCO° (20 mL) and brine (20 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1 :2 to 0: 1) which was further separated by SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 urn); mobile phase: [Neu-MeOH]; B %: 30 %- 30 %, 9 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide Isomer 1 (81.44 mg, 142.17 umol, 13.41% yield, 98.8% purity) as a white solid. MS (ESI) m/z 566.0 [M+H]+.
346
SUBSTITUTE SHEET RULE 26 [000670] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.87 (br s, 1H), 9.62 (br d, J= 5.5 Hz, 1H), 9.54 - 9.48 (m, 1H), 8.87 (s, 1H), 8.32 - 8.25 (m, 1H), 8.03 (d, J= 7.4 Hz, 1H), 7.77 (t, J= 7.8 Hz, 1H), 7.31 (br s, 1H), 4.34 (br d, J= 4.4 Hz, 1H), 4.31 - 4.22 (m, 1H), 3.96 - 3.89 (m, 1H), 3.87 - 3.81 (m, 1H), 3.68 - 3.55 (m, 1H), 3.25 - 3.18 (m, 3H), 1.63 - 1.57 (m, 1H), 1.39 (d, J = 7.5 Hz, 1H), 1.17 - 1.11 (m, 3H), 1.04 - 0.94 (m, 3H), 0.89 - 0.82 (m, 3H).
[000671] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (85.37 mg, 144.96 umol, 13.68% yield, 96.1% purity) was obtained as a white solid. MS (ESI) m/z 566.0 [M+H]+.
[000672] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.02 - 9.73 (m, 1H), 9.51 (s, 1H), 9.49 - 9.44 (m, 1H), 8.86 (s, 1H), 8.29 (dd, J= 1.0, 8.1 Hz, 1H), 8.06 (dd, J= 1.1, 7.5 Hz, 1H), 7.77 (t, J = 7.9 Hz, 1H), 7.31 (br d, J= 3.7 Hz, 1H), 4.36 (d, J= 8.6 Hz, 1H), 4.24 (s, 1H), 3.98 - 3.91 (m, 1H), 3.85 - 3.79 (m, 1H), 3.70 - 3.62 (m, 1H), 3.25 - 3.21 (m, 3H), 1.57 (dd, J= 5.3, 7.3 Hz, 1H), 1.29 - 1.24 (m, 1H), 1.15 (d, J = 6.2 Hz, 3H), 0.98 - 0.95 (m, 3H), 0.84 (s, 3H).
Example 84: Synthesis of (lR,2S,5S)-N-[cyano-(l-oxido-l,6-naphthyridin-l-ium-8- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo [3.1.0] hexane-2-carboxamide
Figure imgf000348_0001
Step 1: 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile
347
SUBSTITUTE SHEET RULE 26 [000673] A solution of l,6-naphthyridine-8-carbaldehyde (1 g, 6.32 mmol, 1 eq) in NHa/MeOH (20 mL) was added with NH4CI (676.44 mg, 12.65 mmol, 2 eq) stirred at 25 °C for 2 h. TMSCN (1.25 g, 12.65 mmol, 1.58 mL, 2 eq) was then added at 0 °C, and then the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue, then diluted with H2O (50 mL) and extracted with ethyl acetate (50 mL * 4). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether : ethyl acetate=5: 1 to 1 : 1) to give 2- amino-2-(l,6-naphthyridin-8-yl)acetonitrile (549 mg, 2.83 mmol, 44.69% yield, 94.8% purity) as a brown solid. MS (ESI) m/z 185.1 [M+H]+
Step 2: (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide
[000674] A mixture of 2-amino-2-(l,6-naphthyridin-8-yl)acetonitrile (549 mg, 2.98 mmol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.09 g, 2.98 mmol, 1 eq) in ACN (30 mL) was added with 1 -methylimidazole (734.13 mg, 8.94 mmol, 712.75 uL, 3 eq) and TCFH (1.67 g, 5.96 mmol, 2 eq), and then the mixture was stirred at 25°C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove ACN. The residue was extracted with EA 120 mL (30 mL * 4). The combined organic layers were washed with brine 20 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give (1R,2S,5S)-N- [cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (1.7 g, crude) as a white solid. MS (ESI) m/z 531.2 [M+H]+
Step 3: (lR,2S,5S)-N-[cyano-(l-oxido-l,6-naphthyridin-l-ium-8-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000675] A solution of (lR,2S,5S)-N-[cyano(l,6-naphthyridin-8-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide (1.15 g, 2.17 mmol, 1 eq) in CHCh (20 mL) was added 3-
348
SUBSTITUTE SHEET RULE 26 chlorobenzenecarboperoxoic acid (561.08 mg, 3.25 mmol, 1.5 eq). The mixture was stirred at 50°C for 16 h. Upon completion, the reaction mixture was diluted with DCM (20 mL) and washed with NaHCCh (aqueous solution) until pH=7, then extracted with DCM (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HC1 condition; column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water(HCl)-ACN]; B%: 30%- 60%, 20 min ) to afford (lR,2S,5S)-N-[cyano-(l-oxido-l,6-naphthyridin-l-ium-8-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (200 mg, 362.28 umol, 16.71% yield, 99% purity) as a white solid. MS (ESI) m/z 547.2 [M+H]+.
[000676] 1HNMR (400 MHz, DMSO-t/e) 5 = 9.68 - 9.45 (m, 1H), 9.38 (br s, 1H), 9.09 (t, J = 2.2 Hz, 1H), 9.03 (dd, J= 1.1, 4.3 Hz, 1H), 8.53 (dd, J= 1.4, 11.8 Hz, 1H), 8.38 (d, J = 8.5 Hz, 1H), 7.77 (ddd, J= 1.1, 4.3, 8.5 Hz, 1H), 6.79 (dd, J= 1.8, 12.5 Hz, 1H), 4.44 - 4.28 (m, 2H), 3.89 (dd, J= 5.4, 10.3 Hz, 1H), 3.70 (dd, J= 4.3, 10.6 Hz, 1H), 1.61 - 1.49 (m, 1H), 1.27 - 1.22 (m, 1H), 1.07 - 0.80 (m, 15H).
Example 85: Synthesis of (lR,2S,5S)-N-[cyano-(2-triisopropylsilylthiazolo[4,5-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 252)
349
SUBSTITUTE SHEET RULE 26
Figure imgf000351_0001
Step 1: 3-bromo-5-nitro-pyridine-4-thiol
[000677] A solution of 3-bromo-4-chloro-5-nitro-pyridine (90 g, 379.05 mmol, 1 eq) in MeOH (450 mL) was added sulfanylsodium (42.50 g, 758.09 mmol, 2 eq) and stirred at 20 °C for 16 h. Upon completion, NaOH aq. (10%, 1 L) was added and was filtered. The filtrate was acidified with HC1 (12 M) to pH<2, and the solid was filtered, the cake was concentrated under reduced pressure to give 3-bromo-5-nitro-pyridine-4-thiol (89 g, crude) as a red solid. MS (ESI) m/z 235.0 [M+H]+.
Step 2: 3-amino-5-bromo-pyridine-4-thiol
[000678] A solution of 3-bromo-5-nitro-pyridine-4-thiol (89 g, 340.77 mmol, 90% purity, 1 eq) in H2O (900 mL) and HC1 (300 mL, 36% purity) was added dichlorotin (129.23 g, 681.53 mmol, 17.68 mL, 2 eq) and stirred at 20 °C for 3 h. Upon completion, the reaction was filtered and concentrated under reduced pressure to give a residue to give 3-amino-5-bromo-pyridine-4- thiol (78 g, crude) as a yellow solid. MS (ESI) m/z 205.0 [M+H]+.
350
SUBSTITUTE SHEET RULE 26 Step 3: 7-bromothiazolo[4,5-c]pyridine
[000679] A mixture of 3-amino-5-bromo-pyridine-4-thiol (78 g, 342.31 mmol, 90% purity, 1 eq) in HCOOH (400 mL) was added Zn (13.09 g, 200.18 mmol, 0.585 eq) slowly and then stirred at 100 °C for 1 h. Upon completion, the reaction was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, petroleum ether: ethyl acetate = 1: 1 to 0: 1) to give 15 g crude as a yellow solid. The crude (12 g, 55.80 mmol, 1 eq) was further purified by H2O (500 mL) and extracted with EA (500 ml * 3). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to to give 7-bromothiazolo[4,5-c]pyridine (7.2 g, 33.48 mmol, 60.00% yield) as a white soild. MS (ESI) m/z 215.0 [M+H]+.
Step 4: (7-bromothiazolo[4,5-c]pyridin-2-yl)-triisopropyl-silane
[000680] A solution of 7-bromothiazolo[4,5-c]pyridine (7.2 g, 33.48 mmol, 1 eq) in THF (50 mL) was added LDA (2 M, 18.41 mL, 1.1 eq) under N2. The mixture was stirred at -78°C for 0.5 h. Then TIPSC1 (7.10 g, 36.83 mmol, 7.88 mL, 1.1 eq) in THE (20 mL) was added dropwise and stirred at -78 °C for 2 h. Upon completion, the reaction mixture was quenched with solution of NH4CI (50 mL) and extracted with EA (50 mL * 3). The combined organic layers were washed by brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 100: 1 to 10: 1) to give (7-bromothiazolo[4,5-c]pyridin-2-yl)-triisopropyl-silane (5.3 g, 11.84 mmol, 35.38% yield, 83% purity) as a yellow solid. MS (ESI) m/z 373.2 [M+H]+.
Step 5: triisopropyl-(7-vinylthiazolo[4,5-c]pyridin-2-yl)silane
[000681] A solution of (7-bromothiazolo[4,5-c]pyridin-2-yl)-triisopropyl-silane (6.1 g, 16.42 mmol, 1 eq) in toluene (60 mL) and H2O (15 mL) was added potassium trifluoro(vinyl)boranuide (2.64 g, 19.71 mmol, 1.2 eq), K3PO4 (6.97 g, 32.85 mmol, 2 eq), and ditert- butyl(cyclopentyl)phosphane; dichloropalladium; iron (1.07 g, 1.64 mmol, 0.1 eq) under N2. The mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (50 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 100: 1 to
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SUBSTITUTE SHEET RULE 26 10:1) to give triisopropyl-(7-vinylthiazolo[4,5-c]pyridin-2-yl)silane (3.4 g, 10.67 mmol, 64.99% yield) as a yellow solid. MS (ESI) m/z 319.3 [M+H]+.
Step 6: 2-triisopropylsilylthiazolo[4,5-c]pyridine-7-carbaldehyde
[000682] A solution of triisopropyl-(7-vinylthiazolo[4,5-c]pyridin-2-yl)silane (3.4 g, 10.67 mmol, 1 eq) in THF (30 mL) and H2O (5 mL) was added K2OSO4.2H2O (393.26 mg, 1.07 mmol, 0.1 eq) and stirred for 30 min, then NalCh (6.85 g, 32.02 mmol, 1.77 mL, 3 eq) was added. The mixture was stirred at 20 °C for 5.5 h. Upon completion, the reaction mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (50 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 100: 1 to 5: 1) to give 2-triisopropylsilylthiazolo[4,5-c]pyridine-7-carbaldehyde (2.4 g, 6.74 mmol, 63.14% yield, 90% purity) as a yellow solid. MS (ESI) m/z 321.3 [M+H]+.
Step 7: benzyl N-[(lS)-l-[[2-[[(lS)-2-amino-2-oxo-l-[[(6R)-5-oxo-4-azaspiro[2.4]heptan-6- yl]methyl]ethyl]amino]-2-oxo-l-(trimethylsilylmethyl)ethyl]carbamoyl]-2,2-dimethyl- propyl] carbamate
[000683] A solution of 2-triisopropylsilylthiazolo[4,5-c]pyridine-7-carbaldehyde (1 g, 3.12 mmol, 1 eq), Ti(i-PrO)4 (1.33 g, 4.68 mmol, 1.38 mL, 1.5 eq), and NH3/MeOH (7 M, 1.78 mL, 4 eq) in DCM (10 mL) was stirred at 20 °C for 1 h. Then, TMSCN (928.57 mg, 9.36 mmol, 1.17 mL, 3 eq) was added, and the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (10 mL) and then extracted with EtOAc (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(2-triisopropylsilylthiazolo[4,5- c]pyridin-7-yl)acetonitrile (1 g, crude) as a yellow solid. MS (ESI) m/z 347.3 [M+H]+.
Step 8: (lR,2S,5S)-N-[cyano-(2-triisopropylsilylthiazolo[4,5-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000684] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
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SUBSTITUTE SHEET RULE 26 (925.18 mg, 2.54 mmol, 1.1 eq), 2-amino-2-(2-triisopropylsilylthiazolo[4,5-c]pyridin-7- yl)acetonitrile (1 g, 2.31 mmol, 80% purity, 1 eq), and 1 -methylimidazole (568.55 mg, 6.93 mmol, 551.99 uL, 3 eq) in ACN (10 mL) was added with TCFH (1.30 g, 4.62 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Xtimate Cl 8 lOu 250mm * 80 mm; mobile phase: [water( NH4HCO3)-ACN]; B%: 60%-95%, 20 mm) to give (lR,2S,5S)-N-[cyano-(2- triisopropylsilylthiazolo[4,5-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.41 mmol, 61.27% yield, 98% purity) as a yellow solid. Then the soild (500 mg, 721.60 umol, 1 eq) was further purified by SFC (column: DAI CEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-MeOH]; B%: 24%-24%, 8 min) to give (lR,2S,5S)-N-[cyano-(2- triisopropylsilylthiazolo[4,5-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (170 mg, 245.34 umol, 34.00% yield) as a white soild. MS (ESI) m/z 693.3 [M+H]+.
[000685] 'H NMR (400 MHz, DMSO-de) 5 = 9.59 (dd, I = 3.2, 7.6 Hz, 1H), 9.52 (d, I = 6.7 Hz, 1H), 9.41 - 9.25 (m, 1H), 8.69 (d, I = 5.0 Hz, 1H), 6.70 - 6.57 (m, 1H), 4.37 (br dd, I = 7.8, 12.2 Hz, 1H), 4.28 (d, I = 8.7 Hz, 1H), 3.90 (ddd, 1 = 5.5, 10.4, 16.1 Hz, 1H), 3.67 (dd, 1 = 10.9, 12.4 Hz, 1H), 1.60 - 1.44 (m, 4H), 1.27 (dd, 1 = 7.6, 17.3 Hz, 1H), 1.19 - 1.11 (m, 18H), 1.06 - 0.90 (m, 12H), 0.89 - 0.80 (m, 3H).
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SUBSTITUTE SHEET RULE 26 Example 86: Synthesis of (lR,2S,5S)-N-[(8-chloro-4-deuterio-phthalazin-l-yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 253)
Figure imgf000355_0001
Step 1: N-tert-butyl-2-chloro-benzamide
[000686] A solution of 2-chlorobenzoyl chloride (10 g, 57.14 mmol, 8.40 mL, 1 eq) in DCM (100 mL) was cooled to 0 °C, and a solution of 2-methylpropan-2-amine (4.18 g, 57.14 mmol, 6.00 mL, 1 eq) and TEA (14.45 g, 142.85 mmol, 19.88 mL, 2.5 eq) in DCM (50 mL) was added dropwise. The mixture was stirred at 20 °C for 3 h. Upon completion, the reaction was diluted with NaHCCh (200 mL). Then was extracted with DCM (100 mL * 2). The organic layer was separated and washed with aqueous citric acid solution (100 mL), then was washed by brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, petroleum ehter: ethyl acetate = 100:0 to 80:20) to give N- tert-butyl-2-chloro-benzamide (12.1 g, 51.44 mmol, 90.03% yield, 90% purity) as a white solid. MS (ESI) m/z 212.1 [M+H]+.
Step 2: 2-tert-buty l-7-chloro-3 -deuterio-3 -hydroxy-isoindolin- 1 -one
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SUBSTITUTE SHEET RULE 26 [000687] A solution of N-tert-butyl-2-chloro-benzamide (6 g, 25.51 mmol, 90% purity, 1 eq) in THF (60 mL) was added N,N,N',N' -tetramethylethane- 1,2-diamine (9.46 g, 81.37 mmol, 12.28 mL, 3.19 eq), and then added sec-butyllithium (1.3 M, 62.60 mL, 3.19 eq) dropwise at -78 °C and then stirred at -78 °C for 2.5 h under N2. Then t77-N,N-dimethylformamide (6.52 g, 81.37 mmol, 6.33 mL, 3.19 eq) was added at -78 °C and stirred at 20 °C for 2.5 h. Upon completion, the reaction was quenched with NH4CI (aq. saturated, 50 mL). Then was extracted with ethyl acetate (50 mL * 2). The organic layer was separated and washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, petroleum ether: ethyl acetate = 90: 10 to 50:50) to give 2-tert-butyl-7- chloro-3-deuterio-3-hydroxy-isoindolin-l-one (6.03 g, 24.05 mmol, 94.28% yield, 96% purity) as a yellow solid. MS (ESI) m/z 241.2 [M+H]+.
Step 3: 8-chloro-4-deuterio-2H-phthalazin-l-one
[000688] 2 -Ter/-butyl-7-chloro-3-deuterio-3-hydroxy-isoindolin-l-one (6.03 g, 25.05 mmol,
1 eq) was suspended in AcOH (24 mL) under nitrogen. The resulting thick slurry was heated to 90 °C. At approximately 80 °C, a homogeneous solution was obtained. Hydrazine hydrate (3.8 g, 75.91 mmol, 3.69 mL, 3.03 eq) was added dropwise (exotherm) at 90 °C. The mixture was stirred at 90 °C for 4 h. As the conversion of starting material was monitored by LCMS (<1%). Water (50 mL) preheated to 80 °C was added, maintaining the mixture at 80-90 °C followed by ramping down to 20 °C over approximately 3 h. Upon completion, the reaction mixture was extracted with Ethyl acetate (50 mL * 2). The organic layer was separated and washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by purified by column (SiCh, petroleum ether: ethyl acetate = 1 :0 to 0: 1) to give 8-chloro-4-deuterio-2H-phthalazin-l-one (6 g, crude) as a yellow solid. The crude was further purified by column (SiCh, petroleum ether : ethyl acetate = 1:0 to 0:1) to give 8-chloro-4- deuterio-2H-phthalazin-l-one (2 g, 33.04 mmol, 1 eq) as a yellow solid. MS (ESI) m/z 182.1 [M+H]+.
Step 4: 4-bromo-5-chloro-l-deuterio-phthalazine
[000689] A mixture of 8-chloro-4-deuterio-2H-phthalazin-l-one (700 mg, 3.85 mmol, 1 eq) and POBn (5.53 g, 19.27 mmol, 1.96 mL, 5 eq) was stirred at 130 °C for 0.5 h. Upon
355
SUBSTITUTE SHEET RULE 26 completion, the residue was diluted with THF (500 mL), the filter cake was evaporated under reduced pressure to to give 4-bromo-5-chloro-l-deuterio-phthalazine (800 mg, crude) as a brown solid.
Step 5: 2-(benzhydrylideneamino)-2-(8-chloro-4-deuterio-phthalazin- 1 -yl)acetonitrile
[000690] A mixture of 2-(benzhydrylideneamino)acetonitrile (720.73 mg, 3.27 mmol, 1 eq) in THF (32 mL) was added LiHMDS (1 M, 8.18 mL, 2.5 eq) at -65 °C under N2, then the mixture was stirred at -65°C for 1 h, then was added 4-bromo-5-chloro-l-deuterio-phthalazine (800 mg, 3.27 mmol, 1 eq) in THF (32 mL) dropwised to the mixture, then the mixture was stirred at -65 °C for 0.5 h, then the mixture was warmed to 20 °C, the mixture was stirred at 20 °C for 3.5 h. Upon completion, the reaction mixture was diluted with NH4CI (200 mL) and extracted with DCM (50 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 100: 1 to Petroleum ether:DCM = 0:1) to give 2-(benzhydrylideneamino)-2-(8-chloro-4-deuterio-phthalazin-l-yl)acetonitrile (550 mg, 1.00 mmol, 30.65% yield, 70% purity) as a red solid.
Step 6: 2-amino-2-(8-chloro-4-deuterio-phthalazin-l -yl)acetonitrile
[000691] A mixture of 2-(benzhydrylideneamino)-2-(8-chloro-4-deuterio-phthalazin-l- yl)acetonitrile (500 mg, 1.30 mmol, 1 eq) in DCM (4 mL) was added HCI (3 M, 4 mL, 9.21 eq) at 20 °C, and the mixture was stirred at 20 °C for 36 h. Upon completion, the reaction liquid was blow-dried with nitrogen. The residue was purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water(HCl)-ACN]; B%: 5%-30%, 8 min) to give 2-amino-2- (8-chloro-4-deuterio-phthalazin-l-yl)acetonitrile (180 mg, 696.57 umol, 53.48% yield, 85% purity) as yellow solid.
Step 7: (lR,2S,5S)-N-[(8-chloro-4-deuterio-phthalazin-l-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000692] To a solution of(lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
356
SUBSTITUTE SHEET RULE 26 (248.82 mg, 682.91 umol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (112.14 mg, 1.37 mmol, 108.87 uL, 2 eq) and TCFH (383.22 mg, 1.37 mmol, 2 eq) at 20 °C. 2-Amino-2-(8- chloro-4-deuterio-phthalazin-l-yl)acetonitrile (150 mg, 682.91 umol, 1 eq) was added, and the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (30 mL) and extracted with DCM (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 8 min) to give (lR,2S,5S)-N-[(8-chloro-4- deuterio-phthalazin-l-yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (0.15 g, 257.07 umol, 37.64% yield, 97% purity) as a light yellow solid. MS (ESI) m/z 565.2 [M+H]+.
[000693] 1H NMR (400 MHz, DMSO-t76) 5 = 9.54 (dd, J= 7.2, 10.8 Hz, 1H), 9.41 - 9.31 (m, 1H), 8.33 - 8.26 (m, 1H), 8.25 - 8.14 (m, 1H), 8.10 - 8.01 (m, 1H), 7.36 (t, J= 6.7 Hz, 1H), 4.40 (dd, J= 8.6, 10.3 Hz, 1H), 4.29 (d, J= 10.6 Hz, 1H), 3.92 (ddd, J= 2.6, 5.3, 7.6 Hz, 1H), 3.77 - 3.65 (m, 1H), 1.58 (ddd, J= 5.4, 7.4, 17.0 Hz, 1H), 1.29 (dd, J= 7.6, 18.7 Hz, 1H), 1.06 - 0.95 (m, 12H), 0.79 (d, J = 3.5 Hz, 3H).
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SUBSTITUTE SHEET RULE 26 Example 87: Synthesis of (lR,2S,5S)-N-[(8-bromo-4-deuterio-phthalazin-l-yl)-cyano- methyl]-3-[(2S)-3, 3-dimethyl-2- [(2,2,2- trifluoroacetyl)amino] butanoyl] -6, 6-dimethyl-3- azabicyclo [3.1.0] hexane-2-carboxamide
Figure imgf000359_0001
Step 1: 7-bromo-3,3-dideuterio-isobenzofuran-l-one
[000694] A solution of 7-bromo-3H-isobenzofuran-l-one (4 g, 18.78 mmol, 1 eq) in THF (400 mL) was cooled to -50 °C. LiHMDS (1 M, 93.88 mL, 5 eq) was then added dropwise at - 50 °C and then stirred at -50 °C for 0.5 h under N2. Then D2O (7.52 g, 375.54 mmol, 20 eq) was added dropwise at -50 °C and stirred for 0.5 h. Upon completion, the reaction was quenched with added NH4CI (aq. saturated, 200 mL) slowly at 0~5 °C and stirred at 0~5 °C for 15 min. The resulting mixture was extracted with ethyl acetate (200 mL*2). The organic layer was separated and washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, petroleum ehter: ethyl acetate = 100:0 to 75:25), repeat this work for 3 times. To give 7-bromo-3,3-dideuterio- isobenzofuran-l-one (1.5 g, 6.98 mmol, 68.18% yield) as a yellow solid. MS (ESI) m/z 215.0&217.0 [M+H]+.
Step 2: 3,7-dibromo-3-deuterio-isobenzofuran-l-one
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SUBSTITUTE SHEET RULE 26 [000695] A mixture of 7-bromo-3,3-dideuterio-isobenzofuran-l-one (1.8 g, 8.37 mmol, 1 eq) in CHCh (19 mL) was added AIBN (68.73 mg, 418.53 umol, 0.05 eq), NBS (2.23 g, 12.56 mmol, 1.5 eq), and then stirred at 70 °C for 14 h under N2. Upon completion, the reaction was quenched with H2O (10 mL) at 20 °C. Then was extracted with DCM (20 mL * 2). The organic layer was separated and washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, petroleum ether: ethyl acetate =10:1 to 3:1) to give 3,7-dibromo-3-deuterio- isobenzofuran-l-one (1.43 g, 4.39 mmol, 52.49% yield, 90% purity) as a yellow solid. MS (ESI) m/z 291.8&293.8&295.8 [M+H]+.
Step 3: 8-bromo-4-deuterio-2H-phthalazin-l-one
[000696] 3,7 -Dibromo-3-deuterio-isobenzofuran-l-one (1.43 g, 4.88 mmol, 1 eq) was suspended in AcOH (6 mL) under nitrogen. The resulting thick slurry was heated to 90 °C. At approximately 80 °C, ahomogeneous solution was obtained. Hydrazine hydrate (740 mg, 14.78 mmol, 718.45 uL, 3.03 eq) was added dropwise (exotherm) at 90 °C. Over approximately 4 h. The resulting suspension was continued to stir at 90 °C. As the conversion of starting material was monitored by LCMS (<1%). Water (12 mL) preheated to 80 °C was added, maintaining the mixture at 80-90 °C followed by ramping down to 20 °C over approximately 3 h. Upon completion, the reaction was quenched with water (20 mL). Then was extracted with ethyl acetate (20 mL * 2). The organic layer was separated and washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, DCM:MeOH =100:0 to 96:4) to give 8-bromo-4-deuterio-2H- phthalazin-l-one (1.1 g, 4.38 mmol, 89.71% yield, 90% purity) as a yellow solid. MS (ESI) m/z 226.1&228.1 [M+H]+.
Step 4: 4,5-dibromo-l-deuterio-phthalazine
[000697] A mixture of 8-bromo-4-deuterio-2H-phthalazin-l-one (700 mg, 3.10 mmol, 1 eq) and POBn (4.44 g, 15.49 mmol, 1.574 mL, 5 eq) was stirred at 130 °C for 0.5 h. Upon completion, the residue was diluted with THF 10 mL, then filtered to give the crude product. The crude product was washed with THF (50 mL) to give the desired product. 4,5-Dibromo-l-
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SUBSTITUTE SHEET RULE 26 deuterio-phthalazine (820 mg, 2.70 mmol, 91.54% yield, 95.4% purity) was obtained as a brown solid. MS (ESI) m/z 329.1 [M+H+41]+.
Step 5: 2-(benzhydrylideneamino)-2-(8-bromo-4-deuterio-phthalazin-l-yl)acetonitrile
[000698] A mixture of 2-(benzhydrylideneamino)acetonitrile (602.28 mg, 2.74 mmol, 1 eq) in THF (18 mL) was added with LiHMDS (1 M, 6.84 mL, 2.5 eq) at -65 °C under N2, and then the mixture was stirred at -65 °C for 0.5 h. 4,5-Dibromo-l-deuterio-phthalazine (790.0 mg, 2.74 mmol, 1 eq) in THF (18 mL) drop wised to the mixture, and then the mixture was stirred at - 65 °C for 0.5 h, then the mixture was warmed to 25 °C. The mixture was stirred at 25 °C for 4 h. Upon completion, the reaction mixture was diluted with water (50 mL) and extracted with DCM (50 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 100: 1 to petroleum ether:DCM = 0:1). Compound 2- (benzhydrylideneamino)-2-(8-bromo-4-deuterio-phthalazin-l-yl)acetonitrile (480 mg, 1.121 mmol, 40.92% yield, 89.0% purity) was obtained as a yellow solid. MS (ESI) m/z 428.2 [M+H]+.
Step 6: 2-amino-2-(8-bromo-4-deuterio-phthalazin-l-yl)acetonitrile
[000699] A mixture of 2-(benzhydrylideneamino)-2-(8-bromo-4-deuterio-phthalazin-l- yl)acetonitrile (460 mg, 1.074 mmol, 1 eq) in HC1 (3 M, 8.0 mL, 42.83 eq) and DCM (8 mL) was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water (HCl)-ACN]; B%: 1%-15%, 8 min.). 2-Amino-2-(8- bromo-4-deuterio-phthalazin-l-yl)acetonitrile (150 mg, 499.07 umol, 53.1% yield, 95.0% purity, HC1) was obtained as a yellow solid. MS (ESI) m/z 264.0 [M+H]+.
Step 7: (lR,2S,5S)-N-[(8-bromo-4-deuterio-phthalazin-l-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000700] To a mixture of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (181.83 mg, 499.07 umol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (122.93 mg, 1.497
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SUBSTITUTE SHEET RULE 26 mmol, 119.34 uL, 3 eq) and TCFH (280.05 mg, 998.13 umol, 2 eq) at 0 °C. Then, 2-amino-2-(8- bromo-4-deuterio-phthalazin-l-yl)acetonitrile (80 mg, 499.07 umol, 1 eq, HC1) was added and the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL) at 25 °C, and then extracted with DCM (20 mb * 2). The combined organic layers were washed with brine (40 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 75 * 30 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 8 min). Compound (lR,2S,5S)-N-[(8-bromo-4-deuterio-phthalazin-l-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (70 mg, 112.03 umol, 24.62% yield, 97.7% purity) was obtained as a white solid. MS (ESI) m/z 610.1 [M+H]+.
[000701] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.55 (dd, J= , 19.7 Hz, 1H), 9.44 - 9.21 (m, 1H), 8.43 (dd, J= 7.7, 15.2 Hz, 1H), 8.37 - 8.30 (m, 1H), 7.96 (dt, J= 3.8, 7.8 Hz, 1H), 7.53 - 7.49 (m, 1H), 4.41 (br d, .7 = 8.0 Hz, 1H), 4.31 (d, J= 5.2 Hz, 1H), 3.91 (br dd, J = 5.5, 10.7 Hz, 1H), 3.72 (br dd, J = 10.4, 17.4 Hz, 1H), 1.66 - 1.49 (m, 1H), 1.34 (dd, J= 4.5, 7.6 Hz, 1H), 1.05 - 0.99 (m, 12H), 0.86 - 0.72 (m, 3H).
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SUBSTITUTE SHEET RULE 26 Example 88: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6- dimethyl-3- [2-( l-methylcyclopropyl)-2- [(2,2,2-trifluoroacetyl)amino] acetyl] -3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 257)
Figure imgf000363_0001
Step 1: methyl (lR,2S,5S)-3-[2-(benzyloxycarbonylamino)-2-(l-methylcyclopropyl)acetyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000702] A solution of 2-(benzyloxycarbonylamino)-2-(l-methylcyclopropyl)acetic acid (1.45 g, 5.51 mmol, 1 eq) in ACN (20 mL) was added methyl (lR,2S,5S)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (1.47 g, 7.16 mmol, 1.3 eq, HC1) and 1 -methylimidazole (1.81 g, 22.03 mmol, 1.76 mL, 4 eq). Then, TCFH (1.85 g, 6.61 mmol, 1.2 eq) was added to the mixture and the mixture was sitrred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL) at 0°C, and extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (30 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate=l/O to 3/1) to give methyl (lR,2S,5S)-3-[2- (benzyloxycarbonylamino)-2-(l-methylcyclopropyl)acetyl]-6,6-dimethyl-3-
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SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxylate (2.4 g, 4.75 mmol, 86.21% yield, 82% purity) as yellow solid. MS (ESI) m/z 415.3 [M+H]+.
Step 2: (lR,2S,5S)-3-[2-(benzyloxycarbonylamino)-2-(l -methylcyclopropyl)acetyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000703] A solution of methyl (lR,2S,5S)-3-[2-(benzyloxycarbonylamino)-2-(l- methylcyclopropyl)acetyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (2.4 g, 5.79 mmol, 1 eq) in THF (12 mL) and H2O (3 mL) was added with LiOH.FEO (728.93 mg, 17.37 mmol, 3 eq). The mixture was was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) and adjusted pH = about 3, then was extracted with ethyl acetate (30 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give (lR,2S,5S)-3-[2- (benzyloxycarbonylamino)-2-(l-methylcyclopropyl)acetyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.7 g, crude) as a colorless oil. MS (ESI) m/z 401.3 [M+H]+.
Step 3: (lR,2S,5S)-3-[2-amino-2-(l-methylcyclopropyl)acetyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000704] A solution of (lR,2S,5S)-3-[2-(benzyloxycarbonylamino)-2-(l- methylcyclopropyl)acetyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.7 g, 4.25 mmol, 1 eq) in isopropanol (20 mL) was added Pd/C (1.7 g, 10% purity) and the mixture was stirred under H2 (15 PSI) at 20 °C for 3 h. Upon completion, the reaction was filtered and concentrated under reduced pressure to give (lR,2S,5S)-3-[2-amino-2-(l- methylcyclopropyl)acetyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.1 g, crude) as a colorless oil. MS (ESI) m/z 267.3 [M+H]+.
Step 4: (lR,2S,5S)-6,6-dimethyl-3-[2-(l -methylcy cl opr opyl)-2- [(2,2,2- trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000705] A solution of (lR,2S,5S)-3-[2-amino-2-(l-methylcyclopropyl)acetyl]-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.10 g, 4.13 mmol, 1 eq) in methyl 2,2,2- trifluoroacetate (23.28 g, 181.85 mmol, 18.33 mL, 44.03 eq) was added E N (1.25 g, 12.39
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SUBSTITUTE SHEET RULE 26 mmol, 1.72 mL, 3 eq), and the solution was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove solvent. The residue was diluted with H2O (15 mL) and ajusted to pH = about 3 and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue (1.1 g, crude). The residue (100 mg, crude) was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3) - ACN]; B%: 10% - 50%, 8 min) to give (lR,2S,5S)-6,6-dimethyl-3-[2-(l-methylcyclopropyl)-2-[(2,2,2-trifluoroacetyl)amino]acetyl]-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (20 mg, 55.20 umol, 1.34% yield, 100% purity) as a white solid. MS (ESI) m/z 363.2 [M+H]+.
[000706] 'H NMR (400 MHz, DEUTERIUM OXIDE) 5 = 4.41 - 4.28 (m, 1H), 4.17 - 4.07 (m, 1H), 4.01 - 3.49 (m, 2H), 1.61 - 1.53 (m, 1H), 1.51 - 1.40 (m, 1H), 1.14 - 0.99 (m, 6H), 0.99 - 0.89 (m, 3H), 0.86 - 0.81 (m, 1H), 0.79 - 0.70 (m, 1H), 0.57 - 0.50 (m, 1H), 0.49 - 0.38 (m, 1H).
Step 5: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-[2-(l- methylcyclopropyl)-2-[(2,2,2-trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000707] A solution of (lR,2S,5S)-6,6-dimethyl-3-[2-(l-methylcyclopropyl)-2-[(2,2,2- trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1 g, 2.76 mmol, 1 eq) in ACN (15 mL) was added with 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (660.75 mg, 3.04 mmol, 1.1 eq) and 1 -methylimidazole (906.36 mg, 11.04 mmol, 879.96 uL, 4 eq). And then TCFH (929.21 mg, 3.31 mmol, 1.2 eq) was added to the mixture and the mixture was sitrred at 20 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL), and then extracted with ethyl acetate (30 mL * 3). The combined organic layers were washed with brine (50 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether : ethyl acetate = 3: 1) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-[2-(l- methylcyclopropyl)-2-[(2,2,2-trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (600 mg, 896.83 umol, 32.50% yield, 84% purity) was obtained as a white solid. MS (ESI) m/z 562.2 [M+H]+.
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SUBSTITUTE SHEET RULE 26 Step 6: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-[2-(l- methylcyclopropyl)-2-[(2,2,2-trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000708] The (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-[2-(l- methylcyclopropyl)-2-[(2,2,2-trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (600 mg, 1.07 mmol, 1 eq) was separated by SFC (column: ChiralPak IH, 250 * 30 mm, 10 um; mobile phase: [Neu-ETOH]; B%: 25% - 25%, 6 min) to give (lR,2S,5S)-N-[(5- chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-[2-(l-methylcyclopropyl)-2-[(2,2,2- trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1.1 (76 mg, 126.17 umol, 11.82% yield, 93.3% purity) as a white solid. MS (ESI) m/z 562.2 [M+H]+.
[000709] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.65 (br d, J= 6.4 Hz, 2H), 9.49 (s, IH), 8.86 (s, IH), 8.32 - 8.23 (m, IH), 8.09 - 7.98 (m, IH), 7.76 (t, J= 7.8 Hz, IH), 7.31 (br d, J= 1.6 Hz, IH), 4.37 (d, J = 6.4 Hz, IH), 4.27 (s, IH), 3.87 - 3.78 (m, IH), 3.71 (d, J= 10.5 Hz, IH), 1.63 (dd, J = 5.3, 7.4 Hz, IH), 1.36 (d, J= 7.5 Hz, IH), 1.05 (d, J= 2.3 Hz, 6H), 0.91 (s, 3H), 0.85 - 0.77 (m, IH), 0.76 - 0.69 (m, IH), 0.34 - 0.20 (m, 2H).
[000710] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-[2-(l- methylcyclopropyl)-2-[(2,2,2-trifluoroacetyl)amino]acetyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (72 mg, 124.53 umol, 11.66% yield, 97.2% purity) was obtained as a white solid. MS (ESI) m/z 562.2 [M+H]+.
[000711] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.68 (br d, J= 6.3 Hz, IH), 9.54 - 9.44 (m, 2H), 8.86 (s, IH), 8.33 - 8.23 (m, IH), 8.06 (dd, J = 1.0, 7.6 Hz, IH), 7.77 (t, J= 7.8 Hz, IH), 7.30 (br d, J= 5.4 Hz, IH), 4.34 (br d, J= 6.1 Hz, IH), 4.21 (s, IH), 3.84 (dd, J= 5.3, 10.4 Hz, IH), 3.69 (d, J= 10.6 Hz, IH), 1.64 - 1.58 (m, IH), 1.25 (d, J= 7.5 Hz, IH), 1.09 (s, 3H), 0.98 (s, 3H), 0.88 (s, 3H), 0.85 - 0.75 (m, 2H), 0.40 - 0.26 (m, 2H).
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SUBSTITUTE SHEET RULE 26 Example 89: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2- [ [3-(trifluoromethyl)- l,2,4-oxadiazol-5-yl] amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 259)
Figure imgf000367_0001
Step 1: 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile
[000712] To a solution of 5-chloroisoquinoline-4-carbaldehyde (150 mg, 782.83 umol, 1 eq) in DCM (3 mL) was added tetraisopropoxytitanium (333.74 mg, 1.17 mmol, 346.56 uL, 1.5 eq) and NEb/MeOH (7 M, 447.33 uL, 4 eq), and the mixture was stirred at 20 °C for 1 h. Then, trimethylsilylformonitrile (232.98 mg, 2.35 mmol, 293.80 uL, 3 eq) was added at 0 °C. The mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was diluted by addition H2O (20 mL), and then was filtered and the filtrate was extracted with DCM (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated in vacuum to give 2-amino-2-(5-chloro-4-isoquinolyl) acetonitrile (150 mg, crude) as a yellow gum. MS (ESI) m/z 218.0 [M+H]+.
Step 2: tert-butyl (lR,2S,5S)-3-[(2S)-2-(cyanoamino)-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2- carboxylate
[000713] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (800 mg, 2.47 mmol, 1 eq) in DCM (15 mL)
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SUBSTITUTE SHEET RULE 26 was added NaHCCh (621.39 mg, 7.40 mmol, 287.68 uL, 3 eq), and then was added carbononitridic bromide (522.33 mg, 4.93 mmol, 362.73 uL, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 4 h. Upon completion, the mixture was quenched by water (60 mL) and then was extracted with DCM (20 mL * 3), the organic phase was concentrated in vacuum to obtained tert-butyl (lR,2S,5S)-3-[(2S)-2-(cyanoamino)-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (1 g, crude) as a white solid. MS (ESI) m/z 350.2 [M+H]+.
Step 3: tert-butyl (lR,2S,5S)-3-[(2S)-2-[[(tert-butoxycarbonylamino)oxycarbonimidoyl]amino]- 3,3-dimethyl-butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000714] To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-(cyanoamino)-3,3-dimethyl- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (1 g, 2.86 mmol, 1 eq) and tertbutyl N-hydroxy carbamate (761.99 mg, 5.72 mmol, 2 eq) in THF (20 mL) was added ZnCh (1 M, 6.30 mL, 2.2 eq) dropwised under N2 at 20 °C, and the mixture was stirred at 20 °C for 48 h. Upon completion, the mixture was concentrated in vacuum and was extracted with ethyl acetate (100 mL * 2) and washed with sat.NEhCl (100 mL), then the organic phase was concentrated in vacuum to obtained tert-butyl (lR,2S,5S)-3-[(2S)-2-[[(tert- butoxycarbonylamino)oxycarbonimidoyl]amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxylate (1.4 g, crude) as a yellow gum. MS (ESI) m/z 483.4 [M+H]+.
Step 4: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifhroromethyl)-l,2,4-oxadiazol-5- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000715] Batch 1: To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-[[(tert- butoxycarbonylamino)oxycarbonimidoyl]amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (800 mg, 1.66 mmol, 1 eq) in DCM (8 mL) was added TFA (1.32 g, 11.60 mmol, 859.11 uL, 7 eq) and TEAA (731.13 mg, 3.48 mmol, 484.19 uL, 2.1 eq) dropwised at 0 °C, the mixture was stirred at 20 °C for 24 h. Upon completion, the mixture was concentrated in vacuum and was purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water (HCl)-ACN]; B%: 45%-65%, 8 min) to obtain (1R,2S,5S)- 3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-
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SUBSTITUTE SHEET RULE 26 dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (25 mg, 55.64 umol, 3.36% yield, 100% purity) as a white solid. MS (ESI) m/z 405.1 [M+H]+.
[000716] 'H NMR (400 MHz, DMSO-de) 5 = 12.72 (s, 1H), 9.35 (d, J = 8.4 Hz, 1H), 4.23 (d, J = 8.6 Hz, 1H), 4.14 (s, 1H), 4.00 - 3.79 (m, 2H), 1.62 - 1.52 (m, 1H), 1.43 (d, J = 7.6 Hz, 1H), 1.15 - 0.80 (m, 15H).
[000717] Batch 2: To a solution of tert-butyl (lR,2S,5S)-3-[(2S)-2-[[(tert- butoxycarbonylamino)oxycarbonimidoyl]amino]-3,3-dimethyl-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (1.4 g, 2.90 mmol, 1 eq) in DCM (14 m ) was added TFA (2.32 g, 20.31 mmol, 1.50 mL, 7 eq) and TFAA (1.28 g, 6.09 mmol, 847.33 uL, 2.1 eq) dropwised at 0 °C, and the mixture was stirred at 20 °C for 24 h. Upon completion, the mixture was concentrated in vacuum and was purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water (HCl)-ACN]; B%: 45%-65%, 8 min) to obtain (1R,2S,5S)- 3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (37 mg, 86.92 umol, 3.00% yield, 95% purity) as a white solid. MS (ESI) m/z 405.1 [M+H]+.
Step 5: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[000718] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifhroromethyl)-l,2,4- oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (50 mg, 123.65 umol, 1 eq) and 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (40.37 mg, 185.47 umol, 1.5 eq) in MeCN (1 mL) was added TCFH (69.38 mg, 247.29 umol, 2 eq) and 1- methylimidazole (30.46 mg, 370.94 umol, 29.57 uL, 3 eq), and the mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum and was purified by prep- TLC (SiCh, ethyl acetate) to obtain (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3- [(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (70 mg, 107.78 umol, 87.17% yield, 93% purity) as a yellow solid. MS (ESI) m/z 604.2 [M+H]+
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SUBSTITUTE SHEET RULE 26 Step 6: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[000719] The (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2- [[3-(trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide was separated by SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-MeOH]; B%: 5%-25%, 15 min) to obtained (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (20 mg, 32.22 umol, 27.80% yield, 97.3% purity) as a white solid. MS (ESI) m/z 604.1 [M+H]+.
[000720] 'H NMR (400 MHz, DMSO-de) 5 = 9.62 (d, J = 6.2 Hz, 1H), 9.49 (s, 1H), 9.37 - 9.19 (m, 1H), 8.88 (s, 1H), 8.27 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 6.8 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.44 - 7.10 (m, 1H), 4.32 (s, 1H), 4.21 (s, 1H), 4.00 - 3.89 (m, 1H), 3.83 (br d, J = 10.4 Hz, 1H), 1.62 (dd, J = 5.6, 7.4 Hz, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.07 - 0.95 (m, 12H), 0.85 (s, 3H).
[000721] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (17 mg, 30.98 umol, 26.74% yield, 98.5% purity) was obtained as a white solid. MS (ESI) m/z 604.2 [M+H]+
[000722] 'H NMR (400 MHz, DMSO-de) 5 = 9.51 (s, 1H), 9.49 (s, 1H), 9.30 (br s, 1H), 8.91
- 8.81 (m, 1H), 8.33 - 8.23 (m, 1H), 8.10 - 7.96 (m, 1H), 7.80 - 7.70 (m, 1H), 7.29 (br s, 1H), 4.35 - 4.14 (m, 2H), 3.99 - 3.88 (m, 1H), 3.82 (br d, J = 10.4 Hz, 1H), 1.65 - 1.55 (m, 1H), 1.29 - 1.23 (m, 1H), 1.09 - 0.94 (m, 12H), 0.86 - 0.79 (m, 3H).
[000723] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[[3- (trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (14 mg, 30.98 umol, 98.5% purity) was re-separated by SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu- MeOH]; B%: 5%-25%, 15 min) to obtain (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[[3-(trifluoromethyl)-l,2,4-oxadiazol-5-yl]amino]butanoyl]-6,6-
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SUBSTITUTE SHEET RULE 26 dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (8 mg, 12.82 umol, 55.31% yield, 96.8% purity) as a white solid. MS (ESI) m/z 604.2 [M+H]+.
[000724] 'H NMR (400 MHz, DMSO-de) 5 = 9.54 - 9.49 (m, 1H), 9.49 - 9.42 (m, 1H), 9.36 - 9.25 (m, 1H), 8.87 (s, 1H), 8.33 - 8.25 (m, 1H), 8.06 (dd, J = 1.0, 7.6 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.34 - 7.24 (m, 1H), 4.31 - 4.16 (m, 2H), 3.99 - 3.87 (m, 1H), 3.82 (br d, J = 10.6 Hz, 1H), 1.64 - 1.56 (m, 1H), 1.27 (br d, J = 7.8 Hz, 1H), 1.05 (s, 9H), 0.97 (s, 3H), 0.79 (s, 3H).
Example 90: Synthesis of (lR,2S,5S)-N-((3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridin-7- yl)(cyano)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 261)
Figure imgf000371_0001
Step 1: 7-bromo-3-chloro-lH-pyrrolo[3,2-c]pyridine
[000725] To a mixture of AcOH (15 mL) and 7-bromo-lH-pyrrolo[3,2-c]pyridine (1 g, 5.08 mmol, 1 eq) at 0 °C was added NCS (1.02 g, 7.61 mmol, 1.5 eq) slowly and the reaction mixture was allowed to warm to 25 °C and stirred for 16 h. Upon completion, the mixture was adjust pH to 7 by NaOH (70 mL), and extracted with ethyl acetate (50 mL * 2). The combined organic layers were washed with brine (50 mL * 2), dried over Na2SO4, filtered and concentrated under
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SUBSTITUTE SHEET RULE 26 reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate=3:l to 0: 1) to give 7-bromo-3-chloro-lH-pyrrolo[3,2-c]pyridine (1.1 g, 4.75 mmol, 93.63% yield) as a yellow solid.
Step 2 : 7-bromo-3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridine
[000726] To an open solution of 7-bromo-3-chloro-lH-pyrrolo[3,2-c]pyridine (525 mg, 2.27 mmol, 1 eq) in DMF (10 mL) was added NaH (136.07 mg, 3.40 mmol, 60% purity, 1.5 eq). The flask was fitted with an argon balloon and stirred for 0.5 h underargon flow. The reaction vessel was cooled to 0 °C and dimethyl sulfate (286.07 mg, 2.27 mmol, 215.09 uL, 1 eq) was added dropwise. The stirring was continued at 25 °C for 1 hr. Upon completion, the reaction mixture was quenched by NH4CI (40 ml), extracted with EtOAc (30 ml * 3). The combined organic layers were washed with brine (30 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to 1 :1) to give 7-bromo-3-chloro-l-methyl-lH-pyrrolo[3,2- c]pyridine (700 mg, 2.85 mmol, 62.86% yield) as a brown solid.
Step 3 : 3-chloro-l-methyl-7-vinyl-lH-pyrrolo[3,2-c]pyridine
[000727] To a solution of 7-bromo-3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridine (700 mg, 2.85 mmol, 1 eq) in Tol. (7 mL) and H2O (1.4 mL) was added K3PO4 (1.21 g, 5.70 mmol, 2 eq) and potassium trifluoro (vinyl)borate (458.31 mg, 3.42 mmol, 1.2 eq). The mixture was added ditert-butyl (cyclopentyl)phosphane dichloropalladium;iron (185.83 mg, 285.13 umol, 0.1 eq) under N2. The mixture was stirred for 16 h at 80 °C. Upon completion, the resulting solution was poured into H2O (30 mL), and then extracted with ethyl acetate (30 mL * 2), the combined organic phase was washed brine (30 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 3:1 to 0: 1) to give 3-chloro-l-methyl-7-vinyl-lH-pyrrolo[3,2-c]pyridine (470 mg, 2.44 mmol, 85.57% yield) as a brown solid.
Step 4 : 3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridine-7-carbaldehyde
[000728] To a solution of 3-chloro-l-methyl-7-vinyl-lH-pyrrolo[3,2-c]pyridine (420 mg, 2.18 mmol, 1 eq) in THF (5 mL) and H2O (1 mL) was added dipotassium dioxido(dioxo)osmium;dihydrate (80.33 mg, 218.02 umol, 0.1 eq) stirred for 0.5 h, then sodium
371
SUBSTITUTE SHEET RULE 26 periodate (1.40 g, 6.54 mmol, 362.43 uL, 3 eq) was added. The mixture was stirred at 25 °C for 12 h. Upon completion, the reaction was poured into H2O (20 mL) and then extracted with ethyl acetate (30 mL * 7). The combined organic phase was washed with brine (30mL * 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate=3:l to 1:1) to give 3 -chloro- 1 -methyl- 1H- pyrrolo[3,2-c]pyridine-7-carbaldehyde (250 mg, 1.28 mmol, 58.92% yield) as a yellow solid.
Step5 : 2-amino-2-(3-chloro-l -methyl- lH-pyrrolo[3,2-c]pyridin-7-yl)acetonitrile
[000729] A solution of 3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridine-7-carbaldehyde (240 mg, 1.23 mmol, 1 eq) in NHVMeOH (3 mL) was added with NH4CI (197.89 mg, 3.70 mmol, 3 eq) and the mixture was stirred at 25 °C for 1 h. TMSCN (367.03 mg, 3.70 mmol, 462.84 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 3 h. LCMS showed a little starting material was remained, then the mixture was stirred for another 2 h at 25 °C. Upon completion, the resulting solution was poured into brine (10 mL), and then extracted with ethyl acetate (10 mL * 2), the combined organic phase was washed brine (20 mL * 3), dried over Na2SO4, filtered and concentrated to give 2-amino-2-(3-chloro-l-methyl-lH-pyrrolo[3,2- c]pyridin-7-yl)acetonitrile (230 mg, crude) as a yellow solid.
Step 6 : (lR,2S,5S)-N-((3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridin-7-yl)(cyano)methyl)-3- ((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[000730] To a solution of 2-amino-2-(3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridin-7- yl)acetonitrile (230 mg, 1.04 mmol, 1 eq) in ACN (3 mL) was added (lR,2S,5S)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxylic acid (455.74 mg, 1.25 mmol, 1.2 eq), 1 -methylimidazole (299.52 mg, 3.65 mmol, 290.79 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (350.95 mg, 1.25 mmol, 1.2 eq), and then the mixture was stirred at 25 °C for 16 h. Upon completion, the resulting solution was poured into brine (10 mL), and then extracted with ethyl acetate (10 mL * 2). The combined organic phase was washed brine (10 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. The residue was purified by prep-HPLC (column: Phenomenex C 18 80 * 40 mm * 3 um; mobile
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SUBSTITUTE SHEET RULE 26 phase: [water (NH-iHCCh)-ACN]; B%: 40%-60%, 8 min), which was further separated by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-ETOH];
B%: 18%-l 8%, 7 min) to give (lR,2S,5S)-N-((3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridin-7- yl)(cyano)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (124.74 mg, 220.00 umol, 21.11% yield, 100% purity) as a white solid. MS (ESI) m/z 567.1 [M+H]+.
[000731] 'H NMR (400 MHz, DMSO-t/r,) 5= 9.57 (d, J= Hz, 1H), 9.41 - 9.25 (m, 1H), 8.92 - 8.80 (m, 1H), 8.56 - 8.38 (m, 1H), 7.86 - 7.54 (m, 1H), 7.01 - 6.76 (m, 1H), 4.40 (s, 1H), 4.23 - 4.14 (m, 1H), 4.00 - 3.92 (m, 1H), 3.92 - 3.86 (m, 3H), 3.78 - 3.68 (m, 1H), 1.66 - 1.60 (m, 1H), 1.33 - 1.28 (m, 1H), 1.07 - 0.98 (m, 12H), 0.86 - 0.79 (m, 3H).
[000732] (lR,2S,5S)-N-((3-chloro-l-methyl-lH-pyrrolo[3,2-c]pyridin-7-yl)(cyano)methyl)- 3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (83.06 mg, 146.49 umol, 14.05% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 567.2[M+H]+.
[000733] 'H NMR (400 MHz, DMSO-t/r,) 5= 9.41 (br d, J= 8.2 Hz, 1H), 9.26 (d, J= 7.7 Hz, 1H), 8.90 - 8.80 (m, 1H), 8.50 (s, 1H), 7.68 (s, 1H), 6.87 - 6.75 (m, 1H), 4.35 - 4.29 (m, 1H), 4.22 - 4.17 (m, 1H), 3.98 - 3.94 (m, 1H), 3.93 - 3.89 (m, 3H), 3.65 - 3.57 (m, 1H), 1.60 - 1.52 (m, 1H), 1.28 - 1.23 (m, 1H), 1.00 - 0.96 (m, 3H), 0.88 - 0.81 (m, 9H), 0.81 - 0.76 (m, 3H).
Example 91: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 253)
Figure imgf000374_0001
373
SUBSTITUTE SHEET RULE 26 Step 1: 7-bromo-l-methyl-pyrazolo[4,3-c]pyridine
[000734] A solution of 7-bromo-lH-pyrazolo[4,3-c]pyridine (700 mg, 3.53 mmol, 1 eq) in DMF (10 mL) was added with NaH (212.08 mg, 5.30 mmol, 60% purity, 1.5 eq) at 0 °C and the mixture was stirred for 0.5 h. Then, CH3I (752.63 mg, 5.30 mmol, 330.10 uL, 1.5 eq) was added to the mixture, and the mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) at 0 °C then extracted with ethyl acetate (15mL * 4). The combined organic layers were washed with brine (30 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 10 / 1 to 4 / 1) to give 7-bromo-l-methyl- pyrazolo[4,3-c]pyridine (470 mg, 2.22 mmol, 62.70% yield, 100% purity) as a white solid. MS (ESI) m/z 212.0 [M+H]+.
Step 2: l-methylpyrazolo[4,3-c]pyridine-7-carbaldehyde
[000735] A solution of butyllithium (2.5 M, 1.06 mL, 1.2 eq) in THF (8 mL) was stirred at - 60 °C, and then 7-bromo-l-methyl-pyrazolo[4,3-c]pyridine (470 mg, 2.22 mmol, 1 eq) in THF (4 mL) was added to the mixture, and the mixture was stirred at -60 °C for 1 h. Then, DMF (486.04 mg, 6.65 mmol, 511.62 uL, 3 eq) was added to the mixture, and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with NHiCl(aq) (20 mL) at 0 °C then extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (30 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 10/1 to 0/1) to give l-methylpyrazolo[4,3-c]pyridine-7-carbaldehyde (270 mg, 1.52 mmol, 68.78% yield, 91% purity) as a white solid. MS (ESI) m/z 162.1 [M+H]+.
Step 3: 2-amino-2-(l-methylpyrazolo[4,3-c]pyridin-7-yl)acetonitrile
[000736] A solution of l-methylpyrazolo[4,3-c]pyridine-7-carbaldehyde (270 mg, 1.68 mmol, 1 eq) in NFh/MeOH (5 mL) was added with NH4CI (179.23 mg, 3.35 mmol, 2 eq) and the solution was stirred at 0 °C for 1.5 h. Then, TMSCN (332.41 mg, 3.35 mmol, 419.18 uL, 2 eq) was added to the mixture, and the mixture was stirred at 25 °C for 14.5 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was diluted with H2O (5 mL) and extracted with EA (5 mL * 4). The combined organic
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SUBSTITUTE SHEET RULE 26 layers were washed with brine (8 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, EA: MeOH = 40:1) to give 2-amino-2-(l-methylpyrazolo[4,3-c]pyridin-7-yl)acetonitrile (180 mg, 875.00 umol, 52.23% yield, 91% purity) as a yellow solid. MS (ESI) m/z 188.2 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000737] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (311.42 mg, 854.70 umol, 1 eq) in ACN (5 mL) was added with TCFH (287.77 mg, 1.03 mmol, 1.2 eq and 1 -methylimidazole (280.68 mg, 3.42 mmol, 272.51 uL, 4 eq), and then the mixture was stirred at 25 °C for 5 min. Then, 2-amino-2-(l-methylpyrazolo[4,3-c]pyridin-7- yl)acetonitrile (160 mg, 854.70 umol, 1 eq was added to the mixture and stirred at 25 °C for 55 min. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with EA (8 mL * 3). The combined organic layers were washed with brine (15 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water(NH4HCO3)- ACN]; B%: 40%-60%, 10 min) to give (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 329.87 umol, 38.59% yield, 80% purity) as a white solid. MS (ESI) m/z 534.3 [M+H]+
Step 4: (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 and Isomer 2
[000738] (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (220 mg, 412.34 umol, 1 eq was separated by SFC (column: DAI CEL CHIRALPAK IC(250 mm * 30 mm, 10 um);mobile phase: [Neu-MeOH]; B%: 10%-10%,14 min) to give (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-
375
SUBSTITUTE SHEET RULE 26 dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (54.89 mg, 102.88 umol, 24.95% yield, 100% purity) as a white solid MS (ESI) m/z 534.2 [M+H]+.
[000739] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.62 - 9.40 (m, 1H), 9.27 (d, J= 7.7 Hz, 1H), 9.22 - 9.13 (m, 1H), 8.58 - 8.51 (m, 1H), 8.40 - 8.33 (m, 1H), 6.97 - 6.83 (m, 1H), 4.43 - 4.31 (m, 1H), 4.21 (s, 1H), 4.18 - 4.09 (m, 3H), 3.95 (dd, J= 5.5, 10.3 Hz, 1H), 3.77 - 3.58 (m, 1H), 3.32 (br s, 2H), 1.66 - 1.55 (m, 1H), 1.32 - 1.23 (m, 1H), 1.06 - 1.00 (m, 9H), 0.99 (s, 3H)
[000740] (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (36.28 mg, 67.32 umol, 16.33% yield, 99% purity) as a whitle solid MS (ESI) m/z 534.2 [M+H]+.
[000741] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.62 - 9.38 (m, 1H), 9.37 - 9.24 (m, 1H), 9.21 - 9.14 (m, 1H), 8.57 - 8.52 (m, 1H), 8.40 - 8.34 (m, 1H), 6.98 - 6.81 (m, 1H), 4.42 - 4.30 (m, 1H), 4.24 - 4.19 (m, 1H), 4.18 - 4.11 (m, 3H), 3.96 (br dd, J= 5.4, 10.0 Hz, 1H), 3.77 - 3.57 (m, 1H), 1.67 - 1.54 (m, 1H), 1.36 - 1.23 (m, 1H), 1.05 - 0.83 (m, 5H), 0.82 - 0.76 (m, 9H).
Example 92: Synthesis of (1R,2S,5S)-N- [cyano- [4-(trifhioromethyl)pyrazolo[l,5-a]pyridin- 3-yl] methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 265)
Figure imgf000377_0001
376
SUBSTITUTE SHEET RULE 26 Step 1: 3-(trifluoromethyl)pyridin-l-ium-l-amine
[000742] Amino 2,4,6-trimethylbenzenesulfonate (45 g, 135.88 mmol, 65% purity, 1 eq) was dissolved in DCM (400 mL) and extracted to get the organic layer, then dried over Na2SO4. The mixture was filtered. To the filtrate was added 3-(trifluoromethyl)pyridine (35.58 g, 241.91 mmol, 1.78 eq) and the resulting mixture was stirred at 25 °C for 16 h. Upon completion, 3- (trifluoromethyl)pyridin-l-ium-l -amine (36 g, crude) was obtained as yellow solid. MS (ESI) m/z 163.1 [M+H]+.
Step 2: methyl 4-(trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carboxylate
[000743] To a solution of 3-(trifluoromethyl)pyridin-l-ium-l-amine (36 g, 220.70 mmol, 1 eq) in DMF (350 mL) was added K2CO3 (45.75 g, 331.04 mmol, 1.5 eq), followed by methyl prop-2-ynoate (22.27 g, 264.84 mmol, 22.05 mL, 1.2 eq). After the addition, the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was quenched by addition H2O (10 mL) at 25 °C, and then diluted with saturated brine (300 mL) and extracted with ethyl acetate (50 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by MPLC (SiCh, petroleum ether: ethyl acetate = 1 :0 to 95:5) to get methyl 4-(trifluoromethyl)pyrazolo[l,5-a]pyridine-3- carboxylate (2.5 g, 9.93 mmol, 4.50% yield, 97% purity) as yellow solid. MS (ESI) m/z 245.1 [M+H]+.
Step 3: 4-(trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carboxylic acid
[000744] To a solution of methyl 4-(trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carboxylate (2.5 g, 10.24 mmol, 1 eq) in THE (10 mL)and H2O (10 mL) was added LiOH.H2O (859.31 mg, 20.48 mmol, 2 eq). The mixture was stirred at 30 °C for 16 h. The mixture was stirred at 30 °C for 16 h. Upon completion, the mixture was added H2O (100 mL) and extracted with ethyl acetate (100 mL * 2). The water layer was acified by 10% citric acid solution to pH=3~4, then extracted with ethyl acetate (50 mL * 3). The organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 4- (trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carboxylic acid (1.75 g, crude) as yellow solid. MS (ESI) m/z 231.2 [M+H]+.
377
SUBSTITUTE SHEET RULE 26 Step 4: imidazol-l-yl-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methanone
[000745] A mixture of 4-(trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carboxylic acid (500 mg, 2.17 mmol, 1 eq) in THF (15 mL) was cooled to 0 °C, and then CDI (528.42 mg, 3.26 mmol, 1.5 eq) was added drop- wise at 0 °C. The mixture was stirred at 20 °C for 16 h, and upon completion, the mixture imidazol-l-yl-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3- yl]methanone (1.2 g, crude) was obtained as a yellow liquid and used for next step without further purification. MS (ESI) m/z 281.0 [M+H]+.
Step 5: [4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methanol
[000746] A solution of imidazol-l-yl-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3- yl]methanone (600 mg, 2.14 mmol, 1 eq) in THF (15 mL) was stirred at 0 °C, then NaBHt (405.05 mg, 10.71 mmol, 5 eq) in H2O (7.5 mL) was added at 0 °C under N2. The mixture was stirred at 20 °C for 1 h. (batch 2). Upon completion, the reaction mixture was quenched by addition IM HC1 (15 mL) and stirred for 30 min, the solution was added aq. NaHCCh to pH = 8 and extracted with EA (30 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, ethyl acetate: petroleum ether = 1 :0 to 3: 1) to give [4- (trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methanol (600 mg, 1.39 mmol, 64.81% yield) as a yellow solid. MS (ESI) m/z 217.0 [M+H]+.
Step 6: 4-(trifluoromethyl)pyrazolo[l ,5-a]pyridine-3-carbaldehyde
[000747] A mixture of [4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methanol (700 mg, 3.24 mmol, 1 eq) in CHCh (30 mL) was added with MnCh (4.22 g, 48.58 mmol, 15 eq), and the mixture was stirred at 70 °C for 3 h. Upon completion, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, ethyl acetate: petroleum ether = 1 :0 to 0: 1) to give 4- (trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carbaldehyde (500 mg, 2.33 mmol, 72.10% yield) as a yellow solid. MS (ESI) m/z 214.9 [M+H]+.
Step 7: 2-amino-2-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]acetonitrile
378
SUBSTITUTE SHEET RULE 26 [000748] To a solution of 4-(trifluoromethyl)pyrazolo[l,5-a]pyridine-3-carbaldehyde (500 mg, 2.33 mmol, 1 eq) in NFh/MeOH (50 mL) was added NH4CI (374.69 mg, 7.00 mmol, 3 eq). After 1 h, TMSCN (926.54 mg, 9.34 mmol, 1.17 mL, 4 eq) was added drop-wise, the mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove MeOH (50 mL). The residue was diluted with H2O (30 mL) and extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2- [4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]acetonitrile (400 mg, crude) as a yellow oil.
Step 8: (lR,2S,5S)-N-[cyano-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide
[000749] To a solution of 2-amino-2-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3- yl]acetonitrile (400 mg, 1.67 mmol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (788.84 mg, 2.17 mmol, 1.3 eq) in ACN (10 mL) was added NMI (546.94 mg, 6.66 mmol, 531.01 uL, 4 eq) at 0 °C, and then TCFH (560.73 mg, 2.00 mmol, 1.2 eq) was added. The mixture was stirred at 0 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (40 mL) and extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250 * 50 mm * 10 um; mobile phase: [water( NH4HCO3) - ACN]; B%: 35% - 75%, 10 min) and by separated SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Hexane-IPA]; B%: 20% - 20%, 7 min, Isomer 1 : RT= 3.701; Isomer 2: RT=2.917) to give (lR,2S,5S)-N-[cyano-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide Isomer 1 (71 mg, 121.05 umol, 7.27% yield, 100% purity) as a white solid. MS (ESI) m/z 587.3 [M+H]+.
[000750] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.48 (d, J= 6.9 Hz, 1H), 9.32 (br s, 1H), 9.10 (d, J= 7.0 Hz, 1H), 8.52 (s, 1H), 7.92 (d, J= 7.3 Hz, 1H), 7.16 (t, J= 7.1 Hz, 1H), 6.10 (d, J =
379
SUBSTITUTE SHEET RULE 26 6.9 Hz, 1H), 4.38 (br s, 1H), 4.29 (s, 1H), 3.95 - 3.84 (m, 1H), 3.71 (d, J= 10.4 Hz, 1H), 1.62 -
1.53 (m, 1H), 1.35 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 0.95 (s, 9H), 0.84 (s, 3H)
[000751] (lR,2S,5S)-N-[cyano-[4-(trifluoromethyl)pyrazolo[l,5-a]pyridin-3-yl]methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (65 mg, 110.82 umol, 6.65% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 587.3 [M+H]+.
[000752] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.45 (d, J= 7.3 Hz, 1H), 9.37 (br d, J= 5.0 Hz, 1H), 9.10 (d, J = 7.0 Hz, 1H), 8.52 (s, 1H), 7.93 (d, J= 7.3 Hz, 1H), 7.17 (t, J= 7.1 Hz, 1H), 6.08 (d, J= 7.4 Hz, 1H), 4.58 - 4.38 (m, 1H), 4.26 (s, 1H), 3.95 - 3.85 (m, 1H), 3.70 (d, J= 10.5 Hz, 1H), 1.59 - 1.37 (m, 1H), 1.25 - 1.14 (m, 1H), 1.10 - 0.89 (m, 12H), 0.88 - 0.79 (m, 3H).
Example 93: Synthesis of (lR,2S,5S)-N-(cyano(4-ethynylpyrazolo[l,5-a]pyridin-3- yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 266)
Figure imgf000381_0001
Step 1: l-amino-3-(hydroxymethyl)pyridin-l-ium
[000753] O-(mesitylsulfonyl) hydroxylamine (16.69 g, 50.40 mmol, 65% purity, 1.1 eq) was dissolved in DCM (100 m ) and dried over Na2SO4. The mixture was filtered. To the filtrate was added pyridin-3-ylmethanol (5 g, 45.82 mmol, 4.39 m , 1 eq) and the resulting mixture was
380
SUBSTITUTE SHEET RULE 26 stirred at 20 °C for 16 h. Upon completion, the reaction l-amino-3 -(hydroxymethyl) pyridin-1- ium (5.73 g, crude) as brown oil in theory was used for next step directly without further purificaion.
Step 2 : methyl 4-(hydroxymethyl)pyrazolo[l,5-a]pyridine-3-carboxylate
[000754] To a solution of l-amino-3-(hydroxymethyl) pyridin-l-ium (5.7 g, 45.55 mmol, 1 eq) in DMF (100 mL) was added K2CO3 (9.44 g, 68.32 mmol, 1.5 eq), followed by methyl propiolate (4.60 g, 54.66 mmol, 4.55 mL, 1.2 eq). After the addition, the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with brine (500 mL) and extracted with EtOAc (200 mL * 2). The organic layers were separated, dried over Na2SO4, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 0:1) to afford methyl 4- (hydroxymethyl)pyrazolo[l,5-a]pyridine-3-carboxylate (2.8 g, 10.62 mmol, 23.31% yield, 78.2% purity) and methyl 4-(hydroxymethyl)pyrazolo[l,5-a]pyridine-3 -carboxylate (1.2 g, 4.89 mmol, 10.75% yield, 84.1% purity) as a yellow solid.
Step 3 : methyl 4-formylpyrazolo[l,5-a]pyridine-3-carboxylate
[000755] To a solution of methyl 4-(hydroxymethyl)pyrazolo[l,5-a]pyridine-3-carboxylate (2 g, 9.70 mmol, 1 eq) in ACN (40 mL) was added (l,l-diacetoxy-3-oxo-lX5,2-benziodoxol-l-yl) acetate (4.94 g, 11.64 mmol, 3.60 mL, 1.2 eq) at 0 °C, and the reaction mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched with aq NaHCO3:Na2S2O3(l: l, 70 mL) and extracted with ethyl acetate (3 * 50 mL). The combined organic phase was washed brine (40 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 5: 1 to 0: 1) to give methyl 4-formylpyrazolo[l,5-a]pyridine-3-carboxylate (1.59 g, 7.79 mmol, 80.28% yield) as a yellow solid.
Step 4 : methyl 4-ethynylpyrazolo[l,5-a]pyridine-3-carboxylate
[000756] To a solution of methyl 4-formylpyrazolo[l,5-a]pyridine-3-carboxylate (1.59 g, 2.45 mmol, 1 eq) in MeOH (5 mL) was added dimethyl (l-diazo-2-oxopropyl)phosphonate (658.61 mg, 3.43 mmol, 1.4 eq) and K2CO3 (676.90 mg, 4.90 mmol, 2 eq) at 0 °C, and then the
381
SUBSTITUTE SHEET RULE 26 mixture was stirred for 12 h at 25 °C. Upon completion, the resulting solution was poured into brine (20 mL), and then extracted with ethyl acetate (20 mL * 2), the combined organic phase was washed brine (15 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 3: 1 to 0:1) to give methyl 4-ethynylpyrazolo[l,5-a]pyridine-3-carboxylate (1.03 g, 1.53 mmol, 62.62% yield) as a white solid.
Step 5 : (4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methanol
[000757] To a solution of methyl 4-ethynylpyrazolo[l,5-a]pyridine-3-carboxylate (1.03 g, 5.13 mmol, 1 eq) in THF (10 mL) was added DIBAL-H (1 M, 25.65 mL, 5 eq) at 0 °C, and then the mixture was stirred for 1 h at 25 °C. Upon completion, the resulting solution was added the sat.seignettesalt (100 mL), and then extracted with ethyl acetate (100 mL * 3), the combined organic phase was washed with brine (100 mL * 3), dried over Na2SO4, filtered and concentrated to give (4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methanol (820 mg, crude) as a yellow solid.
Step 6 : 4-ethynylpyrazolo[l,5-a]pyridine-3-carbaldehyde
[000758] To a solution of (4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methanol (820 mg, 4.76 mmol, 1 eq) in ACN (8 mL) was added (l,l-diacetoxy-3-oxo-lX5,2-benziodoxol-l-yl) acetate (2.42 g, 5.71 mmol, 1.77 mL, 1.2 eq) at 0 °C, and the reaction mixture was stirred at 25 °C for 2 h. (l,l-Diacetoxy-3-oxo-lX5,2-benziodoxol-l-yl) acetate (605.98 mg, 1.43 mmol, 442.32 uL, 0.3 eq) was added, the mixture was stirred for another 1 h at 25 °C. Upon completion, the reaction mixture was quenched with aq NaHCO3:Na2S2O3(l : 1, 30 mL) and extracted with ethyl acetate (30 * 3 mL), the combined organic phase was washed brine (30 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 3: 1 to 0:1) to give 4-ethynylpyrazolo[l,5- a]pyridine-3-carbaldehyde (640 mg, 3.76 mmol, 78.97% yield) as a yellow solid.
Step 7 : 2-amino-2-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[000759] A solution of 4-ethynylpyrazolo[l,5-a]pyridine-3-carbaldehyde (640 mg, 3.76 mmol, 1 eq) in NHs/MeOH (7 mL) was added with NH4CI (603.53 mg, 11.28 mmol, 3 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (1.12 g, 11.28 mmol, 1.41 mL, 3 eq) was
382
SUBSTITUTE SHEET RULE 26 added at 0 °C, the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL), and then extracted with ethyl acetate (30 mL * 3). The combined organic layers were washed with brine (20 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-ethynylpyrazolo[l,5-a]pyridin-3- yl)acetonitrile (530 mg, crude) as a yellow solid.
Step 8 : (lR,2S,5S)-N-(cyano(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000760] To a solution of 2-amino-2-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile (520 mg, 2.65 mmol, 1 eq) in ACN (7 mL) was added (lR,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxylic acid (965.64 mg, 2.65 mmol, 1 eq), 1 -methylimidazole (761.55 mg, 9.28 mmol, 739.37 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (892.33 mg, 3.18 mmol, 1.2 eq), and then the mixture was stirred at 25 °C for 50 min. Upon completion, the resulting solution was concentrated in vacuum. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3) -ACN]; B%: 30%-60%, 8 min), which was further separated by SFC (column: REGIS(S,S)WHELK-O1 (250mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 33%-33%, 6 min) to give (lR,2S,5S)-N-(cyano(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide Isomer 1 (68.43 mg, 122.42 umol, 4.62% yield, 97.06% purity) as a white solid. MS (ESI) m/z 543.2[M+H]+.
[000761] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.36 (br s, 1H), 9.29 (d, J= 6.8 Hz, 1H), 8.84 (d, J= 6.8 Hz, 1H), 8.30 (s, 1H), 7.58 (d, J= 7.0 Hz, 1H), 7.01 (t, J= 7.1 Hz, 1H), 6.52 (d, J = 6.8 Hz, 1H), 4.75 (s, 1H), 4.41 (s, 1H), 4.28 (s, 1H), 3.91 (dd, J= 5.3, 10.2 Hz, 1H), 3.71 (d, J = 10.4 Hz, 1H), 1.53 (dd, J= 5.5, 7.5 Hz, 1H), 1.32 (d, J= 7.6 Hz, 1H), 1.01 (s, 9H), 0.98 (s, 3H), 0.83 (s, 3H).
[000762] To give (lR,2S,5S)-N-(cyano(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl)-3-((S)- 3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
383
SUBSTITUTE SHEET RULE 26 carboxamide Isomer 2 (69.15 mg, 125.04 umol, 4.72% yield, 98.11% purity) as a white solid.
MS (ESI) m/z 543.2[M+H]+.
[000763] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.46 - 9.31 (m, 2H), 8.83 (d, J= 7.0 Hz, 1H), 8.30 (s, 1H), 7.57 (d, J= 7.1 Hz, 1H), 7.01 (t, J= 7.1 Hz, 1H), 6.54 (d, J= 6.5 Hz, 1H), 4.59 (s, 1H), 4.41 (s, 1H), 4.33 (s, 1H), 3.90 (dd, J= 5.3, 10.3 Hz, 1H), 3.72 (d, J= 10.5 Hz, 1H), 1.61 - 1.54 (m, 1H), 1.41 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 0.96 (s, 9H), 0.84 (s, 3H).
Example 94: Synthesis of (lR,2S,5S)-N-[cyano-[5-(trifluoromethyl)imidazo[l,2-a]pyridin-3- yl] methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 268)
Figure imgf000385_0001
Step 1: 5-(trifluoromethyl) imidazo [1, 2-a] pyridine-3-carbaldehyde
[000764] A solution of 6-(trifluoromethyl)pyridin-2-amine (500 mg, 3.08 mmol, 1 eq), 2- bromopropanedial (1.40 g, 9.25 mmol, 3 eq) in EtOH (20 mL) was added NaHCCh (259.11 mg, 3.08 mmol, 119.96 uL, 1 eq) and the mixture was stirred at 90 °C for 14 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove EtOH. The residue was diluted with H2O (40 mL) and extracted with ethyl acetate (35 mL * 3). The combined organic layers were washed with brine (70 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 77:23) and TLC (Platel, SiO2, Petroleum ether:Ethylacetate = 1: 1) to give a 5-(trifluoromethyl) imidazo [1, 2-a] pyridine-3-carbaldehyde (338 mg, 1.29 mmol, 41.96% yield, 82% purity) as a yellow solid. MS (ESI) m/z 215.0 [M+H]+.
384
SUBSTITUTE SHEET RULE 26 Step 2: 2-amino-2-[5-(trifluoromethyl)imidazo[l , 2-a]pyri din-3 -yl]acetonitrile
[000765] A solution of 5-(trifluoromethyl)imidazo[l,2-a]pyridine-3-carbaldehyde (300 mg, 1.40 mmol, 1 eq), NHVMeOH (7 M, 4 mL, 19.99 eq) and Ti(i-PrO)4 (597.24 mg, 2.10 mmol, 620.18 uL, 1.5 eq) in DCM (6 mL) was stirred at 20 °C for 1 h, and then added TMSCN (416.96 mg, 4.20 mmol, 525.80 uL, 3 eq) at 0 °C, and the mixture was stirred at 20 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O (25 mL), and then extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a 2-amino-2-[5- (trifluoromethyl)imidazo[l,2-a]pyri din-3 -yl]acetonitrile (326 mg, crude) as a brown solid. MS (ESI) m/z 241.1 [M+H]+.
Step 3: (lR,2S,5S)-N-[cyano-[5-(trifluoromethyl)imidazo[l,2-a]pyridin-3-yl]methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide
[000766] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (544.00 mg, 1.49 mmol, 1.1 eq), 2-amino-2-[5-(trifluoromethyl)imidazo[l,2-a]pyri din-3 - yl]acetonitrile (326 mg, 1.36 mmol, 1 eq) in ACN (20 mL) was added with 1 -methylimidazole (334.32 mg, 4.07 mmol, 324.58 uL, 3 eq) and [chloro (dimethylamino) methylene] -dimethylammonium hexafluorophosphate (761.65 mg, 2.71 mmol, 2 eq), and the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (40 mL), and then extracted with EA (35 mL * 3). The combined organic layers were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80 * 40 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 8 min) to give a (lR,2S,5S)-N-[cyano-[5- (trifluoromethyl)imidazo[l,2-a]pyridin-3-yl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (135 mg, 229.94 umol, 16.94% yield, 99.9% purity) as a white solid. MS (ESI) m/z 587.2 [M+H]+.
[000767] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.57 (dd, J= 6.8, 19.8 Hz, 1H), 9.34 (br s, 1H), 8.17 (d, J= 2.7 Hz, 1H), 8.09 (d, J= 9.0 Hz, 1H), 7.78 (t, J= 6.6 Hz, 1H), 7.53 (t, J= 7.7 Hz,
385
SUBSTITUTE SHEET RULE 26 1H), 6.29 (t, J= 6.7 Hz, 1H), 4.39 (s, 1H), 4.26 (d, J= 6.7 Hz, 1H), 3.90 (dd, J= 5.4, 10.2 Hz, 1H), 3.74 - 3.66 (m, 1H), 1.57 (ddd, J= 5.4, 7.5, 15.3 Hz, 1H), 1.37 - 1.15 (m, 1H), 1.04 - 0.95 (m, 12H), 0.82 (d, J= 2.3 Hz, 3H).
[000768] 'H NMR (400 MHz, DMSO-t/r,) 5 = 8.14 (s, 1H), 8.06 (d, J= 9.0 Hz, 1H), 7.77 (t, J = 6.1 Hz, 1H), 7.53 (t, .7= 7,9 Hz, 1H), 6.27 (d, J= 2.1 Hz, 1H), 4.36 (d, J = 2.2 Hz, 1H), 4.24 (d, J= 8.6 Hz, 1H), 3.89 (dd, J= 5.5, 10.4 Hz, 1H), 3.68 (dd, J= 7.5, 10.2 Hz, 1H), 1.56 (ddd, J = 5.4, 7.6, 14.9 Hz, 1H), 1.33 - 1.11 (m, 1H), 1.06 - 0.90 (m, 12H), 0.80 (d, J= 3.1 Hz, 3H).
Example 95: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 269)
Figure imgf000387_0001
Step 1: 5-bromoimidazo[l,2-a]pyridine-3-carbaldehyde
[000769] To a solution of 5-bromoimidazo[l,2-a]pyridine (3 g, 15.23 mmol, 1 eq) in AcOH (30 mL) was added HMTA (4.27 g, 30.45 mmol, 2 eq) at 0 °C under N2, the mixture was stirred at 85 °C for 16 h. Upon completion, the mixture was concentrated under reduced pressure to remove AcOH, and poured into 30% aq.NaOH (60 mL) at 20 °C to adjust pH = 9, and then extracted with EA (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 10: 1 to 1 :1) to give the product 5-bromoimidazo[l,2-a]pyridine-3- carbaldehyde (650 mg, 2.60 mmol, 17.07% yield, 90% purity) as white solid. MS (ESI) m/z 225.0, 227.0 [M+H]+.
Step 2: 5-(2-trimethylsilylethynyl)imidazo[l ,2-a]pyridine-3-carbaldehyde
386
SUBSTITUTE SHEET RULE 26 [000770] To a solution of 5-bromoimidazo[l,2-a]pyridine-3-carbaldehyde (550 mg, 2.44 mmol, 1 eq) in dioxane (10 mL) was added TEA (741.92 mg, 7.33 mmol, 1.02 mL, 3 eq), and then Cui (186.18 mg, 977.60 umol, 0.4 eq) and Pd(PPhs)4 (564.84 mg, 488.80 umol, 0.2 eq), ethynyl(trimethyl)silane (288.05 mg, 2.93 mmol, 406.28 uL, 1.2 eq) was added to the mixture slowly under N2. The mixture was stirred at 25 °C for 16 h. Upon completion, the mixture was diluted with H2O (30 mL) and filtered through celite then was extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum to give the product 5-(2-trimethylsilylethynyl)imidazo[l,2-a]pyridine-3-carbaldehyde (200 mg, 701.47 umol, 28.70% yield, 85% purity) as yellow solid. MS (ESI) m/z 243.1 [M+H]+
Step 3: 5-ethynylimidazo[l,2-a]pyridine-3-carbaldehyde
[000771] To a solution of 5-(2-trimethylsilylethynyl)imidazo[l,2-a]pyridine-3-carbaldehyde (200 mg, 825.26 umol, 1 eq) in MeOH (5 mL) was added K2CO3 (285.15 mg, 2.06 mmol, 2.5 eq), and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was filtered to give the product 5-ethynylimidazo[l,2-a]pyridine-3-carbaldehyde (140 mg, crude) in yellow liquid (solution in MeOH). MS (ESI) m/z 171.1 [M+H]+.
Step 4: 2-amino-2-(5-ethynylimidazo[l,2-a]pyridin-3-yl)acetonitrile
[000772] To a solution of 5-ethynylimidazo[l,2-a]pyridine-3-carbaldehyde (140 mg, 822.72 umol, 1 eq) in DCM (5 mL) was added Ti(i-PrO)4 (350.74 mg, 1.23 mmol, 364.21 uL, 1.5 eq) and NHs/MeOH (5 mL), and the mixture was stirred at 25 °C for 1 h. TMSCN (244.87 mg, 2.47 mmol, 308.78 uL, 3 eq) was added at 0°C, and the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) and then was extracted with DCM (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum to give 2-amino-2-(5-ethynylimidazo[l,2-a]pyridin-3-yl)acetonitrile (96 mg, crude) as a yellow solid. MS (ESI) m/z 197.2 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
387
SUBSTITUTE SHEET RULE 26 [000773] To a solution of 2-amino-2-(5-ethynylimidazo[l,2-a]pyridin-3-yl)acetonitrile (96 mg, 489.28 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (178.27 mg, 489.28 umol, 1 eq) in ACN (6 mL) was added NMI (120.51 mg, 1.47 mmol, 117.00 uL, 3 eq) and TCFH (274.56 mg, 978.55 umol, 2 eq), the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (20 mL) and then extracted with ethyl acetate (7 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-TLC (SiCh, Petroleum ether:Ethyl acetate = 0: 1) to give the product (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (110 mg, 141.92 umol, 29.01% yield, 70% purity) as yellow solid. MS (ESI) m/z 543.2 [M+H]+.
Step 6: (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000774] The (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-
2-carboxamide (110 mg, 202.75 umol, 1 eq) was separated by SFC (column: (s, s) WHELK-01 (250 mm * 30 mm, 5 um); mobile phase: [Neu - IP A]; B%: 35% - 35%, 9 min) to get product (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide Isomer 1 (13.73 mg, 24.05 umol, 11.86% yield, 95.024% purity) as white solid. MS (ESI) m/z 543.2 [M+H]+
[000775] 'H NMR (400 MHz, DMSO-t/r,) 5 ppm 9.36 (d, J= 6.6 Hz, 2H), 7.90 (s, 1H), 7.79 (dd, J= 3.6, 6.6 Hz, 1H), 7.39 - 7.30 (m, 2H), 6.86 (br d, J= 6.4 Hz, 1H), 5.06 - 4.93 (m, 1H), 4.40 (br d, J= 6.4 Hz, 1H), 4.25 (s, 1H), 3.91 (dd, J= 5.4, 10.4 Hz, 1H), 3.71 (br d, J= 10.4 Hz, 1H), 1.59 - 1.50 (m, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.04 - 0.92 (m, 12H), 0.81 (s, 3H).
[000776] To get product (lR,2S,5S)-N-[cyano-(5-ethynylimidazo[l,2-a]pyridin-3-yl)methyl]-
3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-
388
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (2.16 mg, 3.84 umol, 1.89% yield, 96.495% purity) as white solid. MS (ESI) m/z 543.2 [M+H]+
[000777] 'H NMR (400 MHz, DMSO-t/r,) 5 ppm 9.70 - 9.21 (m, 2H), 7.90 (s, 1H), 7.83 - 7.72 (m, 1H), 7.42 - 7.27 (m, 2H), 6.89 (br d, J= 4.2 Hz, 1H), 4.74 (br s, 1H), 4.42 (br d, J= 7.4 Hz, 1H), 4.32 (s, 1H), 3.90 (dd, J= 5.4, 10.4 Hz, 1H), 3.72 (d, J= 10.6 Hz, 1H), 1.62 - 1.50 (m, 1H), 1.41 (br d, J= 7.6 Hz, 1H), 1.05 - 0.94 (m, 12H), 0.87 - 0.80 (m, 3H).
Example 96: Synthesis of (lR,2S,5S)-N-[(4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)- cyano-methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 270)
Figure imgf000390_0001
Step 1: amino 2,4,6-trimethylbenzenesulfonate
[000778] TFA (225.87 g, 1.98 mol, 146.67 mL, 14.20 eq) was cooled to 0 °C and (tertbutoxycarbonylamino) 2,4,6-trimethylbenzenesulfonate (44 g, 139.51 mmol, 1 eq) was added portionwsie over 10 min. After the addition, the mixture was stirred at 0 °C for 1.5 h. Upon completion, the reaction mixture was quenched by ice and water (450 mL) and maintained intertemperature below 5 °C. The mixture was stirred for 15 min to form a slurry. The slurry was
389
SUBSTITUTE SHEET RULE 26 filtered and the filter cake was washed with water (240 mL 3) and air dried to afford amino 2,4,6-trimethylbenzenesulfonate (45 g, 135.88 mmol, 97.39% yield, 65% purity) as white solid, which contained about 35 % water. MS (ESI) m/z 216.0 [M+H]+.
Step 2: 3-bromo-5-fluoro-pyridin-l-ium-l-amine
[000779] Amino 2,4,6-trimethylbenzenesulfonate (45 g, 135.88 mmol, 65% purity, 1.2 eq) was dissolved in DCM (500 mL) and dried over Na2SO4, and the mixture was filtered. To the filtrate was added 3-bromo-5-fluoro-pyridine (20 g, 113.65 mmol, 1 eq) and the resulting mixture was stirred at 20 °C for 24 h. Upon completion, the reaction mixture was filtered, the filter cake was washed with DCM (100 mL * 2) and dried in air to afford residue was 3-bromo- 5-fluoro-pyridin-l-ium-l -amine (47 g, crude) as white solid, which was used for next step without further purification. MS (ESI) m/z 191.1 [M+H]+.
Step 3: methyl 4-bromo-6-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate
[000780] To a mixture of 3-bromo-5-fluoro-pyridin-l-ium-l-amine (47 g, 127.04 mmol, 51.9% purity, 1 eq) in DMF (200 mL) was added K2CO3 (26.34 g, 190.56 mmol, 1.5 eq) and methyl prop-2-ynoate (12.82 g, 152.45 mmol, 12.69 mL, 1.2 eq) in turn. After the addition, the reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was diluted with brine (600 mL), and extracted with EtOAc (200 mL * 2). The organic layers were separated, dried over Na2SO4, concentrated in a vacuum to afford a residue. The residue was purified by prep-HPLC (column: Phenomenex luna c 18 250 mm * 100 mm * 15 um; mobile phase: [water (LA)-ACN]; B%: 30%-60%, 20 min) to give methyl 4-bromo-6-fluoro- pyrazolo[l,5-a]pyridine-3-carboxylate (2.67 g, 8.83 mmol, 12.05% yield, 90.29% purity) as a yellow solid. MS (ESI) m/z 273.0 [M+H]+.
Step 4: (4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)methanol
[000781] To a solution of methyl 4-bromo-6-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate (1.3 g, 4.76 mmol, 1 eq) in THE (25 mL) was added DIBAL-H (1 M, 19.04 mL, 4 eq) under N2 at -60°C. The mixture was stirred at 20 °C for 16 h. Two batches were set up in parallel. Upon completion, the reaction mixture was quenched by addition potassium sodium tartrate (30 mL), and then extracted with EtOAc (30mL *3). The combined organic layers were concentrated
390
SUBSTITUTE SHEET RULE 26 under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 5: 1 to 4:1) to give (4-bromo-6-fluoro-pyrazolo[l,5- a]pyri din-3 -yl)methanol (1.9 g, 6.86 mmol, 72.04% yield, 88.464% purity) as a yellow solid. MS (ESI) m/z 245.0 [M+H]+.
Step 5: 4-bromo-6-fluoro-pyrazolo[l,5-a]pyridine-3-carbaldehyde
[000782] To a solution of (4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)methanol (1.2 g, 4.90 mmol, 1 eq) in DCM (20 mL) was added Dess-Martin (3.53 g, 8.32 mmol, 2.58 mL, 1.7 eq). The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with DCM (30mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 85:15 to 60:40) to give 4-bromo-6-fluoro-pyrazolo[l,5-a]pyridine-3- carbaldehyde (1.3 g, 2.67 mmol, 54.62% yield, 50% purity) as a yellow solid. MS (ESI) m/z 243.0 [M+H]+.
Step 6: 2-amino-2-(4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[000783] To a solution of 4-bromo-6-fluoro-pyrazolo[l,5-a]pyridine-3-carbaldehyde (435 mg, 1.79 mmol, 1 eq) in DCM (5 mL) was added Ti(i-PrO)4 (763.07 mg, 2.68 mmol, 792.39 uL, 1.5 eq) and NH3/MeOH (7 M, 1.02 mL, 4 eq) stirred 1 h at 20 °C, then TMSCN (532.70 mg, 5.37 mmol, 671.76 uL, 3 eq) was added at 20 °C. The mixture was stirred at 20 °C for 15 h. Two batches were set up in parallel. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to 1 :1) to give 2-amino-2-(4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3- yl)acetonitrile (800 mg, 2.02 mmol, 56.48% yield, 68% purity) as a yellow solid. MS (ESI) m/z 252 [M+H]+.
Step 4: (lR,2S,5S)-N-[(4-bromo-6-fhioro-pyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
391
SUBSTITUTE SHEET RULE 26 [000784] A mixture of 2-amino-2-(4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)acetonitrile (800 mg, 2.02 mmol, 68% purity, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (736.65 mg, 2.02 mmol, 1 eq) and 1 -methylimidazole (497.98 mg, 6.07 mmol, 483.48 uL, 3 eq) in ACN (10 mb) was added with [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (1.13 g, 4.04 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate Cl 8 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 44%-74%, 20 min) to give (lR,2S,5S)-N-[(4-bromo-6-fluoro- pyrazolo[ 1 , 5-a] pyridin-3 -yl)-cyano-methyl] -3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (630 mg, 1.01 mmol, 49.89% yield, 98.53% purity) as a white solid. MS (ESI) m/z 615.2 [M+H]+.
Step 5: (lR,2S,5S)-N-[(4-bromo-6-fhioro-pyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
[000785] (lR,2S,5S)-N-[(4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (600 mg) was separated by SEC (column: REGIS(S,S)WHELK-O1 (250mm * 25 mm, 10 um); mobile phase: [Neu-IPA];B%: 23%-23%, 5 min) to give (lR,2S,5S)-N-[(4-bromo-6-fluoro-pyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (231.02 mg, 371.26 umol, 38.08% yield, 98.9% purity) as a white solid. MS (ESI) m/z 615.2 [M+H]+.
[000786] 'H NMR (400 MHz, DMSO-t#) 5 = 9.36 (d, J= 6.9 Hz, 2H), 9.23 (dd, J= 1.9, 4.3 Hz, 1H), 8.33 (s, 1H), 8.02 (dd, J= 1.9, 8.1 Hz, 1H), 6.56 (d, J = 6.9 Hz, 1H), 4.40 (d, J= 6.0 Hz, 1H), 4.27 (s, 1H), 3.91 (dd, J= 5.4, 10.3 Hz, 1H), 3.70 (d, J= 10.4 Hz, 1H), 1.59 - 1.49 (m, 1H), 1.32 (d, J = 7.6 Hz, 1H), 1.03 - 0.97 (m, 12H), 0.83 (s, 3H).
392
SUBSTITUTE SHEET RULE 26 [000787] To give ( 1 R,2S, 5 S)-N- [(4-bromo-6-fluoro-pyrazolo [ 1 , 5 -a] pyndin-3 -yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (276.55 mg, 443.53 umol, 45.49% yield, 98.7% purity) as a white solid. MS (ESI) m/z 615.2 [M+H]+.
[000788] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.44 (d, J= 6.4 Hz, 1H), 9.37 (d, J= 7.5 Hz, 1H), 9.23 (dd, J= 2.0, 4.4 Hz, 1H), 8.33 (s, 1H), 8.01 (dd, J= 2.0, 8.2 Hz, 1H), 6.55 (d, J= 6.4 Hz, 1H), 4.41 (d, J= 7.5 Hz, 1H), 4.31 (s, 1H), 3.90 (dd, J= 5.4, 10.3 Hz, 1H), 3.72 (d, J= 10.5 Hz, 1H), 1.58 (dd, J= 5.3, 7.5 Hz, 1H), 1.36 (d, J= 7.6 Hz, 1H), 1.05 - 1.02 (m, 3H), 0.97 (s, 9H), 0.84 (s, 3H).
Example 97: Synthesis of (lR,2S,5S)-N-[cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 272)
Figure imgf000394_0001
Step 1: (Z)-l-(3,5-dichlorophenyl)-N-(2,2-dimethoxyethyl)methanimine
393
SUBSTITUTE SHEET RULE 26 [000789] A solution of 3,5-dichlorobenzaldehyde (3 g, 17.14 mmol, 1 eq) in toluene (50 mL) was added with 2, 2-dimethoxy ethanamine (1.80 g, 17.14 mmol, 1.87 mL, 1 eq). The mixture was stirred at 130 °C for 8 h. Upon completion, the mixture was concentrated under the reduced pressure to give (Z)-l-(3,5-dichlorophenyl)-N-(2,2-dimethoxyethyl)methanimine (4.5 g, crude) as a yellow oil.
Step 2: 5,7-dichloroisoquinoline
[000790] A solution of (Z)-l-(3,5-dichlorophenyl)-N-(2,2-dimethoxyethyl)methanimine (4.5 g, 17.17 mmol, 1 eq) in H2SO4 (45 mL) was stirred at 140 °C for 2 h. Upon completion, the mixture was added to the sat. NaOH (1000 mL) with dropwise under 0 - 10 °C to adjust pH to 7 - 10, and extracted with EA (300 mL * 2). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered and concentratedunder reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 95:5 to 1: 1) to give the product. MS (ESI) m/z 198.0 [M+H]+
Step 3: 4-bromo-5,7-dichloro-isoquinoline
[000791] A solution of 5,7-dichloroisoquinoline (1 g, 5.05 mmol, 1 eq) in HOAc (120 mL) was added NBS (1.35 g, 7.57 mmol, 1.5 eq). The mixture was stirred at 110 °C for 2 h. Upon completion, the mixture was added to the sat. NaHCCh (300 mL) with dropwise under 0 - 10 °C, and extracted with ethyl acetate (100 mL * 2). The combined organic layers were washed with brine (100 mL * 2), dried over Na2SO4, filtered and concentratedunder reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 99:l to 1: 1) to give the product 4-bromo-5,7-dichloro-isoquinoline (500 mg, 1.81 mmol, 35.76% yield) as a yellow solid. MS (ESI) m/z 277.9 [M+H]+
Step 4: 5,7-dichloroisoquinoline-4-carbaldehyde
[000792] To a solution of 4-bromo-5,7-dichloro-isoquinoline (500 mg, 1.81 mmol, 1 eq) in THF (10 mL) was added morpholine-4-carbaldehyde (623.57 mg, 5.42 mmol, 542.24 uL, 3 eq) at -78 °C, n-BuLi (2.5 M, 2.89 mL, 4 eq) was added to the above solution at -78 °C, the mixture was stirred at-78 °C for 2 h. Upon completion, the mixture was added to the sat. NH4CI (20 mL) with dropwise under 0 - 10 °C to adjust pH to 7 - 8, and extracted with ethyl acetate (10 mL * 2).
394
SUBSTITUTE SHEET RULE 26 The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentratedunder reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum etherethyl acetate = 2: 1) to give the product 5,7-dichloroisoquinoline-4- carbaldehyde (220 mg, 973.20 umol, 53.90% yield) as a yellow solid. MS (ESI) m/z 226.0 [M+H]+
Step 5: 2-amino-2-(5,7-dichloro-4-isoquinolyl)acetonitrile
[000793] To a solution of 5,7-dichloroisoquinoline-4-carbaldehyde (300 mg, 1.33 mmol, 1 eq) in DCM (3 mL) was added NH3/MeOH (7 M, 758.34 uL, 4 eq) and Ti(Oz-Pr)4 (565.77 mg, 1.99 mmol, 587.51 uL, 1.5 eq), and then the mixture was stirred at 0 °C for 1 h. TMSCN (394.97 mg, 3.98 mmol, 498.07 uL, 3 eq) was added, and the mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was diluted by addition H2O (30 mL) then filtered and the filtrate was extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10:1 to 0: 1) to give 2-amino-2-(5,7-dichloro-4-isoquinolyl)acetonitrile (250 mg, 991.67 umol, 74.73% yield) as a yellow solid. MS (ESI) m/z 251.9 [M+H]+
Step 6: (lR,2S,5S)-N-[cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000794] To a solution of 2-amino-2-(5,7-dichloro-4-isoquinolyl)acetonitrile (250 mg, 991.67 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (433.59 mg, 1.19 mmol, 1.2 eq) in ACN (10 mL) was added 1 -methylimidazole (244.26 mg, 2.98 mmol, 237.15 uL, 3 eq) and TCEH (556.49 mg, 1.98 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted by addition H2O (30 mL) then extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10:1 to 0: 1) to give the product (lR,2S,5S)-N-[cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-
395
SUBSTITUTE SHEET RULE 26 trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (300 mg, 501.30 umol, 50.55% yield) as a yellow solid. MS (ESI) m/z 598.2 [M+H]+
Step 7: (lR,2S,5S)-N-[cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000795] (lR,2S,5S)-N-[cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (0.3 g, 501.30 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-MeOH]; B%: 20% - 20%, 9 min) to give (1R,2S,5S)-N- [cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer
1 (120 mg, 195.91 umol, 39.08% yield, 97.7% purity) as a white solid.
[000796] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (br d, J= 5.5 Hz, 1H), 9.47 (s, 1H), 9.35 (br s, 1H), 8.90 (s, 1H), 8.46 (d, J= 2.1 Hz, 1H), 8.16 (d, J= 2.1 Hz, 1H), 7.24 (br d, J= 1.5 Hz, 1H), 4.39 (br s, 1H), 4.30 (s, 1H), 3.91 (dd, J= 5.4, 10.3 Hz, 1H), 3.72 (d, J= 10.5 Hz, 1H), 1.59 (dd, J= 5.4, 7.4 Hz, 1H), 1.37 (d, J= 7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.83 (s, 3H).
[000797] (lR,2S,5S)-N-[cyano-(5,7-dichloro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (110 mg, 182.71 umol, 36.45% yield, 99.4% purity) was obtained as a white solid.
[000798] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.48 (s, 1H), 9.45 - 9.41 (m, 1H), 9.36 (br s, 1H), 8.89 (s, 1H), 8.47 (d, J= 2.1 Hz, 1H), 8.20 (d, J= 2.3 Hz, 1H), 7.27 (br s, 1H), 4.38 (br s, 1H), 4.25 (s, 1H), 3.91 (br d, J= 4.8 Hz, 1H), 3.68 (d, J= 10.5 Hz, 1H), 1.60 - 1.53 (m, 1H), 1.26 (d, J= 7.6 Hz, 1H), 0.97 (s, 12H), 0.81 (s, 3H).
Example 98: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylpyrazolo[4,3-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 273)
396
SUBSTITUTE SHEET RULE 26
Figure imgf000398_0001
Step 1: 4-bromo-6-fluoro-isoquinoline
[000799] A solution of 6-fluoroisoquinoline (2 g, 13.59 mmol, 1 eq) in AcOH (20 mL) was added NBS (3.63 g, 20.39 mmol, 1.5 eq) and stirred at 60 °C for 16 h. Upon completion, the reaction mixture was concentrated in a vacuum to remove AcOH to afford a residue. The combined residue was diluted with EtOAc (20 mL) and washed with staturedaqueous NaHCOs solution (10 mL) at 0 °C. Then the organic layer was seprated and the aqueous layer was extracted with EtOAc (20 ml * 2). The combined orangic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 1 / 0 to 20 / 1) to give 4-bromo-6-fluoro- isoquinoline (1 g, 4.34 mmol, 31.90% yield, 98% purity) as a yellow solid. MS (ESI) m/z 226.0 [M+H]+.
Step 2: 6-fluoro-4-vinyl-isoquinoline
[000800] A solution of 4-bromo-6-fluoro-isoquinoline (980 mg, 4.34 mmol, 1 eq) and potassium trifluoro(vinyl)boranuide (696.87 mg, 5.20 mmol, 1.2 eq) in H2O (2 mL) and toluene
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SUBSTITUTE SHEET RULE 26 (8 mL), was added ditert-butyl(cyclopentyl)phosphane dichloropalladium iron (282.56 mg, 433.54 umol, 0.1 eq) and CS2CO3 (4.24 g, 13.01 mmol, 3 eq) under N2 atmosphere. The mixture was stirred at 80 °C for 2 h. Upon completion, the reaction mixture was filtered to give the filtrate then the filtrate was diluted with H2O (20 mL) and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 20/1 to 0/1) to give 6-fluoro-4-vinyl- isoquinoline (730 mg, 3.96 mmol, 91.39% yield, 94% purity) as a brown solid.. MS (ESI) m/z 174.1 [M+H]+.
Step 3: 6-fluoroisoquinoline-4-carbaldehyde
[000801] A solution of 6-fluoro-4-vinyl-isoquinoline (650 mg, 3.75 mmol, 1 eq) in dioxane (10 mL) and H2O (2 mL) was added wtih dipotassium dioxido(dioxo)osmium;dihydrate (276.58 mg, 750.64 umol, 0.2 eq) and 2,6-dimethyl(l 15N)pyridine (811.79 mg, 7.51 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h, and then sodium periodate (4.01 g, 18.77 mmol, 1.04 mL, 5 eq) was added to the mixture at 0 °C, and the mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S 1O2, petroleum ether/ethyl acetate = 20/1 to 0/1). to give . 6- fluoroisoquinoline-4-carbaldehyde (400 mg, 2.10 mmol, 55.98% yield, 92% purity) as a yellow solid. MS (ESI) m/z 176.1 [M+H]+.
Step 4: 2-amino-2-(6-fluoro-4-isoquinolyl)acetonitrile
[000802] A solution of 6-fluoroisoquinoline-4-carbaldehyde (400 mg, 2.28 mmol, 1 eq) in NEb/MeOH (5 mL) was added with NH4CI (244.30 mg, 4.57 mmol, 2 eq) and stirred at 0 °C for 1.5 h. Then, TMSCN (453.12 mg, 4.57 mmol, 571.40 uL, 2 eq) was added to the mixture, and the mixture was stirred at 25 °C for 4.5 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue, then the residue was diluted with H2O (6 mL) and extracted with ethyl acetate (4 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a
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SUBSTITUTE SHEET RULE 26 residue. The residue was purified by column chromatography (S1O2, petroleum ether/ethyl acetate = 20/1 to 0/1) to give 2-amino-2-(6-fluoro-4-isoquinolyl)acetonitrile (220 mg, 984.10 umol, 43.09% yield, 90% purity) as a yellow solid. MS (ESI) m/z 202.1 [M+H]+
Step 5: (lR,2S,5S)-N-[cyano-(6-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000803] A solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (398.41 mg, 1.09 mmol, 1 eq) in ACN (7 mL) then TCFH (306.80 mg, 1.09 mmol, 1 eq) and 1- methylimidazole (359.10 mg, 4.37 mmol, 348.64 uL, 4 eq) was added to the mixture and stirred at 25 °C for 5 min. Then, 2-amino-2-(6-fluoro-4-isoquinolyl)acetonitrile (220 mg, 1.09 mmol, 1 eq) was added to the mixture and stirred at 25 °C for 55 min. Upon completion, the reaction mixture was diluted with H2O (15 mL) and extracted with EA (10 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 20/1 to 1/1) to give (lR,2S,5S)-N-[cyano-(6-fluoro-4- isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (400 mg, 613.65 umol, 56.12% yield, 84% purity) as a light yellow oil. MS (ESI) m/z 548.3 [M+H]+
Step 6: (lR,2S,5S)-N-[cyano-(6-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000804] (lR,2S,5S)-N-[cyano-(6-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (400 mg, 730.54 umol, 1 eq) was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150 * 40 mm * 10 um; mobile phase: [water( NH4HCO3)-ACN];B%: 45%-75%,8 min). Then the product was further separated by SFC (column: DAICEL CHIRALPAK IC(250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 33%-33%, 7 min) to give (lR,2S,5S)-N-[cyano- (6-fhioro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (63.98 mg, 116.26 umol, 15.92% yield, 99.5% purity) as a white solid. MS (ESI) m/z 548.2 [M+H]+.
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SUBSTITUTE SHEET RULE 26 [000805] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.60 (d, J= 8.2 Hz, 1H), 9.42 (s, 1H), 9.32 (br s, 1H), 8.74 (s, 1H), 8.36 (dd, J= 5.7, 9.0 Hz, 1H), 7.74 - 7.64 (m, 2H), 6.95 - 6.87 (m, 1H), 4.38 (br s, 1H), 4.18 (s, 1H), 3.93 (dd, J= 5.4, 10.3 Hz, 1H), 3.71 (d, J= 10.4 Hz, 1H), 1.64 - 1.56 (m, 1H), 1.34 (d, J = 7.6 Hz, 1H), 1.03 (s, 3H), 0.97 (s, 9H), 0.82 (s, 3H).
[000806] (lR,2S,5S)-N-[cyano-(6-fluoro-4-isoquinolyl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (64.58 mg, 113.11 umol, 15.48% yield, 95.9% purity) as a white solid. MS (ESI) m/z 548.2 [M+H]+.
[000807] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.49 (d, J= 7.9 Hz, 1H), 9.41 (s, 2H), 8.72 (s, 1H), 8.37 (dd, J= 5.9, 8.9 Hz, 1H), 7.78 - 7.66 (m, 2H), 6.87 - 6.81 (m, 1H), 4.35 (br s, 1H), 4.23 (s, 1H), 3.93 (dd, <7= 5.6, 10.3 Hz, 1H), 3.65 (d, .7= 10.4 Hz, 1H), 1.55 (dd, J= 5.6, 7.3 Hz, 1H), 1.14 (d, J = 7.6 Hz, 1H), 0.94 (s, 3H), 0.87 (s, 9H), 0.80 (s, 3H).
Example 99: Synthesis of (lR,2S,5S)-3-[2,2-dimethyl-l-[(2,2,2- trifluoroacetyl)amino] cyclopropan ecarbonyl] -6, 6-dimethyl-3-azab icy clo [3.1.0] hexane-2- carboxylic acid
Figure imgf000401_0001
Step 1: methyl (lR,2S,5S)-3-[l-(tert-butoxycarbonylamino)-2,2-dimethyl- cyclopropanecarbonyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate
[000808] To a solution of 1 -(tert-butoxycarbonylamino)-2,2-dimethyl- cyclopropanecarboxylic acid (100 mg, 436.16 umol, 1 eq) and methyl (lR,2S,5S)-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxylate (98.68 mg, 479.78 umol, 1.1 eq, HC1) in DCM (2 mL) was added DMAP (106.57 mg, 872.33 umol, 2 eq), and then EDCI (167.23 mg, 872.33 umol, 2
400
SUBSTITUTE SHEET RULE 26 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (50 mL), and then extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-TLC (SiCh, Petroleum ether:Ethyl acetate = 2: 1) to give the product methyl (lR,2S,5S)-3-[l-(tert-butoxycarbonylamino)-2,2- dimethyl-cyclopropanecarbonyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate (100 mg, 262.83 umol, 60.26% yield) as a yellow oil. MS (ESI) m/z 381.2 [M+H]+.
Step 2: (lR,2S,5S)-3-[l-(tert-butoxycarbonylamino)-2,2-dimethyl-cyclopropanecarbonyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000809] A solution of methyl (lR,2S,5S)-3-[l-(tert-butoxycarbonylamino)-2,2-dimethyl- cyclopropanecarbonyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (100 mg, 262.83 umol, 1 eq) in THF (1.5 mL) and H2O (0.5 mL) was added LiOH.H O (33.09 mg, 788.48 umol, 3 eq). The mixture was stirred at 50 °C for 16 h. Upon completion, the mixture was diluted by addition H2O (10 mL), and then added aq. HCL (1 M) to adjust the pH = 3-4, and extracted with EA (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the product (lR,2S,5S)-3-[l- (tert-butoxycarbonylamino)-2,2-dimethyl-cyclopropanecarbonyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (90 mg, crude) as a white solid. MS (ESI) m/z 367.2 [M+H]+
Step 3: (lR,2S,5S)-3-(l-amino-2,2-dimethyl-cyclopropanecarbonyl)-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[000810] A solution of (lR,2S,5S)-3-[l-(tert-butoxycarbonylamino)-2,2-dimethyl- cyclopropanecarbonyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (90 mg, 245.60 umol, 1 eq) in HCl/dioxane (4 M, 1.80 mL, 29.32 eq) was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated under the reduced pressure to give (lR,2S,5S)-3-(l- amino-2,2-dimethyl-cyclopropanecarbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (70 mg, crude, HC1) as a white solid. MS (ESI) m/z 267.2 [M+H]+.
Step 4: (lR,2S,5S)-3-[2,2-dimethyl-l-[(2,2,2-trifluoroacetyl)amino]cyclopropanecarbonyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
401
SUBSTITUTE SHEET RULE 26 [000811] A solution of (lR,2S,5S)-3-(l-amino-2,2-dimethyl-cyclopropanecarbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (70 mg, 231.18 umol, 1 eq, HC1) in methyl 2,2,2-trifluoroacetate (296.02 mg, 2.31 mmol, 233.09 uL, 10 eq) was added TEA (70.18 mg, 693.54 umol, 96.53 uL, 3 eq). The mixture was stirred at 20 °C for 16 h. Upon completion, the mixture was concentrated under the reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex C 18 75 * 30 mm * 3 um; mobile phase: [water (NH4HCO3) - ACN]; B%: l%-20%, 8 min) to give the product (lR,2S,5S)-3-[2,2-dimethyl-l- [(2,2,2-trifluoroacetyl)amino]cyclopropanecarbonyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (7.26 mg, 19.31 umol, 8.35% yield, 96.4% purity) as a white solid. MS (ESI) m/z 363.1 [M+H]+.
[000812] 1H NMR (400 MHz, D20) 5 = 4.41 - 4.13 (m, 1H), 4.01 (dd, J = 5.5, 10.8 Hz, 1H),
3.64 - 3.47 (m, 1H), 1.67 - 1.53 (m, 1H), 1.52 - 1.43 (m, 1H), 1.38 (d, J = 7.6 Hz, 1H), 1.28 - 1.21 (m, 3H), 1.07 - 0.96 (m, 6H), 0.89 - 0.70 (m, 4H).
Example 100: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 278)
Figure imgf000403_0001
Step 1: 3-bromothieno [2, 3-c] pyridine-4-carbaldehyde
[000813] A solution of thieno[2,3-c]pyridine-4-carbaldehyde (1 g, 6.13 mmol, 1 eq) in lfcSC (20 mL) was added l,3-dibromo-5,5-dimethyl-imidazolidine-2, 4-dione (2.10 g, 7.35 mmol, 1.2 eq) at 0 °C, and the mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture
402
SUBSTITUTE SHEET RULE 26 was quenched by addition H2O 50 mL, the aqueous phase was added Na2CCh to pH = 10 and extracted with EA (100 mL * 3). The combined organic layers were washed with brine 200 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a 3-bromothieno [2,
3-c] pyridine-4-carbaldehyde (930 mg, crude) as a yellow solid. MS (ESI) m/z 241.9 [M+H]+.
Step 2: 3-(2-trimethylsilylethynyl) thieno [2, 3-c] pyridine-4-carbaldehyde
[000814] A solution of 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (880 mg, 3.63 mmol, 1 eq), i-PnNH (1.10 g, 10.90 mmol, 1.54 mL, 3 eq) in DMSO (20 mL) was added Cui (276.91 mg, 1.45 mmol, 0.4 eq), Pd(PPhs)4 (840.09 mg, 727.00 umol, 0.2 eq) and ethynyl(trimethyl)silane (714.04 mg, 7.27 mmol, 1.01 mL, 2 eq). The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was quenched by addition H2O 40 mL, and then extracted with ethyl acetate (35 mL * 3). The combined organic layers were washed with brine (70 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum ether: ethyl acetate = 1 :1) and TLC (Platel, SiCh, Petroleum ether: Ethylacetate = 1: 1) to give a 3-(2-trimethylsilylethynyl) thieno [2, 3-c] pyridine-
4-carbaldehyde (300 mg, 983.04 umol, 27.04% yield, 85% purity) as a yellow solid. MS (ESI) m/z 260.0 [M+H]+.
Step 3: 3-ethynylthieno [2, 3-c] pyridine-4-carbaldehyde
[000815] A solution of 3-(2-trimethylsilylethynyl) thieno [2, 3-c] pyridine-4-carbaldehyde (300 mg, 1.16 mmol, 1 eq) in MeOH (5 mL) was added K2CO3 (399.60 mg, 2.89 mmol, 2.5 eq), and the mixture was stirred at 25 °C for 1 h. Upon completion, to give 3-ethynylthieno [2, 3-c] pyridine-4-carbaldehyde (217 mg, crude) in MeOH as a yellow liquid and used to next step. MS (ESI) m/z 188.0 [M+H]+.
Step 4: 2-amino-2-(3-ethynylthieno [2, 3-c] pyridin-4-yl) acetonitrile
[000816] A solution of 3-ethynylthieno[2,3-c]pyridine-4-carbaldehyde (217 mg, 1.16 mmol, 1 eq) in DCM (7 mL) was added NHs/MeOH (7 M, 6 mL, 36.24 eq) and Ti(i-PrO)4 (494.14 mg, 1.74 mmol, 513.13 uL, 1.5 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (344.96 mg, 3.48 mmol, 435.01 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL), and then
403
SUBSTITUTE SHEET RULE 26 extracted with EA (15 mL * 3). The combined organic layers were washed with brine 40 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a 2-amino-2-(3- ethynylthieno [2, 3-c] pyridin-4-yl) acetonitrile (200 mg, crude) as a yellow solid. MS (ESI) m/z 214.0 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000817] A solution of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (200 mg, 937.83 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (375.88 mg, 1.03 mmol, 1.1 eq) in ACN (12 mL) was added 1 -methylimidazole (231.00 mg, 2.81 mmol, 224.27 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (526.27 mg, 1.88 mmol, 2 eq), and the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (30 mL), and then extracted with DCM (25 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%-65%, 8 min) to give desired compound as a white solid, which was further separated by SEC (column: (s,s) WHELK-01 (250mm * 30 mm, 5 um); mobile phase: [Neu-IPA]; B%: 10%-35%, 15 min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3- c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (93 mg, 166.19 umol, 17.72% yield, 100% purity) as a white solid. MS (ESI) m/z 560.2 [M+H]+.
[000818] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.46 - 9.26 (m, 3H), 8.71 (s, 1H), 8.62 (s, 1H), 7.00 (br d, J= 6.6 Hz, 1H), 4.52 (s, 1H), 4.40 (s, 1H), 4.27 (s, 1H), 3.91 (dd, J= 5.4, 10.1 Hz, 1H), 3.70 (d, J= 10.4 Hz, 1H), 1.57 - 1.50 (m, 1H), 1.36 (d, J= 7.7 Hz, 1H), 1.04 - 0.96 (m, 12H), 0.82 (s, 3H).
[000819] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
404
SUBSTITUTE SHEET RULE 26 carboxamide Isomer 2 (69.5 mg, 124.20 umol, 13.24% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 560.2 [M+H]+.
[000820] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.71 (br d, J= 3.5 Hz, 1H), 9.41 (s, 2H), 8.73 (s, 1H), 8.62 (s, 1H), 7.10 (br d, J= 2.4 Hz, 1H), 4.44 (s, 1H), 4.41 - 4.34 (m, 2H), 3.89 (dd, J = 5.4, 10.3 Hz, 1H), 3.71 (d, J= 10.5 Hz, 1H), 1.60 - 1.55 (m, 1H), 1.47 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 0.92 (s, 9H), 0.85 (s, 3H).
Example 101: Synthesis of (lR,2S,5S)-N-[cyano-(l-cyclopropylpyrrolo[3,2-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 280)
Figure imgf000406_0001
Step 1: 7-bromo-l-cyclopropyl-pyrrolo[3,2-c]pyridine
[000821] A mixture of 7-bromo-lH-pyrrolo[3,2-c]pyridine (1 g, 5.08 mmol, 1 eq), cyclopropylboronic acid (523.15 mg, 6.09 mmol, 1.2 eq), bipy (792.68 mg, 5.08 mmol, 1 eq), CU(OAC)2 (921.85 mg, 5.08 mmol, 1 eq) and Na2CCh (1.08 g, 10.15 mmol, 2 eq) in ACN (50 mL) was stirred at 60 °C for 12 h. Upon completion, the reaction mixture was quenched by addition H2O (30 mL), extracted with ethyl acetate (40 mL * 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 99:1 to 0: 100) to give 7-bromo-l-cyclopropyl-pyrrolo[3,2-c]pyridine (600 mg, 2.53 mmol, 49.86% yield, 100% purity) as a white solid.
Step 2: l-cyclopropyl-7-vinyl-pyrrolo[3,2-c]pyridine
405
SUBSTITUTE SHEET RULE 26 [000822] To a solution of 7-bromo-l-cyclopropyl-pyrrolo[3,2-c]pyridine (500 mg, 2.11 mmol, 1 eq) and trifluoro-potassio-vinyl-boron(l-) (367.22 mg, 2.74 mmol, 1.3 eq) in H2O (2 mL) and Tol. (8 mb) was added K3PO4 (895.29 mg, 4.22 mmol, 2 eq) and ditert- butyl(cyclopentyl)phosphane;dichloropalladium iron (137.44 mg, 210.89 umol, 0.1 eq) under N2. The mixture was stirred at 80 °C for 16 hr. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), extracted with ethyl acetate (20 mL * 2). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1: 1) to afford l-cyclopropyl-7-vinyl-pyrrolo[3,2-c]pyridine (360 mg, 1.74 mmol, 82.47% yield, 89% purity) as a yellow solid.
Step 3: l-cyclopropylpyrrolo[3,2-c]pyridine-7-carbaldehyde
[000823] To a solution of l-cyclopropyl-7-vinyl-pyrrolo[3,2-c]pyridine (340 mg, 1.85 mmol, 1 eq) in THF (10 mL) and H2O (2 mL) was added dipotassium dioxido(dioxo)osmium dihydrate (68.00 mg, 184.55 umol, 0.1 eq) and stirred for 0.5 h, then added sodium periodate (1.18 g, 5.54 mmol, 306.78 uL, 3 eq). The mixture was stirred at 20 °C for 12 h. Upon completion, the reaction was poured into H2O (50 mL) and then extracted with ethyl acetate (100 mL * 7). The combined organic phase was washed with brine (300 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:0 to 4:1) to afford l-cyclopropylpyrrolo[3,2-c]pyridine-7- carbaldehyde (160 mg, 807.69 umol, 43.77% yield, 94% purity) as a yellow solid.
Step 4: 2-amino-2-( 1 -cyclopropylpyrrolo[ 3, 2-c ]pyridin- 7-yl)acetonitrile
[000824] To a mixture of l-cyclopropylpyrrolo[3,2-c]pyridine-7-carbaldehyde (160 mg, 859.25 umol, 1 eq) in DCM (10 mL) was added tetraisopropoxytitanium (366.31 mg, 1.29 mmol, 380.38 uL, 1.5 eq) and NH3/MeOH (7 M, 477.50 uL, 3.89 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (255.74 mg, 2.58 mmol, 322.49 uL, 3 eq) was added, and the mixture was continued to stirred at 25 °C for 15 h. Upon completion, the mixture was quenched with water (20 mL) and ethyl acetate (50 mL), filtered with diatomite, theorganic phase was separated, washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under
406
SUBSTITUTE SHEET RULE 26 reduced pressure to give 2-amino-2-(l-cyclopropylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile (150 mg, crude) as a yellow oil.
Step 5: 2-amino-2-(l-cyclopropylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile
[000825] To a solution of 2-amino-2-(l-cyclopropylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile (150 mg, 706.71 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (257.50 mg, 706.71 umol, 1 eq) in ACN (10 mb) was added [chloro(dimethylamino)methylene]- dimethyl-ammonium hexafluorophosphate (237.95 mg, 848.06 umol, 1.2 eq) and 1- methylimidazole (232.08 mg, 2.83 mmol, 225.33 uL, 4 eq) at 0°C. The mixture was stirred at 25°C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and then extracted with ethyl acetate (20 mL *3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by by prep-HPLC (column: C18 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-60%, 10 min), then further separated by SFC (column: DAI CEL CHIRALPAK IC(250mm * 30 mm, 10 um); mobile phase: [Neu-ETOH]; B%: 5%-20%, 14 min) to give (lR,2S,5S)-N-[cyano-(l-cyclopropylpyrrolo[3,2-c]pyridin-7- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo Isomer 1 (85 mg, 152.17 umol, 21.53% yield, 100% purity) as a white solid. MS (ESI) m/z 559.1 [M+H]+
[000826] 'H NMR (400 MHz, DMSO-de) 5 = 9.51 (br d, J= 6.1 Hz, 1H), 9.38 - 9.26 (m, 1H), 8.98 - 8.83 (m, 1H), 8.51 - 8.41 (m, 1H), 7.47 (d, J= 3.1 Hz, 1H), 7.02 - 6.93 (m, 1H), 6.71 - 6.61 (m, 1H), 4.48 - 4.33 (m, 1H), 4.28 - 4.19 (m, 1H), 3.93 (br dd, J= 5.3, 10.1 Hz, 1H), 3.87 - 3.79 (m, 1H), 3.76 - 3.60 (m, 1H), 1.67 - 1.51 (m, 1H), 1.30 (d, J= 7.5 Hz, 1H), 1.27 - 1.06 (m, 3H), 1.05 - 0.95 (m, 12H), 0.90 - 0.78 (m, 4H)
[000827] To give (lR,2S,5S)-N-[cyano-(l-cyclopropylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo Isomer 2 (21 mg, 37.59 umol, 5.32% yield, 100% purity) as a white solid. MS (ESI) m/z 559.1 [M+H] [000828] 1 H NMR (400 MHz, DMSO-de) 5 = 9.45 - 9.36 (m, 1 H), 9.28 (d, J = 7.5 Hz, 1 H),
8.87 (s, 1H), 8.49 - 8.40 (m, 1H), 7.46 (d, J= 3.4 Hz, 1H), 7.17 - 7.03 (m, 1H), 6.63 (d, J= 3.4
407
SUBSTITUTE SHEET RULE 26 Hz, 1H), 4.38 - 4.32 (m, 1H), 4.26 (s, 1H), 3.93 (dd, J= 5.6, 10.3 Hz, 1H), 3.71 (tt, J= 3.5, 6.9 Hz, 1H), 3.63 (d, J= 10.4 Hz, 1H), 1.54 (dd, J= 5.7, 7.3 Hz, 1H), 1.23 (d, J= 7.6 Hz, 1H), 1.19 - 1.07 (m, 4H), 0.98 (s, 3H), 0.95 - 0.87 (m, 9H), 0.80 (s, 3H).
Example 102: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7- yl)methyl]-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 282)
Figure imgf000409_0001
Step 1: 7-bromo-l-methyl-pyrrolo[3,2-c]pyridine
[000829] To a solution of 7-bromo-lH-pyrrolo[3,2-c]pyridine (800 mg, 4.06 mmol, 1 eq) in DMF (5 mL) was added NaH (417.35 mg, 10.43 mmol, 60% purity, 2.57 eq) at 0 °C. The mixture was stirred at 0 °C for 0.5 h. Mel (691.57 mg, 4.87 mmol, 303.32 uL, 1.2 eq) was added, and the mixture was stirred at 25 °C for 1.5 h. Upon completion, the reaction mixture was diluted with ice water (60 mL) and filtered, the mother liquer was extracted with ethyl acetate (40 mL * 3). The combined organic layers were washed with brine (50 mL * 2), dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10:1 to 3: 1) to give 7-bromo-l-methyl- pyrrolo[3,2-c]pyridine (500 mg, 2.37 mmol, 58.35% yield) as a white solid. MS (ESI) m/z 211.0 [M+H]+.
Step 2: l-methylpyrrolo[3,2-c]pyridine-7-carbaldehyde
408
SUBSTITUTE SHEET RULE 26 [000830] To a solution of 7-bromo-l-methyl-pyrrolo[3,2-c]pyridine (500 mg, 2.37 mmol, 1 eq) in THF (20 mL) was added morpholine-4-carbaldehyde (2.18 g, 18.95 mmol, 1.90 mL, 8 eq) at 25 °C, and then n-BuLi (2.5 M, 3.79 mL, 4 eq) was added at -60 °C. The mixture was stirred at -60 °C under N2 atmosphere for 2 h. Upon completion, the reaction mixture was quenched by water (30 mL) at 0 °C, and the mother liquer was extracted with ethyl acetate (20 mL * 3). The combined organic layers were washed with brine (20 mL* 2), dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, petroleum etherethyl acetate = 0: 1) to afford l-methylpyrrolo[3,2-c]pyridine-7- carbaldehyde (165 mg, 1.03 mmol, 43.48% yield) as a white solid MS (ESI) m/z 161.1 [M+H]+
Step 3: 2-amino-2-(l-methylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile
[000831] To a solution of l-methylpyrrolo[3,2-c]pyridine-7-carbaldehyde (165 mg, 1.03 mmol, 1 eq) in DCM (5 mL) was added NHVMeOH (7 M, 588.65 uL, 4 eq) and Ti(i-PrO)4 (439.17 mg, 1.55 mmol, 20.07 uL, 1.5 eq) at 25 °C. After the addition, the mixture was stirred at 25 °C for 0.5 h. And then TMSCN (306.59 mg, 3.09 mmol, 386.62 uL, 3 eq) was added. The resulting mixture was stirred at 25 °C for 15.5 h. Upon completion, the combined was diluted with water (8 mL), extracted with ethyl acetate (5 mL), and then filtered to give a solution. The solution was extracted with ethyl acetate (5mL * 2), and the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(l- methylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile (140 mg, crude) as a yellow solid. MS (ESI) m/z 187.2 [M+H]+
Step 4: (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-(5-cyclopropyl-lH- pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000832] To a solution of 2-amino-2-(l-methylpyrrolo[3,2-c]pyridin-7-yl)acetonitrile (140 mg, 751.83 umol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-(5-cyclopropyl-lH-pyrrole-2- carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (216.78 mg, 751.83 umol, 1 eq) and 1 -methylimidazole (185.18 mg, 2.26 mmol, 179.79 uL, 3 eq). Then, [Chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (421.89 mg, 1.50 mmol, 2 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by prep-HPLC
409
SUBSTITUTE SHEET RULE 26 (column: Phenomenex Luna Cl 8 75 * 30 mm * 3 um; mobile phase: [water(FA)-ACN]; B%: 30%-60%, 8 min) to give (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3- (5-cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 481.89 umol, 64.10% yield) as a white solid. MS (ESI) m/z 457.2 [M+H]+.
Step 5: (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-(5-cyclopropyl-lH- pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[000833] The (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-(5- cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 481.89 umol, 1
Figure imgf000411_0001
separated by SFC (column: DAI CEL CHIRALPAK IC(250mm
* 30 mm, 10 um); mobile phase: [Neu-MeOH]; B%: 48%-48%, 9 min) to give Isomer 1 (74 mg, 162.09 umol, 33.64% yield) and Isomer 2 (24 mg, 52.57 umol, 10.91% yield), chiral purity is not good and further separated again by SFC (column: DAICEL CHIRALPAK IC(250mm * 30 mm, 10 um); mobile phase: [Neu-MeOH]; B%: 45%-45%, 10 min) to give (lR,2S,5S)-N-[cyano-(l- methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-(5-cyclopropyl-lH-pyrrole-2-carbonyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (36.07 mg, 79.01 umol, 16.40% yield) as a white solid. MS (ESI) m/z 457.3 [M+H]+.
[000834] 'H NMR (400 MHz, METHANOL-d-i) 5 = 9.20 - 9.05 (m, 1H), 8.66 - 8.52 (m, 1H), 7.67 (d, J= 3.3 Hz, 1H), 7.08 (d, J= 3.3 Hz, 1H), 6.98 (s, 1H), 6.67 (d, J= 3.9 Hz, 1H), 5.84 (d, J= 3.9 Hz, 1H), 4.51 (s, 1H), 4.20 - 4.07 (m, 4H), 3.84 (d, J= 10.4 Hz, 1H), 1.93 - 1.84 (m, 1H), 1.76 (dd, J = 5.6, 7.3 Hz, 1H), 1.45 (d, J= 7.6 Hz, 1H), 1.11 (s, 3H), 0.95 - 0.88 (m, 5H), 0.68 - 0.61 (m, 2H).
[000835] (lR,2S,5S)-N-[cyano-(l-methylpyrrolo[3,2-c]pyridin-7-yl)methyl]-3-(5- cyclopropyl-lH-pyrrole-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (24.57 mg, 53.82 umol, 11.17% yield) was obtained as a white solid. MS (ESI) m/z 457.3 [M+H]+.
[000836] 'H NMR (400 MHz, METHANOL-d4) 5 = 9.03 - 8.89 (m, 1H), 8.60 - 8.45 (m, 1H), 7.54 - 7.41 (m, 1H), 6.96 - 6.80 (m, 2H), 6.70 - 6.57 (m, 1H), 5.89 - 5.75 (m, 1H), 4.67 - 4.52 (m, 1H), 4.17 - 4.05 (m, 4H), 3.85 - 3.76 (m, 1H), 1.92 - 1.82 (m, 1H), 1.78 - 1.65 (m, 1H), 1.53 - 1.40 (m, 1H), 1.11 - 1.06 (m, 3H), 0.93 - 0.87 (m, 5H), 0.67 - 0.60 (m, 2H)
410
SUBSTITUTE SHEET RULE 26 Example 103: Synthesis of (lR,2S,5S)-N-[cyano-(l-methylimidazo[4,5-c]pyridin-7- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 283)
Figure imgf000412_0001
Step 1: l-methyl-7-vinyl-imidazo[4,5-c]pyridine
[000837] A mixture of 7-bromo-l-methyl-imidazo[4,5-c]pyridine (1.9 g, 8.96 mmol, 1 eq), potassium;trifluoro(vinyl)boranuide (3.60 g, 26.88 mmol, 3 eq) and CS2CO3 (5.84 g, 17.92 mmol, 2 eq) in dioxane (15 mL) and H2O (5 mL) was added Pd(dppf)Ch (655.63 mg, 896.03 umol, 0.1 eq) under N2. The mixture was stirred at 90 °C for 16 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL *3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM:MeOH = 1:0 to 20: 1) to give l-methyl-7-vinyl-imidazo[4,5- c]pyridine (1.12 g, 6.96 mmol, 77.66% yield, 98.9% purity) as a yellow solid.
Step 2: l-methylimidazo[4,5-c]pyridine-7-carbaldehyde
[000838] To a solution of l-methyl-7-vinyl-imidazo[4,5-c]pyridine (1.12 g, 7.04 mmol, 1 eq) in THF (9 mL) and H2O (3 mL) was added K2OSO4.2H2O (259.23 mg, 703.57 umol, 0.1 eq).
The reaction mixture was stirred for 30 min, and then NaIO4 (4.51 g, 21.11 mmol, 1.17 mL, 3 eq) was added. The mixture was stirred at 20 °C for 5.5 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30mL * 3). The combined
411
SUBSTITUTE SHEET RULE 26 organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give l-methylimidazo[4,5-c]pyridine-7-carbaldehyde (1.13 g, crude) as a yellow solid. MS (ESI) m/z 162.0 [M+H]+.
Step 3: 2-amino-2-(l-methylimidazo[4,5-c]pyridin-7-yl)acetonitrile
[000839] To a solution of l-methylimidazo[4,5-c]pyridine-7-carbaldehyde (1.13 g, 7.01 mmol, 1 eq) in DCM (10 mL) was added Ti(i-PrO)4 (2.99 g, 10.52 mmol, 3.10 mL, 1.5 eq) and NEb/MeOH (7 M, 4.01 mL, 4 eq). The reaction mixture was stirred 1 h at 20 °C, and then TMSCN (2.09 g, 21.03 mmol, 2.63 mL, 3 eq) was added at 20°C. The mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3)-ACN]; B%: l%-20%, 10 min) to give 2- amino-2-(l-methylimidazo[4,5-c]pyridin-7-yl)acetonitrile (700 mg, 3.51 mmol, 50.10% yield, 93.95% purity) as a white solid. MS (ESI) m/z 188.1 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(l-methylimidazo[4,5-c]pyridin-7-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000840] A mixture of 2-amino-2-(l-methylimidazo[4,5-c]pyridin-7-yl)acetonitrile (645 mg, 3.45 mmol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.26 g, 3.45 mmol, 1 eq) and 1- methylimidazole (848.67 mg, 10.34 mmol, 823.95 uL, 3 eq) in ACN (15 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (1.93 g, 6.89 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water( NH4HCCh)-ACN]; B%: 40%-60%, 8 min) to give (lR,2S,5S)-N-[cyano-(l- methylimidazo[4,5-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-
412
SUBSTITUTE SHEET RULE 26 trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (71.3 mg, 129.90 umol, 3.77% yield, 97.208% purity) as a white solid. MS (ESI) m/z 534.3 [M+H]+.
[000841] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.67 - 9.16 (m, 2H), 9.03 (d, J= 14.7 Hz, 1H), 8.53 (d, J= 4.8 Hz, 1H), 8.37 (d, J= 18.7 Hz, 1H), 6.97 - 6.69 (m, 1H), 4.45 - 4.28 (m, 1H), 4.20 (s, 1H), 4.02 - 3.89 (m, 4H), 3.78 - 3.56 (m, 1H), 1.68 - 1.52 (m, 1H), 1.29 (t, J= 7.9 Hz, 1H), 0.94 (d, J = 6.8 Hz, 15H).
Example 104: Synthesis of (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)- cyano-methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 291)
Figure imgf000414_0001
Step 1: ethyl 5-bromo-[l,2,4]triazolo[4,3-a]pyridine-3-carboxylate
[000842] To a solution of (6-bromo-2-pyridyl)hydrazine (8 g, 42.55 mmol, 1 eq) in MeOH (120 mL) was added ethyl 2-oxoacetate (17.37 g, 85.10 mmol, 50% purity, 2 eq), and the mixture was stirred at 60 °C for 2 h. The reaction was concentrated in vacuo. The mixture of residue and [acetoxy(phenyl)-X3-iodanyl] acetate (15.07 g, 46.80 mmol, 1.1 eq) were dissolved in DCM (100 mL) and stirred at 20 °C for 16 h. Upon completion, the reaction was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate=10/l to 0/1) to give ethyl 5-bromo-[l,2,4]triazolo[4,3- a]pyridine-3-carboxylate (7.8 g, 25.99 mmol, 61.09% yield, 90% purity) as a yellow solid. MS (ESI) m/z 270.0 [M+H]+.
413
SUBSTITUTE SHEET RULE 26 Step 2: (5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)methanol
[000843] A solution of ethyl 5-bromo-[l,2,4]triazolo[4,3-a]pyridine-3-carboxylate (2 g, 7.41 mmol, 1 eq) in THF (100 mL) was added DIBAL-H (1 M, 29.62 mb, 4 eq) at 0 °C and then the reaction mixture was stirred for 2 h. Upon completion, the mixture was quenched with water (2 mL), 15% NaOH aqueous solution (2 mL), water (5 mL) and DCM (200 mL) at 0 °C. The resulting mixture was moved to 20 °C and stirred for 15 min, then added anhydrous Na2SO4, stirred further for 15 min, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate=10/l to 0/1) to give (5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)methanol (330 mg, 1.45 mmol, 9.77% yield) as a yellow solid. MS (ESI) m/z 230.1 [M+H]+.
Step 3: 5-bromo-[l,2,4]triazolo[4,3-a]pyridine-3-carbaldehyde
[000844] To a solution of (5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)methanol (330 mg, 1.45 mmol, 1 eq) in DCM (20 mL) was added Dess-Martin (736.52 mg, 1.74 mmol, 537.61 uL, 1.2 eq) at 0 °C, and then the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (30 mL) extracted with DCM (40 mL). The combined organic layers were washed with NaHCCh (30 mL * 2) and brine (30 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, DCM/MeOH = 10/1) to give 5-bromo-[l,2,4]triazolo[4,3- a]pyridine-3-carbaldehyde (230 mg, 1.02 mmol, 70.32% yield) as a yellow solid. MS (ESI) m/z 228.0 [M+H]+.
Step 4: 2-amino-2-(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)acetonitrile
[000845] A solution of 5-bromo-[l,2,4]triazolo[4,3-a]pyridine-3-carbaldehyde (230 mg, 1.02 mmol, 1 eq) in NHLMeOH (5 mL) was added NH4CI (108.86 mg, 2.04 mmol, 2 eq) and stirred at 20 °C for 1 h. Then TMSCN (201.90 mg, 2.04 mmol, 254.60 uL, 2 eq) was added to the mixture, the mixture was stirred at 20 °C for 15 h. Upon completion, the reaction was concentrated under reduced pressure to give 2-amino-2-(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3- yl)acetonitrile (160 mg, crude) as a green solid. MS (ESI) m/z 254.0 [M+H]+.
414
SUBSTITUTE SHEET RULE 26 Step 5: (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000846] A solution of 2-amino-2-(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)acetonitrile (150 mg, 595.07 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (216.82 mg, 595.07 umol, 1 eq) in MeCN (8 mL, 93% purity) was added 1 -methylimidazole (195.43 mg, 2.38 mmol, 189.74 uL, 4 eq) at 0 °C. Then, [chloro(dimethylamino)methylene]- dimethyl-ammonium;hexafluorophosphate (200.36 mg, 714.09 umol, 1.2 eq) was added at 0 °C. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O 10 mL, and then extracted with ethyl acetate (20 mL).The combined organic layers were washed with brine (15 mL * 2), dried over Na2SO4 filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 100 * 30 mm * 10 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 30%- 60%, 8 min) to give (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)-cyano-methyl]- 3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 155.41 umol, 26.12% yield, 93% purity) was obtained as a white solid. MS (ESI) m/z 598.3 [M+H]+.
Step 6: (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000847] The (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)-cyano-methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (50 mg, 83.55 umol, 1 eq) was separated by SFC (column: (s,s) WHELK-01 (250 mm * 30 mm, 5 um); mobile phase: [Neu-IPA]; B%: 15%-50%, 16 min) to give (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)-cyano-methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (15 mg, 24.97 umol, 29.88% yield, 99.6% purity) as a white solid. MS (ESI) m/z 598.1 [M+H]+.
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SUBSTITUTE SHEET RULE 26 [000848] 'H NMR (400 MHz, DMSO-d/r,) 5 = 9.78 - 9.58 (m, 1H), 9.45-9.26 (m, 1H), 8.01 - 7.89 (m, 1H), 7.45 - 7.34 (m, 2H), 7.00 (d, J= 6.9 Hz, 1H), 4.39 (s, 1H), 4.24 (s, 1H), 3.93 (dd, J = 5.4, 10.3 Hz, 1H), 3.75 - 3.66 (m, 1H), 1.63 - 1.52 (m, 1H), 1.43 - 1.34 (m, 1H), 1.04 - 0.95 (m, 12H), 0.83 - 0.79 (m, 3H)
[000849] (lR,2S,5S)-N-[(5-bromo-[l,2,4]triazolo[4,3-a]pyridin-3-yl)-cyano-methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide Isomer 2 (15 mg, 24.31 umol, 29.10% yield, 97.0% purity) was obtained as a white solid. MS (ESI) m/z 598.1 [M+H]+.
[000850] 'H NMR (400 MHz, DMSO-de) 5 = 9.75 (d, J= 6.1 Hz, 1H), 9.48 - 9.20 (m, 1H), 7.95 (dd, J= 1.1, 8.9 Hz, 1H), 7.45 - 7.34 (m, 2H), 7.02 (d, J= 6.1 Hz, 1H), 4.41 (s, 1H), 4.27 (s, 1H), 3.92 (dd, J= 5.4, 10.3 Hz, 1H), 3.73 (d, J= 10.5 Hz, 1H), 1.60 (dd, J = 5.5, lA z, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.06 - 1.00 (m, 12H), 0.82 (s, 3H)
Example 105: Synthesis of (lR,2S,5S)-N-[cyano(5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 298)
Figure imgf000417_0001
Step 1: 3-vinyl-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole
[000851] To a solution of 3-bromo-5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole (950 mg, 5.08 mmol, 1 eq) in Tol. (10 mL) and H2O (2.5 mL) was added K3PO4 (2.16 g, 10.16 mmol, 2 eq) and potassium trifluoro (vinyl) boranuide (816.43 mg, 6.10 mmol, 1.2 eq). The mixture was added ditert-butyl (cyclopentyl) phosphane dichloropalladium iron (331.04 mg, 507.92 umol, 0.1 eq)
416
SUBSTITUTE SHEET RULE 26 under N2. The mixture was stirredat 80 °C for 4 hr under N2. Upon completion, the reaction mixture was cooled to 25 °C and concentrated to get the reaidue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 4/1 to 3/1) to give 3-vinyl-5,6- dihydro-4H-pyrrolo[l,2-b]pyrazole (467 mg, 3.27 mmol, 64.41% yield, 94% purity) as a brown oil.
Step 2: 5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole-3-carbaldehyde
[000852] To a solutionof 3 -vinyl-5, 6-dihydro-4H-pyrrolo[l,2-b]pyrazole (467 mg, 3.48 mmol, 1 eq) in THF (10 mL) and H2O (2 mL) was added dipotassium dioxide (dioxo)osmium dihydrate (128.24 mg, 348.05 umol, 0.1 eq), and the resulting mixture was stirred for 0.5 h. Sodium periodate (2.23 g, 10.44 mmol, 578.58 uL, 3 eq) was added, and the mixture was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was filtered through Celite, pour into brine (30mL) and then extracted with ethyl acetate (10 mL * 20). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (S1O2, petroleum ether/ethyl acetate = 3/1 to 7/3). To give 5,6- dihydro-4H-pyrrolo[l,2-b]pyrazole-3-carbaldehyde (350 mg, 2.31 mmol, 66.47% yield, 90% purity) as a yellow solid
Step 3: 2-amino-2-(5,6-dihydro-4H-pyrrolo[l ,2-b]pyrazol-3-yl)acetonitrile
[000853] A solution of 5,6-dihydro-4H-pyrrolo[l,2-b]pyrazole-3-carbaldehyde (350 mg, 2.57 mmol, 1 eq) in DCM (5 mL) was added Ti(i-PrO)4 (1.10 g, 3.86 mmol, 1.14 mL, 1.5 eq) and NFU/MeOH (7 M, 1.47 mL, 4 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (765.11 mg, 7.71 mmol, 964.83 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was filtered and the filter was concentrated and then extracted with ethyl acetate (10 mL * 3). The combined organiclayers were washed with brine (lOmL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give aresidue. To give 2-amino-2-(5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)acetonitrile (250 mg, crude) as a yellow oil.
Step 4: (lR,2S,5S)-N-[cyano(5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
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SUBSTITUTE SHEET RULE 26 [000854] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (561.62 mg, 1.54 mmol, 1 eq) and 2-amino-2-(5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3- yl)acetonitrile (250 mg, 1.54 mmol, 1 eq) in ACN (5 mb) was added TCFH (864.97 mg, 3.08 mmol, 2 eq) and 1 -methylimidazole (442.92 mg, 5.39 mmol, 430.02 uL, 3.5 eq), and then the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 8 min) which was further separated by SFC (column: DAICEL CHIRALPAK AD (250mm * 30mm, 10 um); mobile phase: [Neu-ETOH]; B%: 16%-16%, 5 min). To give (lR,2S,5S)-N-[cyano(5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)methyl]-3-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (62.4 mg, 110.43 umol, 7.17% yield, 90% purity) as a white solid. MS (ESI) m/z 509.1 [M+H]+.
[000855] 'H NMR (400 MHz, DMSO-d6) 5 = 9.44 - 9.32 (m, 1H), 9.20 (d, J= 7.6 Hz, 1H), 7.47 (s, 1H), 6.03 (d, J= 7.5 Hz, 1H), 4.46 - 4.38 (m, 1H), 4.25 (s, 1H), 4.05 (t, J= 7.4 Hz, 2H), 3.90 (dd, J= 5.4, 10.1 Hz, 1H), 3.72 (d, J= 10.4 Hz, 1H), 2.86 (t, J= 7.3 Hz, 2H), 2.58 - 2.54 (m, 2H), 1.61 - 1.49 (m, 1H), 1.32 (d, J = 7.6 Hz, 1H), 1.04 - 0.98 (m, 12H), 0.86 - 0.81 (m, 3H),
[000856] (lR,2S,5S)-N-[cyano(5,6-dihydro-4H-pyrrolo[l,2-b]pyrazol-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide Isomer 2 (62.03 mg, 121.98 umol, 7.92% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 509.1 [M+H]+.
[000857] 'H NMR (400 MHz, DMSO-de) 5 = 9.36 (br s, 1H), 9.13 (d, J= 7.6 Hz, 1H), 7.46 (s, 1H), 5.92 (d, J= 7.4 Hz, 1H), 4.40 (br s, 1H), 4.25 (s, 1H), 4.05 (t, J= 7.3 Hz, 2H), 3.89 (dd, J= 5.4, 10.3 Hz, 1H), 3.69 (br d, J= 10.3 Hz, 1H), 2.97 - 2.79 (m, 2H), 2.56 (br d, J= 7.3 Hz, 2H), 1.56 - 1.47 (m, 1H), 1.24 (d, J= 7.6 Hz, 1H), 1.00 (s, 12H), 0.83 (s, 3H).
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SUBSTITUTE SHEET RULE 26 Example 106: Synthesis of (lR,2S,5S)-N-(cyano(l-cyclopropyl-5-methyl-lH-pyrazol-4- yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 300)
Figure imgf000420_0001
Step 1 : l-cyclopropyl-4-iodo-5-methyl-lH-pyrazole
[000858] To a solution of l-cyclopropyl-4-iodo-pyrazole (2 g, 8.55 mmol, 1 eq) in THF (40 mL) was added LDA (2 M, 5.13 mL, 1.2 eq) at -76 °C. After 1 h at -76 °C, CH3I (1.38 g, 9.74 mmol, 606.48 pL, 1.14 eq) was added dropwise to the mixture. The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction was quenched with a saturated solution of NH4CI (30 mL) and extracted with EtOAc 90 mL (30 mL * 3). The combined organic phases were dried, concentrated. Then the residue was purified by column chromatography (SiCh, petroleum etherethyl acetate = 99:1 to 94:6) to give l-cyclopropyl-4-iodo-5-methyl-pyrazole (1.9 g, 7.66 mmol, 89.63% yield, 100% purity) as a yellow oil.
Step 2 : l-cyclopropyl-5-methyl-lH-pyrazole-4-carbaldehyde
[000859] A solution of l-cyclopropyl-4-iodo-5-methyl-pyrazole (900 mg, 3.63 mmol, 1 eq) and morpholine-4-carbaldehyde (1.25 g, 10.88 mmol, 1.09 mL, 3 eq) in THF (10 mL) was cooled to -78 °C, and then n-BuLi (2.5 M, 5.80 mL, 4 eq) was added. The reaction was stirred at -78 °C for 2 h. Upon completion, the reaction mixture was quenched with NH4CI 30 mL and
419
SUBSTITUTE SHEET RULE 26 extracted with EtOAc (30 mL * 8). The combined organic layers were washed with brine (30 * 4 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Then the residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate=3: l to 0: 1) to give l-cyclopropyl-5-methyl-pyrazole-4-carbaldehyde (670 mg, 1.87 mmol, 51.65% yield, 42% purity) as a yellow solid.
Step 3 : 2-amino-2-(l-cyclopropyl-5-methyl-lH-pyrazol-4-yl)acetonitrile
[000860] To a solution of l-cyclopropyl-5-methyl-pyrazole-4-carbaldehyde (10 mg, 66.59 umol, 1 eq) in DCM (1 mL) was added Ti(i-PrO)4 (28.39 mg, 99.88 umol, 29.48 uL, 1.5 eq) and NEh/MeOH (7 M, 38.05 uL, 4 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (19.82 mg, 199.76 umol, 24.99 uL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHa/MeOH. The reaction mixture was quenched by addition H2O (5 mL), and then extracted with EA (5 mL * 5). The combined organic layers were washed with brine (5 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(l-cyclopropyl-5- methyl-pyrazol-4-yl)acetonitrile (2 mg, crude) as yellow oil.
Step 4 : (lR,2S,5S)-N-(cyano(l-cyclopropyl-5-methyl-lH-pyrazol-4-yl)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000861] To a solution of 2-amino-2-(l-cyclopropyl-5-methyl-pyrazol-4-yl)acetonitrile (210 mg, 1.19 mmol, 1 eq) in ACN (4 mL) was added (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (434.21 mg, 1.19 mmol, 1 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (668.74 mg, 2.38 mmol, 2 eq), and then 1 -methylimidazole (293.52 mg, 3.58 mmol, 284.97 uL, 3 eq) was added. The mixture was stirred for 50 min at 25 °C. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250 * 50 mm * lOum; mobile phase: [water( NH4HCO3)-ACN]; B%: 35%-65%, 10 min), which was further separated by SEC (DAICEL CHIRALPAK IC(250 mm * 30 mm, 10 um);mobile phase: [Neu-IPA];B%: 5%-25%, 15 min) to give (lR,2S,5S)-N-[cyano-(l-cyclopropyl-5-methyl- pyrazol-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-
420
SUBSTITUTE SHEET RULE 26 dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (159.54 mg, 303.99 umol, 25.51% yield, 99.57% purity) as a white solid. MS (ESI) m/z 523.3 [M+H]+.
[000862] 'H NMR (400 MHz, DMSO-t76) 5 = 9.34 (br s, 1H), 9.15 (d, J = 7.4 Hz, 1H), 7.43 (s, 1H), 6.02 (d, J = 7.4 Hz, 1H), 4.41 (br s, 1H), 4.24 (s, 1H), 3.91 (dd, J = 5.5, 10.3 Hz, 1H), 3.72 (d, J = 10.4 Hz, 1H), 3.57 - 3.47 (m, 1H), 2.30 (s, 3H), 1.57 (dd, J = 5.4, 7.3 Hz, 1H), 1.31 (d, J = 7.6 Hz, 1H), 1.03 (s, 3H), 1.02 - 0.98 (m, 12H), 0.88 (s, 1H), 0.84 (s, 3H).
[000863] (lR,2S,5S)-N-[cyano-(l-cyclopropyl-5-methyl-pyrazol-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (155.52 mg, 297.61 umol, 24.97% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 523.3 [M+H]+.
[000864] 1H NMR (400 MHz, DMSO-t76) 5 = 9.37 (br s, 1H), 9.11 (d, J = 7.5 Hz, 1H), 7.41 (s, 1H), 5.93 (d, J = 7.4 Hz, 1H), 4.40 (s, 1H), 4.24 (s, 1H), 3.90 (dd, J = 5.4, 10.3 Hz, 1H), 3.68 (d, J = 10.5 Hz, 1H), 3.53 - 3.47 (m, 1H), 2.33 (s, 3H), 1.52 (dd, J = 5.5, 7.2 Hz, 1H), 1.20 (d, J = 7.6 Hz, 1H), 1.01 - 0.98 (m, 15H), 0.97 - 0.90 (m, 1H), 0.83 (s, 3H).
Example 107: Synthesis of (lR,2S,5S)-N-[cyano(6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazin- 3-yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 301)
421
SUBSTITUTE SHEET RULE 26
Figure imgf000423_0002
Figure imgf000423_0001
Step 1: 2-(4-bromopyrazol-l-yl)ethoxy-tert-butyl-dimethyl-silane
[000865] 4 -bromo- IH-pyrazole (5 g, 34.02 mmol, 1 eq) was added to ACN (40 mL) and the mixture was stirred in 0 °C. To the solution was added CS2CO3 (16.63 g, 51.03 mmol, 1.5 eq), followed by a solution of 2-bromoethoxy-tert-butyl-dimethyl-silane (8.95 g, 37.42 mmol, 1.1 eq) in ACN (5 mL) dropwise. The reaction mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was filtered through Celite, washed with MTBE (50 mL). The filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 98:2) to give 2-(4-bromopyrazol-l-yl)ethoxy-tert-butyl-dimethyl- silane (10 g, 32.76 mmol, 96.29% yield) as a off-white oil.
Step 2: 4-bromo-2-[2-[tert-butyl(dimethyl)silyl]oxyethyl]pyrazole-3-carbaldehyde
[000866] To a solution of 2-(4-bromopyrazol-l-yl)ethoxy-tert-butyl-dimethyl-silane (3 g, 9.83 mmol, 1 eq) in THF (20 mL) was added dropwise LDA (2 M, 7.37 mL, 1.5 eq) at -78°C. After addition, the mixture was stirred at this temperature for 30 min, and then DMF (1.29 g, 17.69 mmol, 1.36 mL, 1.8 eq) was added dropwise at -78 °C. The resulting mixture was stirred at -78 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL),
422
SUBSTITUTE SHEET RULE 26 extracted with MTBE (3 x 30 mL) and washed with brine (50 mL), the combined organics were dried with Na2SO4 and concentrated to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 99: 1 to 90: 10) to give 4-bromo-2-[2-[tert- butyl(dimethyl)silyl]oxyethyl]pyrazole-3-carbaldehyde (3.1 g, 7.63 mmol, 77.61% yield, 82% purity) as a white solid.
Step 3: 3-bromo-6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazine
[000867] To a solution of 4-bromo-2-[2-[tert-butyl(dimethyl)silyl]oxyethyl]pyrazole-3- carbaldehyde (2.9 g, 8.70 mmol, 1 eq) in DCM (87 mL) was added (dropwise) EbSiH (3.04 g, 26.10 mmol, 4.17 mL, 3 eq) and TMSOTf (11.60 g, 52.21 mmol, 9.43 mL, 6 eq). The mixture was stirred at -78 °C for Ih. Then, the mixture was first stirred in 0 °C for 2 h and at 20 °C for 14 h. Upon completion, the reaction mixture was quenched by addition NaHCCh (50 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 99:1 to 20:80) to give 3-bromo-6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazine (1.56 g, 7.68 mmol, 88.30% yield, N/A purity) as a white solid.
Step 4: 3-vinyl-6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazine
[000868] To a solution of potassium trifluoro(vinyl)boranuide (1.24 g, 9.22 mmol, 1.2 eq) in H2O (5 mL) and toluene (20 mL) was added 3-bromo-6,7-dihydro-4H-pyrazolo[5,l- c][ 1,4] oxazine (1.56 g, 7.68 mmol, 1 eq) then ditert-butyl(cyclopentyl)phosphane dichloropalladium iron (500.76 mg, 768.34 umol, 0.1 eq). The mixture was added K3PO4 (3.26 g, 15.37 mmol, 2 eq) under N2 and stirred at 80 °C for 12 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL), extracted with ethyl acetate (30 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography by (SiCh, Petroleum ether:Ethyl acetate = 99: 1 to 30:70) to afford 3-vinyl-6,7-dihydro-4H- pyrazolo[5,l-c][l,4]oxazine (800 mg, 4.99 mmol, 64.96% yield, 93.7% purity) was obtained as a yellow solid.
Step 5: l-cyclopropylpyrrolo[3,2-c]pyridine-7-carbaldehyde
423
SUBSTITUTE SHEET RULE 26 [000869] To a solution of 3-vinyl-6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazine (800 mg, 5.33 mmol, 1 eq) in THF (30 mL) and H2O (6 mL) was added dipotassium dioxido(dioxo)osmium dihydrate (196.28 mg, 532.70 umol, 0.1 eq) and stirred for 0.5 h. Sodium periodate (3.42 g, 15.98 mmol, 885.55 uL, 3 eq) was added, and the mixture was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was filtered through Celite, pour into brine (30 mL) and then extracted with Ethyl acetate (50 mL * 3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 99: 1 to 3:7) to give 6,7-dihydro-4H-pyrazolo[5,l- c][l,4]oxazine-3-carbaldehyde (660 mg, 3.95 mmol, 74.10% yield, 91% purity) was obtained as a yellow solid.
Step 6: 2-amino-2-(6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazin-3-yl)acetonitrile
[000870] To a mixture of 6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazine-3-carbaldehyde (620 mg, 4.07 mmol, 1 eq) in NHVMeOH (7 M, 10 mL, 17.18 eq) was added NH4CI (653.92 mg, 12.22 mmol, 3 eq), and the mixture was stirred at 20 °C for 1 h. TMSCN (1.21 g, 12.22 mmol, 1.53 mL, 3 eq) was added, and the mixture was continued to stirred at 20 °C for 15 h. Upon completion, the mixture was quenched with water (10 mL) and extracted with ethyl acetate (20 mL*2), filtered with diatomite, the organic phase was separated, washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(6,7- dihydro-4H-pyrazolo[5,l-c][l,4]oxazin-3-yl)acetonitrile (570 mg, crude) as a yellow oil.
Step 7: (lR,2S,5S)-N-[cyano(6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
[000871] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (1.17 g, 3.20 mmol, 1 eq) and 2-amino-2-(6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazin-3-yl)acetonitrile (570 mg, 3.20 mmol, 1 eq) in ACN (10 mL) was added [chloro(dimethylamino)methylene]- dimethyl-ammonium hexafluorophosphate (1.08 g, 3.84 mmol, 1.2 eq) and 1 -methylimidazole (1.05 g, 12.80 mmol, 1.02 mL, 4 eq) at 0 °C. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and then extracted
424
SUBSTITUTE SHEET RULE 26 with ethyl acetate (20 mL *3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by by prep-HPLC (column: Agela DuraShell Cl 8 250 * 70 mm * 10 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 35%-65%, 18 min), then further separated by SFC (column: DAI CEL CHIRALPAK IG (250mm * 30 mm, 10 um); mobile phase: [Neu-MeOH]; B%: 30%-30%, 4 min) to give (lR,2S,5S)-N-[cyano(6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazin- 3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (291.14 mg, 552.16 umol, 20.69% yield, 99.48% purity) was obtained as white solid. MS (ESI) m/z 525.2 [M+H]+
[000872] 'H NMR (400 MHz, DMSO-de) 5 = 9.36 (br s, 1H), 9.24 (d, J= 7.7 Hz, 1H), 7.56 - 7.50 (m, 1H), 6.14 (s, 1H), 4.82 - 4.76 (m, 2H), 4.40 (s, 1H), 4.21 (s, 1H), 4.12 - 4.08 (m, 2H), 4.06 - 3.95 (m, 2H), 3.90 (dd, J= 5.5, 10.4 Hz, 1H), 3.71 (d, J= 10.5 Hz, 1H), 1.56 (dd, J= 5.4, 7.5 Hz, 1H), 1.31 (d, J= 7.6 Hz, 1H), 1.04 - 1.02 (m, 3H), 1.01 - 0.98 (m, 9H), 0.83 (s, 3H).
[000873] (lR,2S,5S)-N-[cyano(6,7-dihydro-4H-pyrazolo[5,l-c][l,4]oxazin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (251.81 mg, 480.06 umol, 17.99% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 525.2 [M+H]
[000874] 1 H NMR (400 MHz, DMSO-de) 5 = 9.46 - 9.30 (m, 1 H), 9.17 (d, J = 7.5 Hz, 1 H),
7.53 (s, 1H), 6.00 - 5.94 (m, 1H), 4.94 - 4.87 (m, 1H), 4.83 - 4.76 (m, 1H), 4.40 (s, 1H), 4.23 (s, 1H), 4.14 - 4.08 (m, 2H), 4.07 - 4.01 (m, 2H), 3.93 - 3.86 (m, 1H), 3.72 - 3.65 (m, 1H), 1.54 (dd, J = 5.5, 7.5 Hz, 1H), 1.24 (d, J= 7.6 Hz, 1H), 1.02 - 0.96 (m, 12H), 0.83 (s, 3H).
Example 108: Synthesis of (lR,2S,5S)-N-[cyano(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin- 3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 302)
425
SUBSTITUTE SHEET RULE 26
Figure imgf000427_0001
Step 1: 6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine
[000875] A solution of l,2-dihydropyrazol-3-one (20 g, 237.88 mmol, 1 eq), K2CCh(l 18.35 g, 856.36 mmol, 3.6 eq) in DMF (920 mL) was stirred 1 h, and then was added dropwise 1,3- dibromopropane (62.43 g, 309.24 mmol, 31.53 mL, 1.3 eq) at 130 °C. The mixture was stirred at this temperature for 2 h. Upon completion, the mixture was cooled to 20 °C, then the reaction mixture was poured into FLO (3000 mL) at 20 °C, and then extracted with ethyl acetate (1000 mL * 3). The combined organic layers were washed with brine (1500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1 :0 to 5: 1) to give 6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine (15 g, 120.83 mmol, 50.80% yield) as a yellow oil. MS (ESI) m/z 125.2 [M+H]+
Step 2: 3-bromo-6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine
[000876] A mixture of 6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine (13 g, 104.72 mmol, 1 eq) in ACN (130 mL) was added with NBS (18.64 g, 104.72 mmol, 1 eq) at 0 °C and stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with FEO (200 mL) and extracted with ethyl acetate (150 mL * 3). The combined organic layers were washed with brine (200 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the
426
SUBSTITUTE SHEET RULE 26 crude product. The crude was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10:1 to 1: 1) to give 3-bromo-6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine(17 g, 75.36 mmol, 71.96% yield, 90% purity) as a yellow solid. MS (ESI) m/z 203.1 [M+H]+
Step 3: 3-vinyl-6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine
[000877] To a mixture of 3-bromo-6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine (17 g, 83.73 mmol, 1 eq) in H2O (40 m ) and Tol.(160 mL) was added potassium trifluoro(vinyl)boranuide (13.46 g, 100.47 mmol, 1.2 eq) and KsPCh (35.55 g, 167.46 mmol, 2 eq) under N2. Ditert- butyl(cyclopentyl)phosphane dichloropalladium;iron (5.46 g, 8.37 mmol, 0.1 eq) was added to the mixture under N2 and stirred at 80 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (150 mL) and extracted with ethyl acetate (150 mL * 3). The combined organic layers were washed with brine (200 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, petroleum etherethyl acetate = 10:1 to 1:3) to give 3-vinyl-6,7-dihydro-5H-pyrazolo[5,l- b][ 1,3] oxazine (8.72 g, 52.26 mmol, 62.41% yield, 90% purity) as a brown oil. MS (ESI) m/z 151.2 [M+H]+
Step 4: 6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine-3-carbaldehyde
[000878] A mixture of 3-vinyl-6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine (8.72 g, 52.26 mmol, 90% purity, 1 eq) in THE (200 mL) and H2O (40 mL) was added K2OSO4.2H2O (1.93 g, 5.23 mmol, 0.1 eq) and stirred at 25 °C for 0.5 h, then NalCh (33.53 g, 156.77 mmol, 8.69 mL, 3 eq) was added to the mixture at 0 °C and stirred at 25 °C for 15.5 h. Upon completion, the reaction mixture was diluted with H2O (150 mL) and extracted with ethyl acetate 2 L (200 mL * 10). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 1 :5) to give 6,7-dihydro-5H-pyrazolo[5,l- b][l,3]oxazine-3-carbaldehyde (5.46 g, 32.30 mmol, 61.80% yield, 90% purity) as a brown solid. MS (ESI) m/z 153.2 [M+H]+
Step 5: 2-amino-2-(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3-yl)acetonitrile
427
SUBSTITUTE SHEET RULE 26 [000879] To a solution of 6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazine-3-carbaldehyde (2.73 g, 17.94 mmol, 1 eq) in DCM (40 mL) was added Ti(i-PrO)4 (7.65 g, 26.91 mmol, 7.94 mL, 1.5 eq) and NHa/MeOH (7 M, 10.25 mL, 4 eq), and the mixture was stirred at 25 °C for 0.5 h. TMSCN (5.34 g, 53.83 mmol, 6.73 mL, 3 eq) was dropwised to the solution at 0 °C and the mixture was stirred at 25 °C for 0.5 h. Upon completion, the reaction mixture was quenched by addition H2O (60 mL) and filtered, then extracted with DCM (50 mL * 3). The combined organic layers were washed with brine (70 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3- yl)acetonitrile (2.2 g, crude) as a brown oil. MS (ESI) m/z 162.1 [M+H]+
Step 6: (lR,2S,5S)-N-[cyano(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide
[000880] A mixture of 2-amino-2-(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3- yl)acetonitrile (2.2 g, 4.32 mmol, 35% purity, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxylic acid (2.36 g, 6.48 mmol, 1.5 eq) in ACN (40 mL) was added 1 -methylimidazole (1.06 g, 12.96 mmol, 1.03 mL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (2.42 g, 8.64 mmol, 2 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (40 mL) and extracted with ethyl acetate (40 mL * 3). The combined organic layers were washed with brine (60 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-65%, 20 min) to give (1R,2S,5S)-N- [cyano(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (1.4 g, 2.40 mmol, 55.59% yield, 90% purity) as a yellow solid. MS (ESI) m/z 525.2 [M+H]+
Step 7: (lR,2S,5S)-N-[cyano(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide
428
SUBSTITUTE SHEET RULE 26 [000881] (lR,2S,5S)-N-[cyano(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.5 g, 953.23 umol, 1 eq) was purified by SFC (condition: column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu- ETOH]; B%: 30%-30%, 5 min ) to give (lR,2S,5S)-N-[cyano(6,7-dihydro-5H-pyrazolo[5,l- b][l,3]oxazin-3-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (0.18 g, 343.16 umol, 36.00% yield, 100% purity) as a white solid. MS (ESI) m/z 525.2 [M+H]+
[000882] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.35 (br d, J = 7.4 Hz, 1H), 9.08 (d, J = 6.9 Hz, 1H), 7.44 - 7.37 (m, 1H), 5.68 (d, J = 6.9 Hz, 1H), 4.41 (br d, J = 7.5 Hz, 1H), 4.38 - 4.31 (m, 2H), 4.27 (s, 1H), 4.08 (t, J = 6.1 Hz, 2H), 3.89 (dd, J = 5.4, 10.3 Hz, 1H), 3.71 (d, J = 10.4 Hz, 1H), 2.18 (quin, J = 5.5 Hz, 2H), 1.55 (dd, J = 5.5, 7.3 Hz, 1H), 1.29 (d, J = 7.5 Hz, 1H), 1.08 - 0.78 (m, 15H)
[000883] (lR,2S,5S)-N-[cyano(6,7-dihydro-5H-pyrazolo[5,l-b][l,3]oxazin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 0.14 g, 266.90 umol, 28.00% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 525.2 [M+H]+
[000884] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.36 (br d, J = 8.0 Hz, 1H), 9.09 (d, J = 7.4 Hz, 1H), 7.37 (s, 1H), 5.69 (d, J = 7.4 Hz, 1H), 4.40 (br d, J = 7.9 Hz, 1H), 4.37 - 4.31 (m, 2H), 4.26 (s, 1H), 4.07 (t, J = 6.1 Hz, 2H), 3.88 (dd, J = 5.4, 10.3 Hz, 1H), 3.69 (d, J = 10.5 Hz, 1H), 2.21 - 2.14 (m, 2H), 1.52 (dd, J = 5.5, 7.4 Hz, 1H), 1.23 (d, J = 7.6 Hz, 1H), 1.04 - 0.97 (m, 12H), 0.83 (s, 3H)
Example 109: Synthesis of (lR,2S,5S)-N-[cyano-(3-cyclopropylthieno[2,3-c]pyridin-4- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifhioroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 305)
429
SUBSTITUTE SHEET RULE 26
Figure imgf000431_0001
Step 1: 3-bromothieno[2,3-c]pyridine-4-carbaldehyde
[000885] A solution of thieno[2,3-c]pyridine-4-carbaldehyde (450 mg, 2.76 mmol, 1 eq) in H2SO4 (10 mb) was added l,3-dibromo-5,5-dimethyl-imidazolidine-2, 4-dione (946.08 mg, 3.31 mmol, 1.2 eq) at 0 °C, the mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O 20 mL at 0 °C, and then used Na2CCh absolution adjust pH=12 and extracted with ethyl acetate 120 mL (40 mL * 3). The combined organic layers were washed with brine 120 mL (120 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to afford 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (400 mg, 1.44 mmol, 52.07% yield, 86.9% purity). MS (ESI) m/z 241.9 [M+H]+
Step 2: 3-cyclopropylthieno[2,3-c]pyridine-4-carbaldehyde
[000886] A mixture of 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (200 mg, 826.13 umol, 1 eq), potassium;cyclopropyl(trifluoro)boranuide (183.37 mg, 1.24 mmol, 1.5 eq), CS2CO3 (807.51 mg, 2.48 mmol, 3 eq), ditert-butyl(cyclopentyl)phosphane dichloropalladium iron (53.84 mg, 82.61 umol, 0.1 eq) in toluene (10 mL) and H2O (3 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80 °C for 16 h under N2 atmosphere. Upon completion, the reaction mixture was diluted with H2O 10 mL and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give 3-cyclopropylthieno[2,3- c]pyridine-4-carbaldehyde (147 mg, 571.34 umol, 69.16% yield, 79% purity) as a light yellow solid. MS (ESI) m/z 204.0 [M+H]+.
430
SUBSTITUTE SHEET RULE 26 Step 3: 2-amino-2-(3-cyclopropylthieno[2,3-c]pyridin-4-yl)acetonitrile
[000887] A solution of 3-cyclopropylthieno[2,3-c]pyridine-4-carbaldehyde (142 mg, 698.61 umol, 1 eq) in NEb/MeOH (8 mL) was added with NH4CI (74.74 mg, 1.40 mmol, 2 eq) for Ih and then TMSCN (138.61 mg, 1.40 mmol, 174.80 uL, 2 eq) was added at 0°C, the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHs/MeOH. The residue was diluted with H2O (10 mL) and extracted with EA (10 mL * 3). The combined organic layers were washed with brine (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(3- cyclopropylthieno[2,3-c]pyridin-4-yl)acetonitrile (162 mg, crude) as a light yellow solid. MS (ESI) m/z 230.1 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(3-cyclopropylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000888] A mixture of 2-amino-2-(3-cyclopropylthieno[2,3-c]pyridin-4-yl)acetonitrile (162 mg, 706.50 umol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (257.42 mg, 706.50 umol, 1 eq) in ACN (6 mL) was added 1 -methylimidazole (174.02 mg, 2.12 mmol, 168.95 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (396.46 mg, 1.41 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove ACN. The residue was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (30mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (neutral condition; column: Waters Xbridge Prep OBD C18 150 * 40 mm * 10 um; mobile phase: [water (NH3H2O+NH4HCO3)-ACN]; B%: 30%-60%, 8 min) to afford (1R,2S,5S)- N-[cyano-(3-cyclopropylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (75 mg, 121.69 umol, 43.78% yield, 93.4% purity) as a white solid. MS (ESI) m/z 576.2 [M+H]+
431
SUBSTITUTE SHEET RULE 26 [000889] 'H NMR (400 MHz, DMSO-t76) 5 = 9.40 - 9.30 (m, 2H), 8.73 (s, 1H), 7.93 - 7.83 (m, 1H), 7.16 - 7.05 (m, 1H), 4.36 (s, 1H), 4.31 - 4.20 (m, 1H), 3.92 (br dd, J= 5.4, 10.4 Hz, 1H), 3.64 (br d, J= 10.4 Hz, 1H), 3.30 (s, 1H), 2.16 - 2.06 (m, 1H), 1.58 - 1.50 (m, 1H), 1.20 (d, J= 7.3 Hz, 1H), 1.04 - 0.94 (m, 6H), 0.91 (s, 9H), 0.81 (s, 3H), 0.76 - 0.70 (m, 1H).
[000890] (lR,2S,5S)-N-[cyano-(3-cyclopropylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (70 mg, 116.25 umol, 41.83% yield, 95.6% purity) was obtained as a white solid. MS (ESI) m/z 576.2 [M+H]+
[000891] 'H NMR (400 MHz, DMSO-t76) 5 = 9.51 (d, J= 6.1 Hz, 1H), 9.44 - 9.26 (m, 1H), 8.72 (s, 1H), 7.97 - 7.84 (m, 1H), 7.10 - 6.99 (m, 1H), 4.39 (br s, 1H), 4.20 (s, 1H), 3.92 (br dd, J = 5.3, 10.0 Hz, 1H), 3.71 (br d, J= 10.1 Hz, 1H), 3.29 (s, 1H), 2.26 - 2.15 (m, 1H), 1.65 - 1.59 (m, 1H), 1.32 (d, J= 7.6 Hz, 1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.80 (s, 6H), 0.66 - 0.57 (m, 1H).
Step 5: ( 1R, 2S, 5S)-N-[ cyano- (3-cyclopropylthieno[ 2, 3-c]pyridin-4-yl)methyl]-3-[ (2S)-3, 3- dimethyl-2-[ (2, 2, 2-trifluoroacetyl)amino]butanoyl]-6, 6-dimethyl-3-azabicyclo[ 3.1.0 Jhexane-2- carboxamide
[000892] (lR,2S,5S)-N-[cyano-(3-cyclopropylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (75 mg, 121.69 umol, 43.78% yield, 93.4% purity) was separated by SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um);mobile phase: [Neu-MeOH]; B%: 5%- 20%, 15 min) to give the product (lR,2S,5S)-N-[cyano-(3-cyclopropylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (63 mg, 103.42 umol, 79.38% yield, 94.5% purity) as a white solid. MS (ESI) m/z 576.2 [M+H]+
[000893] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.37 (br d, J= 7.4 Hz, 1H), 9.26 (s, 1H), 8.73 (s, 1H), 7.94 - 7.86 (m, 1H), 7.12 (br d, J= 7.3 Hz, 1H), 4.36 (s, 1H), 4.25 (s, 1H), 3.92 (dd, J = 5.5, 10.3 Hz, 1H), 3.64 (d, J= 10.4 Hz, 1H), 3.32 (s, 1H), 2.16 - 2.05 (m, 1H), 1.55 (dd, J= 5.7, 7.2 Hz, 1H), 1.30 - 1.17 (m, 1H), 1.03 - 0.94 (m, 6H), 0.91 (s, 9H), 0.81 (s, 3H), 0.76 - 0.70 (m, 1H).
432
SUBSTITUTE SHEET RULE 26 Example 110: Synthesis of (lR,2S,5S)-N-(cyano(3-(prop-l-yn-l-yl)thieno[2,3-c]pyridin-4- yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 306)
Figure imgf000434_0001
Step 1: ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate
[000894] Ethyl 2-sulfanylacetate (34.02 g, 283.12 mmol, 30.93 mL, 1 eq) and CS2CO3 (119.92 g, 368.06 mmol, 1.3 eq) were added to a solution of 3,5-dibromopyridine-4- carbaldehyde (75 g, 283.12 mmol, 1 eq) in THF (750 mL). The mixture was stirred at 60 °C for 3 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate (160 g, crude) as yellow solid. MS (ESI) m/z 285.9 [M+H]+.
Step 2: 4-bromothieno[2,3-c]pyridine-2-carboxylic acid
433
SUBSTITUTE SHEET RULE 26 [000895] To a solution of ethyl 4-bromothieno[2,3-c]pyridine-2-carboxylate (84 g, 293.56 mmol, 1 eq) in THF (850 mL) and MeOH (340 mL) was added NaOH (7 M, 168.00 mL, 4.01 eq). The mixture was stirred at 20 °C for 6 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove THF and MeOH, H2O (1500 mL) was added to the residue, IM HC1 was added to pH = 3, solid was precipitated and filtered out. The solid was washed with H2O (500 mL), dired under vacuum to give a 4-bromothieno[2,3-c]pyridine-2- carboxylic acid (75 g, crude) as white solid. MS (ESI) m/z 255.9 [M-H]+.
Step 3: 4-bromothieno[2,3-c]pyridine
[000896] A solution of 4-bromothieno[2,3-c]pyridine-2-carboxylic acid (75 g, 290.59 mmol, 1 eq), copper(I) oxide (41.58 g, 290.59 mmol, 29.70 mL, 1 eq) and 1 , 10-phenanthroline (41.89 g, 232.48 mmol, 0.8 eq) in NMP (760 mL) and H2O (15.2 mL), and quinoline (228.22 g, 1.77 mol, 209.38 mL, 6.08 eq) was stirred at 160 °C for 4 h. Upon completion, the reaction was poured into H2O (1200 mL) and then extracted with ethyl acetate (400 mL * 5). The combined organic phase was washed with brine (900 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (neutral condition; column: Phenomenex luna C 18 (250 * 70 mm, 15 um); mobile phase: [water (NH4HCCh)-ACN]; B%: 15%-50%, 20 min ) to give 4-bromothieno[2,3-c]pyridine (33 g, 154.15 mmol, 53.05% yield) as yellow solid. MS (ESI) m/z 213.9 [M+H]+.
Step 4: 4-vinylthieno[2,3-c]pyridine
[000897] To a solution of 4-bromothieno[2,3-c]pyridine (33 g, 154.15 mmol, 1 eq) in Tol. (400 mL) and H2O (100 mL) was added K3PO4 (65.44 g, 308.29 mmol, 2 eq) and potassium trifluoro(vinyl)boranuide (24.78 g, 184.98 mmol, 1.2 eq). The mixture was added ditert- butyl(cyclopentyl)phosphane dichloropalladium;iron (10.05 g, 15.41 mmol, 0.1 eq) under N2. The mixture was stirred at 80 °C for 16 h under N2. Upon completion, the reaction was poured into H2O (1000 mL) and then extracted with ethyl acetate (500 mL * 4). The combined organic phase was washed with brine (1500 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 30/1 to 0/1) to give 4-vinylthieno[2,3-c]pyridine (24 g, 148.86 mmol, 96.57% yield) as black brown oil. MS (ESI) m/z 162.1 [M+H]+.
434
SUBSTITUTE SHEET RULE 26 Step 5: thieno[2,3-c]pyridine-4-carbaldehyde
[000898] To a solution of 4-vinylthieno[2,3-c]pyridine (24 g, 148.86 mmol, 1 eq) in THF (400 mL) and H2O (80 mL) was added K2OSO4.2H2O (5.48 g, 14.89 mmol, 0.1 eq) and stirred at 25 °C for 0.5 h. Then NalCh (95.52 g, 446.59 mmol, 24.75 mL, 3 eq) at 0 °C. The mixture was stirred at 25 °C for 13.5 h. Upon completion, the mixture reaction was filtered to remove the insoluble. The reaction was quenched by H2O (1500 mL) slowly and then extracted with ethyl acetate (400 mL * 7). The combined organic phase was washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 20/1 to 0/1) to give thieno[2,3-c]pyridine- 4-carbaldehyde (12 g, 73.53 mmol, 49.40% yield) as yellow solid. MS (ESI) m/z 164.0 [M+H]+.
Step 6: 3-bromothieno[2,3-c]pyridine-4-carbaldehyde
[000899] To a solution of thieno[2,3-c]pyridine-4-carbaldehyde (11.5 g, 70.47 mmol, 1 eq) in H2SO4 (150 mL) was added l,3-dibromo-5,5-dimethyl-imidazolidine-2, 4-dione (22.16 g, 77.51 mmol, 1.1 eq) at 0 °C. The mixture was stirred at 25 °C for 14 h. Upon completion, the reaction solution is then poured onto crushed ice. Then was adjusted to pH~8 with NaHCCh aq, then was extrated with ethyl acetate (500 mL * 8), the combined orhanic phase was washed with brine(1000 mL), dried over Na2SO4 and concentrated in vacuum. The crude product was triturated with (petroleum ether: ethyl acetate = 20: 1) to give 3-bromothieno[2,3-c]pyridine-4- carbaldehyde (12 g, crude) as yellow solid. MS (ESI) m/z 241.9 [M+H]+.
Step 7: (3-bromothieno[2,3-c]pyridin-4-yl)methanol
[000900] To a solution of 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (9.3 g, 38.42 mmol, 1 eq) in MeOH (100 mL) was added NaBH4 (2.18 g, 57.62 mmol, 1.5 eq) at 0 °C. The mixture was stirred at 0 °C for 1 h. Upon completion, the residue was poured into NH4CI aq (400 mL). The aqueous phase was extracted with ethyl acetate (150 mL * 7). The combined organic phase was washed with brine (500 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum (3-bromothieno[2,3-c]pyridin-4-yl)methanol (7.3 g, crude) as yellow solid. MS (ESI) m/z 243.9 [M+H]+.
Step 8: 3-bromo-4-(((tert-butyldimethylsilyl)oxy)methyl)thieno[2,3-c]pyridine
435
SUBSTITUTE SHEET RULE 26 [000901] To a solution of (3-bromothieno[2,3-c]pyridin-4-yl)methanol (7 g, 28.68 mmol, 1 eq) in DMF (70 mL) was added TBSC1 (6.48 g, 43.01 mmol, 5.27 mL, 1.5 eq) and imidazole (3.90 g, 57.35 mmol, 2 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the residue was poured into H2O (500 mL). The aqueous phase was extracted with ethyl acetate (200 mL * 3). The combined organic phase was washed with brine (200 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 1/0 to 5/1) to give (3-bromothieno[2,3- c]pyridin-4-yl)methoxy-tert-butyl-dimethyl-silane (9 g, 25.11 mmol, 87.58% yield) as yellow oil. MS (ESI) m/z 358.0 [M+H]+.
Step 9: 4-(((tert-butyldimethylsilyl)oxy)methyl)-3 -(prop- 1 -yn- 1 -yl)thieno[2,3-c]pyridine
[000902] To a solution of 4,4,5,5-tetramethyl-2-prop-l-ynyl-l,3,2-dioxaborolane (1.39 g, 8.37 mmol, 3 eq) and (3-bromothieno[2,3-c]pyridin-4-yl)methoxy-tert-butyl-dimethyl-silane (1 g, 2.79 mmol, 1 eq) in toluene (12 mL) and H2O (2.4 mL) was added CS2CO3 (1.82 g, 5.58 mmol, 2 eq) and ditert-butyl(cyclopentyl)phosphane dichloropalladium iron (181.86 mg, 279.04 umol, 0.1 eq) under N2. The mixture was stirred at 80 °C for 4 h. Upon completion, the residue was poured into H2O (200 mL). The aqueous phase was extracted with ethyl acetate (80 mL * 3). The combined organic phase was washed with brine (80 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 1/0 to 5/1) to give tert-butyl-dimethyl-[(3-prop-l-ynylthieno[2,3- c]pyridin-4-yl)methoxy] silane (5.39 g, 14.60 mmol, 74.74% yield, 86% purity) as yellow oil. MS (ESI) m/z 318.0 [M+H]+.
Step 10: (3-(prop-l-yn-l-yl)thieno[2,3-c]pyridin-4-yl)methanol
[000903] A solution of tert-butyl-dimethyl-[(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methoxy] silane (5.39 g, 14.60 mmol, 86% purity, 1 eq) in HCl/dioxane (50 mL) (4M) was stirred at 25 °C for 1 h. Upon completion, the reaction mixture concentrated under reduced pressure to give a residue. The residue was poured into H2O (300 mL) and added Na2CCh aq adjust to pH = 8. The aqueous phase was extracted with ethyl acetate (200 mL * 5). The combined organic phase was washed with brine (400 mL), dried over anhydrous Na2SO4, filtered
436
SUBSTITUTE SHEET RULE 26 and concentrated in vacuum to give (3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methanol (3.45 g, crude) as yellow solid. MS (ESI) m/z 204.1 [M+H]+.
Step 11: 3-(prop-l-yn-l-yl)thieno[2,3-c]pyridine-4-carbaldehyde
[000904] To a solution of (3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methanol (3.45 g, 16.97 mmol, 1 eq) in DCM (50 mL) was added MnCh (29.51 g, 339.47 mmol, 20 eq). The mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a 3-prop-l-ynylthieno[2,3-c]pyridine-4-carbaldehyde (3 g, crude) as yellow solid. MS (ESI) m/z 202.0 [M+H]+.
Step 12: 2-amino-2-(3-(prop-l-yn-l-yl)thieno[2,3-c]pyridin-4-yl)acetonitrile
[000905] A mixture of 3-prop-l-ynylthieno[2,3-c]pyridine-4-carbaldehyde (3 g, 14.91 mmol, 1 eq) and Ti(i-PrO)4 (6.36 g, 22.36 mmol, 6.60 mL, 1.5 eq), NH3/MeOH (7 M, 13.80 mL, 6.48 eq) in DCM (15 mL) was stirred at 25 °C for 1 h. Then was added TMSCN (4.44 g, 44.72 mmol, 5.59 mL, 3 eq) at 0 °C. The mixture was stirred at 25 °C for 11 h. Upon completion, the reaction was pour into H2O (100 mL) and then extracted with DCM (80 mL * 7). The combined organic phase was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give a 2-amino-2-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)acetonitrile (3 g, crude) as yellow solid.
Figure imgf000438_0001
Step 13: (lR,2S,5S)-N-(cyano(3-(prop-l-yn-l-yl)thieno[2,3-c]pyridin-4-yl)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000906] To a solution of 2-amino-2-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)acetonitrile (400 mg, 1.58 mmol, 90% purity, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (577.11 mg, 1.58 mmol, 1 eq) in ACN (15 mL) was added TCEH (666.62 mg, 2.38 mmol, 1.5 eq) and NMI (260.08 mg, 3.17 mmol, 252.50 uL, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was pour into H2O (20 mL) and then extracted with ethyl acetate (15 mL * 3). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (neutral
437
SUBSTITUTE SHEET RULE 26 condition; column: Waters Xbridge BEH C 18 100 * 30 mm * 10 um; mobile phase: [water (NH4HCO3) - ACN]; B%: 55%-85%, 8 min) to give desired compound (550 mg,) as a white solid, which was further separated by SFC (condition: column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA];B%: 33%-33%, 10 min) to give a (1R,2S,5S)- N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (85 mg, 148.18 umol, 9.36% yield, 100% purity) as white solid. MS (ESI) m/z 574.2 [M+H]+.
[000907] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.42 - 9.34 (m, 2H), 9.28 (d, J= 6.6 Hz, 1H), 8.70 (s, 1H), 8.40 (s, 1H), 7.05 (d, J= 6.2 Hz, 1H), 4.41 (s, 1H), 4.28 (s, 1H), 3.92 (dd, J= 5.4, 10.3 Hz, 1H), 3.72 (d, J= 10.4 Hz, 1H), 2.06 (s, 3H), 1.56 (dd, J= 5.3, 7.5 Hz, 1H), 1.34 (d, J = 7.6 Hz, 1H), 1.03 - 0.96 (m, 12H), 0.81 (s, 3H).
[000908] ( 1 R,2S , 5 S)-N - [cyano-(3 -prop- 1 -yny lthieno[2, 3 -c]pyridin-4-yl)methy 1] -3 - [(2S)-3 , 3 - dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (85 mg, 142.25 umol, 8.98% yield, 96% purity) was obtained as white solid. MS (ESI) m/z 574.2 [M+H]+.
[000909] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.48 - 9.32 (m, 3H), 8.76 - 8.69 (m, 1H), 8.42 - 8.36 (m, 1H), 7.28 - 7.02 (m, 1H), 4.42 (s, 1H), 4.37 - 4.26 (m, 1H), 3.89 (dd, J= 5.3, 10.4 Hz, 1H), 3.76 - 3.70 (m, 1H), 2.10 - 2.06 (m, 3H), 1.61 - 1.55 (m, 1H), 1.45 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 1.00 - 0.92 (m, 9H), 0.86 - 0.81 (m, 3H).
438
SUBSTITUTE SHEET RULE 26 Example 111: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 310)
Figure imgf000440_0001
Step 1: 3-bromo-5-fluoro-pyridine-4-carboxylic acid
[000910] To a solution of LDA (2 M, 184.67 mL, 1.3 eq) in THF (500 mL) was added 3- bromo-5-fluoro-pyridine (50 g, 284.11 mmol, 1 eq) at -60 °C, and the mixture was stirred at - 60 °C for 1 h. The reaction mixture was then poured into a mixture of dry ice (12.50 g, 284.11 mmol, 1 eq). The resulting mixture was stirred for 1 hr at 20 °C. Upon completion, the reaction mixture was poured into H2O (1000 mL) and the pH value of the solution was adjusted to 3 with hydrogen chloride (1 mol/L), then extracted with DCM (500 mL * 3). The organic layer was washed by brine (300 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by (SiCh, PE:EA = 100:1 to 0:1) to give 3-bromo-5- fluoro-pyridine-4-carboxylic acid (40 g, 163.64 mmol, 57.60% yield, 90% purity) as a brown solid.
439
SUBSTITUTE SHEET RULE 26 Step 2: 3-bromo-5-fluoro-pyridine-4-carboxamide
[000911] To a mixture of 3-bromo-5-fluoro-pyridine-4-carboxylic acid (32 g, 145.46 mmol, 1 eq) and (COC1)2 (35.45 g, 279.28 mmol, 24.45 mb, 1.92 eq) in DCM (500 mL) at 0 °C, catalytic amount of DMF (1.06 g, 14.55 mmol, 1.12 mL, 0.1 eq) was added, and the mixture was stirred at 25 °C for 1 h, then concentrated in vacum. The residue in DCM (320 mL) was added dropwise into an NIL/MeOH (7 M, 320.00 mL, 15.40 eq) at 0 °C. The mixture was stirred at 0 °C for 1 h. Upon completion, the mixture was treated with water (800 mL) and extracted with DCM (200 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 3-bromo-5-fluoro-pyridine-4-carboxamide (30 g, crude) as a black brown solid.
Step 3: 3-benzylsulfanyl-5-bromo-pyridine-4-carboxamide
[000912] 3 -Bromo-5-fluoro-pyridine-4-carboxamide (30 g, 136.98 mmol, 1 eq), BnSH (17.86 g, 143.83 mmol, 16.85 mL, 1.05 eq) and lUCCh (20.83 g, 150.68 mmol, 1.1 eq) were suspended in i-PrOH (1640 mL) and heated at 88 °C for 3 h. Upon completion, the reaction was diluted with water (2000 mL) and extracted with DCM (1000 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, PE:EA = 100:0 to 0:1) to give 3-benzylsulfanyl-5-bromo- pyridine-4-carboxamide (30 g, 74.26 mmol, 54.21% yield, 80% purity) as a yellow solid.
Step 4: 4-bromoisothiazolo[5,4-c]pyridin-3-one
[000913] To a solution of 3-benzylsulfanyl-5-bromo-pyridine-4-carboxamide (30 g, 92.82 mmol, 1 eq) in DCE (800 mL) was added dropwise sulfuryl chloride (35.08 g, 259.91 mmol, 25.66 mL,2.8 eq) in DCM (260 mL) to the solution, the solution was stirred for 2 h at 20 °C. Upon completion, the reaction mixture was poured into H2O (400 mL) and extracted with DCM (100 mL * 3). The combined organic layers was filtered and concentrated under reduced pressure to give 4-bromoisothiazolo[5,4-c]pyridin-3-one (30 g, crude) as a yellow solid.
Step 5: (4-bromoisothiazolo[5,4-c]pyridin-3-yl) trifluoromethanesulfonate
[000914] 4-bromoisothiazolo[5,4-c]pyridin-3-one (8 g, 34.62 mmol, 1 eq) was added dropwise to a solution of pyridine (4.11 g, 51.93 mmol, 4.19 mL, 1.5 eq) and Tf2O (14.65 g,
440
SUBSTITUTE SHEET RULE 26 51.93 mmol, 8.57 mL, 1.5 eq) in DCM (40 mL) at 20 °C. The reaction was allowed to stir for 2 h at 20 °C. The reaction mixture was was diluted with ethyl acetate (50 mL) and washed sequentially with water (100 mL) and brine (50 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 100:1 to 0: 1) to give (4-bromoisothiazolo[5,4-c]pyridin-3-yl) trifluoromethanesulfonate (4 g, 9.36 mmol, 27.04% yield, 85% purity) as a brown solid.
Step 6: 2-(4-bromoisothiazolo[5,4-c]pyridin-3-yl)ethynyl-trimethyl-silane
[000915] To a solution of ethynyl(trimethyl)silane (1.62 g, 16.52 mmol, 2.29 mL, 1.5 eq) in THF (70 mL) was added LiHMDS (1 M, 16.52 mL, 1.5 eq) at -60 °C for 0.5 h, and then (4- bromoisothiazolo[5,4-c]pyridin-3-yl) trifluoromethanesulfonate (4 g, 11.02 mmol, 1 eq) was added the solution. The reaction was stirred for 0.5 h at -60 °C. Upon completion, the reaction mixture was cooled and diluted with H2O (100 mL) and extracted with DCM (50 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-(4-bromoisothiazolo[5,4-c]pyridin-3-yl)ethynyl-trimethyl-silane (1.5 g, crude) as a yellow solid.
Step 7: trimethyl-[2-(4-vinylisothiazolo[5,4-c]pyridin-3-yl)ethynyl]silane
[000916] To a solution of 2-(4-bromoisothiazolo[5,4-c]pyridin-3-yl)ethynyl-trimethyl-silane (1.2 g, 3.86 mmol, 1 eq) in dry toluene (20 mL) was added tributyl(vinyl)tin (1.83 g, 5.78 mmol, 1.68 mL, 1.5 eq) and the reaction was mixture was purged with argon for 0.5 h. Pd(PPU)4 (222.74 mg, 192.76 umol, 0.05 eq) was added and the mixture heated at 110 °C for 11.5 hr. Upon completion, the reaction mixture was diluted with FLO (100 mL) and extracted with ethyl acetate (30 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give trimethyl-[2-(4-vinylisothiazolo[5,4-c]pyridin-3- yl)ethynyl] silane (300 mg, crude) as a yellow solid.
Step 8: 3-ethynylisothiazolo[5,4-c]pyridine-4-carbaldehyde
[000917] To a solution of trimethyl-[2-(4-vinylisothiazolo[5,4-c]pyridin-3-yl)ethynyl]silane (300 mg, 1.16 mmol, 1 eq) in THF (10 mL) and H2O (2 mL) was added K2OSO4.2H2O (42.78
441
SUBSTITUTE SHEET RULE 26 mg, 116.09 umol, 0.1 eq , and then the mixture was stirred 0.5 h at 20 °C. NalCh (744.93 mg, 3.48 mmol, 192.99 uL, 3 eq was added, and the solution was stirred at 20 °C for 11.5 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL), and then extracted with ethyl acetate (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- TLC(SiO2, petroleum etherethyl acetate = 2: 1) to give 3-ethynylisothiazolo[5,4-c]pyridine-4- carbaldehyde (200 mg, 956.40 umol, 82.38% yield, 90% purity) as a white solid.
Step 9: 2-amino-2-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)acetonitrile
[000918] To a solution of 3-ethynylisothiazolo[5,4-c]pyridine-4-carbaldehyde (200 mg, 1.06 mmol, 1 eq} in DCM (8 mL) was added NHVMeOH (7 M, 607.24 uL, 4 eq} and Ti(i-PrO)4 (453.03 mg, 1.59 mmol, 470.44 uL, 1.5 eq). After stirring 0.5 h at 20 °C, TMSCN (316.28 mg, 3.19 mmol, 398.84 uL, 3 eq) was added at 0 °C, and then the solution was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was quenched by addition H2O (20 mL), and then extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(3-ethynylisothiazolo[5,4- c]pyridin-4-yl)acetonitrile (200 mg, crude) as a yellow solid.
Step 10: (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000919] To a solution of 2-amino-2-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)acetonitrile (200 mg, 933.51 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (340.13 mg, 933.51 umol, 1 eq) in ACN (10 mL) was added NMI (153.28 mg, 1.87 mmol, 148.82 uL, 2 eq) and TCFH (523.85 mg, 1.87 mmol, 2 eq), and the solution was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (15 mL) and extracted with DCM (8mL * 3). The combined organic layers was filtered and concentrated under reduced pressure to give a residue. The crude was purified by prep-HPLC(column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 urn; mobile phase: [water (NH4HCO3)- ACN] ; B%: 30%-70%, 8 min) to give (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-
442
SUBSTITUTE SHEET RULE 26 dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (300 mg, 508.39 umol, 54.46% yield, 95% purity) as a white solid. MS (ESI) m/z 560.2 [M+H]+.
Step 10: (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000920] The (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 535.15 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 16%-16%, 8 min) to give (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (70 mg, 124.87 umol, 23.33% yield, 100% purity). MS (ESI) m/z 560.2 [M+H]+.
[000921] 'H NMR (400 MHz, DMSO-de) 5 = 9.75 (s, 1H), 9.41 (d, J= 6.7 Hz, 1H), 9.37 (br d, J= 6.6 Hz, 1H), 8.80 (s, 1H), 6.99 (d, J= 6.6 Hz, 1H), 4.92 (s, 1H), 4.40 (br d, J= 6.9 Hz, 1H), 4.25 (s, 1H), 3.92 (dd, J= 5.4, 10.3 Hz, 1H), 3.70 (d, J= 10.5 Hz, 1H), 1.56 (dd, J= 5.4, 7.5 Hz, 1H), 1.36 (d, J= 7.6 Hz, 1H), 1.00 (s, 9H), 0.98 - 0.93 (m, 3H), 0.81 (s, 3H).
[000922] The (lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 535.15 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 16%-16%, 8 min) to give lR,2S,5S)-N-[cyano-(3-ethynylisothiazolo[5,4-c]pyridin-4- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (70 mg, 124.87 umol, 23.33% yield, 100% purity). MS (ESI) m/z 560.2 [M+H]+.
[000923] 'H NMR (400 MHz, DMSO-de) 5 = 9.74 (s, 1H), 9.69 (br d, J= 6.1 Hz, 1H), 9.38 (br s, 1H), 8.85 - 8.80 (m, 1H), 7.05 (br d, J= 4.6 Hz, 1H), 4.77 (s, 1H), 4.40 (br s, 1H), 4.34 (s,
443
SUBSTITUTE SHEET RULE 26 1H), 3.89 (dd, J= 5.4, 10.5 Hz, 1H), 3.79 - 3.65 (m, 1H), 1.62 - 1.54 (m, 1H), 1.47 (d, J= 7.6 Hz, 1H), 1.04 (s, 3H), 1.00 - 0.93 (m, 9H), 0.85 - 0.81 (m, 3H).
Example 112: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 311)
Figure imgf000445_0001
Step 1: 4-vinyl-lH-pyrazolo [3, 4-c] pyridine
[000924] A solution of 4-bromo-lH-pyrazolo[3,4-c]pyridine (15 g, 75.75 mmol, 1 eq), potassium;trifluoro(vinyl)boranuide (12.18 g, 90.90 mmol, 1.2 eq) in H2O (45 mL) and dioxane (180 mL) was added CS2CO3 (49.36 g, 151.50 mmol, 2 eq) and Pd(dppf)Ch (5.54 g, 7.57 mmol, 0.1 eq) under N2, and the mixture was stirred at 100 °C for 14 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove dioxane. The reaction mixture was quenched by addition H2O (100 mL), and then extracted with ethyl acetate (80 mL * 3). The combined organic layers were washed with brine (130 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:0 to 20:45) and TLC(Platel, SiCh, Petroleum ether: Ethylacetate = 0:1) to give a 4-vinyl-lH-pyrazolo [3, 4-c] pyridine (5.7 g, 34.55 mmol, 45.62% yield, 88% purity) as a yellow solid. MS (ESI) m/z 146.1 [M+H]+.
444
SUBSTITUTE SHEET RULE 26 Step 2: 3-iodo-4-vinyl-lH-pyrazolo [3, 4-c] pyridine
[000925] A solution of 4-vinyl-lH-pyrazolo[3,4-c]pyridine (5.7 g, 39.27 mmol, 1 eq) in DMF (600 mL) and ACN (600 mL) was added NIS (13.25 g, 58.90 mmol, 1.5 eq), and the mixture was stirred at 80 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove ACN. The combined reaction mixture was poured into water (350 mL) and extracted with ethyl acetate (300 mL * 3). The combined organic phase was concentrated in vacuo to give desired compound. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1:0 to 46:54) and TLC(Platel, SiCh, petroleum ether: ethyl acetate = 0:1) to give a 3-iodo-4-vinyl-lH-pyrazolo [3, 4-c] pyridine (3.6 g, 10.63 mmol, 27.06% yield, 80% purity) as a yellow solid. MS (ESI) m/z 272.1 [M+H]+.
Step 3: 3-iodo-l-methyl-4-vinyl-pyrazolo [3, 4-c] pyridine
[000926] A solution of 3-iodo-4-vinyl-lH-pyrazolo [3, 4-c] pyridine (3.6 g, 13.28 mmol, 1 eq) in DMF (50 mL) was added NaH (531.25 mg, 13.28 mmol, 60% purity, 1 eq) at 0 °C under N2 stirred for 0.5 h, added dimethyl sulfate (1.68 g, 13.28 mmol, 1.26 mL, 1 eq) was drop-wise, the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by NH4CI, and then extracted with ethyl acetate (80 mL * 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Titank Cis Bulk 250 *150 mm 10 u; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-60%, 30 min) to give a 3-iodo-l-methyl-4-vinyl-pyrazolo [3, 4-c] pyridine (1.47 g, 5.05 mmol, 38.05% yield, 98% purity) as a yellow solid. MS (ESI) m/z 286.0 [M+H]+.
Step 4: 3-iodo-l-methyl-pyrazolo [3, 4-c] pyridine-4-carbaldehyde
[000927] A solution of 3-iodo-l-methyl-4-vinyl-pyrazolo[3,4-c]pyridine (1.47 g, 5.16 mmol, 1 eq), K2OSO4.2H2O (379.98 mg, 1.03 mmol, 0.2 eq) in THF (75 mL) and H2O (15 mL) was stirred at 25 °C for 0.5 h, then added NalCh (3.31 g, 15.47 mmol, 857.18 uL, 3 eq), the mixture was stirred at 25 °C for 14 h. Upon completion, the reaction mixture was quenched by addition H2O (100 mL), and then extracted with EA (50 mL * 3). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum
445
SUBSTITUTE SHEET RULE 26 etherethyl acetate = 1 :0 to 77:23) and TLC (Platel, SiCh, petroleum ether: ethylacetate = 0: 1) to give a 3-iodo-l-methyl-pyrazolo [3, 4-c] pyridine-4-carbaldehyde (820 mg, 2.60 mmol, 50.41% yield, 91% purity) as a yellow solid. MS (ESI) m/z 288.0 [M+H]+.
Step 5: 1 -methyl-3-(2-trimethylsilylethynyl)pyrazolo[3,4-c]pyridine-4-carbaldehyde
[000928] A solution of 3-iodo-l-methyl-pyrazolo[3,4-c]pyridine-4-carbaldehyde (400 mg, 1.39 mmol, 1 eq) in DMSO (25 mL) was added Cui (106.15 mg, 557.38 umol, 0.4 eq), Pd(PPhs)4 (322.04 mg, 278.69 umol, 0.2 eq), N-isopropylpropan-2-amine (423.01 mg, 4.18 mmol, 590.80 uL, 3 eq and ethynyl(trimethyl)silane (273.72 mg, 2.79 mmol, 386.07 uL, 2 eq under N2. The mixure was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (60 mL), and then extracted with ethyl acetate (45 mL * 3). The combined organic layers were washed with brine (90 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, petroleum ether: ethyl acetate = 1:0 to 64:36) and TLC (Platel, SiCh, petroleum ether: ethylacetate = 0:1) to give a l-methyl-3-(2-trimethylsilylethynyl)pyrazolo[3,4-c]pyridine-4-carbaldehyde (477 mg, 1.02 mmol, 36.58% yield, 55% purity) as a yellow solid. MS (ESI) m/z 258.1 [M+H]+.
Step 6: 1 -methyl-3-(2-trimethylsilylethynyl)pyrazolo[3,4-c]pyridine-4-carbaldehyde
[000929] A solution of l-methyl-3-(2-trimethylsilylethynyl)pyrazolo[3,4-c]pyridine-4- carbaldehyde (427 mg, 995.48 umol, 60% purity, 1 eq in MeOH (5 mL) was added K2CO3 (343.95 mg, 2.49 mmol, 2.5 eq), the mixture was stirred at 25 °C for 20 min. Upon completion, to give a 3-ethynyl-l-methyl-pyrazolo [3, 4-c] pyridine-4-carbaldehyde (184 mg, 993.62 umol, 99.81% yield, N/A purity) in MeOH (5 mL) as a yellow liquid and used to next step.
Step 7: 2-amino-2-(3-ethynyl-l -methyl-pyrazolo[3,4-c]pyridin-4-yl)acetonitrile
[000930] A solution of 3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridine-4-carbaldehyde (184 mg, 993.62 umol, 1 eq in DCM (7 mL) was added Ti(i-PrO)4 (423.60 mg, 1.49 mmol, 439.88 uL, 1.5 eq) and NHLMeOH (7 M, 5.52 mL, 38.89 eq was stirred at 25 °C for 1 h, and then TMSCN (295.72 mg, 2.98 mmol, 372.91 uL, 3 eq) was added at 0 °C. The mixture was stirred at 25 °C for 2 h. The reaction mixture was quenched by addition H2O (30 mL), and then extracted with EA (25 mL * 3). The combined organic layers were washed with brine (60 mL), dried over
446
SUBSTITUTE SHEET RULE 26 Na2SO4, filtered and concentrated under reduced pressure to give a 2-amino-2-(3-ethynyl-l- methyl-pyrazolo[3,4-c]pyridin-4-yl)acetonitrile (266 mg, crude) as a yellow solid. MS (ESI) m/z 212.1 [M+H]+.
Step 8: (lR,2S,5S)-N-[cyano-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4-yl)methyl]-3-[(2S)- 3,3-dirnethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
[000931] A solution of 2-amino-2-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4- yl)acetonitrile (266 mg, 1.26 mmol, 1 eq), (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (504.74 mg, 1.39 mmol, 1.1 eq) in ACN (12 mL) was added [chloro(dimethylamino)methylene]- dimethyl-ammonium;hexafluorophosphate (706.69 mg, 2.52 mmol, 2 eq) and 1 -methylimidazole (310.19 mg, 3.78 mmol, 301.15 uL, 3 eq), and the mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove ACN. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN) to give desired compound as a white solid, which was further separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA];B%: 30%-30%, 8 min) to give (lR,2S,5S)-N-[cyano-(3-ethynyl-l-methyl- pyrazolo[3,4-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 1 (95 mg, 170.38 umol, 13.53% yield, 100% purity) as a white solid. MS (ESI) m/z 558.2 [M+H]+.
[000932] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.44 - 9.31 (m, 3H), 8.49 (s, 1H), 6.58 (d, J = Hz, 1H), 4.62 (s, 1H), 4.41 (br s, 1H), 4.30 - 4.22 (m, 4H), 3.91 (dd, J= 5.4, 10.2 Hz, 1H), 3.71 (d, J= 10.4 Hz, 1H), 1.55 (dd, J= 5.4, 7.5 Hz, 1H), 1.39 (d, J= 7.8 Hz, 1H), 1.04 - 0.96 (m, 12H), 0.82 (s, 3H).
[000933] To give (lR,2S,5S)-N-[cyano-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (75 mg, 133.17 umol, 10.57% yield, 99% purity) as a white solid. MS (ESI) m/z 558.2 [M+H]+.
447
SUBSTITUTE SHEET RULE 26 [000934] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (d, J= 6.6 Hz, 1H), 9.43 - 9.29 (m, 2H), 8.51 (s, 1H), 6.59 (d, J= 6.6 Hz, 1H), 4.58 (s, 1H), 4.43 - 4.34 (m, 2H), 4.24 (s, 3H), 3.89 (dd, J = 5.3, 10.4 Hz, 1H), 3.72 (br d, J= 10.6 Hz, 1H), 1.60 - 1.46 (m, 2H), 1.04 (s, 3H), 0.94 (s, 9H), 0.87 - 0.82 (m, 3H).
Example 113: Synthesis of (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrrolo[2,3- c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 318)
Figure imgf000449_0001
Step 1: 4-vinyl-lH-pyrrolo[2,3-c]pyridine
[000935] To a solution of 4-bromo-lH-pyrrolo[2,3-c]pyridine (25 g, 126.88 mmol, 1 eq) in H2O (80 mL) and Tol. (400 mL) was added potassium trifluoro(vinyl)boranuide (20.40 g, 152.26 mmol, 1.2 eq) and then ditert-butyl(cyclopentyl)phosphane;dichloropalladium iron (4.13 g, 6.34 mmol, 0.05 eq). The mixture was added CS2CO3 (124.02 g, 380.65 mmol, 3 eq) under N2 and stirred at 80 °C for 12 h. Upon completion, the reaction mixture was quenched by addition H2O (200 mL), extracted with ethyl acetate (500 mL * 3). The combined organic layers were washed with brine (1000 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with ethyl acetate at 20 °C to give 4-vinyl-lH- pyrrolo[2,3-c]pyridine (16 g, 110.98 mmol, 87.46% yield, n/a purity) as a yellow solid.
448
SUBSTITUTE SHEET RULE 26 Step 2: 3-bromo-4-vinyl-lH-pyrrolo[2,3-c]pyridine
[000936] To a solution of 4-vinyl-lH-pyrrolo[2,3-c]pyridine (16 g, 71.03 mmol, 64% purity, 1 eq) in DCM (250 mL) was added 1 -bromopyrrolidine-2, 5 -di one (15.17 g, 85.23 mmol, 1.2 eq) at 0°C. The mixture was stirred at 20 °C for 3 h. Upon completion, the reaction mixture was quenched with NaHCCh (40 mL), and extracted with DCM (50 mL*3). The organic layer was washed with brine (80 mL) dried over Na2SO4, and concentrated under reduced pressure to give 3-bromo-4-vinyl-lH-pyrrolo[2,3-c]pyridine (16 g, crude) as a yellow solid.
Step 3: 3-bromo-l-methyl-4-vinyl-pyrrolo[2,3-c]pyridine
[000937] To a solution of 3-bromo-4-vinyl-lH-pyrrolo[2,3-c]pyridine (12 g, 53.80 mmol, 1 eq) in DMF (300 mL) was added NaH (2.15 g, 53.80 mmol, 60% purity, 1 eq) at 0 °C for 15 min under N2, then dimethyl sulfate (9.97 g, 79.05 mmol, 7.50 mL, 1.47 eq) added at 0 °C. The mixture was stirred at 0 °C for 1 h. Upon completion, the reaction mixture was quenched by addition sat. NH4CI (300 mL) at 0°C, extracted with ethyl acetate ( 300mL * 3). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%- 55%, 20 min) to give 3-bromo-l-methyl-4-vinyl-pyrrolo[2,3-c]pyridine (3.1 g, 13.07 mmol, 24.31% yield, 100% purity) as a yellow solid.
Step 4: 3-bromo-l-methyl-pyrrolo[2,3-c]pyridine-4-carbaldehyde
[000938] To a solution of 3-bromo-l-methyl-4-vinyl-pyrrolo[2,3-c]pyridine (2.5 g, 10.54 mmol, 1 eq) in THF (30 mL) and H2O (6 mL) was added dipotassium dioxido(dioxo)osmium dihydrate (388.51 mg, 1.05 mmol, 0.1 eq) stirred for 0.5 h, then added sodium periodate (6.77 g, 31.63 mmol, 1.75 mL, 3 eq). The mixture was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was filtered through Celite, filtered and concentrated in vacuo. The residue was triturated with DMF : H2O =3: 1 at 20 °C, then the filterliquor was purified by prep-HPLC (column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 15%-45%, 20 min) to give 3-bromo-l-methyl-pyrrolo[2,3-c]pyridine-4-carbaldehyde (1.7 g, 7.11 mmol, 67.44% yield, 100% purity) as a green solid.
449
SUBSTITUTE SHEET RULE 26 Step 5: l-methyl-3-prop-l-ynyl-pyrrolo[2,3-c]pyridine-4-carbaldehyde
[000939] A mixture of 3-bromo-l-methyl-pyrrolo[2,3-c]pyridine-4-carbaldehyde (500 mg, 2.09 mmol, 1 eq), 4,4,5,5-tetramethyl-2-prop-l-ynyl-l,3,2-dioxaborolane (1.04 g, 6.27 mmol, 3 eq), CS2CO3 (1.36 g, 4.18 mmol, 2 eq) , ditert-butyl(cyclopentyl)phosphane;dichloropalladium iron (136.31 mg, 209.15 umol, 0.1 eq) in dioxane (15 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80 °C for 3 h under N2 atmosphere. Upon completion, the reaction mixture was filtered, then the residue concentrated under reduced pressure to remove dioxane. The residue was purified by prep-HPLC (column: Welch Xtimate C18 250 * 100 mm # 10 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 10%-40%, 20 min) to give l-methyl-3-prop-l-ynyl-pyrrolo[2,3-c]pyridine-4-carbaldehyde (270 mg, 1.36 mmol, 21.71% yield, 100% purity) was obtained as a yellow solid.
Step 6: 2-amino-2-( 1 -methyl-3 -prop- 1 -ynyl-pyrrolo[2,3 -c]pyridin-4-yl)acetonitrile
[000940] To a mixture of l-methyl-3-prop-l-ynyl-pyrrolo[2,3-c]pyridine-4-carbaldehyde (270 mg, 1.36 mmol, 1 eq) in DCM (10 mL) was added tetraisopropoxytitanium (580.69 mg, 2.04 mmol, 603.00 uL, 1.5 eq) and NFU/MeOH (7 M, 756.95 uL, 3.89 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (405.41 mg, 4.09 mmol, 511.23 uL, 3 eq) was added, and the mixture was continued to stirred at 25 °C for 15 h. Upon completion, the mixture was quenched with water (10 mL) and ethyl acetate (20 mL), filtered with diatomite, the organic phase was separated, and extracted with Ethyl acetate (20 mL * 3), washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(l-methyl-3-prop-l- ynyl-pyrrolo[2,3-c]pyridin-4-yl)acetonitrile (270 mg, crude) as a yellow oil.
Step 7: (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrrolo[2,3-c]pyridin-4-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[000941] To a solution of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (438.67 mg, 1.20 mmol, 1 eq) and 2-amino-2-(l-methyl-3-prop-l-ynyl-pyrrolo[2,3-c]pyridin-4- yl)acetonitrile (270 mg, 1.20 mmol, 1 eq) in DCM (10 mL) was added DMAP (367.72 mg, 3.01 mmol, 2.5 eq) and EDCI (461.60 mg, 2.41 mmol, 2 eq). The mixture was stirred at 20 °C for 2 h.
450
SUBSTITUTE SHEET RULE 26 Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and then extracted with Ethyl acetate (20 mL *3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate=50/50 to 1/99), then purified by prep-TLC (SiCh, Ethyl acetate:Methanol=10: 1), which was further separated by SEC (column: REGIS(S,S)WHELK-Ol(250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 35%-35%, 8 min) to give (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl- pyrrolo[2,3-c]pyridin-4-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (120 mg, 210.30 umol, 17.47% yield, 100% purity) as a white solid. MS (ESI) m/z 571.2 [M+H]+
[000942] 'H NMR (400 MHz, DMSO-de) 5 = 9.37 (br d, J = 8.2 Hz, 1H), 9.22 (d, J = 6.8 Hz, 1H), 8.97 (s, 1H), 8.38 (s, 1H), 7.87 (s, 1H), 6.69 (d, J = 6.8 Hz, 1H), 4.42 (d, J = 8.1 Hz, 1H), 4.30 (s, 1H), 3.94 - 3.89 (m, 4H), 3.72 (d, J = 10.6 Hz, 1H), 2.01 (s, 3H), 1.54 (dd, J = 5.3, 7.6 Hz, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.03 (s, 9H), 0.96 (s, 3H), 0.81 (s, 3H).
[000943] To give ( 1 R,2S, 5 S)-N- [cyano-( 1 -methyl-3 -prop- 1 -ynyl-pyrrolo [2,3-c]pyridin-4- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (140 mg, 245.35 umol, 20.38% yield, 100% purity) as a white solid. MS (ESI) m/z 571.2 [M+H]+
451
SUBSTITUTE SHEET RULE 26 Example 114: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino] butanoyl] -3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 321)
Figure imgf000453_0001
Step 1: 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[000944] To a solution of 5-bromoisoquinoline-4-carbaldehyde (2 g, 8.47 mmol, 1 eq) in DMSO (30 mb) was added i-PnNH (2.57 g, 25.42 mmol, 3.59 mL, 3 eq), then was added Cui (645.42 mg, 3.39 mmol, 0.4 eq) and Pd(PPhs)4 (979.02 mg, 847.23 umol, 0.1 eq). After addition, ethynyl(trimethyl)silane (1.66 g, 16.94 mmol, 2.35 mL, 2 eq) was added to slowly under N2, the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with H2O (60 mL) and celite filtered then extracted with EtOAc (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 96:4 to 91 :9) to give 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (1.69 g, 5.78 mmol, 68.25% yield, 86.69% purity) as a yellow solid. MS (ESI) m/z 309.2 [M+H]+.
Step 2: 5-ethynylisoquinoline-4-carbaldehyde
[000945] To a solution of 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (1.69 g, 6.67 mmol, 1 eq) in MeOH (8 mL) was added K2CO3 (2.30 g, 16.68 mmol, 2.5 eq). The mixture was stirred at 20 °C for 40 min. Upon completion, the mixture was filtered to give 5-
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SUBSTITUTE SHEET RULE 26 ethynylisoquinoline-4-carbaldehyde (1.21 g, crude) as a yellow liquid, which was used for next step directly.
Step 3: 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile
[000946] To a solution of 5-ethynylisoquinoline-4-carbaldehyde (1.21 g, 6.68 mmol, 1 eq) in DCM (10 mL) was added Ti(i-PrO)4 (2.85 g, 10.02 mmol, 2.96 mL, 1.5 eq) and NH3/MeOH (7 M, 10 mL, 10.48 eq) stirred 1 h at 20 °C, then TMSCN (1.99 g, 20.03 mmol, 2.51 mL, 3 eq) was added at 20°C. The mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO-i, filtered and concentrated under reduced pressure to give 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (740 mg, crude) as a yellow oil. MS (ESI) m/z 208.1 [M+H]+.
Step 4: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2- carboxamide
[000947] A mixture of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3. L0]hexane-2-carboxylic acid (330 mg, 812.02 umol, 1 eq), 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (185.10 mg, 893.22 umol, 1.1 eq) and 1 -methylimidazole (200.01 mg, 2.44 mmol, 194.18 uL, 3 eq) in ACN (5 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphate (455.67 mg, 1.62 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C 18 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 8 mm) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4- isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2-carboxamide (400 mg, 671.58 umol, 82.70% yield) as a yellow oil. MS (ESI) m/z 596.3 [M+H]+.
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SUBSTITUTE SHEET RULE 26 Step 5: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000948] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3- (l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (400 mg) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 6 min) to give (lR,2S,5S)-N-[cyano-(5- ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (114.45 mg, 192.16 umol, 28.61% yield, 100% purity) as a white solid. MS (ESI) m/z 596.3 [M+H]+.
[000949] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.82 (s, 1H), 9.52 - 9.41 (m, 2H), 8.81 (s, 1H), 8.34 (d, J= 8.1 Hz, 1H), 8.14 (d, J= 6.5 Hz, 1H), 7.79 (t, .7= 7,8 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 4.70 (s, 1H), 4.37 (d, J= 7.3 Hz, 1H), 4.25 (s, 1H), 3.98 - 3.88 (m, 2H), 3.67 (d, J= 10.5 Hz, 1H), 1.53 (dd, J= 5.4, 7.4 Hz, 1H), 1.38 - 1.32 (m, 4H), 1.20 (d, J= 6.4 Hz, 3H), 0.97 (s, 3H), 0.82 - 0.75 (m, 4H), 0.70 - 0.62 (m, 1H), 0.42 - 0.30 (m, 2H).
[000950] To give (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (105.74 mg, 177.53 umol, 26.44% yield, 100% purity) as a white solid. MS (ESI) m/z 596.3 [M+H]+.
[000951] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.88 - 9.76 (m, 1H), 9.72 (d, J= 7.1 Hz, 1H), 9.46 (s, 1H), 8.82 (s, 1H), 8.33 (d, J= 7.4 Hz, 1H), 8.14 (d, J= 7.1 Hz, 1H), 7.79 (t, .7= 7,8 Hz, 1H), 7.68 (d, J= l.Q Hz, 1H), 4.66 (s, 1H), 4.37 - 4.29 (m, 2H), 3.94 - 3.83 (m, 2H), 3.71 - 3.63 (m, 1H), 1.58 (dd, J= 5.5, 7.3 Hz, 1H), 1.47 (d, J= 7.5 Hz, 1H), 1.27 (s, 3H), 1.09 (d, J= 6.1 Hz, 3H), 1.04 (s, 3H), 0.85 (s, 3H), 0.75 - 0.69 (m, 1H), 0.62 - 0.52 (m, 1H), 0.32 - 0.25 (m, 2H).
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SUBSTITUTE SHEET RULE 26 Example 115: Synthesis of (lR,2S,5S)-N-[cyano-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin- 7-yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 322)
Figure imgf000456_0001
Step 1: ethyl 3-bromo-5-chloro-pyridine-4-carboxylate
[000952] To a solution of LDA (2 M, 77.95 mL, 1.2 eq) in THF (96 mL) under N2 at - 65 °C was added a solution of 3-bromo-5-chloro-pyridine (25 g, 129.91 mmol, 1 eq) in THF (192 mL) at - 65 °C. The mixture was stirred at -65 °C for 0.5 h, then ethyl carbonochloridate (145.49 g, 1.34 mol, 127.63 mL, 10.32 eq) was added to the former mixture under N2 at -65 °C and stirred for 0.5 h. Upon completion, the mixture was quenched by aq.NaHCCh (600 mL) at 0 °C. And then extracted with EA (300 mL * 3). The combined organic layers were washed with brine (450 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1 :0 to 60: 1) to give ethyl 3-bromo-5-chloro-pyridine-4-carboxylate (15.5 g, 52.74 mmol, 40.60% yield, 90% purity) as a colorless oil. MS (ESI) m/z 264.1 [M+H]+
Step 2: ethyl 3-chloro-5-methyl-pyridine-4-carboxylate
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SUBSTITUTE SHEET RULE 26 [000953] To a mixture of ethyl 3-bromo-5-chloro-pyridine-4-carboxylate (10.6 g, 40.08 mmol, 1 eq) in dioxane (200 mb) and H2O (20 mL) was added methylboronic acid (3.60 g, 60.11 mmol, 1.5 eq) and dipotassium carbonate (11.08 g, 80.15 mmol, 2 eq) under N2, cyclopentyl(diphenyl)phosphane;dichloropalladium iron (2.93 g, 4.01 mmol, 0.1 eq) was added to the mixture and stirred at 100 °C for 16 h under N2. Upon completion, the reaction mixture was quenched by addition H2O (400 mL) and filtered, then extracted with ethyl acetate (200 mL * 3). The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1 :0 to 50:1) to give ethyl 3- chloro-5-methyl-pyridine-4-carboxylate (6 g, 27.05 mmol, 67.50% yield, 90% purity) as a colorless oil. MS (ESI) m/z 200.1 [M+H]+
Step 3: ethyl 3-(bromomethyl)-5-chloro-pyridine-4-carboxylate
[000954] To a mixture of ethyl 3-chloro-5-methyl-pyridine-4-carboxylate (4.25 g, 21.29 mmol, 1 eq) in CCI4 (150 mL) was added NBS (7.58 g, 42.58 mmol, 2 eq), followed by AIBN (349.58 mg, 2.13 mmol, 0.1 eq), and then the mixture was stirred at 90 °C for 16 h under N2. Upon completion, the reaction mixture was diluted by H2O (300 mL), then extracted with DCM (300 mL * 3), washed by brine (450 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, petroleum etherethyl acetate = 100: 1 to 50: 1) to give crude product 7 g, then the crude product was purified by prep-TLC (SiCh, PE:EA = 5: 1) to give ethyl 3-(bromomethyl)-5-chloro- pyridine-4-carboxylate (2.5 g, 8.53 mmol, 40.05% yield, 95% purity) as a yellow oil. MS (ESI) m/z 278.1 [M+H]+
Step 4: 7-chloro-2-methyl-3H-pyrrolo[3,4-c]pyridin-l-one
[000955] To a solution of ethyl 3-(bromomethyl)-5-chloro-pyridine-4-carboxylate (0.9 g, 3.23 mmol, 1 eq) in THF (27 mL) was added CS2CO3 (4.42 g, 13.57 mmol, 4.2 eq) and methanamine (654.50 mg, 9.69 mmol, 3 eq, HC1), the mixture was stirred at 25 °C for 5 h. Upon completion, the reaction mixture was diluted with THF (30 mL), then the filtrate was concentrated under reduced pressure to give 7-chloro-2-methyl-3H-pyrrolo[3,4-c]pyridin-l-one (0.6 g, crude) as a yellow solid. MS (ESI) m/z 183.2 [M+H]+.
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SUBSTITUTE SHEET RULE 26 Step 5: 2-methyl-7-vinyl-3H-pyrrolo[3,4-c]pyridin-l-one
[000956] To a mixture of 7-chloro-2-methyl-3H-pyrrolo[3,4-c]pyridin-l-one (1.6 g, 8.76 mmol, 1 eq) in dioxane (32 mL) and H2O (6.4 mL) was added CS2CO3 (8.56 g, 26.29 mmol, 3 eq) and potassium trifluoro(vinyl)boranuide (1.17 g, 8.76 mmol, 1 eq) under N2, ditert- butyl(cyclopentyl)phosphane dichloropalladium;iron (571.06 mg, 876.20 umol, 0.1 eq) was added to the former mixture under N2 and stirred at 80 °C for 4 h. Upon completion, the reaction mixture was diluted with H2O (100 mL) and extracted with (EA:MeOH = 10: 1) (40 mL * 5). The combined organic layers were dried over Na2SO4, then concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, petroleum etherethyl acetate = 10:1 to EA:MeOH = 10:1) to give 2-methyl-7-vinyl-3H-pyrrolo[3,4- c]pyridin-l-one (1.2 g, 6.54 mmol, 74.69% yield, 95% purity) as a brown solid. MS (ESI) m/z 175.3 [M+H]+
Step 6: 2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridine-7-carbaldehyde
[000957] To a mixture of 2-methyl-7-vinyl-3H-pyrrolo[3,4-c]pyridin-l-one (1.2 g, 6.89 mmol, 1 eq) in dioxane (40 mL) and H2O (8 mL) was added K2OSO4.2H2O (507.63 mg, 1.38 mmol, 0.2 eq) and 2,6-dimethylpyridine (1.48 g, 13.78 mmol, 1.60 mL, 2 eq), the mixture was stirred at 25 °C for 0.5 h, then NalCh (7.37 g, 34.44 mmol, 1.91 mL, 5 eq) was added to the mixture at 0 °C and stirred at 25 °C for 16 h. Upon completion, the reaction mixture was quenched by H2O (70 mL) and stirred for 15 min, then filtered and the filtrate was extracted with (EA:THF = 10:1) (60 mL * 15). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to EA:THF = 10: 1) to give 2- methyl-l-oxo-3H-pyrrolo[3,4-c]pyridine-7-carbaldehyde (0.88 g, 4.50 mmol, 65.26% yield, 90% purity) as a yellow solid. MS (ESI) m/z 177.2 [M+H]+
Step 7: 2-amino-2-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7-yl)acetonitrile
[000958] To a solution of 2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridine-7-carbaldehyde (0.88 g, 4.50 mmol, 90% purity, 1 eq) in DCM (15 mL) was added NEb/MeOH (7 M, 1.93 mL, 3 eq) and Ti(i-PrO)4 (1.92 g, 6.74 mmol, 1.99 mL, 1.5 eq). The mixture was stirred at 25 °C for 0.5 h, then TMSCN (891.99 mg, 8.99 mmol, 1.12 mL, 2 eq) was added to the former mixture at 0 °C and
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SUBSTITUTE SHEET RULE 26 stirred at 25 °C for 0.5 h. Upon completion, the reaction mixture was quenched by addition H2O (30 mL) at 25 °C and stirred for 5 min, then filtered and the filtrate was extracted with (DCM:MeOH = 10:1) (20 mL * 5). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(2-methyl-l-oxo-3H- pyrrolo[3,4-c]pyridin-7-yl)acetonitrile (870 mg, crude) as a brown oil. MS (ESI) m/z 203.3 [M+H]+.
Step 8: (lR,2S,5S)-N-[cyano-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
[000959] A mixture of (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.57 g, 4.30 mmol, 1 eq) and 2-amino-2-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7-yl)acetonitrile (0.87 g, 4.30 mmol, 1 eq) in ACN (25 mL) was added 1 -methylimidazole (1.06 g, 12.91 mmol, 1.03 mL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (2.41 g, 8.60 mmol, 2 eq), the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by H2O (40 mL) at 25 °C, and then extracted with EA (30 mL * 3). The combined organic layers were washed with brine (45 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 20 min) to give (1R,2S,5S)-N- [cyano-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (1.5 g, 2.71 mmol, 62.92% yield, 99% purity) as a yellow solid. MS (ESI) m/z 549.3 [M+H]+
Step 9: (lR,2S,5S)-N-[cyano-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
[000960] (lR,2S,5S)-N-[cyano-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.5 g, 911.48 umol, 1 eq) was separated by
458
SUBSTITUTE SHEET RULE 26 SFC(condition:column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 35%-35%, 5 min) to give (lR,2S,5S)-N-[cyano-(2-methyl-l-oxo-3H- pyrrolo[3,4-c]pyridin-7-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 1 (0.16 g, 291.67 umol, 32.00% yield, 100% purity) as a white solid. MS (ESI) m/z 549.3 [M+H]+.
[000961] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.67 (d, J = 7.7 Hz, 1H), 9.43 (br d, J = 7.5 Hz, 1H), 8.97 - 8.94 (m, 1H), 8.89 (s, 1H), 6.81 (d, J = 7.7 Hz, 1H), 4.63 (s, 2H), 4.41 - 4.35 (m, 1H), 4.31 (s, 1H), 3.89 (dd, J = 5.4, 10.4 Hz, 1H), 3.68 (d, J = 10.5 Hz, 1H), 3.13 (s, 3H), 1.56 (dd, J = 5.6, 7.6 Hz, 1H), 1.42 (d, J = 7.7 Hz, 1H), 1.07 - 0.82 (m, 15H).
[000962] To give (lR,2S,5S)-N-[cyano-(2-methyl-l-oxo-3H-pyrrolo[3,4-c]pyridin-7- yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (0.2 g, 364.59 umol, 40.00% yield, 100% purity) as a white solid. MS (ESI) m/z 549.3 [M+H]+.
[000963] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.53 (d, J = 7.9 Hz, 1H), 9.48 - 9.38 (m, 1H), 8.99 - 8.92 (m, 1H), 8.89 - 8.83 (m, 1H), 6.90 - 6.79 (m, 1H), 4.63 (s, 2H), 4.42 - 4.36 (m, 1H), 4.33 - 4.25 (m, 1H), 3.89 (br dd, J = 5.5, 10.3 Hz, 1H), 3.68 (d, J = 10.5 Hz, 1H), 3.12 (s, 3H), 1.57 - 1.49 (m, 1H), 1.31 - 1.23 (m, 1H), 1.03 - 0.83 (m, 15H).
Example 116: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-
459
SUBSTITUTE SHEET RULE 26 trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 324)
Figure imgf000461_0001
Step 1: 2-amino-2-(4-bromopyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
[000964] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (500 mg, 2.22 mmol, 1 eq), Ti(i-PrO)4 (947.21 mg, 3.33 mmol, 983.60 uL, 1.5 eq), and NH3/MeOH (7 M, 1.27 mL, 4 eq) in DCM (5 mL) was stirred at 20 °C for 1 h, and then TMSCN (661.25 mg, 6.67 mmol, 833.86 uL, 3 eq) was added. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (5 mL) at 20 °C and then extracted with EtOAc (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3- yl)acetonitrile (400 mg, crude) as a yellow solid. MS (ESI) m/z 236.1 [M+H]+.
Step 2: ((lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[000965] A solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (498.02 mg, 1.23 mmol, 1 eq), 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (400 mg, 1.59 mmol, 1.3 eq), 1 -methylimidazole (301.83 mg, 3.68 mmol, 293.04 uL, 3 eq) in ACN (5 mL)
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SUBSTITUTE SHEET RULE 26 was added TCFH (687.68 mg, 2.45 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was poured into H2O (5 mL) and then extracted with EtOAc (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75 * 30 mm * 3 um; mobile phase: [water(FA)-ACN]; B%: 40%-80%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 445.69 umol, 36.37% yield, 95% purity) as a yellow solid. MS (ESI) m/z 639.0 [M+H]+.
Step 3: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[000966] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5 -a] pyri din-3 -yl)-cyano-methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 469.14 umol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK AD (250mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 16%-16%, 6 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (80.47 mg, 125.84 umol, 26.82% yield) as a white solid. MS (ESI) m/z 639.0 [M+H]+.
[000967] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.85 (s, 1H), 9.33 (d, J= l.Q Hz, 1H), 8.84 (d, J= 7.0 Hz, 1H), 8.32 (s, 1H), 7.70 (d, J= 7.2 Hz, 1H), 6.95 (t, J= 7.1 Hz, 1H), 6.59 (d, J= 6.8 Hz, 1H), 4.41 - 4.30 (m, 1H), 4.22 (s, 1H), 3.98 - 3.88 (m, 2H), 3.67 (d, J= 10.3 Hz, 1H), 1.54 (dd, J= 5.7, 7.5 Hz, 1H), 1.35 - 1.31 (m, 4H), 1.20 - 1.13 (m, 3H), 0.97 (s, 3H), 0.82 - 0.79 (m, 3H), 0.77 - 0.75 (m, 2H), 0.39 - 0.31 (m, 2H)
[000968] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-6,6-dimethyl- 3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (81.49 mg, 127.44 umol, 27.16% yield) was obtained as a white solid. MS (ESI) m/z 639.0 [M+H]+.
461
SUBSTITUTE SHEET RULE 26 [000969] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.80 (br s, 1H), 9.35 (d, J= 6.5 Hz, 1H), 8.84 (d, J= Hz, 1H), 8.30 (s, 1H), 7.69 (d, J= 7.3 Hz, 1H), 6.95 (t, J= 7.2 Hz, 1H), 6.57 (d, J = 6.5 Hz, 1H), 4.35 (br d, J= 7.1 Hz, 1H), 4.27 (s, 1H), 3.95 - 3.82 (m, 2H), 3.68 (br d, J= 10.5 Hz, 1H), 1.57 (br dd, J= 5.4, 7.2 Hz, 1H), 1.38 (d, J= 7.6 Hz, 1H), 1.30 (s, 3H), 1.15 (d, J= 6.1 Hz, 3H), 1.03 (s, 3H), 0.82 (s, 3H), 0.75 (br d, J= 11.1 Hz, 1H), 0.64 - 0.57 (m, 1H), 0.35 - 0.27 (m, 2H)
Example 117: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 325)
Figure imgf000463_0001
Step 1: (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol
[000970] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carboxylic acid (2.7 g, 11.20 mmol, 1 eq) in THF (20 mL) was added with CDI (2.72 g, 16.80 mmol, 1.5 eq) under N2 at 20 °C for 17.5 h. Then the solution was added to a solution of NaBHi (2.12 g, 56.01 mmol, 5 eq) in H2O (20 mL) and stirred at 20°C for 30 min. Upon completion, the reaction mixture was diluted with IM HC1 (20 mL) and the solution was neutralised with saturated aqueous NaHCCh, then extracted with EA (40 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 100:1 to 50: 1) to give (4-
462
SUBSTITUTE SHEET RULE 26 bromopyrazolo[l,5-a]pyridin-3-yl)methanol (2 g, 8.10 mmol, 72.35% yield, 92% purity) as a white solid. MS (ESI) m/z 229.1 [M+H]+.
Step 2: 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde
[000971] A solution of (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol (2 g, 8.81 mmol, 1 eq) in DCM (30 mL) was added with MnCh (15.32 g, 176.17 mmol, 20 eq), and then the mixture was stirred at 20 °C for 12 h. Upon completion, the mixture was filtered and concentrated under reduced pressure to give 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (1.8 g, crude) as a white solid. MS (ESI) m/z 225.0 [M+H]+.
Step 3: 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)a cetonitrile
[000972] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (550 mg, 2.44 mmol, 1 eq), Ti(i-PrO)4 (1.04 g, 3.67 mmol, 1.08 mL, 1.5 eq), NH3/MeOH (7 M, 1.40 mL, 4 eq) in DCM (5 mL) was stirred for 1 h at 20 °C, then TMSCN (727.38 mg, 7.33 mmol, 917.25 uL, 3 eq) was added. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (5 mL) at 20 °C, and then extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (400 mg, crude) as a yellow solid. MS (ESI) m/z 226.0 [M+H]+.
Step 4: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3-tert- butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000973] A solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (487.98 mg, 1.19 mmol, 1 eq), 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (300 mg, 1.19 mmol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (294.29 mg, 3.58 mmol, 285.71 uL, 3 eq) and TCFH (670.49 mg, 2.39 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC
463
SUBSTITUTE SHEET RULE 26 (column: Phenomenex C18 80 * 40 mm * 3 um; mobile phase: [water(NH4HCO3)-ACNJ; B%: 40%-60%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 252.54 umol, 21.14% yield, 90% purity) as a white solid. MS (ESI) m/z 665.2 [M+H]+.
Step 5: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3-tert- butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000974] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5-a] pyri din-3 -yl)-cyano-methyl]-3-
[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 280.60 umol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK IC (250mm * 30 mm, 10 um); mobile phase: [Neu-ETOH]; B%: 24%-24%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (64.57 mg, 100.66 umol, 35.87% yield, 100% purity) as a white solid. MS (ESI) m/z 665.2 [M+H]+.
[000975] 'H NMR (400 MHz, DMSO-t/e) 5 = 9.64 (br s, 1H), 9.25 (d, J= 6.8 Hz, 1H), 8.84 (d, J= 6.8 Hz, 1H), 8.32 (s, 1H), 7.70 (d, J= 7.5 Hz, 1H), 6.94 (t, J= 7.3 Hz, 1H), 6.60 (d, J = 7.0 Hz, 1H), 4.48 - 4.34 (m, 1H), 4.23 (s, 1H), 4.12 - 3.72 (m, 2H), 3.65 (d, J= 10.5 Hz, 1H), 1.60 - 1.50 (m, 1H), 1.34 (d, J= 7.5 Hz, 1H), 1.20 - 0.78 (m, 18H).
[000976] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (65.85 mg, 102.65 umol, 36.58% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 665.2 [M+H]+.
[000977] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.64 (br s, 1H), 9.26 (d, J= 6.6 Hz, 1H), 8.84 (d, J= 6.8 Hz, 1H), 8.32 (s, 1H), 7.69 (d, J= 7.5 Hz, 1H), 6.94 (t, J = 7.1 Hz, 1H), 6.57 (d, J = 6.6 Hz, 1H), 4.44 - 4.33 (m, 1H), 4.27 (s, 1H), 3.96 (dd, J= 5.5, 10.7 Hz, 1H), 3.91 - 3.80 (m, 1H), 3.66 (d, J= 10.5 Hz, 1H), 1.69 - 1.48 (m, 1H), 1.40 (d, J= 7.7 Hz, 1H), 1.16 - 0.75 (m, 18H)
464
SUBSTITUTE SHEET RULE 26 Example 118: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl] -3- [(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6, 6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 326)
Figure imgf000466_0001
Step 1: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000978] A solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (500 mg, 1.36 mmol, 1 eq) in ACN (10 mL) was added with 2-amino-2-(4-bromopyrazolo[l,5- a]pyri din-3 -yl)acetonitrile (342.70 mg, 1.36 mmol, 1 eq), and 1 -methylimidazole (336.17 mg, 4.09 mmol, 326.38 uL, 3 eq), and then TCFH (765.92 mg, 2.73 mmol, 2 eq) was added. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250 * 70 mm * 10 um; mobile phase: [water(NH4HCO3)-ACN]; B%: 30%-60%, 20 min) and then further purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water(FA)-ACN]; B%: 30%-70%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 345.01 umol, 25.28% yield, 94% purity) as a white solid. MS (ESI) m/z 601.1 [M+H]+.
465
SUBSTITUTE SHEET RULE 26 Step 2: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000979] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5-a] pyri din-3 -yl)-cyano-methyl]-3-
[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 367.03 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 25%-25%, 5 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (70.53 mg, 117.67 umol, 32.06% yield) as a white solid. MS (ESI) m/z 601.1 [M+H]+.
[000980] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.86 (br s, 1H), 9.35 (d, J= 7.0 Hz, 1H), 8.84 (d, J= 6.9 Hz, 1H), 8.32 (s, 1H), 7.70 (d, J= 7.3 Hz, 1H), 6.94 (t, J= 7.2 Hz, 1H), 6.58 (d, J = 6.9 Hz, 1H), 4.36 (br d, J= 7.5 Hz, 1H), 4.23 (s, 1H), 4.01 - 3.87 (m, 1H), 3.86 - 3.80 (m, 1H), 3.71 - 3.61 (m, 1H), 3.25 (s, 3H), 1.55 (dd, J= 5.4, 7.4 Hz, 1H), 1.33 (d, J= 7.6 Hz, 1H), 1.17 (d, J= 6.2 Hz, 3H), 0.98 (s, 3H), 0.85 (s, 3H)
[000981] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (67.09 mg, 111.93 umol, 30.50% yield) was obtained as a white solid. MS (ESI) m/z 601.1 [M+H]+.
[000982] 'H NMR (400 MHz, DMSO-d6) 5 = 9.85 (br s, 1H), 9.40 (d, J= 6.5 Hz, 1H), 8.83 (d, J= 6.9 Hz, 1H), 8.31 (s, 1H), 7.68 (d, J= 7.4 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 6.56 (d, J = 6.4 Hz, 1H), 4.35 (br d, J= 8.1 Hz, 1H), 4.28 (s, 1H), 3.98 - 3.87 (m, 1H), 3.87 - 3.79 (m, 1H), 3.66 - 3.55 (m, 1H), 3.22 (s, 3H), 1.58 (dd, J= 5.4, 7.3 Hz, 1H), 1.38 (d, J= 7.6 Hz, 1H), 1.15 (d, J= 6.1 Hz, 3H), 1.04 (s, 3H), 0.86 (s, 3H)
466
SUBSTITUTE SHEET RULE 26 Example 119: Synthesis of (3S,3aS,6aR)-2-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxylic acid
Figure imgf000468_0001
Step 1: ethyl (3S,3aS,6aR)-2-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]- 3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylate
[000983] To a solution of ethyl (3S,3aS,6aR)-l,2,3,3a,4,5,6,6a- octahydrocyclopenta[c]pyrrole-3-carboxylate (1 g, 4.55 mmol, 1 eq, HC1) and (2S)-2-(tert- butoxycarbonylamino)-3,3-dimethyl-butanoic acid (1.05 g, 4.55 mmol, 1 eq) in DCM (12 mL) was added DMAP (1.11 g, 9.10 mmol, 2 eq and EDCI (1.75 g, 9.10 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with water (50 mL) and extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 50:1 to 10:1) to afford ethyl (3S,3aS,6aR)-2-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylate (1.4 g, 3.46 mmol, 76.02% yield, 98% purity) as a yellow oil.
Step 2: (3S,3aS,6aR)-2-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]- 3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid
[000984] To a solution of ethyl (3S,3aS,6aR)-2-[(2S)-2-(tert-butoxycarbonylamino)-3,3- dimethyl-butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylate (1.4 g, 3.53
467
SUBSTITUTE SHEET RULE 26 mmol, 1 eq) in THF (10 mL), H2O (3 mL) and MeOH (3 mL) was added LiOH.IUO (296.30 mg, 7.06 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was acide with aq. HC1 (IM) to adjust pH=3, and then extracted with EA (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give (3S,3aS,6aR)-2-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]- 3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1.2 g, crude) as a white solid.
Step 3: (3S, 3aS,6aR)-2-[(2S)-2-amino-3,3-dimethyl-butanoyl]-3, 3a, 4,5,6, 6a- hexahydro- 1 Iley clopenta[c]pyrrole-3-carboxylic acid
[000985] A solution of (3S,3aS,6aR)-2-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (0.1 g, 271.40 umol, 1 eq) in HCl/dioxane (4 M, 2 mL, 29.48 eq) was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure, and then diluted with DCM (5 mL) and extracted concentrated under reduced pressure to give (3S,3aS,6aR)-2-[(2S)-2-amino- 3,3-dimethyl-butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (0.08 g, crude, HC1) as a white oil.
Step 4: (3S,3aS,6aR)-2-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid
[000986] A solution of (3S,3aS,6aR)-2-[(2S)-2-amino-3,3-dimethyl-butanoyl]-3,3a,4,5,6,6a- hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (0.04 g, 131.23 umol, 1 eq, HC1) in methyl 2,2,2-trifluoroacetate (508.00 mg, 3.97 mmol, 400.00 uL, 30.23 eq) was added with TEA (39.84 mg, 393.69 umol, 54.80 uL, 3 eq), and the solution was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NELHCOsj-ACN]; B%: 10%-40%, 8 min) to give (3S,3aS,6aR)-2- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH- cyclopenta[c]pyrrole-3-carboxylic acid (22.91 mg, 61.62 umol, 46.96% yield, 98% purity) as a white solid. MS (ESI) m/z 365.1 [M+H]+.
468
SUBSTITUTE SHEET RULE 26 [000987] 'H NMR (400 MHz, D2O) 5 = 4.65 - 4.44 (m, 1H), 4.34 - 4.03 (m, 1H), 3.92 - 3.72 (m, 1H), 3.70 - 3.27 (m, 1H), 2.80 - 2.56 (m, 2H), 2.09 - 1.76 (m, 2H), 1.74 - 1.50 (m, 3H), 1.47 - 1.33 (m, 1H), 1.13 - 0.90 (m, 9H).
Example 120: Synthesis of (3S,3aS,6aR)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-2-[(2S)- 3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -3, 3a, 4,5,6, 6a-hexahydro- 1H- cyclopenta[c] pyrrole- 3-carboxamide (Compound 330)
Figure imgf000470_0001
Step 1: 5-bromoisoquinoline-4-carbaldehyde
[000988] To a mixture of cone. H2SO4 (100 mL) and isoquinoline-4-carbaldehyde (10 g, 63.63 mmol, 1 eq) at 0 °C was added NBS (14.04 g, 78.87 mmol, 1.24 eq) slowly and the reaction mixture was allowed to come to 25 °C was stirred for 16 h. Upon completion, the mixture was adjust pH to 7 by NaOH (1000 mL), and extracted with ethyl acetate (800 mL * 2). The combined organic layers were washed with brine (1000 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 3: 1 to 0: 1) and re-purified by prep-HPLC (column: Welch Xtimate C18 250 * 70 mm # 10 um; mobile phase: [water (NHiHCChj-ACN]; B%: 25%-55%, 20 min) to give 5-bromoisoquinoline-4-carbaldehyde (2 g, 8.47 mmol, 13.32% yield) as yellow solid.
Step 2: 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile
469
SUBSTITUTE SHEET RULE 26 [000989] To a solution of 5-bromoisoquinoline-4-carbaldehyde (0.5 g, 2.12 mmol, 1 eq) in NHa/MeOH (10 mL) was added NH4CI (339.89 mg, 6.35 mmol, 3 eq). After stirring at 20 °C for 1 h, TMSCN (630.40 mg, 6.35 mmol, 794.96 uL, 3 eq) was added drop-wise at 0 °C, the solution was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was diluted with water (20 mL) and extracted with DCM (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 1 :0 to 8:1) to give 2- amino-2-(5-bromo-4-isoquinolyl)acetonitrile (0.35 g, 1.20 mmol, 56.74% yield, 90% purity) as a yellow oil.
Step 3: (3S,3aS,6aR)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-2-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxamide
[000990] To a solution of (3S,3aS,6aR)-2-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (681.16 mg, 1.87 mmol, 1.4 eq) and 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (0.35 g, 1.34 mmol, 1 eq) in ACN (10 mL) was added TCFH (749.34 mg, 2.67 mmol, 2 eq) and 1- methylimidazole (219.27 mg, 2.67 mmol, 212.89 uL, 2 eq), the solution was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with water (30 mL) and extracted with DCM (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters X bridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 40%-70%, 8 min) to give (3S,3aS,6aR)-N-[(5-bromo-4-isoquinolyl)- cyano-methyl]-2-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a- hexahydro-lH-cyclopenta[c]pyrrole-3-carboxamide (0.5 g, 821.76 umol, 61.54% yield) as a yellow oil.
Step 4: (3S,3aS,6aR)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-2-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxamide
470
SUBSTITUTE SHEET RULE 26 [000991] (3S,3aS,6aR)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-2-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxamide (0.5 g, 821.76 umol, 1 eq) was separated by SFC (column: ChiralPak IH, 250 * 30 mm, lOum; mobile phase: [Neu-IPA]; B%: 25%-25%, 8 min) to give (3S,3aS,6aR)-N-[(5- bromo-4-isoquinolyl)-cyano-methyl]-2-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxamide Isomer 1 (66.87 mg, 109.90 umol, 13.37% yield, 100% purity) as a white solid. MS (ESI) m/z 608.1 [M+H]+.
[000992] 'H NMR (400 MHz, DMSO-de) 5 = 9.54 (br d, J = 6.2 Hz, IH), 9.50 (s, IH), 9.28 (br s, IH), 8.90 (s, IH), 8.32 (d, J = 7.8 Hz, IH), 8.27 (d, J = 7.2 Hz, IH), 7.67 (t, J = 7.8 Hz, IH), 7.44 (br d, J = 6.1 Hz, IH), 4.50 (br s, IH), 4.28 (d, J = 4.0 Hz, IH), 3.84 (dd, J = 7.6, 10.2 Hz, IH), 3.64 (dd, J = 3.1, 10.6 Hz, IH), 2.82 - 2.70 (m, IH), 2.56 (br dd, J = 3.9, 7.5 Hz, IH), 1.94 - 1.73 (m, 2H), 1.70 - 1.48 (m, 3H), 1.39 (br dd, J = 6.2, 12.7 Hz, IH), 0.95 (s, 9H).
[000993] (3S,3aS,6aR)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-2-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxamide Isomer 2 (63.6 mg, 102.44 umol, 12.47% yield, 98% purity) was obtained as a white solid. MS (ESI) m/z 608.1 [M+H]+.
[000994] 1 H NMR (400 MHz, DMSO-de) 5 = 9.57 - 9.48 (m, IH), 9.42 (br d, J = 7.1 Hz, IH),
9.27 (br s, IH), 8.89 (s, IH), 8.33 (d, J = 7.9 Hz, IH), 8.28 (d, J = 7.5 Hz, IH), 7.73 - 7.62 (m, IH), 7.43 (br d, J = 6.7 Hz, IH), 4.50 (br d, J = 4.5 Hz, IH), 4.17 (d, J = 4.8 Hz, IH), 3.82 (dd, J = 7.5, 10.3 Hz, IH), 3.64 (dd, J = 2.7, 10.5 Hz, IH), 2.73 - 2.61 (m, IH), 2.56 (dt, J = 3.8, 7.5 Hz, IH), 1.86 - 1.67 (m, 2H), 1.67 - 1.44 (m, 3H), 1.43 - 1.29 (m, IH), 1.05 - 0.95 (m, 9H).
471
SUBSTITUTE SHEET RULE 26 Example 121: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 339)
Figure imgf000473_0001
Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[000995] A mixture of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (0.5 g, 1.52 mmol, 65% purity, 1 eq) and (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (558.28 mg, 1.52 mmol, 1 eq) in ACN (10 mL) was added 1 -methylimidazole (375.36 mg, 4.57 mmol, 364.42 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (855.19 mg, 3.05 mmol, 2 eq), and the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (20 mL * 5), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Welch Xtimate C 18 250 * 70 mm # lOum; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 20 min) to give (1R,2S,5S)- N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (0.52 g, 925.97 umol, 60.76% yield, 100% purity) as a yellow solid. MS (ESI) m/z 562.1 [M+H]+
[000996] 'H NMR (400 MHz, DMSO-de) 5 = 9.86 (br t, J = 7.2 Hz, 1H), 9.74 - 9.39 (m, 2H), 8.76 - 8.68 (m, 1H), 8.62 (s, 1H), 7.21 - 6.93 (m, 1H), 4.55 - 4.42 (m, 1H), 4.41 - 4.21 (m, 2H),
472
SUBSTITUTE SHEET RULE 26 3.96 - 3.79 (m, 2H), 3.71 - 3.50 (m, 1H), 3.28 - 3.17 (m, 3H), 1.62 - 1.51 (m, 1H), 1.49 - 1.34 (m, 1H), 1.20 - 0.97 (m, 6H), 0.86 (d, J = 16.0 Hz, 3H)
Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[000997] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (0.4 g, 712.28 umol, 1 eq) was purified by SFC (condition: column: REGIS (s,s) WHELK-01 (250 mm * 30 mm, 5um) mobile phase: [Heptane-EtOH];B%: 50%-50%, 10 min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (0.12 g, 209.84 umol, 29.46% yield, 98.2% purity) as a white solid. MS (ESI) m/z 562.1 [M+H]+
[000998] 'H NMR (400 MHz, DMSO-de) 5 = 9.87 (br s, 1H), 9.49 - 9.39 (m, 2H), 8.71 (s, 1H), 8.62 (s, 1H), 7.02 (br d, J = 6.5 Hz, 1H), 4.52 (s, 1H), 4.37 (br d, J = 8.4 Hz, 1H), 4.24 (s, 1H), 3.96 - 3.88 (m, 1H), 3.86 - 3.79 (m, 1H), 3.73 - 3.62 (m, 1H), 3.26 (s, 3H), 1.55 (dd, J = 5.3, 7.4 Hz, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.18 (d, J = 6.2 Hz, 3H), 0.98 (s, 3H), 0.84 (s, 3H)
[000999] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (0.12 g, 209.20 umol, 29.37% yield, 97.9% purity) was obtained as a white solid. MS (ESI) m/z 562.1 [M+H]+
[0001000] 'H NMR (400 MHz, DMSO-de) 5 = 9.85 (br d, J = 6.2 Hz, 1H), 9.70 (br d, J = 5.7 Hz, 1H), 9.41 (s, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.13 (br s, 1H), 4.47 (s, 1H), 4.39 - 4.28 (m, 2H), 3.94 - 3.87 (m, 1H), 3.86 - 3.80 (m, 1H), 3.63 - 3.53 (m, 1H), 3.19 (s, 3H), 1.58 (dd, J = 5.3, 7.4 Hz, 1H), 1.47 (d, J = 7.6 Hz, 1H), 1.14 - 1.02 (m, 6H), 0.88 (s, 3H)
473
SUBSTITUTE SHEET RULE 26 Example 122: Synthesis of (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-
1(11), 4(12), 5,7, 9-pentaen-3-yl)-6,6-dimethyl- 3-[(2S, 3R)-3-(l-methylcyclopropoxy)-2-[(2, 2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 343)
Figure imgf000475_0001
Step 1: methyl isoquinoline-4-carboxylate
[0001001] To a solution of 4-bromoisoquinoline (55 g, 264.35 mmol, 1 eq) in MeOH (600 mL) was added TEA (267.50 g, 2.64 mol, 367.95 mL, 10 eq), Xantphos (30.59 g, 52.87 mmol, 0.2 eq) and Pd(OAc)2 (5.93 g, 26.44 mmol, 0.1 eq). After the addition, the resulting mixture was stirred at 75 °C under CO (264.35 mmol, 1 eq) (30 psi) for 48 h. Two batches were set up in parallel. Upon completion, the combined reaction mixture was filtered. The filtrate was concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 10: 1 to 2: 1) to afford methyl isoquinoline-4-carboxylate (81 g, 393.76 mmol, 74.48% yield, 91% purity) as white solid and other cur de product (55 g) which need further purification.
Step 2: methyl 5-bromoisoquinoline-4-carboxylate
474
SUBSTITUTE SHEET RULE 26 [0001002] To a mixture of methyl isoquinoline-4-carboxylate (81 g, 432.71 mmol, 1 eq) in H2SO4 (560 mL) at 0 °C was added NBS (110.90 g, 623.10 mmol, 1.44 eq) slowly in portions. After the addition, the resulting mixture was stirred at 25 °C for 12 h. To the reaction mixture was added ice (300 g) and EtOAc (1 L). Upon completion, the resulting mixture was adjusted pH to 7-8 with sat.aqueous NaHCCh. The organic layer was separated, dried over Na2SO4, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 10:1 to 6: 1) to afford methyl 5- bromoisoquinoline-4-carboxylate (80 g, 269.68 mmol, 62.32% yield, 89.7% purity) as a yellow solid. MS (ESI) m/z 266.0 [M+H]+.
Step 3: methyl 5-allylisoquinoline-4-carboxylate
[0001003] To a solution of methyl 5-bromoisoquinoline-4-carboxylate (20 g, 75.16 mmol, 1 eq), PPhs (1.97 g, 7.52 mmol, 0.1 eq) and allyl(tributyl)stannane (37.33 g, 112.74 mmol, 34.57 mL, 1.5 eq) in toluene (200 mL) under N2 was added Pd2(dba)3 (6.88 g, 7.52 mmol, 0.1 eq). After the addition, the mixture was stirred at 110 °C for 12 h. Upon completion, the reaction mixture cooled to 25 °C, diluted with EtOAc (1000 mL) and water (1000 mL). The mixture was extracted with EtOAc (1000 mL * 3). The combined organic layer was dried over Na2SO4, filtered, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 20: 1 to 3:1) to afford methyl 5- allylisoquinoline-4-carboxylate (26 g, 93.81 mmol, 62.41% yield, 82% purity) as yellow oil. MS (ESI) m/z 228.1 [M+H]+.
Step 4: methyl 5-(2-methoxy-2-oxo-ethyl)isoquinoline-4-carboxylate
[0001004] Methyl 5-allylisoquinoline-4-carboxylate (28 g, 123.21 mmol, 1 eq) in anhydrous DCM (1 L) was cooled to -60 °C. Then, 250 mL 2.5 M NaOH/MeOH was added. After the addition, ozone was bubbled pass through the reaction solution at 15 psi for 6 h. The reaction mixture was quenched by (500 mL) 10% aqueous citric acid, extracted with DCM (1 L * 2). Upon completion, the combined organic layer was dried over Na2SO4, fitlered, conentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 20: 1 to 8: 1) to afford methyl 5-(2-methoxy-2-oxo-
475
SUBSTITUTE SHEET RULE 26 ethyl)isoquinoline-4-carboxylate (6.8 g, 16.58 mmol, 13.45% yield, 63.2% purity). MS (ESI) m/z 260.1 [M+H]+.
Step 5: methyl 3-oxo-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4,6,8(12),9-pentaene-2- carboxylate
[0001005] A mixture of methyl 5-(2-methoxy-2-oxo-ethyl)isoquinoline-4-carboxylate (3.4 g, 13.11 mmol, 1 eq) and t-BuOK (4.41 g, 39.34 mmol, 3 eq) in toluene (40 mL) was stirred at 50 °C for 2 h. Two batches were set up in parallel. Upon completion, the reaction mixture was concentrated in a vacuum to afford methyl 3-oxo-6-azatricyclo[6.3.1.04,12]dodeca- 1(1 l),4,6,8(12),9-pentaene-2-carboxylate (6 g, crude) as red solid, which was used for next step without further purification. MS (ESI) m/z 228.0 [M+H]+.
Step 6: 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one
[0001006] A solution of methyl 3-oxo-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4, 6, 8(12), 9- pentaene-2-carboxylate (3 g, 13.20 mmol, Crude purity, 1 eq) in 6 M HC1 (60 mL) was strried at 110 °C for 1 h. Two batches were set up in parallel. Upon completion, the reaction mixture was cooled to 25 °C, poured into sat.aq.NaHCCh (1.5 L), diluted with EtOAc (1 L). The resulting mixture was filtered and the filter cake was wahsed with EtOAc (500 mL). The combined filtrate was extracted with EtOAc (1.5 L * 2). The organic layer was separated, dried over Na2SO4, filtered, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 3: 1 to 5: 1) to give 6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one (2.36 g, 13.95 mmol, 47.20% yield) as a yellow oil. MS (ESI) m/z 170.0 [M+H]+.
Step 7: 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one
[0001007] To a solution of 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one (1.5 g, 8.87 mmol, 1 eq) and Ti(i-PrO)4 (3.78 g, 13.30 mmol, 3.93 mL, 1.5 eq) in DCM (15 mL) was added NEh/MeOH (7 M, 5.07 mL, 4 eq), and the resulting mixture was stirred 1 h at 25 °C. TMSCN (2.64 g, 26.60 mmol, 3.33 mL, 3 eq) was added at 25 °C. The mixture was stirred at 25 °C for 15 h. The reaction mixture was poured into H2O (30 mL) at 25 °C, and then extracted with EtOAc (30 mL * 3). Upon completion, the combined organic layers were washed with brine
476
SUBSTITUTE SHEET RULE 26 (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 5: 1 to 0: 1) to give 3-amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaene-3- carbonitrile (600 mg, 2.70 mmol, 30.50% yield, 88% purity) as a black brown oil. MS (ESI) m/z 196.2 [M+H]+.
Step 8: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4(12), 5,7, 9-pentaen-3- yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001008] A mixture of 3-amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4(12), 5,7, 9-pentaene-
3 -carbonitrile (300 mg, 1.54 mmol, 1 eq), (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (749.43 mg, 1.84 mmol, 1.2 eq) and 1 -methylimidazole (378.51 mg, 4.61 mmol, 367.49 uL, 3 eq) in ACN (10 mL) was added [chloro(dimethylamino)methylene]-dimethyl- ammonium hexafluorophosphate (862.35 mg, 3.07 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was poured into H2O 30 mL at 20 °C, and then extracted with EtOAC (30 mL * 3). Upon completion, the combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 10 min) to give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (410 mg, 636.78 umol, 41.44% yield, 90.64% purity) as a yellow solid. MS (ESI) m/z 584.3 [M+H]+.
Step 9: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001009] (lR,2S,5S)-N-(3-cyano-6-azatncyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (600 mg) was
477
SUBSTITUTE SHEET RULE 26 separated by SFC (column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 35%-35%, 7 min) to give (lR,2S,5S)-N-(3-cyano-6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (121.74 mg, 208.60 umol, 29.69% yield, 100% purity) as a white solid. MS (ESI) m/z 584.3 [M+H]+.
[0001010] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.77 (s, 1H), 9.50 (s, 1H), 9.41 (s, 1H), 8.88 (s, 1H), 8.05 (d, J= 8.1 Hz, 1H), 7.82 - 7.76 (m, 1H), 7.74 - 7.70 (m, 1H), 4.36 (d, J= 8.2 Hz, 1H), 4.28 (d, J= 18.3 Hz, 1H), 4.19 (s, 1H), 3.99 - 3.91 (m, 2H), 3.78 (d, J= 18.3 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 1.60 (dd, J= 5.3, 7.4 Hz, 1H), 1.38 (s, 1H), 1.36 - 1.34 (m, 3H), 1.22 (d, J= 6.2 Hz, 3H), 1.01 (s, 3H), 0.80 (s, 3H), 0.77 (br d, J= 4.6 Hz, 1H), 0.71 - 0.64 (m, 1H), 0.42 - 0.30 (m, 2H).
[0001011] (lR,2S,5S)-N-(3-cyano-6-azatncyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (141.72 mg, 241.74 umol, 34.41% yield, 99.55% purity) was obtained as a white solid. MS (ESI) m/z 584.3 [M+H]+.
[0001012] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.78 (d, J= 7.0 Hz, 1H), 9.50 (s, 1H), 9.38 (s, 1H), 8.82 (s, 1H), 8.03 (d, J= 8.1 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.75 - 7.70 (m, 1H), 4.36 (s, 1H), 4.26 (d, J= 18.2 Hz, 1H), 4.22 (s, 1H), 3.97 - 3.89 (m, 3H), 3.69 (d, J= 10.4 Hz, 1H), 1.57 (dd, J = 5.3, 7.4 Hz, 1H), 1.42 (d, J= 7.6 Hz, 1H), 1.35 (s, 3H), 1.22 (d, J= 6.1 Hz, 3H), 1.01 - 0.97 (m, 3H), 0.81 (s, 3H), 0.77 (d, J= 4.8 Hz, 1H), 0.69 (d, J= 5.1 Hz, 1H), 0.40 - 0.32 (m, 2H).
Example 123: Synthesis of (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- l(ll),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 344)
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SUBSTITUTE SHEET RULE 26
Figure imgf000480_0001
Step 1: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001013] A mixture of 3-amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4(12), 5,7, 9-pentaene- 3 -carbonitrile (300 mg, 1.54 mmol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (675.54 mg, 1.84 mmol, 1.2 eq) and 1 -methylimidazole (378.50 mg, 4.61 mmol, 367.47 uL, 3 eq) in ACN (1 mL) was added [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (862.35 mg, 3.07 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was poured into H2O (30 mL) at 20 °C, and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 10 min) to give (lR,2S,5S)-N-(3-cyano-6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (404 mg, 709.54 umol, 46.17% yield, 95.46% purity) as a yellow solid. MS (ESI) m/z 544.2 [M+H]+.
Step 2: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001014] (lR,2S,5S)-N-(3-cyano-6-azatncyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (600 mg) was separated by SFC (column: DAICEL
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SUBSTITUTE SHEET RULE 26 CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 35%-35%, 11 min) to give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (109.08 mg, 200.69 umol, 27.00% yield, 100% purity) as a white solid. MS (ESI) m/z 544.2 [M+H]+.
[0001015] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.82 (br s, 1H), 9.51 (s, 1H), 9.41 (s, 1H), 8.88 (s, 1H), 8.04 (d, J= 8.1 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.74 - 7.69 (m, 1H), 4.36 (br d, .7= 8.2 Hz, 1H), 4.27 (d, J= 18.3 Hz, 1H), 4.19 (s, 1H), 3.96 (dd, J= 5.4, 10.3 Hz, 1H), 3.85 - 3.74 (m, 2H), 3.70 - 3.63 (m, 1H), 3.28 (s, 3H), 1.62 (dd, J= 5.4, 7.5 Hz, 1H), 1.36 (d, J= 7.6 Hz, 1H), 1.22 (d, J= 6.2 Hz, 3H), 1.02 (s, 3H), 0.84 (s, 3H).
[0001016] (lR,2S,5S)-N-(3-cyano-6-azatncyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (138.4 mg, 245.21 umol, 32.99% yield, 96.3% purity) was obtained as a white solid. MS (ESI) m/z 544.2 [M+H]+.
[0001017] 1H NMR (400 MHz, DMSO-t/6) 5 = 9.81 (s, 1H), 9.51 (s, 1H), 9.37 (s, 1H), 8.81 (s, 1H), 8.03 (d, J= 8.1 Hz, 1H), 7.83 - 7.75 (m, 1H), 7.75 - 7.70 (m, 1H), 4.36 (d, J= 8.2 Hz, 1H), 4.31 - 4.21 (m, 2H), 3.99 - 3.82 (m, 3H), 3.70 - 3.60 (m, 1H), 3.27 (s, 3H), 1.59 (dd, J= 5.4, 7.4 Hz, 1H), 1.42 (d, J= 7.6 Hz, 1H), 1.22 (d, J = 6.1 Hz, 3H), 1.00 (s, 3H), 0.85 (s, 3H).
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SUBSTITUTE SHEET RULE 26 Example 124: Synthesis of N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide (Compound 345)
Figure imgf000482_0001
Step 1: methyl 6-azaspiro[3.4]octane-7-carboxylate
[0001018] A solution of 6-tert-butoxycarbonyl-6-azaspiro[3.4]octane-7-carboxylic acid (7 g, 27.42 mmol, 1 eq) in HCI/MeOH (4M, 70 mb) was stirred at 60 °C for 18 h. Upon completion, the mixture was concentrated in vacuum to obtained methyl 6-azaspiro[3.4]octane-7-carboxylate (5.5 g, crude, HC1) as a black brown gum.
Step 2: methyl 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6- azaspiro[3.4]octane-7-carboxylate
[0001019] To a solution of methyl 6-azaspiro[3.4]octane-7-carboxylate (6.5 g, 31.60 mmol, 1 eq, HC1) and (2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoic acid (8.04 g, 34.76
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SUBSTITUTE SHEET RULE 26 mmol, 1.1 eq) in DCM (20 mL) was added DMAP (11.58 g, 94.81 mmol, 3 eq) and EDCI (12.12 g, 63.20 mmol, 2 eq), and the reaction was stirred at 20 °C for 2 h. Upon completion, the mixture was adjusted pH~l with aq. HC1 (IM) and then was extracted with DCM (100 mL * 3), then the organic phase was adjusted pH~7 with sat.NaHCCh, then the organic phase was dried with Na2SO4, filtered and concentrated in vacuum and the crude product was purified by column (SiCh, PE:EA = 1:0 to 5:1) to obtained methyl 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3- dimethyl-butanoyl]-6-azaspiro[3.4]octane-7-carboxylate (10 g, 23.53 mmol, 74.46% yield, 90% purity) as a yellow oil. MS (ESI) m/z 383.2 [M+H]+
Step 3: 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7- carboxylic acid
[0001020] To a solution of methyl 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6-azaspiro[3.4]octane-7-carboxylate (10 g, 26.14 mmol, 1 eq) in THF (60 mL) and H2O (20 mL) was added LiOH.IUO (3.29 g, 78.43 mmol, 3 eq) at 20 °C, and the mixture was stirred at 20 °C for 6 h. Upon completion, the mixture was adjusted pH~l with aq. HC1 (IM) and then was extracted with EA (50 mL * 3), then the organic phase was dried with Na2SO4, filtered and concentrated in vacuum to obtained 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3- dimethyl-butanoyl]-6-azaspiro[3.4]octane-7-carboxylic acid (9.5 g, crude) as a light yellow gum. MS (ESI) m/z 367.1 [M-H]+
Step 4: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifhroromethyl)-l,2,4-oxadiazol-5- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[0001021] A solution of 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6- azaspiro[3.4]octane-7-carboxylic acid (9.5 g, 25.78 mmol, 1 eq) in HCl/dioxane (100 mL, 4 M) the mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum to afford 6-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7-carboxylic acid (8 g, crude, HC1) as a yellow gum. MS (ESI) m/z 269.3 [M+H]+
Step 5: 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7- carboxylic acid
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SUBSTITUTE SHEET RULE 26 [0001022] A solution of 6-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7- carboxylic acid (8 g, 26.25 mmol, 1 eq, HC1) in methyl 2,2,2-trifluoroacetate (33.61 g, 262.46 mmol, 26.46 mL, 10 eq) and TEA (7.97 g, 78.74 mmol, 10.96 mL, 3 eq) was stirred at 20 °C for 6 h. Upon completion, the mixture was concentrated in vacuum and then was adjust pH~l with HC1 (1 M), then was extracted with ethyl acetate (50 mL * 3), the organic phase was concentrated in vacuum. The crude product was triturated with the solution (EA:PE = 1 : 30) at 5 °C for 30 min, then the mixture was filtered and the filter cake was obtained 6-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxylic acid (1.9 g, 4.43 mmol, 16.88% yield, 85% purity) as a white solid. The mother liquor was concentrated in vacuum to afford the crude as a yellow gum and was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water (HCl)-ACN]; B%: 32%- 62%, 20 min) to obtained 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6- azaspiro[3.4]octane-7-carboxylic acid (1.3 g, 3.21 mmol, 12.23% yield, 90% purity) as a white solid. MS (ESI) m/z 365.1 [M+H]+
Step 6: 5-bromoisoquinoline-4-carbaldehyde
[0001023] To a mixture of cone. H2SO4 (50 mL) and isoquinoline-4-carbaldehyde (12.8 g, 81.44 mmol, 1 eq) at 0 °C was added NBS (14.50 g, 81.44 mmol, 1 eq) slowly and the reaction mixture was warmed to 25 °C was stirred for 16 h. Upon completion, the mixture was adjust pH~7 by NaOH (1000 mL), and extracted with ethyl acetate (1000 mL * 2). The combined organic layers were washed with brine (1000 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue and was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water (HCl)-ACN]; B%: 15%- 45%, 20 min) to afford 5-bromoisoquinoline-4-carbaldehyde (5.16 g, 18.58 mmol, 22.81% yield, 85% purity) as yellow solid. MS (ESI) m/z 236.1, 238.1 [M+H]+
Step 7: 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[0001024] To a solution of 5-bromoisoquinoline-4-carbaldehyde (2.98 g, 10.73 mmol, 85% purity, 1 eq, Parallel 2 batches) in DMSO (80 mL) was added N-isopropylpropan-2-amine (3.26 g, 32.19 mmol, 4.55 mL, 3 eq), and then was added Cui (817.42 mg, 4.29 mmol, 0.4 eq) and palladium triphenylphosphane (2.48 g, 2.15 mmol, 0.2 eq), ethynyl(trimethyl)silane (2.11 g,
483
SUBSTITUTE SHEET RULE 26 21.46 mmol, 2.97 mL, 2 eq) slowly under N2. The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was filtered through celite and diluted with H2O (500 mL) and then extracted with DCM (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue and was purified by column (SiCh, PE:EA=l:0 to 4:1) to afford 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (5 g, 14.80 mmol, 68.97% yield, 75% purity) as a brown solid. MS (ESI) m/z 254.2 [M+H]+
Step 8: 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[0001025] To a solution of 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (5 g, 13.81 mmol, 75% purity, 1 eq) in MeOH (50 mL) was added K2CO3 (4.77 g, 34.53 mmol, 2.5 eq), and the mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was filtered and the filtrate was used to next step directly. 5-ethynylisoquinoline-4-carbaldehyde (2.5 g, crude) was obtained in a yellow liquid (in MeOH). MS (ESI) m/z 182.3 [M+H]+
Step 9: 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile
[0001026] To a solution of 5-ethynylisoquinoline-4-carbaldehyde (2.5 g, 13.80 mmol, 1 eq) in DCM (50 mL) was added Ti(i-PrO)4 (5.88 g, 20.70 mmol, 6.11 mL, 1.5 eq) and NH3/MeOH (50 mL, 7M). The solution was stirred at 20 °C for 1 h, and then TMSCN (4.11 g, 41.39 mmol, 5.18 mL, 3 eq) was added at 0 °C, and the mixture was stirred at 20 °C for 3 h. Upon completion, the mixture was diluted with H2O (300 mL) and filtered and then extracted with DCM (100 mL * 6). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to obtained 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (2 g, crude) was obtained as a black brown gum. MS (ESI) m/z 208.2 [M+H]+
Step 10: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide
[0001027] To a solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (2 g, 4.83 mmol, 50% purity, 1 eq) and 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6- azaspiro[3.4]octane-7-carboxylic acid (1.95 g, 4.83 mmol, 90% purity, 1 eq) in ACN (60 mL) was added TCFH (1.35 g, 4.83 mmol, 1 eq) and 1 -methylimidazole (1.19 g, 14.48 mmol, 1.15 mL, 3 eq). The resulting mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was
484
SUBSTITUTE SHEET RULE 26 concentrated in vacuum and then was diluted with DCM (100 mL) and then was filtered through celite and then was added water (300 mL) and was extracted with DCM (200 mL * 4), the organic phase was dried with Na2SO4 and then was concentrated in vacuum. The crude product was purified by column (SiCh, PE:EA =1 :0 to 1:1) and was re-purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)- ACN] ; B%: 35%-75%, 10 min) to obtained N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3, 3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide (600 mg, 993.54 umol, 20.59% yield) as a light yellow solid. MS (ESI) m/z 554.2 [M+H]+
Step 11: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide
[0001028] The N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide (600 mg, 95% purity) was separated by SFC (column: REGIS(S,S)WHELK-O1( 250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 34%-34%, 10 min) to give N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 1 (65 mg, 116.83 umol, 10.78% yield, 99.5% purity) as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001029] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.51 - 9.42 (m, 1H), 9.28 (br d, J= 6.6 Hz, 2H), 8.80 (s, 1H), 8.33 (br d, J= 8.0 Hz, 1H), 8.13 (br d, J= l.Q Hz, 1H), 7.79 (t, J= 7.8 Hz, 1H), 7.50 (br d, J= 6.6 Hz, 1H), 4.68 (s, 1H), 4.57 - 4.46 (m, 1H), 4.27 (br t, J= 8.0 Hz, 1H), 3.88 (br d, J= 9.8 Hz, 1H), 3.47 (br d, J= 10.0 Hz, 1H), 2.16 (br dd, J= 7.6, 11.9 Hz, 1H), 2.02 - 1.76 (m, 7H), 1.03 (s, 9H).
[0001030] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 2 (65 mg, 117.42 umol, 10.83% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001031] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.45 (s, 1H), 9.32 (br dd, J= 8.0, 13.4 Hz, 2H), 8.85 - 8.73 (m, 1H), 8.32 (br d, J= 7.8 Hz, 1H), 8.13 (br d, J= Hz, 1H), 7.78 (t, J= 7.6 Hz, 1H), 7.67 (br d, J= 7.2 Hz, 1H), 4.72 (s, 1H), 4.66 (br d, J= 8.0 Hz, 1H), 4.35 (br t, J= 7.4 Hz,
485
SUBSTITUTE SHEET RULE 26 1H), 3.86 (br d, J = 10.2 Hz, 1H), 3.60 (br d, J= 10.0 Hz, 1H), 2.31 (br dd, J= 8.6, 12.4 Hz, 1H), 2.03 - 1.76 (m, 7H), 0.97 (s, 9H).
[0001032] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 3 (63 mg, 113.58 umol, 10.48% yield, 99.8% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001033] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.58 (br s, 1H), 9.51 - 9.43 (m, 1H), 9.37 (br d, J= 7.2 Hz, 1H), 8.86 - 8.71 (m, 1H), 8.32 (br d, .7 = 8,0 Hz, 1H), 8.19 - 8.07 (m, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.64 (br d, .7= 7.2 Hz, 1H), 4.82 - 4.58 (m, 2H), 4.29 (br t, J= 7.6 Hz, 1H), 3.91 (br d, J= 10.1 Hz, 1H), 3.52 (br d, J= 10.1 Hz, 1H), 2.21 (br dd, J= 8.4, 12.2 Hz, 1H), 2.01 - 1.80 (m, 7H), 1.01 - 0.91 (m, 9H).
[0001034] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 4 (65 mg, 117.42 umol, 10.83% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001035] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.56 (br d, J= 7.0 Hz, 1H), 9.45 (s, 1H), 9.29 (br s, 1H), 8.86 (s, 1H), 8.36 - 8.28 (m, 1H), 8.13 (d, J= 6.4 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.57 (d, J= 6.8 Hz, 1H), 4.60 (s, 1H), 4.52 (br d, J= 2.4 Hz, 1H), 4.41 (t, J= 7.8 Hz, 1H), 3.87 (d, J= 10.2 Hz, 1H), 3.53 (d, J= 10.2 Hz, 1H), 2.30 (dd, J= 7.8, 12.4 Hz, 1H), 2.05 - 1.96 (m, 2H), 1.94 - 1.82 (m, 5H), 0.94 (s, 9H).
486
SUBSTITUTE SHEET RULE 26 Example 125: Synthesis of (3S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-2-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-2-azaspiro[4.5]decane-3-carboxamide (Compound 346)
Figure imgf000488_0001
Step 1: methyl 6-azaspiro[3.4]octane-7-carboxylate
[0001036] A solution of 6-tert-butoxycarbonyl-6-azaspiro[3.4]octane-7-carboxylic acid (7 g, 27.42 mmol, 1 eq) in HCl/MeOH (4M, 70 mb) was stirred at 60 °C for 18 h. Upon completion, the mixture was concentrated in vacuum to obtained methyl 6-azaspiro[3.4]octane-7-carboxylate (5.5 g, crude, HC1) as a black brown gum.
Step 2: methyl 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6- azaspiro[3.4]octane-7-carboxylate
[0001037] To a solution of methyl 6-azaspiro[3.4]octane-7-carboxylate (6.5 g, 31.60 mmol, 1 eq, HC1) and (2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoic acid (8.04 g, 34.76
487
SUBSTITUTE SHEET RULE 26 mmol, 1.1 eq) in DCM (20 mL) was added DMAP (11.58 g, 94.81 mmol, 3 eq) and EDCI (12.12 g, 63.20 mmol, 2 eq), and the reaction was stirred at 20 °C for 2 h. Upon completion, the mixture was adjusted pH~l with aq. HC1 (IM) and then was extracted with DCM (100 mL * 3), then the organic phase was adjusted pH~7 with sat.NaHCCh, then the organic phase was dried with Na2SO4, filtered and concentrated in vacuum and the crude product was purified by column (SiCh, PE:EA = 1:0 to 5:1) to obtained methyl 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3- dimethyl-butanoyl]-6-azaspiro[3.4]octane-7-carboxylate (10 g, 23.53 mmol, 74.46% yield, 90% purity) as a yellow oil. MS (ESI) m/z 383.2 [M+H]+
Step 3: 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7- carboxylic acid
[0001038] To a solution of methyl 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl- butanoyl]-6-azaspiro[3.4]octane-7-carboxylate (10 g, 26.14 mmol, 1 eq) in THF (60 mL) and H2O (20 mL) was added LiOH.IUO (3.29 g, 78.43 mmol, 3 eq) at 20 °C, and the mixture was stirred at 20 °C for 6 h. Upon completion, the mixture was adjusted pH~l with aq.HCl (IM) and then was extracted with ethyl acetate (50 mL * 3), then the organic phase was dried with Na2SO4, filtered and concentrated in vacuum to obtained 6-[(2S)-2-(tert-butoxycarbonylamino)- 3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7-carboxylic acid (9.5 g, crude) as a light yellow gum. MS (ESI) m/z 367.1 [M-H]+
Step 4: (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[[3-(trifhroromethyl)-l,2,4-oxadiazol-5- yl]amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[0001039] A solution of 6-[(2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoyl]-6- azaspiro[3.4]octane-7-carboxylic acid (9.5 g, 25.78 mmol, 1 eq) in HCl/dioxane (100 mL, 4 M) was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum to obtained 6-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7-carboxylic acid (8 g, crude, HC1) as a yellow gum. MS (ESI) m/z 269.3 [M+H]+
Step 5: 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7- carboxylic acid
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SUBSTITUTE SHEET RULE 26 [0001040] A solution of 6-[(2S)-2-amino-3,3-dimethyl-butanoyl]-6-azaspiro[3.4]octane-7- carboxylic acid (8 g, 26.25 mmol, 1 eq, HC1) in methyl 2,2,2-trifluoroacetate (33.61 g, 262.46 mmol, 26.46 mL, 10 eq) and TEA (7.97 g, 78.74 mmol, 10.96 mL, 3 eq) was stirred at 20 °C for 6 h. Upon completion, the mixture was concentrated in vacuum and then was adjust pH~l with HC1 (1 M), and then was extracted with ethyl acetate (50 mL * 3). The organic phase was concentrated in vacuum. The crude product was triturated with the solution (EA:PE = 1 : 30) at 5 °C for 30 min, then the mixture was filtered and the filter cake was obtained 6-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxylic acid (1.9 g, 4.43 mmol, 16.88% yield, 85% purity) as a white solid. The mother liquor was concentrated in vacuum to obtained the crude as a yellow gum and was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water (HCl)-ACN]; B%: 32%- 62%, 20 min) to obtained 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6- azaspiro[3.4]octane-7-carboxylic acid (1.3 g, 3.21 mmol, 12.23% yield, 90% purity) as a white solid. MS (ESI) m/z 365.1 [M+H]+
Step 6: 5-bromoisoquinoline-4-carbaldehyde
[0001041] To a mixture of cone. H2SO4 (50 mL) and isoquinoline-4-carbaldehyde (12.8 g, 81.44 mmol, 1 eq) at 0 °C was added NBS (14.50 g, 81.44 mmol, 1 eq) slowly and the reaction mixture was warmed to 25 °C was stirred for 16 h. Upon completion, the mixture was adjust pH~7 by NaOH (1000 mL), and extracted with ethyl acetate (1000 mL * 2). The combined organic layers were washed with brine (1000 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue and was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water (HCl)-ACN]; B%: 15%- 45%, 20 min) to obtained 5-bromoisoquinoline-4-carbaldehyde (5.16 g, 18.58 mmol, 22.81% yield, 85% purity) as yellow solid. MS (ESI) m/z 236.1, 238.1 [M+H]+
Step 7: 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[0001042] To a solution of 5-bromoisoquinoline-4-carbaldehyde (2.98 g, 10.73 mmol, 85% purity, 1 eq, Parallel 2 batches) in DMSO (80 mL) was added N-isopropylpropan-2-amine (3.26 g, 32.19 mmol, 4.55 mL, 3 eq), and then Cui (817.42 mg, 4.29 mmol, 0.4 eq) and palladium triphenylphosphane (2.48 g, 2.15 mmol, 0.2 eq), and ethynyl(trimethyl)silane (2.11 g, 21.46
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SUBSTITUTE SHEET RULE 26 mmol, 2.97 mL, 2 eq) were added to slowly under N2. The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was filtered through celite and diluted with H2O (500 mL) and then extracted with DCM (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue and was purified by column (SiCh, PE:EA=l:0 to 4: 1) to obtained 5-(2-trimethylsilylethynyl)isoquinoline-4- carbaldehyde (5 g, 14.80 mmol, 68.97% yield, 75% purity) was obtained as a brown solid. MS (ESI) m/z 254.2 [M+H]+
Step 8: 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[0001043] To a solution of 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (5 g, 13.81 mmol, 75% purity, 1 eq) in MeOH (50 mL) was added K2CO3 (4.77 g, 34.53 mmol, 2.5 eq), and the mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was filtered and the filtrate was used to next step directly. 5-ethynylisoquinoline-4-carbaldehyde (2.5 g, crude) was obtained in a yellow liquid (in MeOH). MS (ESI) m/z 182.3 [M+H]+
Step 9: 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile
[0001044] To a solution of 5-ethynylisoquinoline-4-carbaldehyde (2.5 g, 13.80 mmol, 1 eq) in DCM (50 mL) was added Ti(i-PrO)4 (5.88 g, 20.70 mmol, 6.11 mL, 1.5 eq) and NH3/MeOH (50 mL, 7M), and the mixture was stirred at 20 °C for 1 h. TMSCN (4.11 g, 41.39 mmol, 5.18 mL, 3 eq) was added at 0 °C, and the mixture was stirred at 20 °C for 3 h. Upon completion, the mixture was diluted with H2O (300 mL) and filtered and then extracted with DCM (100 mL * 6). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to obtained 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (2 g, crude) was obtained as a black brown gum. MS (ESI) m/z 208.2 [M+H]+
Step 10: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide
[0001045] To a solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (2 g, 4.83 mmol, 50% purity, 1 eq) and 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6- azaspiro[3.4]octane-7-carboxylic acid (1.95 g, 4.83 mmol, 90% purity, 1 eq) in ACN (60 mL) was added TCFH (1.35 g, 4.83 mmol, 1 eq) and 1 -methylimidazole (1.19 g, 14.48 mmol, 1.15
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SUBSTITUTE SHEET RULE 26 mL, 3 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was concentrated in vacuum and then was diluted with DCM (100 mL) and then was filtered through celite and then was added water (300 mL) and was extracted with DCM (200 mL * 4), the organic phase was dried with Na2SO4 and then was concentrated in vacuum. The crude product was purified by column (SiCh, PE:EA =1 :0 to 1:1) and was re-purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)- ACN] ; B%: 35%-75%, 10 min) to obtained N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3, 3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide (600 mg, 993.54 umol, 20.59% yield) as a light yellow solid. MS (ESI) m/z 554.2 [M+H]+
Step 11: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide
[0001046] The N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide (600 mg, 95% purity) was separated by SFC (column: REGIS(S,S)WHELK-O1( 250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 34%-34%, 10 min) to give N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 1 (65 mg, 116.83 umol, 10.78% yield, 99.5% purity) as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001047] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.51 - 9.42 (m, 1H), 9.28 (br d, J= 6.6 Hz, 2H), 8.80 (s, 1H), 8.33 (br d, J= 8.0 Hz, 1H), 8.13 (br d, J= 7.0 Hz, 1H), 7.79 (t, J= 1.8 Hz, 1H), 7.50 (br d, J= 6.6 Hz, 1H), 4.68 (s, 1H), 4.57 - 4.46 (m, 1H), 4.27 (br t, J= 8.0 Hz, 1H), 3.88 (br d, J= 9.8 Hz, 1H), 3.47 (br d, J= 10.0 Hz, 1H), 2.16 (br dd, J= 7.6, 11.9 Hz, 1H), 2.02 - 1.76 (m, 7H), 1.03 (s, 9H).
[0001048] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 2 (65 mg, 117.42 umol, 10.83% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001049] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.45 (s, 1H), 9.32 (br dd, J= 8.0, 13.4 Hz, 2H), 8.85 - 8.73 (m, 1H), 8.32 (br d, J= 7.8 Hz, 1H), 8.13 (br d, J= 7.0 Hz, 1H), 7.78 (t, J= 7.6 Hz, 1H), 7.67 (br d, J= 7.2 Hz, 1H), 4.72 (s, 1H), 4.66 (br d, J= 8.0 Hz, 1H), 4.35 (br t, J= 7.4 Hz,
491
SUBSTITUTE SHEET RULE 26 1H), 3.86 (br d, J = 10.2 Hz, 1H), 3.60 (br d, J= 10.0 Hz, 1H), 2.31 (br dd, J= 8.6, 12.4 Hz, 1H), 2.03 - 1.76 (m, 7H), 0.97 (s, 9H).
[0001050] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 3 (63 mg, 113.58 umol, 10.48% yield, 99.8% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001051] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.58 (br s, 1H), 9.51 - 9.43 (m, 1H), 9.37 (br d, J= 7.2 Hz, 1H), 8.86 - 8.71 (m, 1H), 8.32 (br d, .7 = 8,0 Hz, 1H), 8.19 - 8.07 (m, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.64 (br d, .7= 7.2 Hz, 1H), 4.82 - 4.58 (m, 2H), 4.29 (br t, J= 7.6 Hz, 1H), 3.91 (br d, J= 10.1 Hz, 1H), 3.52 (br d, J= 10.1 Hz, 1H), 2.21 (br dd, J= 8.4, 12.2 Hz, 1H), 2.01 - 1.80 (m, 7H), 1.01 - 0.91 (m, 9H).
[0001052] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6-azaspiro[3.4]octane-7-carboxamide Isomer 4 (65 mg, 117.42 umol, 10.83% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 554.2 [M+H]+
[0001053] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.56 (br d, J= 7.0 Hz, 1H), 9.45 (s, 1H), 9.29 (br s, 1H), 8.86 (s, 1H), 8.36 - 8.28 (m, 1H), 8.13 (d, J= 6.4 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.57 (d, J= 6.8 Hz, 1H), 4.60 (s, 1H), 4.52 (br d, J= 2.4 Hz, 1H), 4.41 (t, J= 7.8 Hz, 1H), 3.87 (d, J= 10.2 Hz, 1H), 3.53 (d, J= 10.2 Hz, 1H), 2.30 (dd, J= 7.8, 12.4 Hz, 1H), 2.05 - 1.96 (m, 2H), 1.94 - 1.82 (m, 5H), 0.94 (s, 9H).
492
SUBSTITUTE SHEET RULE 26 Example 126: Synthesis of (lR,2S,5S)-N-[(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3- yl)-cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 349)
Figure imgf000494_0001
Step 1: amino 2,4,6-trimethylbenzenesulfonate
[0001054] TFA (92.40 g, 810.36 mmol, 60.00 mL, 14.20 eq) was cooled to 0 °C and (tertbutoxycarbonylamino) 2,4,6-trimethylbenzenesulfonate (18 g, 57.07 mmol, 1 eq) was added portionwsie over 10 min. After the addition, the mixture was stirred at 0 °C for 1 h. The reaction mixture was quenched by ice and water (180 mL) and maintained inter-temperature below 5 °C. The mixture was stirred for 30 min to form a slurry. The slurry was filtered and the filter cake was washed with water (100 mL * 3) and air dried to afford amino 2,4,6- trimethylbenzenesulfonate (20 g, crude) as a white solid.
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SUBSTITUTE SHEET RULE 26 Step 2: 3-bromo-5-cyclopropyl-pyridin-l-ium-l-amine
[0001055] Amino 2,4,6-trimethylbenzenesulfonate (11.00 g, 51.10 mmol, 1.2 eq) was dissolved in DCM (300 mb) and dried over Na2SO4. The mixture was filtered. To the filtrate was added 3-bromo-5-cyclopropyl-pyridine (8.5 g, 42.92 mmol, 1.01 eq) and the resulting mixture was stirred at 0 °C for 3 h. Upon completion, the reaction mixture was filtered, the filter cake was washed with DCM (lOOmL * 3) and dried in air to afford a residue to give 3-bromo-5- cyclopropyl-pyridin-l-ium-1 -amine (16.36 g, crude) as a white solid.
Step 3: methyl 6-cyclopropyl-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate
[0001056] To a solution of 3-bromo-5-cyclopropyl-pyridin-l-ium-l-amine (16.2 g, 75.67 mmol, 1 eq) in DMF (160 mL) was added K2CO3 (15.69 g, 113.51 mmol, 1.5 eq) and methyl prop-2-ynoate (6.36 g, 75.67 mmol, 6.30 mL, 1 eq), and the reaction mixture was stirred at 20 °C for 12 h. Upon completion, The reaction mixture was diluted with brine (100 mL), and extracted with EtOAc (100 mL *2). The organic layers were separated, dried over Na2SO4, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 4/1 to 3/1) to give methyl 6-cyclopropyl-4-methyl-pyrazolo[l,5- a]pyridine-3-carboxylate (2.1 g, 8.57 mmol, 11.33% yield, 94% purity) as a yellow solid.
Step 4: (4-bromo-6-cyclopropyl-pyrazolo[l ,5-a]pyridin-3-yl)methanol
[0001057] To methyl 4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridine-3-carboxylate (2.1 g, 7.12 mmol, 1 eq) in THF (20 mL) was added DIBAL-H (1 M, 35.58 mL, 5 eq) at 0 °C was stirred for 1 h. The resulting solution was seignettesalt( 30 mL), and then extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed brine (30 mL * 3), dried over Na2SO4, filtered and concentrated to give a crude product. Upon completion, the residue was purified by prep-TLC (SiCh, Petroleum ether/Ethyl acetate = 1/1 to 1/2) to give (4-bromo-6- cyclopropyl-pyrazolo[l,5-a]pyridin-3-yl)methanol (1.8 g, 6.20 mmol, 87.13% yield, 92% purity) as a white solid.
Step 5: 4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde
[0001058] To a solution of (4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3-yl)methanol (1.8 g, 6.74 mmol, 1 eq) in DCM (20 mL) was added MnCh (11.72 g, 134.77 mmol, 20 eq) at 25 °C,
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SUBSTITUTE SHEET RULE 26 the reaction mixture was stirred at 25 °C for 2 h. LCMS showed the reaction was not completed, then the mixture was stirred at 25 °C for 12 h. Upon completion, The mixture was filtered, and concentrated under reduced pressure to give a residue .To give 4-bromo-6-cyclopropyl- pyrazolo[l,5-a]pyridine-3-carbaldehyde (1.7 g, crude) as a light yellow solid.
Step 6: 2-amino-2-(4-bromo-6-cyclopropyl-pyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
[0001059] A solution of 4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde (300 mg, 1.13 mmol, 1 eq), NH4CI (181.60 mg, 3.39 mmol, 3 eq) in NHa/MeOH (3 mL) was stirred at 20°C for 1 h, then TMSCN (336.79 mg, 3.39 mmol, 424.71 uL, 3 eq) was added. The mixture was stirred at 20 °C for 15 h. Upon completion, the mixture was filtered and concentrated under reduced pressure to give 2-amino-2-(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3- yl)acetonitrile (320 mg, crude) as a yellow solid. MS (ESI) m/z 276.2 [M+H]+.
Step 7: (lR,2S,5S)-N-[(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001060] A solution of 2-amino-2-(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3- yl)acetonitrile (280 mg, 961.72 umol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (428.28 mg, 1.06 mmol, 90% purity, 1.1 eq) and NMI (236.87 mg, 2.89 mmol, 229.97 uL, 3 eq). TCFH (539.68 mg, 1.92 mmol, 2 eq) was added, and the mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (3 mL) and extracted with ethyl acetate (3 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex C 18 80 * 40 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 45%-65%, 8 min) to give (lR,2S,5S)-N-[(4-bromo-6-cyclopropyl- pyrazolo[ 1 , 5-a] pyridin-3 -yl)-cyano-methyl] -3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (300 mg, 442.36 umol, 46.00% yield, 94% purity) as a white solid. MS (ESI) m/z 637.4 [M+H]+.
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SUBSTITUTE SHEET RULE 26 Step 8: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001061] (lR,2S,5S)-N-[(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 470.60 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 40%-40%, 5 min) to give (lR,2S,5S)-N-[(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3-yl)- cyano-methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (96.32 mg, 151.09 umol, 32.11% yield, 100% purity) as a white solid. MS (ESI) m/z 637.4 [M+H]+.
[0001062] 'H NMR (400 MHz, DMSO-de) 5 = 9.36 (br s, 1H), 9.31 (d, J = 7.1 Hz, 1H), 8.67 (s, 1H), 8.22 (s, 1H), 7.45 (d, J = 0.9 Hz, 1H), 6.52 (d, J = 7.0 Hz, 1H), 4.40 (br d, J = 4.0 Hz, 1H), 4.26 (s, 1H), 3.90 (dd, J = 5.4, 10.3 Hz, 1H), 3.70 (d, J = 10.4 Hz, 1H), 2.07 - 1.94 (m, 1H), 1.54 (dd, J = 5.4, 7.4 Hz, 1H), 1.31 (d, J = 7.6 Hz, 1H), 1.05 - 0.91 (m, 14H), 0.85 - 0.77 (m, 5H).
[0001063] (lR,2S,5S)-N-[(4-bromo-6-cyclopropyl-pyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (93.12 mg, 146.07 umol, 31.04% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 637.4 [M+H]+.
[0001064] 'H NMR (400 MHz, DMSO-de) 5 = 9.43 - 9.27 (m, 2H), 8.67 (s, 1H), 8.22 (s, 1H), 7.44 (d, J = 1.0 Hz, 1H), 6.52 (d, J = 6.5 Hz, 1H), 4.40 (br d, J = 7.3 Hz, 1H), 4.31 (s, 1H), 3.90 (dd, J = 5.3, 10.3 Hz, 1H), 3.72 (d, J = 10.5 Hz, 1H), 2.10 - 1.93 (m, 1H), 1.57 (dd, J = 5.4, 7.3 Hz, 1H), 1.35 (d, J = 7.5 Hz, 1H), 1.14 - 0.88 (m, 14H), 0.87 - 0.78 (m, 5H).
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SUBSTITUTE SHEET RULE 26 Example 127: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 351)
Figure imgf000498_0001
Step 1: (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol
[0001065] The 4-bromopyrazolo[l,5-a]pyridine-3-carboxylic acid (1.3 g, 5.39 mmol, 1 eq) in THF (15 mL) was added with CDI (1.31 g, 8.09 mmol, 1.5 eq). The mixture was stirred at 20 °C for 17.5 h under N2 atmosphere. Then the mixture was added to a solution of NaBFLi (1.02 g, 26.97 mmol, 5 eq) in H2O (15 mL) and was stirred for 30 min at 20 °C. Upon completion, the mixture was diluted with IM HC1 (20 mL) and the solution was neutralised with saturated aqueous NaHCCh, then extracted with ethyl acetate (40 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by
497
SUBSTITUTE SHEET RULE 26 prep-TLC (SiCh, Petroleum ether:Ethyl acetate = 0: 1) to get product (4-bromopyrazolo[l,5- a]pyri din-3 -yl)methanol (2 g, 7.13 mmol, 81% purity) as white solid. MS (ESI) m/z 227.0 [M+H]+
Step 2: 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde
[0001066] The (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol (0.5 g, 2.20 mmol, 1 eq) in DCM (25 mL) was added MnCh (3.83 g, 44.04 mmol, 20 eq . The mixture was stirred at 20 °C for 48 h. Upon completion, the mixture was filtered and concentrated in vacuum to get crude product 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (1.8 g, crude) as white solid. MS (ESI) m/z 225.0 [M+H]+
Step 3: 2-amino-2-(4-bromopyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
[0001067] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (0.6 g, 2.67 mmol, 1 eq), Ti(i-PrO)4 (1.14 g, 4.00 mmol, 1.18 mL, 1.5 eq), NH3/MeOH (7 M, 1.52 mL, 4 eq) in DCM (6 mL) was stirred at 20 °C for 1 h, and then TMSCN (793.53 mg, 8.00 mmol, 1.00 mL, 3 eq) was added at 0 °C. The mixture was stirred at 20 °C for 2 h. Upon completion, the mixture was poured into H2O (50 mL) at 20 °C, and then extracted with DCM (50 mL * 5). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressur to get crude product 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.63 g, crude) as yellow solid. MS (ESI) m/z 251.0 [M+H]+
Step 4: methyl (2S,3R)-2-amino-3-methoxy-butanoate
[0001068] The (3R)-2-(tert-butoxycarbonylamino)-3-methoxy-butanoic acid (2 g, 8.57 mmol, 1 eq) in HCl/MeOH (4 M, 60 mL, 27.99 eq) was stirred at 60 °C for 6 h. Upon completion, the mixture was concentrated in vacuum to get crude product methyl (2S,3R)-2-amino-3-methoxy- butanoate (1.6 g, crude) as white solid. MS (ESI) m/z 148.2 [M+H]+
Step 5: methyl (2S,3R)-3-methoxy-2-[[l- trifluoromethyl)cyclopropanecarbonyl]amino]butanoate
[0001069] To a solution of methyl (2S,3R)-2-amino-3-methoxy-butanoate (1.6 g, 8.71 mmol, 1 eq, HC1) in DCM (30 mL) was added l-(trifluoromethyl)cyclopropanecarboxylic acid (1.34 g, 8.71 mmol, 1 eq), HATU (6.63 g, 17.43 mmol, 2 eq) and DIEA (3.38 g, 26.14 mmol, 4.55 mL, 3 498
SUBSTITUTE SHEET RULE 26 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition water (30 mL) and extracted with DCM (25 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 9: 1) to get methyl (2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoate (2.29 g, 7.60 mmol, 87.22% yield, 94% purity) as yellow oil. MS (ESI) m/z 284.1 [M+H]+
Step 6: (2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoic acid
[0001070] The methyl (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoate 3.43 g, 12.11 mmol, 1 eq) in THF (15 mL) and H2O (15 mL) was added LiOH.PEO (1.52 g, 36.33 mmol, 3 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted by addition H2O (100 mL), and then added HC1 (1 M, aq.) to adjust the pH = 3 - 4, and extracted with ethyl acetate (100 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoic acid (3.3 g, crude) as yellow oil. MS (ESI) m/z 270.1 [M+H]+
Step 7: methyl (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2- carboxylate
[0001071] To a solution of (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoic acid (2 g, 7.43 mmol, 1 eq) in DCM (10 mL) was added methyl (lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (1.53 g, 7.43 mmol, 1 eq, HC1), HATU (5.65 g, 14.86 mmol, 2 eq) and DIEA (2.88 g, 22.29 mmol, 291.14 uL, 3 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition water (100 mL) and extracted with DCM (80 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 9:1 to 17:3) to give the product methyl (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3-
499
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxylate (1.7 g, 3.68 mmol, 35.86% yield) as white solid. MS (ESI) m/z 421.2 [M+H]+
Step 8: (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[0001072] To a solution of methyl (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (1.7 g, 4.04 mmol, 1 eq) in THF (9 mL) and H2O (9 mL) was added LiOH.FEO (509.00 mg, 12.13 mmol, 3 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was adjusted pH~l with HC1 (I M, aq.) and then extracted with EA (80 mL * 3). The organic phase was dried with Na2SO4, filtered and concentrated in vacuum to give the crude product (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.6 g, crude) as yellow oil. MS (ESI) m/z 407.2 [M+H]+
Step 9: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001073] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.02 g, 2.51 mmol, 1 eq), 2-amino-2-(4- bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.63 g, 2.51 mmol, 1 eq) in ACN (10 mL) was added TCFH (1.41 g, 5.02 mmol, 2 eq) and 1 -methylimidazole (618.03 mg, 7.53 mmol, 600.03 uL, 3 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was quenched by addition water (80 mL) and extracted with DCM (50 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water (FA)- ACN]; B%: 40%-75%, 20 min) to get product (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3- yl)-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[[l-
500
SUBSTITUTE SHEET RULE 26 (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.88 g, 1.38 mmol, 54.85% yield, 100% purity) as white solid. MS (ESI) m/z 639.1 [M+H]+
Step 10: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001074] The (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (0.88 g, 1.38 mmol, 1 eq) was separated by SFC (column: REGIS (S,S) WHELK-01 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 6 min) to afford (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (0.3 g, 467.74 umol, 33.99% yield, 99.7% purity) as white solid. MS (ESI) m/z 639.1 [M+H]+
[0001075] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.31 (d, J= 7.0 Hz, 1H), 8.84 (d, J= 6.9 Hz, 1H), 8.31 (s, 1H), 7.70 (d, J= 7.3 Hz, 2H), 6.94 (t, J= 7.2 Hz, 1H), 6.57 (d, J= Hz, 1H), 4.38 (t, J = '1.3 Hz, 1H), 4.23 (s, 1H), 3.93 - 3.78 (m, 2H), 3.68 - 3.53 (m, 1H), 3.23 (s, 3H), 1.53 (dd, J= 4.7, 7.3 Hz, 1H), 1.33 - 1.20 (m, 5H), 1.10 (d, J = 6.3 Hz, 3H), 1.00 - 0.95 (m, 3H), 0.84 (s, 3H).
[0001076] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (0.31 g, 483.33 umol, 35.12% yield, 99.7% purity) was obtained as white solid. MS (ESI) m/z 639.1 [M+H]+
[0001077] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.54 - 9.05 (m, 1H), 8.83 (d, J = 6.9 Hz, 1H), 8.30 (s, 1H), 7.90 - 7.61 (m, 2H), 6.94 (t, J = 7.2 Hz, 1H), 6.56 (d, J = 6.4 Hz, 1H), 4.36 (t, J = 7.4 Hz, 1H), 4.27 (s, 1H), 3.86 (br d, J = 2.6 Hz, 2H), 3.60 - 3.47 (m, 1H), 3.23 - 3.09 (m, 3H), 1.56 (br d, J = 7.4 Hz, 1H), 1.39 - 1.16 (m, 5H), 1.10 - 0.82 (m, 9H).
501
SUBSTITUTE SHEET RULE 26 Example 128: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 352)
Figure imgf000503_0001
Step 1: 2-amino-2-(4-bromopyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
[0001078] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (0.6 g, 2.67 mmol, 1 eq), Ti(i-PrO)4 (1.14 g, 4.00 mmol, 1.18 mL, 1.5 eq), NH3/MeOH (7 M, 1.52 mL, 4 eq) in DCM (6 mL) was stirred at 20 °C for 1 h, and then TMSCN (793.53 mg, 8.00 mmol, 1.00 mL, 3 eq) was added at 0 °C. The mixture was stirred at 20 °C for 2 h. Upon completion, the mixture was poured into H2O (50 mL) at 20 °C, and then extracted with DCM (50 mL * 5). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to get crude product 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.7 g, crude) as yellow solid.
Step 2: ethyl (3S,3aS,6aR)-2-[(2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylate
502
SUBSTITUTE SHEET RULE 26 [0001079] To a solution of (2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoic acid (1.5 g, 5.49 mmol, 1 eq) and ethyl (3S,3aS,6aR)- l,2,3,3a,4,5,6,6a-octahydrocyclopenta[c]pyrrole-3-carboxylate (1.01 g, 5.49 mmol, 1 eq) in DCM (15 mb) was added with DMAP (2.01 g, 16.46 mmol, 3 eq) and EDCI (2.10 g, 10.98 mmol, 2 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition water (30 mL) and extracted with DCM (25 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 98:2 to 95:5) to afford ethyl (3S,3aS,6aR)-2-[(2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylate (1.41 g, 2.80 mmol, 50.97% yield, 87% purity) as yellow oil. MS (ESI) m/z 439.3 [M+H]+
Step 3: (3S,3aS,6aR)-2-[(2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid
[0001080] To a solution of ethyl (3S,3aS,6aR)-2-[(2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylate (1.41 g, 3.22 mmol, 1 eq) in THF (8 mL) and H2O (8 mL) was added LiOH.TEO (539.62 mg, 12.86 mmol, 3 eq). The mixture was stirred at 20 °C for 3 h. Upon completion, the mixture was adjusted pH~l with HC1 (IM, aq.), and then was extracted with ethyl acetate (40 mL * 4), and then the organic phase was dried with Na2SO4, filtered and concentrated in vacuum to get crude product (3S,3aS,6aR)-2-[(2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1.3 g, crude) as yellow oil. MS (ESI) m/z 411.3 [M+H]+
Step 4: (3S,3aS,6aR)-2-[(2S,3R)-2-amino-3-(l-methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a- hexahydro- lH-cyclopenta[c]pyrrole-3-carboxylic acid
[0001081] The (3S,3aS,6aR)-2-[(2S,3R)-2-(tert-butoxycarbonylamino)-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1.3 g, 3.17 mmol, 1 eq) in HCl/dioxane (4 M, 15.00 mL, 18.95 eq) was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give the
503
SUBSTITUTE SHEET RULE 26 crude product (3S,3aS,6aR)-2-[(2S,3R)-2-amino-3-(l-methylcyclopropoxy)butanoyl]- 3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1 g, crude) as yellow oil. MS (ESI) m/z 311.3 [M+H]+
Step 5: (3S,3aS,6aR)-2-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid
[0001082] To a solution of (3S,3aS,6aR)-2-[(2S,3R)-2-amino-3-(l- methylcyclopropoxy)butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1 g, 3.22 mmol, 1 eq) in methyl 2,2,2-trifluoroacetate (12.38 g, 96.65 mmol, 899.94 uL, 30.78 eq) was added TEA (978.04 mg, 9.67 mmol, 1.35 mL, 3 eq). The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give the crude product (3S,3aS,6aR)-2-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1.36 g, crude) as yellow oil. MS (ESI) m/z 407.1 [M+H]+
Step 6: (3S,3aS,6aR)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-2-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH- cyclopenta[c]pyrrole-3-carboxamide
[0001083] To a solution of (3S,3aS,6aR)-2-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxylic acid (1.36 g, 3.35 mmol, 1.2 eq), 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.7 g, 2.79 mmol, 1 eq) in ACN (1 mL) was added 1 -methylimidazole (686.67 mg, 8.36 mmol, 666.67 uL, 3 eq) and TCFH (1.56 g, 5.58 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (60 mL) and then extracted with ethyl acetate (40 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by prep-HPLC (column: Phenomenex luna C 18 (250 * 70 mm, 15 um); mobile phase: [water (FA)-ACN]; B%: 50%-80%, 20 min) to get product (3S,3aS,6aR)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-2-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-
504
SUBSTITUTE SHEET RULE 26 cyclopenta[c]pyrrole-3-carboxamide (0.57 g, 891.37 umol, 31.97% yield) as white solid. MS (ESI) m/z 639.1 [M+H]+
Step 7: (3S,3aS,6aR)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-2-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH- cyclopenta[c]pyrrole-3-carboxamide
[0001084] The (3S,3aS,6aR)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-2- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a- hexahydro-lH-cyclopenta[c]pyrrole-3-carboxamide (0.88 g, 1.38 mmol, 1 eq) was separated by SFC (column: REGIS (S,S) WHELK-01 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 50%-50%, 5 min) to give product (3S,3aS,6aR)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)- cyano-methyl]-2-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3-carboxamide Isomer 1 (0.22 g, 340.60 umol, 38.21% yield, 99% purity) as white solid. MS (ESI) m/z 639.1 [M+H]+
[0001085] 'H NMR (400 MHz, DMSO-de) 5 = 9.75 (br s, 1H), 9.27 (d, J= Hz, 1H), 8.83 (d, J= 6.7 Hz, 1H), 8.36 - 8.22 (m, 1H), 7.69 (d, J= Hz, 1H), 6.94 (t, J= 7.2 Hz, 1H), 6.56 (d, J= 7.0 Hz, 1H), 4.46 (br d, J= 6.3 Hz, 1H), 4.13 (d, J= 4.3 Hz, 1H), 3.99 - 3.79 (m, 2H), 3.66 - 3.54 (m, 1H), 2.73 - 2.66 (m, 1H), 2.54 (td, J= 4.0, 7.8 Hz, 1H), 1.84 - 1.70 (m, 2H), 1.61 - 1.47 (m, 3H), 1.34 (s, 4H), 1.19 (d, J= 6.2 Hz, 3H), 0.74 (br s, 1H), 0.71 - 0.63 (m, 1H), 0.43 - 0.27 (m, 2H)
[0001086] (3S,3aS,6aR)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-2-[(2S,3R)- 3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro- lH-cyclopenta[c]pyrrole-3-carboxamide Isomer 2 (0.21 g, 325.12 umol, 36.47% yield, 99% purity) was obtained as white solid. MS (ESI) m/z 639.1 [M+H]+
[0001087] 'H NMR (400 MHz, DMSO-de) 5 = 9.72 (br s, 1H), 9.22 (d, J= 6.4 Hz, 1H), 8.84 (d, J= 6.9 Hz, 1H), 8.33 - 8.26 (m, 1H), 7.75 - 7.64 (m, 1H), 6.95 (t, J= 7.2 Hz, 1H), 6.58 - 6.37 (m, 1H), 4.65 - 4.37 (m, 1H), 4.22 (d, J= 3.7 Hz, 1H), 3.95 - 3.78 (m, 2H), 3.56 (dd, J= 3.5, 10.6 Hz, 1H), 2.73 (br d, J= 3.5 Hz, 1H), 2.61 - 2.51 (m, 1H), 1.93 - 1.73 (m, 2H), 1.64 - 1.48 (m, 3H), 1.36 - 1.25 (m, 4H), 1.18 - 1.06 (m, 3H), 0.80 - 0.69 (m, 1H), 0.65 - 0.52 (m, 1H), 0.38 - 0.23 (m, 2H).
505
SUBSTITUTE SHEET RULE 26 Example 129: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 385)
Figure imgf000507_0001
Step 1: 3-(2-trimethylsilylethynyl)thieno[2,3-c]pyridine-4-carbaldehyde
[0001088] A solution of 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (4.5 g, 18.59 mmol, 1 eq) and N-isopropylpropan-2-amine (5.64 g, 55.76 mmol, 7.88 mb, 3 eq) in DMSO (45 mL) was added with Cui (1.42 g, 7.44 mmol, 0.4 eq), palladium triphenylphosphane (4.30 g, 3.72 mmol, 0.2 eq) and ethynyl(trimethyl)silane (3.65 g, 37.18 mmol, 5.15 mL, 2 eq), and the resulting mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was quenched by H2O (20 mL), and then extracted with EtOAc (20 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, PE:EA = 100:1 to 40:1) to give 3-(2-trimethylsilylethynyl)thieno[2,3-c]pyridine-4-carbaldehyde (2 g, 7.71 mmol, 41.48% yield) as a brown oil. MS (ESI) m/z 260.1 [M+H]+.
Step 2: 3-ethynylthieno[2,3-c]pyridine-4-carbaldehyde
[0001089] A solution of 3-(2-trimethylsilylethynyl)thieno[2,3-c]pyridine-4-carbaldehyde (2 g, 7.71 mmol, 1 eq) in MeOH (10 mL) was added K2CO3 (2.66 g, 19.28 mmol, 2.5 eq) and the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was give 3-
506
SUBSTITUTE SHEET RULE 26 ethynylthieno[2,3-c]pyridine-4-carbaldehyde (1.5 g, crude) as a yellow liquid and used to next step directly.MS (ESI) m/z 188.2 [M+H]+.
Step 3: 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile
[0001090] A solution of 3-ethynylthieno[2,3-c]pyridine-4-carbaldehyde (1.5 g, 8.01 mmol, 1 eq) in DCM (5 mL) was added Ti(i-PrO)4 (2.28 g, 8.01 mmol, 2.36 mL, 1 eq) and NH MeOH (7 M, 42 mL, 36.69 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (2.38 g, 24.04 mmol, 3.01 mL, 3 eq) was added and stirred at 25 °C for 15 h. Upon completion, the solution was quenched with H2O (20 mL), and then extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to afford 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4- yl)acetonitrile (1.5 g, crude) as brown oil. MS (ESI) m/z 214.0 [M+H]+.
Step 4: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001091] A mixture of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (500 mg, 2.34 mmol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (957.55 mg, 2.34 mmol, 1 eq), TCEH (986.76 mg, 3.52 mmol, 1.5 eq) and 1 -methylimidazole (577.50 mg, 7.03 mmol, 560.68 uL, 3 eq), and then the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by water (10 mL), and then extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC(column: Waters Xbridge BEH Cl 8 250 * 70 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 20 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (800 mg, 1.33 mmol, 56.52% yield) as yellow solid. MS (ESI) m/z 604.2 [M+H]+.
507
SUBSTITUTE SHEET RULE 26 Step 5: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001092] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (800 mg, 1.33 mmol, 1 eq) was purified by SFC (column: (s,s) WHELK-01 (250 mm * 30 mm, 5 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 10 min) to give (lR,2S,5S)-3- [(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3- c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (110 mg, 182.22 umol, 13.75% yield, 100% purity) as a white solid. MS (ESI) m/z 604.1 [M+H]+.
[0001093] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (br s, 1H), 9.43 (s, 1H), 9.37 (br d, J= 6.6 Hz, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.04 (br d, J= 6.6 Hz, 1H), 4.85 - 4.48 (m, 1H), 4.44 - 4.36 (m, 1H), 4.25 (s, 1H), 4.00 - 3.74 (m, 2H), 3.69 - 3.56 (m, 1H), 1.58 - 1.50 (m, 1H), 1.38 (d, J = 7.6 Hz, 1H), 1.20 - 1.06 (m, 12H), 0.99 - 0.94 (m, 3H), 0.80 (s, 3H)
[0001094] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (87 mg, 144.12 umol, 10.88% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 604.1 [M+H]+.
[0001095] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (br s, 1H), 9.56 - 9.46 (m, 1H), 9.44 - 9.34 (m, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.14 (br s, 1H), 4.51 (s, 1H), 4.38 (br d, J= 7.4 Hz, 1H), 4.34 - 4.23 (m, 1H), 3.95 (dd, J= 5.4, 10.5 Hz, 1H), 3.90 - 3.81 (m, 1H), 3.64 (d, J= 10.6 Hz, 1H), 1.61 - 1.53 (m, 1H), 1.48 (d, J= 7.5 Hz, 1H), 1.10 - 0.97 (m, 15H), 0.85 (s, 3H)
508
SUBSTITUTE SHEET RULE 26 Example 130: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 386)
Figure imgf000510_0001
Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-
(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001096] A solution of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (310.58 mg, 1.02 mmol, 70% purity, 1 eq) in ACN (5 mL) was added with (lR,2S,5S)-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (500 mg, 1.02 mmol, 80% purity, 1 eq) and NMI (251.10 mg, 3.06 mmol, 243.79 uL, 3 eq), followed by TCFH (572.07 mg, 2.04 mmol, 2 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the mixture was diluted with H2O (5 mL) and extracted with EA (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-60%, 10 min) to give (lR,2S,5S)-N-[cyano- (3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (240 mg, 383.93 umol, 37.66% yield, 94% purity) as a white solid. MS (ESI) m/z 588.2 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
509
SUBSTITUTE SHEET RULE 26 [0001097] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (230 mg, 391.41 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 33%-33%, 6 min) to afford (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (63.65 mg, 102.58 umol, 26.21% yield, 94.7% purity) as a white solid. MS (ESI) m/z 588.2 [M+H]+.
[0001098] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.92 - 9.82 (m, 1H), 9.46 (br d, J= 6.1 Hz, 1H), 9.44 (s, 1H), 8.70 (s, 1H), 8.63 (s, 1H), 7.02 (br d, J= 6.4 Hz, 1H), 4.52 (s, 1H), 4.44 - 4.35 (m, 1H), 4.23 (s, 1H), 4.08 - 3.81 (m, 2H), 3.77 (br d, J= 10.3 Hz, 1H), 3.47 - 3.35 (m, 1H), 1.59 - 1.49 (m, 1H), 1.41 - 1.33 (m, 1H), 1.23 (br d, J = 6.1 Hz, 3H), 0.98 (s, 3H), 0.82 (s, 3H), 0.64 - 0.54 (m, 1H), 0.48 - 0.30 (m, 3H)
[0001099] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (67.54 mg, 112.76 umol, 28.81% yield, 98.1% purity) was obtained as a white solid. MS (ESI) m/z 588.2 [M+H]+.
[0001100] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.92 - 9.82 (m, 1H), 9.73 (br s, 1H), 9.42 (s, 1H), 8.71 (s, 1H), 8.63 (s, 1H), 7.21 - 7.06 (m, 1H), 4.49 (s, 1H), 4.41 - 4.33 (m, 1H), 4.31 (s, 1H), 3.90 (br dd, J= 5.0, 10.3 Hz, 1H), 3.85 - 3.69 (m, 2H), 3.30 (br d, .7= 2,9 Hz, 1H), 1.63 - 1.54 (m, 1H), 1.48 (br d, J= 7.5 Hz, 1H), 1.13 (br d, J= 6.1 Hz, 3H), 1.04 (s, 3H), 0.86 (s, 3H), 0.52 - 0.26 (m, 4H)
510
SUBSTITUTE SHEET RULE 26 Example 131: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2- trifluoroacetyl)amino] butanoyl] -N- [cyano-(3-prop- 1-ynylthieno [2,3-c] pyridin-4-yl)methyl] - 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 389)
Figure imgf000512_0001
Step 1: tert-butyl (2S,3R)-2-(benzyloxycarbonylamino)-3-hydroxy-butanoate
[0001101] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (645.09 mg, 1.58 mmol, 1 eq) in ACN (12 mL) was added NMI (389.05 mg, 4.74 mmol, 377.72 uL, 3 eq) and TCFH (886.36 mg, 3.16 mmol, 2 eq), and then 2-amino-2-(3-prop-l- ynylthieno[2,3-c]pyridin-4-yl)acetonitrile (359 mg, 1.58 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction was quenched by addition H2O (20 mL) at 25 °C, and then extracted with DCM (20 mL * 2). The combined organic layers were washed with brine (40 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80 * 40 mm * 3 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 50%-70%, 8 min). Compound (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(3- prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (540 mg, 874.24 umol, 55.34% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 618.3 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
511
SUBSTITUTE SHEET RULE 26 [0001102] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (540 mg, 874.24 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 23%-23%,5 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (261 mg, 422.55 umol, 26.75% yield, 100% purity) as a white solid. MS (ESI) m/z 618.2 [M+H]+.
[0001103] 'H NMR (400 MHz, DMSO-t76) 5 = 9.60 (br s, 1H), 9.41 (s, 1H), 9.21 (d, J= 6.4 Hz, 1H), 8.70 (s, 1H), 8.40 (s, 1H), 7.07 (br d, J= 6.3 Hz, 1H), 4.40 (br s, 1H), 4.25 (s, 1H), 4.04 - 3.85 (m, 2H), 3.67 (d, J= 10.6 Hz, 1H), 2.07 (s, 3H), 1.56 (dd, J= 5.3, 7.5 Hz, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.11 (m, 9H), 1.06 (d, J= 6.1 Hz, 3H), 1.00 - 0.95 (m, 3H), 0.80 (s, 3H).
[0001104] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (265 mg, 429.02 umol, 27.16% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 618.2 [M+H]+.
[0001105] 'H NMR (400 MHz, DMSO-t76) 5 = 9.62 (br s, 1H), 9.39 (s, 1H), 9.34 (d, J= 6.8 Hz, 1H), 8.73 - 8.66 (m, 1H), 8.40 (s, 1H), 7.21 (br d, J= 6.1 Hz, 1H), 4.38 (br s, 1H), 4.29 (s, 1H), 3.95 (dd, J= 5.3, 10.6 Hz, 1H), 3.85 (dd, J= 6.2, 7.9 Hz, 1H), 3.66 (d, J= 10.7 Hz, 1H), 2.08 (s, 3H), 1.59 (dd, J= 5.5, 7.4 Hz, 1H), 1.44 (d, J= 7.6 Hz, 1H), 1.12 - 1.03 (m, 15H), 0.83 (s, 3H).
512
SUBSTITUTE SHEET RULE 26 Example 132: Synthesis of (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 390)
Figure imgf000514_0001
Step 1: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001106] A solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (700 mg, 1.72 mmol, 9.79e-l eq) in DCM (5 mL) was added with 2-amino-2-(3-prop-l-ynylthieno[2,3- c]pyridin-4-yl)acetonitrile (400 mg, 1.76 mmol, 1 eq), DMAP (645.02 mg, 5.28 mmol, 3 eq) and EDCI (674.75 mg, 3.52 mmol, 2 eq), and then the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by H2O (5 mL) and extracted with ethyl acetate (5 mL* 3). The organic layers were washed with HC1 (IM, 5 mL), then wash with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC((column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-70%, 23 min) to give (lR,2S,5S)-N-[cyano- (3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (800 mg, 1.30 mmol, 73.83% yield) as a yellow solid.MS (ESI) m/z 616.3 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
513
SUBSTITUTE SHEET RULE 26 [0001107] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (800 mg, 1.30 mmol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 27%-27%, 6 min) to afford (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (111.96 mg, 180.76 umol, 13.91% yield, 99.4% purity) as a white solid. MS (ESI) m/z 616.3 [M+H]+.
[0001108] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.82 (d, J= 8.2 Hz, 1H), 9.41 (s, 1H), 9.29 (d, J= 6.6 Hz, 1H), 8.70 (s, 1H), 8.41 (s, 1H), 7.06 (br d, J= 6.3 Hz, 1H), 4.37 (t, J= 8.3 Hz, 1H), 4.24 (s, 1H), 4.01 - 3.79 (m, 2H), 3.69 (d, J= 10.5 Hz, 1H), 2.07 (s, 3H), 1.56 (dd, J= 5.3, 7.5 Hz, 1H), 1.36 (s, 1H), 1.35 - 1.31 (m, 3H), 1.21 - 1.06 (m, 3H), 1.01 - 0.93 (m, 3H), 0.83 - 0.74 (m, 4H), 0.71 - 0.62 (m, 1H), 0.41 - 0.29 (m, 2H)
[0001109] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (143.24 mg, 232.66 umol, 17.90% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 616.3 [M+H]+.
[0001110] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.82 (br d, J= 7.7 Hz, 1H), 9.43 (br d, J= 6.7 Hz, 1H), 9.39 (s, 1H), 8.76 - 8.64 (m, 1H), 8.40 (s, 1H), 7.20 (br d, J= 5.5 Hz, 1H), 4.35 (br t, J = 8.2 Hz, 1H), 4.28 (s, 1H), 3.94 - 3.83 (m, 2H), 3.69 (d, J= 10.6 Hz, 1H), 2.07 (s, 3H), 1.58 (dd, J= 5.4, 7.5 Hz, 1H), 1.44 (d, J= 7.6 Hz, 1H), 1.30 (s, 3H), 1.15 (d, J= 6.1 Hz, 3H), 1.08 - 1.01 (m, 3H), 0.82 (s, 3H), 0.77 - 0.70 (m, 1H), 0.65 - 0.57 (m, 1H), 0.37 - 0.25 (m, 2H).
514
SUBSTITUTE SHEET RULE 26 Example 133: Synthesis of (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)inethyl|-6.6-diniethyl-3-|(2S.3R)-3-( l-metliylcyclobutoxy)-2-|(2.2.2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 391)
Figure imgf000516_0001
Step 1: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-
[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001111] A solution of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (310.58 mg, 1.02 mmol, 70% purity, 1 eq) in ACN (5 mL) was added with (lR,2S,5S)-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (500 mg, 1.02 mmol, 80% purity, 1 eq) and NMI (251.10 mg, 3.06 mmol, 243.79 uL, 3 eq), and then was added TCFH (572.07 mg, 2.04 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex C 18 80 * 40 mm * 3 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 35%-65%, 8 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-[(2S,3R)- 3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane- 2-carboxamide (300 mg, 452.60 umol, 47.57% yield, 95% purity) as a white solid. MS (ESI) m/z 630.4 [M+H]+.
515
SUBSTITUTE SHEET RULE 26 Step 2: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-
[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001112] The (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 476.42 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 28%-28%, 7 min) to afford (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (62.73 mg, 96.03 umol, 20.16% yield, 96.4% purity) as a white solid. MS (ESI) m/z 630.2 [M+H]+.
[0001113] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.73 (br d, J= 7.3 Hz, 1H), 9.41 (s, 1H), 9.24 (d, J= 6.6 Hz, 1H), 8.69 (s, 1H), 8.40 (s, 1H), 7.06 (br d, J= 6.3 Hz, 1H), 4.43 (br t, J = 7.5 Hz, 1H), 4.25 (s, 1H), 3.97 (dd, J= 5.4, 10.5 Hz, 1H), 3.93 - 3.84 (m, 1H), 3.69 (d, J= 10.5 Hz, 1H), 2.15 - 2.05 (m, 5H), 1.79 - 1.70 (m, 2H), 1.62 - 1.54 (m, 2H), 1.53 - 1.45 (m, 1H), 1.36 (d, J = 7.5 Hz, 1H), 1.28 (s, 3H), 1.09 (d, J= 6.2 Hz, 3H), 0.98 (s, 3H), 0.80 (s, 3H).
[0001114] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (70.13 mg, 111.37 umol, 23.38% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 630.2 [M+H]+.
[0001115] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.72 (br s, 1H), 9.42 - 9.30 (m, 2H), 8.71 (s, 1H), 8.40 (s, 1H), 7.21 (br d, J= 4.9 Hz, 1H), 4.41 (br d, J= 6.3 Hz, 1H), 4.30 (s, 1H), 3.94 (dd, J= 5.3, 10.4 Hz, 1H), 3.88 - 3.77 (m, 1H), 3.68 (d, J= 10.6 Hz, 1H), 2.11 - 2.06 (m, 3H), 2.06 - 1.96 (m, 2H), 1.75 - 1.62 (m, 2H), 1.61 - 1.50 (m, 2H), 1.49 - 1.40 (m, 2H), 1.23 (s, 3H), 1.11 - 0.97 (m, 6H), 0.84 (s, 3H).
516
SUBSTITUTE SHEET RULE 26 Example 134: Synthesis of (lR,2S,5S)-N-[cyano(3-oxa-9,10-diazatricyclo[7.3.0.02,6]dodeca- l(12),2(6),4,7,10-pentaen-12-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
(Compound 398)
Figure imgf000518_0001
Step 1: (4-bromo-3 -pyridyl) acetate
[0001116] A solution of 4-bromopyri din-3 -ol (10 g, 57.47 mmol, 1 eq) in DCM (100 mL) was added with TEA (23.26 g, 229.89 mmol, 32.00 mL, 4 eq), and then acetyl chloride (9.02 g,
114.95 mmol, 8.20 mL, 2 eq) was added dropwise at 0 °C. The mixture was stirred at 20 °C for 3 h. Upon completion, the reaction mixture was diluted with H2O (100 mL) and extracted with ethyl acetate (100 mL * 3). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 1/0 to 10/1) to give (4-bromo-3 -pyridyl) acetate (9.2 g, 37.05 mmol, 64.47% yield, 87% purity) as a yellow solid. MS (ESI) m/z 216.1 [M+H]+.
Step 2: [4-(2-trimethylsilylethynyl)-3 -pyridyl] acetate
517
SUBSTITUTE SHEET RULE 26 [0001117] A solution of (4-bromo-3 -pyridyl) acetate (9.2 g, 42.59 mmol, 1 eq) in THF (1000 mL) was added ethynyl(trimethyl)silane (10.46 g, 106.47 mmol, 14.75 mL, 2.5 eq) and Et3N (64.64 g, 638.79 mmol, 88.91 mL, 15 eq) under N2. After 10 mm, Pd(PPh3)2Cl2 (1.79 g, 2.56 mmol, 0.06 eq) and Cui (973.27 mg, 5.11 mmol, 0.12 eq) was added to the mixture under N2. The mixture was stirred at 20 °C for 24 h. Upon completion, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give [4-(2-trimethylsilylethynyl)-3- pyridyl] acetate (5 g, crude) as a black brown solid. MS (ESI) m/z 234.1 [M+H]+.
Step 3: furo[2,3-c]pyridine
[0001118] A solution of [4-(2-trimethylsilylethynyl)-3-pyridyl] acetate (5 g, 21.43 mmol, 1 eq) in MeOH (500 mL) was added KF (4.98 g, 85.71 mmol, 2.01 mL, 4 eq). The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 0/1 to 1/0) to give furo[2,3-c]pyridine (800 mg, 5.44 mmol, 25.39% yield, 81% purity) as a yellow solid. MS (ESI) m/z 120.2 [M+H]+.
Step 4: furo[2,3-c]pyridin-6-ium-6-amine
[0001119] A solution of amino 2,4,6-trimethylbenzenesulfonate (1.73 g, 8.06 mmol, 1.2 eq) and furo[2,3-c]pyridine (800 mg, 6.72 mmol, 1 eq) in DCM (20 mL) was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was filtered, the filter cake was washed with DCM (100 mL *3) and dried in air to give furo[2,3-c]pyridin-6-ium-6-amine (1 g, crude) as a white solid. MS (ESI) m/z 135.3 [M]
Step 5: methyl 3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10-pentaene-12- carboxylate
[0001120] A solution of furo[2,3-c]pyridin-6-ium-6-amine (1 g, 7.40 mmol, 1 eq) and methyl prop-2-ynoate (808.74 mg, 9.62 mmol, 800.73 uL, 1.3 eq) in DMF (15 mL) was added K2CO3 (1.53 g, 11.10 mmol, 1.5 eq) and stirred at 20 °C for 12 h. Upon completion, the reaction mixture was quenched by addition H2O (30 mL) at 0 °C, and extracted with ethyl acetate (30 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column
518
SUBSTITUTE SHEET RULE 26 chromatography (SiCh, petroleum ether/ethyl acetate = 1/0 to 1/1) to afford methyl 3-oxa-9,10- diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10-pentaene-12-carboxylate (300 mg, 1.38 mmol, 18.70% yield, 99.7% purity) as a yellow solid MS (ESI) m/z 217.1 [M+H]+
Step 6: 3 -oxa-9, 10-diazatricyclo[7.3.0.02,6] dodeca- 1(12), 2(6), 4, 7, 10-pentaen- 12-ylmethanol
[0001121] A solution of methyl 3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaene-12-carboxylate (250 mg, 1.16 mmol, 1 eq) and THF (5 mL) was added DIBAL-H (1 M, 5.78 mL, 5 eq at 0 °C and stirred for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (10 mL) at 0 °C, and extracted with EA (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaen-12-ylmethanol (190 mg, crude) as a yellow solid.
Step 7 : 3 -oxa-9, 10-diazatricyclo[7.3.0.02,6] dodeca- 1(12), 2(6), 4, 7, 10-pentaene- 12-carbaldehyde
[0001122] A mixture of 3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10-pentaen- 12-ylmethanol (190 mg, 1.01 mmol, 1 eq} in CHCh (30 mL) was added MnCh (1.32 g, 15.14 mmol, 15 eq}, and the mixture was stirred at 70 °C for 2 h. Upon completion, The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether/ethyl acetate = 1/0 to 0/1) to give 3-oxa-9,10-diazatricyclo[7.3.0.02,6]dodeca-l(12),2(6),4,7 ,10-pentaene- 12- carbaldehyde (130 mg, 663.38 umol, 65.70% yield, 95% purity) as a white solid. MS (ESI) m/z 187.2 [M+H]+
Step 8: 2-amino-2-(3 -oxa-9, 10-diazatricy clo[7.3.0.02,6] dodeca- 1 ( 12), 2(6), 4, 7, 10-pentaen- 12- yl)acetonitrile
[0001123] To a solution of 3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaene-12-carbaldehyde (140 mg, 752.01 umol, 1 eq} in NEh/MeOH (20 mL) (7 M) was added NH4CI (120.68 mg, 2.26 mmol, 3 eq). After 1 h, TMSCN (373.04 mg, 3.76 mmol, 470.41 uL, 5 eq) was added drop- wise, the mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove MeOH (20 mL). The residue was diluted with H2O (15 mL) and extracted with DCM (10 mL * 3). The combined
519
SUBSTITUTE SHEET RULE 26 organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca- l(12),2(6),4,7,10-pentaen-12-yl)acetonitrile (130 mg, crude) as a yellow solid. MS (ESI) m/z 213.2 [M+H]+
Step 9: (lR,2S,5S)-N-[cyano(3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaen-12-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001124] To a solution of 2-amino-2-(3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca- l(12),2(6),4,7,10-pentaen-12-yl)acetonitrile (130 mg, 612.61 umol, 1 eq) and (lR,2S,5S)-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (290.17 mg, 796.39 umol, 1.3 eq) in ACN (5 mL) was added NMI (201.19 mg, 2.45 mmol, 195.33 uL, 4 eq) at 0 °C, and then TCFH (206.26 mg, 735.13 umol, 1.2 eq) was added at 0 °C. The mixture was stirred at 20 °C for 12 h. Upon completion, the reaction mixture was diluted with H2O (20 mL) and extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (column: Waters Xbridge Prep OBD Cl 8 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3) - ACN]; B%: 40% - 80%, 8 mm) to give (lR,2S,5S)-N-[cyano(3-oxa-9,10- diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10-pentaen-12-yl)methyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 354.49 umol, 57.87% yield, 90% purity) as a white solid. MS (ESI) m/z 559.3 [M+H]+
Step 10: (lR,2S,5S)-N-[cyano(3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaen-12-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001125] (lR,2S,5S)-N-[cyano(3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaen-12-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (200 mg, 358.07 umol, 1 eq) was separated by SFC. The residue was separated by SFC (column: DAICEL CHIRALPAK IC( 250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 23%-23%, 8 min) to give (lR,2S,5S)-N-[cyano(3-
520
SUBSTITUTE SHEET RULE 26 oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10-pentaen-12-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (63 mg, 112.79 umol, 31.50% yield, 100% purity) as a white solid. MS (ESI) m/z 559.2 [M+H]+
[0001126] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.43 - 9.13 (m, 2H), 8.59 (d, J = 7.3 Hz, 1H), 8.34 - 8.06 (m, 2H), 7.30 (d, J= 7.3 Hz, 1H), 7.14 (d, J= 1.9 Hz, 1H), 6.38 (d, J= 6.1 Hz, 1H), 4.42 (s, 1H), 4.32 (s, 1H), 3.98 - 3.86 (m, 1H), 3.72 (br d, J= 10.5 Hz, 1H), 1.63 - 1.49 (m, 1H),
1.40 (d, J= 7.5 Hz, 1H), 1.09 - 1.02 (m, 3H), 0.97 (s, 9H), 0.82 (s, 3H).
[0001127] (lR,2S,5S)-N-[cyano(3-oxa-9,10-diazatricyclo[7.3.0.02’6]dodeca-l(12),2(6),4,7,10- pentaen-12-yl)methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (63 mg, 112.79 umol, 31.50% yield, 100% purity) as a white solid. MS (ESI) m/z 559.2 [M+H]+
[0001128] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.32 (br d, J= 7.0 Hz, 2H), 8.60 (d, J= 7.2 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.17 (s, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H),
6.40 (d, J= Hz, 1H), 4.42 (s, 1H), 4.27 (s, 1H), 3.98 - 3.86 (m, 1H), 3.71 (d, J= 10.5 Hz, 1H), 1.60 - 1.52 (m, 1H), 1.24 (d, J= 7.6 Hz, 1H), 1.01 (s, 9H), 0.93 (s, 3H), 0.81 (s, 3H)
Example 135: Synthesis of (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 402)
Figure imgf000522_0001
Step 1: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
521
SUBSTITUTE SHEET RULE 26 [0001129] To a solution of 2-amino-2-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)acetonitrile (360 mg, 1.58 mmol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (580.24 mg, 1.58 mmol, 1 eq), TCFH (666.62 mg, 2.38 mmol, 1.5 eq) and NMI (390.14 mg, 4.75 mmol, 378.77 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (0.5 mL) at 25 °C, and then diluted with H2O (10 mL) and extracted with ethyl acetate (5 mL * 2). The combined organic layers were washed with sat. NaCl (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)- ACN] ; B%: 35%-60%, 10 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide (300 mg, 521.19 umol, 1 eq) as white solid. MS (ESI) m/z 576.4 [M+H]+
Step 2: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[0001130] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)- 3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3. L0]hexane-2- carboxamide (300 mg, 521.19 umol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 8 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide Isomer 1 (84 mg, 145.93 umol, 9.21% yield). MS (ESI) m/z 576.2 [M+H]+
[0001131] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.85 (br s, 1H), 9.52 - 9.30 (m, 2H), 8.72 (s, 1H), 8.39 (s, 1H), 7.19 (br d, J= 4.5 Hz, 1H), 4.35 (br d, J = 9.0 Hz, 1H), 4.28 (s, 1H), 3.96 - 3.82 (m, 2H), 3.58 (dd, J= 6.3, 8.9 Hz, 1H), 3.21 (s, 3H), 2.05 (s, 3H), 1.60 (dd, J= 5.2, 7.3 Hz, 1H), 1.43 (d, J= 7.6 Hz, 1H), 1.16 (d, J= 6.1 Hz, 3H), 1.05 (s, 3H), 0.86 (s, 3H)
[0001132] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)- 3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
522
SUBSTITUTE SHEET RULE 26 carboxamide Isomer 2 (89 mg, 154.62 umol, 9.76% yield) was obtained as white solid. MS (ESI) m/z 576.2 [M+H]+
[0001133] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.85 (br s, 1H), 9.50 - 9.30 (m, 2H), 8.72 (s, 1H), 8.39 (s, 1H), 7.27 - 7.12 (m, 1H), 4.40 - 4.15 (m, 2H), 3.97 - 3.76 (m, 2H), 3.58 (dd, J= 6.3, 8.9 Hz, 1H), 3.21 (s, 3H), 2.05 (s, 3H), 1.60 (dd, J = 5.3, 7.3 Hz, 1H), 1.43 (d, J= 7.5 Hz, 1H), 1.16 (d, J = 6.1 Hz, 3H), 1.05 (s, 3H), 0.86 (s, 3H)
Example 136: Synthesis of N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide (Compound 415)
Figure imgf000524_0001
Step 1: (2S)-2-amino-3,3-dimethyl-butanoic acid
[0001134] A mixture of (2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoic acid (10 g, 43.24 mmol, 1 eq) was added with HCl/dioxane (4 M, 100 mL, 9.25 eq) and the mixture was
523
SUBSTITUTE SHEET RULE 26 stirred at 20 °C for 1 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give (2S)-2-amino-3,3-dimethyl-butanoic acid (7.2 g, crude, HC1) as a white solid. MS (ESI) m/z 132.1 [M+H]+.
Step 2: (2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoic acid
[0001135] To a solution of (2S)-2-amino-3,3-dimethyl-butanoic acid (7.2 g, 42.95 mmol, 1 eq, HC1) in TEA (17.38 g, 171.80 mmol, 23.91 mL, 4 eq) was added methyl 2,2,2-trifluoroacetate (55.00 g, 429.51 mmol, 43.31 mL, 10 eq). The mixture was stirred at 20 °C for 4 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give (2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoic acid (9 g, crude) as a yellow oil. MS (ESI) m/z 225.9 [M+H]+.
Step 3: tert-butyl 7-[[cyano-(5-ethynyl-4-isoquinolyl)methyl]carbamoyl]-2,2-difluoro-6- azaspiro[3.4]octane-6-carboxylate
[0001136] A mixture of 6-tert-butoxy carbonyl-2, 2-difluoro-6-azaspiro[3.4]octane-7-carboxylic acid (230 mg, 789.59 umol, 1 eq), 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (179.99 mg, 868.55 umol, 1.1 eq) and 1 -methylimidazole (194.48 mg, 2.37 mmol, 188.82 uL, 3 eq) in ACN (5 mL) was added with [chloro(dimethylamino)methylene]-dimethyl-ammonium hexafluorophosphate (443.08 mg, 1.58 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 5: 1 to 3:1) to afford tert-butyl 7-[[cyano-(5-ethynyl-4-isoquinolyl)methyl]carbamoyl]-2,2-difluoro-6- azaspiro[3.4]octane-6-carboxylate (730 mg, crude) as a yellow oil. MS (ESI) m/z 481.2 [M+H]+.
Step 4: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-2,2-difluoro-6-azaspiro[3.4]octane-7- carboxamide
[0001137] To a solution of tert-butyl 7-[[cyano-(5-ethynyl-4-isoquinolyl)methyl]carbamoyl]- 2,2-difluoro-6-azaspiro[3.4]octane-6-carboxylate (665 mg, 1.38 mmol, 1 eq) in DCM (30 mL)
524
SUBSTITUTE SHEET RULE 26 was added TFA (15.40 g, 135.06 mmol, 10 mL, 97.59 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was adjust PH=8 with NaHCCh, and then extracted with EtOAc (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give N-[cyano-(5-ethynyl-4- isoquinolyl)methyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide (526 mg, crude) as a yellow solid. MS (ESI) m/z 381.2 [M+H]+.
Step 5: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-2,2-difluoro-6-azaspiro[3.4]octane-7- carboxamide
[0001138] To a solution of N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-2,2-difluoro-6- azaspiro[3.4]octane-7-carboxamide (526 mg, 1.38 mmol, 1 eq), (2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoic acid (376.97 mg, 1.66 mmol, 1.2 eq), [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (775.96 mg, 2.77 mmol, 2 eq) in ACN (5 mL) was added 1 -methylimidazole (16.93 g, 206.21 mmol, 16.44 mL,
149.13 eq) at 0 °C. The mixture was stirred at 25 °C for 1 h. The reaction mixture was poured into H2O (30 mL) at 25 °C, and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NELHCChj-ACN]; B%: 30%-60%, 20 min) to give N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide (180 mg,
302.14 umol, 21.85% yield, 98.96% purity) as a white solid. MS (ESI) m/z 590.3 [M+H]+.
Step 6: N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide
[0001139] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide (150 mg) was separated by SEC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 20%-20%, 10 min) to give N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]- 6-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-
525
SUBSTITUTE SHEET RULE 26 azaspiro[3.4]octane-7-carboxamide Isomer 1 (12 mg, 20.35 umol, 8.00% yield, 100% purity) as a white solid. MS (ESI) m/z 590.3 [M+H]+.
[0001140] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.48 - 9.44 (m, 1H), 9.36 (d, J= 7.2 Hz, 2H), 8.93 - 8.73 (m, 1H), 8.32 (dd, J= 1.0, 8.2 Hz, 1H), 8.17 - 8.08 (m, 1H), 7.79 (t, J= 7.7 Hz, 1H), 7.64 (d, J= 7.2 Hz, 1H), 4.67 (s, 1H), 4.62 (s, 1H), 4.44 (dd, J= 5.4, 8.4 Hz, 1H), 3.89 (d, J = 10.4 Hz, 1H), 3.77 (d, J= 10.5 Hz, 1H), 2.69 - 2.54 (m, 4H), 2.40 (dd, J= 8.6, 12.8 Hz, 1H), 2.06 (dd, J= 5.3, 12.9 Hz, 1H), 0.97 (s, 9H).
[0001141] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide Isomer 2 (11 mg, 18.66 umol, 7.33% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 590.3 [M+H]+.
[0001142] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.59 (d, J= 6.9 Hz, 1H), 9.45 (s, 1H), 9.31 (s, 1H), 8.85 (s, 1H), 8.36 - 8.29 (m, 1H), 8.13 (d, J= 6.6 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.57 (d, J= 6.9 Hz, 1H), 4.57 (s, 1H), 4.51 - 4.43 (m, 2H), 3.92 (d, J= 10.4 Hz, 1H), 3.69 (d, J= 10.4 Hz, 1H), 2.73 - 2.54 (m, 4H), 2.38 (dd, J= 8.1, 12.6 Hz, 1H), 2.06 (dd, J= 7.6, 12.6 Hz, 1H), 0.94 (s, 9H).
[0001143] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide Isomer 3 (14 mg, 23.75 umol, 9.33% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 590.3 [M+H]+.
[0001144] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.64 (s, 1H), 9.48 - 9.45 (m, 1H), 9.40 (d, J = 6.9 Hz, 1H), 8.77 (s, 1H), 8.33 (d, J= 7.4 Hz, 1H), 8.14 (dd, J= 1.1, 7.3 Hz, 1H), 7.78 (t, J= 7.7 Hz, 1H), 7.64 (d, J= l.Q Hz, 1H), 4.78 (s, 1H), 4.73 - 4.67 (m, 1H), 4.42 - 4.31 (m, 1H), 3.93 (d, J= 10.5 Hz, 1H), 3.73 (d, J= 10.4 Hz, 1H), 2.72 - 2.53 (m, 4H), 2.34 - 2.26 (m, 1H), 1.99 (dd, J = 5.8, 12.9 Hz, 1H), 0.97 (s, 9H).
[0001145] N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-2,2-difluoro-6-azaspiro[3.4]octane-7-carboxamide Isomer 4 (10 mg, 16.96 umol, 6.67% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 590.3 [M+H]+.
526
SUBSTITUTE SHEET RULE 26 [0001146] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.46 (s, 1H), 9.36 (d, J= 7.4 Hz, 2H), 8.81 (s, 1H), 8.32 (d, J= 7.5 Hz, 1H), 8.13 (d, J = 6.6 Hz, 1H), 7.79 (t, J= 7.7 Hz, 1H), 7.64 (d, J= 7.2 Hz, 1H), 4.69 - 4.58 (m, 2H), 4.44 (dd, J= 5.4, 8.5 Hz, 1H), 3.89 (d, J= 10.4 Hz, 1H), 3.77 (d, J = 10.4 Hz, 1H), 2.68 - 2.52 (m, 4H), 2.43 - 2.34 (m, 1H), 2.06 (dd, J= 5.3, 12.8 Hz, 1H), 0.97 (s, 9H).
Example 137: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide. (Compound 417)
Figure imgf000528_0001
Step 1: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-
[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001147] A solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (700 mg, 1.69 mmol, 50% purity, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (758.76 mg, 1.86 mmol, 1.1 eq) in DCM (30 mL) was added DMAP (619.00 mg, 5.07 mmol, 3 eq), EDCI (647.55 mg, 3.38 mmol, 2 eq), and the mixture was stirred at 25 °C for 1 h. Upon completion. The reaction mixture was quenched by addition H2O (60 mL), and then extracted with DCM (40 mL * 3). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 70 mm * 10 um; mobile
527
SUBSTITUTE SHEET RULE 26 phase: [water (NH4HCCh)-ACN]; B%: 40%-70%, 20 min) to give desired compound as a white solid, which was further separated by SFC (column: REGIS(S,S)WHELK-Ol(250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 40%-40%, 6 min) to give (lR,2S,5S)-3-[(2S,3R)-3-tert- butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (128 mg, 210.97 umol, 12.49% yield, 98.5% purity) as a white solid. MS (ESI) m/z 598.3 [M+H]+.
[0001148] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.62 (br d, J= 8.6 Hz, 1H), 9.49 - 9.45 (m, 1H), 9.41 (br d, J= 6.7 Hz, 1H), 8.81 (s, 1H), 8.34 (d, J = 8.2 Hz, 1H), 8.14 (d, J= 7.3 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.58 (d, J= 6.6 Hz, 1H), 4.70 (s, 1H), 4.41 (br t, J= 8.1 Hz, 1H), 4.26 (s, 1H), 4.00 - 3.88 (m, 2H), 3.65 (br d, J= 10.6 Hz, 1H), 1.57 - 1.51 (m, 1H), 1.36 (d, J= 7.6 Hz, 1H), 1.15 - 1.07 (m, 12H), 0.98 (s, 3H), 0.80 (s, 3H).
[0001149] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (152 mg, 253.32 umol, 15.00% yield, 99.6% purity) was obtained as a white solid. MS (ESI) m/z 598.3 [M+H]+.
[0001150] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.61 (br t, J= 8.8 Hz, 2H), 9.46 (s, 1H), 8.81 (s, 1H), 8.33 (dd, J= 1.0, 8.2 Hz, 1H), 8.14 (dd, J= 1.0, 7.3 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.68 (d, J= Hz, 1H), 4.68 (s, 1H), 4.43 - 4.31 (m, 2H), 3.96 (dd, J= 5.3, 10.6 Hz, 1H), 3.89 - 3.80 (m, 1H), 3.65 (d, J= 10.7 Hz, 1H), 1.63 - 1.54 (m, 1H), 1.48 (d, J= 7.5 Hz, 1H), 1.06 - 0.98 (m, 15H), 0.85 (s, 3H).
Example 138: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 418)
Figure imgf000529_0001
528
SUBSTITUTE SHEET RULE 26 Step 1: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001151] A solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (406.65 mg, 1.18 mmol, 60% purity, 1.1 eq) in ACN (5 mL) was added with (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)- 3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane- 2-carboxylic acid (500 mg, 1.07 mmol, 90% purity, 1 eq) and NMI (263.63 mg, 3.21 mmol, 255.95 uL, 3 eq), and TCFH (600.64 mg, 2.14 mmol, 2 eq) was added. The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, petroleum ether: ethyl acetate = 100:1 to 40: 1) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2- carboxamide (400 mg, 459.29 umol, 38.62% yield, 70% purity) as a white solid. MS (ESI) m/z 610.4 [M+H]+.
Step 2: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2- carboxamide
[0001152] The (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (400 mg, 459.29 umol, 70% purity, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu- IPA]; B%: 40%-40%, 7 min) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (62.23 mg, 98.10 umol, 21.36% yield, 96.1% purity) as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001153] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.86 - 9.72 (m, 1H), 9.53 - 9.40 (m, 2H), 8.81 (s, 1H), 8.37 - 8.29 (m, 1H), 8.14 (dd, J= 1.1, 7.2 Hz, 1H), 7.79 (t, J= 7.7 Hz, 1H), 7.57 (d, J =
529
SUBSTITUTE SHEET RULE 26 6.8 Hz, 1H), 4.71 (s, 1H), 4.43 (br t, J= 7.6 Hz, 1H), 4.25 (s, 1H), 3.96 (dd, J= 5.4, 10.6 Hz, 1H), 3.89 (dd, J= 6.4, 8.1 Hz, 1H), 3.66 (d, J= 10.5 Hz, 1H), 2.14 - 2.04 (m, 2H), 1.79 - 1.70 (m, 2H), 1.60 - 1.46 (m, 3H), 1.36 (d, J= 7.5 Hz, 1H), 1.28 (s, 3H), 1.12 (d, J= 6.1 Hz, 3H), 1.04 - 0.96 (m, 3H), 0.81 (s, 3H).
[0001154] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3- (1 -methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (70.78 mg, 110.30 umol, 24.01% yield, 95% purity) was obtained as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001155] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.86 - 9.70 (m, 1H), 9.66 (d, J= 7.5 Hz, 1H), 9.46 (s, 1H), 8.81 (s, 1H), 8.33 (dd, J= 0.9, 8.1 Hz, 1H), 8.18 - 8.08 (m, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.68 (d, J= 7.0 Hz, 1H), 4.69 (s, 1H), 4.40 (br d, J= 8.1 Hz, 1H), 4.33 (s, 1H), 4.01 - 3.88 (m, 1H), 3.86 - 3.76 (m, 1H), 3.72 - 3.62 (m, 1H), 2.09 - 1.92 (m, 2H), 1.74 - 1.65 (m, 1H), 1.64 - 1.54 (m, 2H), 1.52 - 1.31 (m, 3H), 1.25 - 1.18 (m, 3H), 1.12 - 0.97 (m, 6H), 0.96 - 0.72 (m, 3H).
Example 139: Synthesis of (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3- [(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 434)
Figure imgf000531_0001
Step 1: 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile
530
SUBSTITUTE SHEET RULE 26 [0001156] To a solution of 5-bromoisoquinoline-4-carbaldehyde (1 g, 4.24 mmol, 1 eq) in NHa/MeOH (7 M) (10 mL) was added with NH4CI (679.77 mg, 12.71 mmol, 3 eq), and the mixture was stirred at 25 °C for 1 h. TMSCN (1.26 g, 12.71 mmol, 1.59 mL, 3 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHVMeOH. The reaction mixture was quenched by addition H2O (30 mL), and then extracted with EA (10 mL * 3). The combined organic layers weredried over Na2SO4, filtered and concentrated in vacuum and was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 0:1) to afford 2-amino-2-(5- bromo-4-isoquinolyl)acetonitrile (730 mg, 2.59 mmol, 61.15% yield, 93% purity) as yellow oil. MS (ESI) m/z 262.2, 264.2 [M+H]+
Step 2: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001157] To a solution of 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (700 mg, 2.48 mmol, 93% purity, 1 eq) in ACN (10 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.29 g, 2.98 mmol, 1.2 eq) and NMI (693.31 mg, 8.44 mmol, 673.12 uL, 3 eq). TCFH (1.67 g, 5.96 mmol, 2 eq) was added, and the mixture was stirred at 25 °C for 1 h. Upon completion, the mixture was diluted with H2O (50 mL) and filtered then was extracted with EA (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-HPLC (column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3) - ACN]; B%: 40% - 70%, 10 min) to give the product (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.43 mmol, 57.73% yield, 97% purity) as yellow solid. MS (ESI) m/z 676.2, 678.2 [M+H]+
Step 3: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
531
SUBSTITUTE SHEET RULE 26 [0001158] The (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.48 mmol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 urn); mobile phase: [Neu - IP A]; B%: 26% - 26%, 6 min) to give (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (112.74 mg, 166.65 umol, 11.27% yield, 100% purity) as white solid. MS (ESI) m/z 676.1, 678.1 [M+H]+
[0001159] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.01 - 9.69 (m, 1H), 9.65 - 9.43 (m, 2H), 8.89 (s, 1H), 8.31 (ddd, J= 1.2, 7.8, 18.8 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.51 (br d, J= 6.8 Hz, 1H), 4.40 (br d, J= 8.4 Hz, 1H), 4.24 (s, 1H), 4.12 - 3.99 (m, 1H), 3.94 (dd, J= 5.4, 10.4 Hz, 1H), 3.66 (d, J= 10.6 Hz, 1H), 1.56 (dd, J= 5.4, 7.4 Hz, 1H), 1.48 - 1.32 (m, 2H), 1.26 (d, J = 6.0 Hz, 3H), 1.06 - 0.95 (m, 3H), 0.80 (s, 3H), 0.72 - 0.60 (m, 1H), 0.56 - 0.39 (m, 3H), 0.38 - 0.17 (m, 2H), 0.15 - -0.03 (m, 2H).
[0001160] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (165.93 mg, 244.59 umol, 16.55% yield, 99.723% purity) was obtained as white solid. MS (ESI) m/z 676.1, 678.1 [M+H]+
[0001161] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.78 (s, 1H), 9.69 - 9.45 (m, 2H), 8.89 (s, 1H), 8.30 (dd, J= 1.8, 18.8 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.55 - 7.41 (m, 1H), 4.45 - 4.33 (m, 1H), 4.32 - 4.23 (m, 1H), 4.07 - 3.97 (m, 1H), 3.96 - 3.87 (m, 1H), 3.73 - 3.64 (m, 1H), 1.63 - 1.53 (m, 1H), 1.48 - 1.33 (m, 2H), 1.28 - 1.16 (m, 3H), 1.05 - 1.01 (m, 3H), 0.90 - 0.77 (m, 3H), 0.71 - 0.57 (m, 1H), 0.51 (br s, 3H), 0.31 - 0.13 (m, 2H), 0.12 -0.07 (m, 2H).
532
SUBSTITUTE SHEET RULE 26 Example 140: Synthesis of (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3- [(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 435)
Figure imgf000534_0001
Step 1: 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile
[0001162] A solution of 5-bromoisoquinoline-4-carbaldehyde (1 g, 4.24 mmol, 1 eq) in NHVMeOH (7 M) (10 mL) was added NH4CI (679.77 mg, 12.71 mmol, 3 eq) was stirred at 25 °C for 1 h, and then TMSCN (1.26 g, 12.71 mmol, 1.59 mL, 3 eq) was added at 0 °C. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHa/MeOH. The reaction mixture was quenched by addition H2O (30 mL), and then extracted with ethyl acetate (10 mL * 3). The combined organic layers weredried over Na2SO4, filtered and concentrated in vacuum and was purified by column chromatography (SiCh, petroleum etherethyl acetate = 10:1 to 0: 1) to give 2-amino-2-(5-bromo- 4-isoquinolyl)acetonitrile (730 mg, 2.59 mmol, 61.15% yield, 93% purity) as yellow oil. MS (ESI) m/z 262.2, 264.2 [M+H]+
533
SUBSTITUTE SHEET RULE 26 Step 2: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001163] To a solution of 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (700 mg, 2.48 mmol, 93% purity, 1 eq) in ACN (10 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.29 g, 2.98 mmol, 1.2 eq) and NMI (693.31 mg, 8.44 mmol, 673.12 uL, 3 eq) and then was added TCFH (1.67 g, 5.96 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the mixture was diluted with H2O (50 mL) and filtered then was extracted with ethyl acetate (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-HPLC (column: C18 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3) - ACN]; B%: 40% - 70%, 10 min) to give (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.43 mmol, 57.73% yield, 97% purity) as yellow solid. MS (ESI) m/z 676.2, 678.2 [M+H]+
Step 3: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001164] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.48 mmol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu - IP A]; B%: 26% - 26%, 6 min) to afford (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (112.74 mg, 166.65 umol, 11.27% yield, 100% purity) as white solid. MS (ESI) m/z 676.1, 678.1 [M+H]+
[0001165] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.01 - 9.69 (m, 1H), 9.65 - 9.43 (m, 2H), 8.89 (s, 1H), 8.31 (ddd, J= 1.2, 7.8, 18.8 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.51 (br d, J= 6.8 Hz,
534
SUBSTITUTE SHEET RULE 26 1H), 4.40 (br d, J= 8.4 Hz, 1H), 4.24 (s, 1H), 4.12 - 3.99 (m, 1H), 3.94 (dd, J= 5.4, 10.4 Hz, 1H), 3.66 (d, J= 10.6 Hz, 1H), 1.56 (dd, J= 5.4, 7.4 Hz, 1H), 1.48 - 1.32 (m, 2H), 1.26 (d, J = 6.0 Hz, 3H), 1.06 - 0.95 (m, 3H), 0.80 (s, 3H), 0.72 - 0.60 (m, 1H), 0.56 - 0.39 (m, 3H), 0.38 - 0.17 (m, 2H), 0.15 - -0.03 (m, 2H).
[0001166] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (165.93 mg, 244.59 umol, 16.55% yield, 99.723% purity) was obtained as white solid. MS (ESI) m/z 676.1, 678.1 [M+H]+
[0001167] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.78 (s, 1H), 9.69 - 9.45 (m, 2H), 8.89 (s, 1H), 8.30 (dd, J= 7.8, 18.8 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.55 - 7.41 (m, 1H), 4.45 - 4.33 (m, 1H), 4.32 - 4.23 (m, 1H), 4.07 - 3.97 (m, 1H), 3.96 - 3.87 (m, 1H), 3.73 - 3.64 (m, 1H), 1.63 - 1.53 (m, 1H), 1.48 - 1.33 (m, 2H), 1.28 - 1.16 (m, 3H), 1.05 - 1.01 (m, 3H), 0.90 - 0.77 (m, 3H), 0.71 - 0.57 (m, 1H), 0.51 (br s, 3H), 0.31 - 0.13 (m, 2H), 0.12 -0.07 (m, 2H).
Example 141: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 201A)
Figure imgf000536_0001
[0001168] Step 1: methyl (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(9H-fluoren-9- ylmethoxycarbonylamino) butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2- carboxylate
[0001169] To a mixture of methyl (lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylate (1 g, 4.86 mmol, 1 eq, HC1) and (2S,3R)-3-tert-butoxy-2-(9H-fluoren-9-
535
SUBSTITUTE SHEET RULE 26 ylmethoxycarbonylamino)butanoic acid (1.93 g, 4.86 mmol, 1 eq) in DMF (10 mL) was added DIEA (1.89 g, 14.59 mmol, 2.54 mL, 3 eq), then HATU (3.70 g, 9.72 mmol, 2 eq) was added. The mixture was stirred at 25 °C for 30 min. Upon completion, the reaction mixture was diluted with H2O 40 mL and extracted with EA 80 mL (40 mL * 2). The combined organic layers were washed with brine 80 mL (80 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, Petroleum ether:Ethyl acetate = 15:1 to 8:1). Compound methyl (lR,2S,5S)-3-[(2S,3R)-3-tert- butoxy-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (2.35 g, 4.11 mmol, 84.57% yield, 96% purity) was obtained as a colourless oil. MS (ESI) m/z 493.4 [M+H-56]+.
[0001170] Step 2: (lR,2S,5S)-3-[(2S,3R)-2-amino-3-tert-butoxy-butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxylic acid
[0001171] To a solution of methyl (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-(9H-fluoren-9- ylmethoxycarbonylamino)butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylate (2.3 g, 4.19 mmol, 1 eq) in THE (15 mL) and H2O (5 mL) was added LiOH.EEO (615.63 mg, 14.67 mmol, 3.5 eq). The mixture was stirred at 40 °C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure to give (lR,2S,5S)-3-[(2S,3R)-2-amino-3-tert-butoxy- butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (6.8 g, crude) as a white solid. MS (ESI) m/z 311.3 [M-H]’.
[0001172] Step 3: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[0001173] To a mixture of (lR,2S,5S)-3-[(2S,3R)-2-amino-3-tert-butoxy-butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.3 g, 4.16 mmol, 1 eq) and TEA (1.26 g, 12.48 mmol, 1.74 mL, 3 eq) was added methyl 2,2,2-trifluoroacetate (5.33 g, 41.61 mmol, 4.20 mL, 10 eq). After the addition, the resulting mixture was stirred at 25 °C for 4 h. Upon completion, The reaction mixture was concentrated in a vacuum to remove most of methyl 2,2,2- trifluoroacetate to afford a residue. The combined residue was diluted with EtOAc (20 mL) and washed with 1 M aqueous HC1 (20 mL). The organic layer was seprated and the aqueous layer was extracted with EtOAc (20 mL * 2). The combined orangic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue.
536
SUBSTITUTE SHEET RULE 26 [0001174] The residue was triturated with petroleum ether: ethyl acetate = 20:1 (20 mL) at 25 °C for 15 min. Then filtered to give the crude product. The crude product was washed with petroleum ether: ethyl acetate (20: 1) (200 mL) to give the desired product. Compound (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.08 g, 2.59 mmol, 62.15% yield, 97.8% purity) was obtained as a white solid. MS (ESI) m/z 407.2 [M-H]'.
[0001175] Step 4: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano(4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001176] To a mixture of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (200 mg, 489.70 umol, 1 eq) in DMF (5 mL) was added DIEA (189.87 mg, 1.47 mmol, 255.89 uL, 3 eq), then 2-amino-2-(4-isoquinolyl)acetonitrile (89.72 mg, 489.70 umol, 1 eq) and HATU (372.40 mg, 979.40 umol, 2 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with EA 40 mL (20 mL * 2). The combined organic layers were washed with brine 40 mL (40 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by neutral prep-HPLC (column: Waters Xbridge BEH Cl 8 100 * 30mm * lOum; mobile phase:[water( NH4HCO3)-ACN]; B%:40%-70%, 10 mm). Compound (lR,2S,5S)-3-[(2S,3R)-3- tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano(4-isoquinolyl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (125 mg, 211.82 umol, 43.25% yield, 97.2% purity) was obtained as a white solid. MS (ESI) m/z 574.2 [M+H]+.
[0001177] 'H NMR (400 MHz, DMSO-d6) 5 = 9.71 - 9.56 (m, 1H), 9.47 - 9.34 (m, 2H), 8.77 - 8.67 (m, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.09 - 7.72 (m, 3H), 6.87 (dd, J = 7.8, 13.1 Hz, 1H), 4.44 - 4.32 (m, 1H), 4.22 (d, J = 2.8 Hz, 1H), 4.07 - 3.95 (m, 1H), 3.93 - 3.82 (m, 1H), 3.73 - 3.59 (m, 1H), 1.64 - 1.53 (m, 1H), 1.37 (d, J = 7.5 Hz, 1H), 1.19 - 1.08 (m, 9H), 1.05 - 0.88 (m, 6H), 0.80 (d, J = 8.9 Hz, 3H).
537
SUBSTITUTE SHEET RULE 26 Example 142: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 202A)
[0001178]
Figure imgf000539_0002
trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
Figure imgf000539_0001
[0001179] To a solution of 2-amino-2-(7,8-difluoro-4-isoquinolyl)acetonitrile (100 mg, 456.23 umol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (279.49 mg, 684.34 umol, 1.5 eq), 1- methylimidazole (112.37 mg, 1.37 mmol, 109.10 uL, 3 eq) in ACN (5 mb) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (256.01 mg, 912.45 umol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O 30 mL at 20 °C, and then extracted with EtOAc (35 mL * 3). The combined organic layers were washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)-
538
SUBSTITUTE SHEET RULE 26 ACN];B%: 45%-70%,8min) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-difluoro-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (117.7 mg, 192.31 umol, 42.15% yield, 99.6% purity) as a white solid. MS (ESI) m/z 610.2 [M+H]+.
[0001180] 'H NMR (400 MHz, DMSO-t/r,) 8 = 9.90 - 9.35 (m, 3H), 8.89 - 8.73 (m, 1H), 8.16 -
7.86 (m, 2H), 7.01 - 6.70 (m, 1H), 4.36 (d, J= 7.1 Hz, 1H), 4.18 (d, J= 5.1 Hz, 1H), 3.98 (dd, J = 5.4, 10.4 Hz, 1H), 3.85 (ddd, J= 6.4, 7.6, 13.9 Hz, 1H), 3.62 (t, J= 11.2 Hz, 1H), 1.62 - 1.45 (m, 1H), 1.38 - 1.16 (m, 1H), 1.14 - 0.79 (m, 18H).
Example 143: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-dichloro-4-isoquinolyl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 203A)
Figure imgf000540_0001
[0001181] Step 1: 4-bromo-7,8-dichloro-isoquinoline
[0001182] To a solution of 7,8-dichloroisoquinoline (2 g, 10.10 mmol, 1 eq) in AcOH (20 mL) was added NBS (2.16 g, 12.12 mmol, 1.2 eq). The mixture was stirred at 110 °C for 2 h. Upon completion, the reaction mixture was removed solvent, then poured into H2O 50 mL at 20 °C, NaHCOi was added, adjusted pH to 8, then extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine 45 mL, dried over Na2SO4, filtered and concentrated
539
SUBSTITUTE SHEET RULE 26 under reduced pressure to give 4-bromo-7,8-dichloro-isoquinoline (2.6 g, crude) as a yellow solid. MS (ESI) m/z 278.0 [M+H]+.
[0001183] Step 2: 7,8-dichloro-4-vinyl-isoquinoline
[0001184] To a solution of 4-bromo-7,8-dichloro-isoquinoline (2.4 g, 8.67 mmol, 1 eq) in toluene (24 mL), was added PPh3 (227.30 mg, 866.60 umol, 0.1 eq) and Pd(dba)2 (498.30 mg, 866.60 umol, 0.1 eq), stirred 30 min at 20 °C, then tributyl(vinyl)stannane (4.12 g, 13.00 mmol, 3.78 mL, 1.5 eq) in toluene (3 mL) was added. The mixture was stirred at 110 °C for 15.5 h. Upon completion, the mixture was quenched by addition of H2O 60 mL. Then extracted with EA (60 mL * 3). The combined organic layers were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S 1O2, Petroleum ether:Ethyl acetate = 90: 1 to 30: 1) to give 7,8- dichloro-4-vinyl-isoquinoline (1.4 g, 5.94 mmol, 68.49% yield, 95% purity) as a yellow solid. MS (ESI) m/z 224.1 [M+H]+.
[0001185] Step 3: 7,8-dichloroisoquinoline-4-carbaldehyde
[0001186] To a solution of 7,8-dichloro-4-vinyl-isoquinoline (1.4 g, 6.25 mmol, 1 eq) in THE (15 mL) and H2O (3 mL) was added K2OSO4.2H2O (230.20 mg, 624.76 umol, 0.1 eq), and the resulting mixture was stirred for 30min. Then NaIO4 (4.01 g, 18.74 mmol, 1.04 mL, 3 eq) was added. The mixture was stirred at 20 °C for 5.5 h. Upon completion, the reaction mixture was poured into H2O 45 mL at 20 °C, and then extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 90:1 to 20: 1) to give 7,8-dichloroisoquinoline-4- carbaldehyde (1.2 g, 5.04 mmol, 80.72% yield, 95% purity) as a yellow solid. MS (ESI) m/z 226.0 [M+H]+.
[0001187] Step 4: 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile
[0001188] A solution of 7,8-dichloroisoquinoline-4-carbaldehyde (1.2 g, 5.31 mmol, 1 eq), Ti(i-PrO)4 (2.26 g, 7.96 mmol, 2.35 mL, 1.5 eq), NH3/MeOH (7 M, 3.03 mL, 4 eq) in DCM (12 mL) was stirred 1 h at 20 °C, then TMSCN (1.58 g, 15.93 mmol, 1.99 mL, 3 eq) was added. The mixture was stirred at 20 °C for 5 h. Upon completion, the reaction mixture was poured into H2O
540
SUBSTITUTE SHEET RULE 26 50 mL at 20°C, and then extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine 50 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM:MeOH = 100:0 to 95:5) to give 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile (1.2 g, 4.43 mmol, 83.39% yield, 93% purity) as a yellow solid. MS (ESI) m/z 252.1 [M+H]+.
[0001189] Step 5: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-dichloro-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001190] To a solution of 2-amino-2-(7,8-dichloro-4-isoquinolyl)acetonitrile (90 mg, 357.00 umol, 1 eq , (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (189.55 mg, 464.10 umol, 1.3 eq), 1- methylimidazole (87.93 mg, 1.07 mmol, 85.37 uL, 3 eq in ACN (5 mL), was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (200.33 mg, 714.00 umol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O 25 mL at 20 °C, and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine 25 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um;mobile phase: [water( NH4HCO3)- ACN];B%: 50%-80%,8min) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(7,8-dichloro-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (120 mg, 186.21 umol, 52.16% yield, 99.7% purity) as a white solid. MS (ESI) m/z 642.1 [M+H]+.
[0001191] 'H NMR (400 MHz, DMSO-t/r,) 8 = 9.85 - 9.31 (m, 3H), 9.05 - 8.82 (m, 1H), 8.20 - 7.92 (m, 2H), 6.98 (dd, J= 4.3, 7.9 Hz, 1H), 4.34 (d, J= 19.1 Hz, 1H), 4.16 (d, J= 9.9 Hz, 1H), 3.98 (dd, J= 5.0, 10.1 Hz, 1H), 3.90 - 3.71 (m, 1H), 3.68 - 3.56 (m, 1H), 1.62 - 1.45 (m, 1H), 1.40 - 1.19 (m, 1H), 1.11 - 0.74 (m, 18H).
541
SUBSTITUTE SHEET RULE 26 Example 144: Synthesis of (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3- [(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 204A)
Figure imgf000543_0001
[0001192] Step 1: 5-bromoisoquinoline-4-carbaldehyde
[0001193] A mixture of cone. H2SO4 (50 mL) and isoquinoline-4-carbaldehyde (5 g, 31.81 mmol, 1 eq) at 0 °C was added NBS (7.02 g, 39.45 mmol, 1.24 eq) slowly and the reaction mixture was allowed to warm to 25 °C and stirred for 16 h. Upon completion, the mixture was adjust to pH=7~9 by sat. NaOH (100 mL), and extracted with EA (40 mL * 2). The combined organic layers were washed with brine (40 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 3: 1 to 0: 1) to give the product 5-bromoisoquinoline-4- carbaldehyde (2 g, 8.47 mmol, 26.63% yield) as a yellow solid. MS (ESI) m/z 236.0 [M+H]+
[0001194] Step 2: 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile
[0001195] A solution of 5-bromoisoquinoline-4-carbaldehyde (200 mg, 847.23 umol, 1 eq) in NHa/MeOH (7 M, 5 mL) was added NH4CI (135.96 mg, 2.54 mmol, 3 eq) and stirred at 25 °C for 1 h, then added TMSCN (252.15 mg, 2.54 mmol, 317.97 uL, 3 eq) at 0 °C, the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHa/MeOH. The reaction mixture was quenched by addition H2O (20 mL), and then extracted with EA (20 mL * 3). The combined organic layers were washed with brine 542
SUBSTITUTE SHEET RULE 26 (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE:EA = 0:1) to give the product 2-amino-2-(5- bromo-4-isoquinolyl)acetonitrile (120 mg, 457.83 umol, 54.04% yield) as a yellow oil. MS (ESI) m/z 262.0 [M+H]+
[0001196] Step 3: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert- butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001197] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (80 mg, 195.88 umol, 1 eq) in ACN (3 mL) was added 2-amino-2-(5-bromo-4- isoquinolyl)acetonitrile (51.34 mg, 195.88 umol, 1 eq), then added 1 -methylimidazole (56.29 mg, 685.58 umol, 54.65 uL, 3.5 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium; hexafluorophosphate (65.95 mg, 235.06 umol, 1.2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the residue was quenched by H2O (10 mL) and was extracted with EA (10 mL * 3). The combined organic phase was washed with brine (10 mL), dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 100 * 30 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 8 min) to give the product (1R,2S,5S)- N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (62 mg, 95.02 umol, 48.51% yield) as a white solid. MS (ESI) m/z 654.1 [M+H]+
[0001198] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.75 - 9.17 (m, 3H), 8.94 - 8.82 (m, 1H), 8.37 - 8.21 (m, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.49 (br t, J= 5.8 Hz, 1H), 4.54 - 4.15 (m, 2H), 4.07 - 3.78 (m, 2H), 3.65 (br dd, J= 6.3, 10.3 Hz, 1H), 1.63 - 1.52 (m, 1H), 1.46 - 1.32 (m, 1H), 1.12 - 0.96 (m, 15H), 0.82 (d, J = 13.6 Hz, 3H)
[0001199] Step 4: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert- butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001200] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
543
SUBSTITUTE SHEET RULE 26 (40 mg, 61.30 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-Ol(250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 45%-45%, 10 min) to give the product (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (17.46 mg, 26.76 umol, 43.65% yield) as a white solid. MS (ESI) m/z 654.1 [M+H]+
[0001201] XH NMR (400 MHz, DMSO-r/e) 8 = 9.70 - 9.31 (m, 3H), 8.88 (s, 1H), 8.31 (dd, J = 7.8, 19.0 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.50 (d, J= 6.8 Hz, 1H), 4.44 - 4.34 (m, 1H), 4.24 (s, 1H), 4.03 - 3.86 (m, 2H), 3.64 (d, J= 10.5 Hz, 1H), 1.59 - 1.53 (m, 1H), 1.35 (d, J= 7.7 Hz, 1H), 1.07 (br d, J= 6.1 Hz, 12H), 0.99 - 0.96 (m, 3H), 0.80 (s, 3H)
[0001202] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-tert-butoxy- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (18.89 mg, 28.95 umol, 47.23% yield) as obtained a white solid. MS (ESI) m/z 654.1 [M+H]+
[0001203] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.66 (br d, J= 8.1 Hz, 1H), 9.55 (br d, J= 6.8 Hz, 1H), 9.50 (s, 1H), 8.88 (s, 1H), 8.37 - 8.22 (m, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.48 (d, J= 6.6 Hz, 1H), 4.38 (t, J= 7.9 Hz, 1H), 4.31 (s, 1H), 3.97 (dd, J= 5.4, 10.4 Hz, 1H), 3.92 - 3.81 (m, 1H), 3.66 (d, J= 10.5 Hz, 1H), 1.62 - 1.55 (m, 1H), 1.43 (d, J= 7.5 Hz, 1H), 1.10 - 1.02 (m, 15H), 0.83 (s, 3H)
Example 145: Synthesis of (lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-N-((5-chloroisoquinolin-4-yl)(cyano)methyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 205A)
Figure imgf000545_0001
544
SUBSTITUTE SHEET RULE 26 [0001204] Stepl :(lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-N-((5-chloroisoquinolin-4-yl)(cyano)methyl)-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001205] To a solution of 2-amino-2-(5-chloroisoquinolin-4-yl)acetonitrile (31.98 mg, 146.91 umol, 1 eq) in ACN (3 mL) was added (lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (60 mg, 146.91 umol, 1 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (82.44 mg, 293.82 umol, 2 eq), then 1 -methylimidazole (36.19 mg, 440.73 umol, 35.13 uL, 3 eq) was added, then the mixture was stirred for 1 h at 25 °C. Upon completion, the reaction mixture was quenched by addition H2O (10 mL), and extracted with EA (10 mL * 3). The combined organic layers were washed with aqueous NaCl (10 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE:EA = 0:1), which was further separated by SFC column: DAICEL CHIRALPAK AD(250 mm * 30 mm, 10 urn); mobile phase: [Neu-IPA]; B%: 20%- 20%, 8 min to give (lR,2S,5S)-3-((2S,3R)-3-(tert-butoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-N-((5-chloroisoquinolin-4-yl)(cyano)methyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (18.01 mg, 27.74 umol, 18.89% yield, 93.67% purity) as a white solid. MS (ESI) m/z 608.2 [M+H]+.
[0001206] 'H NMR (400 MHz, DMSO-de) 5 = 9.68 - 9.57 (m, 1H), 9.56 - 9.46 (m, 1H), 9.37 (br d, J = 7.0 Hz, 1H), 8.87 (s, 1H), 8.29 (br d, J = 7.9 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.83 - 7.74 (m, 1H), 7.34 (br s, 1H), 4.39 (br d, J = 7.8 Hz, 1H), 4.24 (s, 1H), 4.00 - 3.85 (m, 2H), 3.64 (br d, J = 10.6 Hz, 1H), 1.60 - 1.51 (m, 1H), 1.30 (br d, J = 7.5 Hz, 1H), 1.16 - 1.09 (m, 9H), 1.02 (br d, J = 6.0 Hz, 3H), 0.99 - 0.95 (m, 3H), 0.80 (s, 3H).
[0001207] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [(5-chloro-4-isoquinolyl)-cyano-methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (31.49 mg, 50.98 umol, 34.70% yield, 98.44% purity) was obtained as a white solid. MS (ESI) m/z 608.1 [M+H]+.
[0001208] 'H NMR (400 MHz, DMSO-de) 5 = 9.74 - 9.58 (m, 1H), 9.50 (br s, 2H), 8.92 - 8.83 (m, 1H), 8.35 - 8.20 (m, 1H), 8.13 - 8.00 (m, 1H), 7.81 - 7.67 (m, 1H), 7.42 - 7.20 (m, 1H), 4.42 -
545
SUBSTITUTE SHEET RULE 26 4.35 (m, 1H), 4.32 - 4.24 (m, 1H), 4.00 - 3.85 (m, 2H), 3.78 - 3.57 (m, 1H), 1.62 - 1.54 (m, 1H),
1.40 (br d, J = 7.3 Hz, 1H), 1.06 (s, 12H), 1.04 - 1.01 (m, 3H), 0.83 (s, 3H)
Example 146: Synthesis of (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo [3.1.0]hexane-2-carboxamide (Compound 208A)
Figure imgf000547_0001
[0001209] Step 1: (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[0001210] To a solution of 2-amino-2-(5-chloro-4-isoquinolyl)acetonitrile (230 mg, 1.06 mmol, 1 eq) and (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (388.31 mg, 1.06 mmol, 1 eq) in ACN (10 mL) and then TCFH (594.83 mg, 2.12 mmol, 2 eq and 1 -methylimidazole (304.59 mg, 3.71 mmol, 295.72 uL, 3.5 eq was added, and then the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (20 mL) and then extracted with ethyl acetate (20 mL * 2). The combined organic layers were washed with 15 % citric acid (20 mL * 2), NaHCCh (20 mL) and brine (20 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, petroleum ether: ethyl acetate = 1 :2 to 0: 1) which was further separated by SFC (column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 urn); mobile phase: [Neu-MeOH]; B %: 30 %- 30 %, 9 min) to give (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (81.44 mg, 142.17 umol, 13.41% yield, 98.8% purity) as a white solid. MS (ESI) m/z 566.0 [M+H]+.
546
SUBSTITUTE SHEET RULE 26 [0001211] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.87 (br s, 1H), 9.62 (br d, J= 5.5 Hz, 1H), 9.54 - 9.48 (m, 1H), 8.87 (s, 1H), 8.32 - 8.25 (m, 1H), 8.03 (d, J= 7.4 Hz, 1H), 7.77 (t, J= 7.8 Hz, 1H), 7.31 (br s, 1H), 4.34 (br d, J= 4.4 Hz, 1H), 4.31 - 4.22 (m, 1H), 3.96 - 3.89 (m, 1H), 3.87 - 3.81 (m, 1H), 3.68 - 3.55 (m, 1H), 3.25 - 3.18 (m, 3H), 1.63 - 1.57 (m, 1H), 1.39 (d, J = 7.5 Hz, 1H), 1.17 - 1.11 (m, 3H), 1.04 - 0.94 (m, 3H), 0.89 - 0.82 (m, 3H).
[0001212] (lR,2S,5S)-N-[(5-chloro-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (85.37 mg, 144.96 umol, 13.68% yield, 96.1% purity) was obtained as a white solid. MS (ESI) m/z 566.0 [M+H]+.
[0001213] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.02 - 9.73 (m, 1H), 9.51 (s, 1H), 9.49 - 9.44 (m, 1H), 8.86 (s, 1H), 8.29 (dd, J= 1.0, 8.1 Hz, 1H), 8.06 (dd, J= 1.1, 7.5 Hz, 1H), 7.77 (t, J = 7.9 Hz, 1H), 7.31 (br d, J= 3.7 Hz, 1H), 4.36 (d, J= 8.6 Hz, 1H), 4.24 (s, 1H), 3.98 - 3.91 (m, 1H), 3.85 - 3.79 (m, 1H), 3.70 - 3.62 (m, 1H), 3.25 - 3.21 (m, 3H), 1.57 (dd, J= 5.3, 7.3 Hz, 1H), 1.29 - 1.24 (m, 1H), 1.15 (d, J = 6.2 Hz, 3H), 0.98 - 0.95 (m, 3H), 0.84 (s, 3H).
Example 147: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 209A)
Figure imgf000548_0001
[0001214] Step 1: (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol
547
SUBSTITUTE SHEET RULE 26 [0001215] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carboxylic acid (2.7 g, 11.20 mmol, 1 eq) in THF (20 mL) was added CDI (2.72 g, 16.80 mmol, 1.5 eq) under N2 at 20 °C for 17.5 h. Then the solution was added to a solution of NaBH4 (2.12 g, 56.01 mmol, 5 eq) in H2O (20 mL) and stirred at 20°C for 30 min. Upon completion, the reaction mixture was diluted with IM HC1 (20 mL) and the solution was neutralised with saturated aqueous NaHCCh, then extracted with EA (40 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 100: 1 to 50: 1) to give (4- bromopyrazolo[l,5-a]pyridin-3-yl)methanol (2 g, 8.10 mmol, 72.35% yield, 92% purity) as a white solid. MS (ESI) m/z 229.1 [M+H]+.
[0001216] Step 2: 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde
[0001217] A solution of (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol (2 g, 8.81 mmol, 1 eq) in DCM (30 mL) was added MnCh (15.32 g, 176.17 mmol, 20 eq) and then the mixture was stirred at 20 °C for 12 h. Upon completion, the mixture was filtered and concentrated under reduced pressure to give 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (1.8 g, crude) as a white solid. MS (ESI) m/z 225.0 [M+H]+.
[0001218] Step 3: 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)a cetonitrile
[0001219] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (550 mg, 2.44 mmol, 1 eq), Ti(i-PrO)4 (1.04 g, 3.67 mmol, 1.08 mL, 1.5 eq), NH3/MeOH (7 M, 1.40 mL, 4 eq) in DCM (5 mL) was stirred for 1 h at 20 °C, then TMSCN (727.38 mg, 7.33 mmol, 917.25 uL, 3 eq) was added. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (5 mL) at 20 °C, and then extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (400 mg, crude) as a yellow solid. MS (ESI) m/z 226.0 [M+H]+.
[0001220] Step 4: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
548
SUBSTITUTE SHEET RULE 26 [0001221] A solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (487.98 mg, 1.19 mmol, 1 eq), 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (300 mg, 1.19 mmol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (294.29 mg, 3.58 mmol, 285.71 uL, 3 eq) and TCFH (670.49 mg, 2.39 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (5 mL) and extracted with EA (5 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80 * 40 mm * 3 um; mobile phase: [water(NH4HCO3)-ACN]; B%: 40%-60%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)- 3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (180 mg, 252.54 umol, 21.14% yield, 90% purity) as a white solid. MS (ESI) m/z 665.2 [M+H]+.
[0001222] Step 5: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001223] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5-a] pyri din-3 -yl)-cyano-methyl]-3- [(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 280.60 umol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK IC (250mm * 30 mm, 10 um); mobile phase: [Neu-ETOH]; B%: 24%-24%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (64.57 mg, 100.66 umol, 35.87% yield, 100% purity) as a white solid. MS (ESI) m/z 665.2 [M+H]+.
[0001224] 'H NMR (400 MHz, DMSO-de) 5 = 9.64 (br s, 1H), 9.25 (d, J = 6.8 Hz, 1H), 8.84 (d, J = 6.8 Hz, 1H), 8.32 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H), 6.60 (d, J = 7.0 Hz, 1H), 4.48 - 4.34 (m, 1H), 4.23 (s, 1H), 4.12 - 3.72 (m, 2H), 3.65 (d, J = 10.5 Hz, 1H), 1.60 - 1.50 (m, 1H), 1.34 (d, J = 7.5 Hz, 1H), 1.20 - 0.78 (m, 18H).
[0001225] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-
549
SUBSTITUTE SHEET RULE 26 carboxamide Isomer 2 (65.85 mg, 102.65 umol, 36.58% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 665.2 [M+H]+.
[0001226] 'H NMR (400 MHz, DMSO-de) 5 = 9.64 (br s, 1H), 9.26 (d, J = 6.6 Hz, 1H), 8.84 (d, J = 6.8 Hz, 1H), 8.32 (s, 1H), 7.69 (d, J = 7.5 Hz, 1H), 6.94 (t, J = 7.1 Hz, 1H), 6.57 (d, J = 6.6 Hz, 1H), 4.44 - 4.33 (m, 1H), 4.27 (s, 1H), 3.96 (dd, J = 5.5, 10.7 Hz, 1H), 3.91 - 3.80 (m, 1H), 3.66 (d, J = 10.5 Hz, 1H), 1.69 - 1.48 (m, 1H), 1.40 (d, J = 7.7 Hz, 1H), 1.16 - 0.75 (m, 18H)
Example 148: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl] -3- [(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (compound 210A)
Figure imgf000551_0001
[0001227] Step 1: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-
[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001228] A solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (500 mg, 1.36 mmol, 1 eq) in ACN (10 mL) was added 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3- yl)acetonitrile (342.70 mg, 1.36 mmol, 1 eq), 1 -methylimidazole (336.17 mg, 4.09 mmol, 326.38 uL, 3 eq) then TCFH (765.92 mg, 2.73 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (10 mL) and extracted with EA (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250 * 70 mm * 10 um; mobile phase: [water(NELiHCO3)- ACN] ; B%: 30%- 60%, 20 min) and then further purified by prep-HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water(FA)-ACN]; B%: 30%-70%, 8 min) to give (lR,2S,5S)-N-[(4-
550
SUBSTITUTE SHEET RULE 26 bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (220 mg, 345.01 umol, 25.28% yield, 94% purity) as a white solid. MS (ESI) m/z 601.1 [M+H]+.
[0001229] Step 2: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001230] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5-a] pyri din-3 -yl)-cyano-methyl] -3 - [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (220 mg, 367.03 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 25%-25%, 5 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (70.53 mg, 117.67 umol, 32.06% yield) as a white solid. MS (ESI) m/z 601.1 [M+H]+.
[0001231] 'H NMR (400 MHz, DMSO-de) 5 = 9.86 (br s, 1H), 9.35 (d, J = 7.0 Hz, 1H), 8.84 (d, J = 6.9 Hz, 1H), 8.32 (s, 1H), 7.70 (d, J = 7.3 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 6.58 (d, J = 6.9 Hz, 1H), 4.36 (br d, J = 7.5 Hz, 1H), 4.23 (s, 1H), 4.01 - 3.87 (m, 1H), 3.86 - 3.80 (m, 1H), 3.71 - 3.61 (m, 1H), 3.25 (s, 3H), 1.55 (dd, J = 5.4, 7.4 Hz, 1H), 1.33 (d, J = 7.6 Hz, 1H), 1.17 (d, J = 6.2 Hz, 3H), 0.98 (s, 3H), 0.85 (s, 3H)
[0001232] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (67.09 mg, 111.93 umol, 30.50% yield) was obtained as a white solid. MS (ESI) m/z 601.1 [M+H]+.
[0001233] 'H NMR (400 MHz, DMSO-d6) 5 = 9.85 (br s, 1H), 9.40 (d, J = 6.5 Hz, 1H), 8.83 (d, J = 6.9 Hz, 1H), 8.31 (s, 1H), 7.68 (d, J = 7.4 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 6.56 (d, J = 6.4 Hz, 1H), 4.35 (br d, J = 8.1 Hz, 1H), 4.28 (s, 1H), 3.98 - 3.87 (m, 1H), 3.87 - 3.79 (m, 1H), 3.66 - 3.55 (m, 1H), 3.22 (s, 3H), 1.58 (dd, J = 5.4, 7.3 Hz, 1H), 1.38 (d, J = 7.6 Hz, 1H), 1.15 (d, J = 6.1 Hz, 3H), 1.04 (s, 3H), 0.86 (s, 3H)
551
SUBSTITUTE SHEET RULE 26 Example 149: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- niethyl|-6.6-dimethyl-3-|(2S.3R)-3-( 1 -metliylcyclopropoxy)-2-|(2.2.2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 211 A)
Figure imgf000553_0001
[0001234] Step 1: 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[0001235] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (500 mg, 2.22 mmol, 1 eq), Ti(i-PrO)4 (947.21 mg, 3.33 mmol, 983.60 uL, 1.5 eq), and NH3/MeOH (7 M, 1.27 mL, 4 eq) in DCM (5 mL) was stirred at 20 °C for 1 h, and then TMSCN (661.25 mg, 6.67 mmol, 833.86 uL, 3 eq) was added. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (5 mL) at 20 °C and then extracted with EtOAc (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3- yl)acetonitrile (400 mg, crude) as a yellow solid. MS (ESI) m/z 236.1 [M+H]+.
[0001236] Step 2: ((lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
552
SUBSTITUTE SHEET RULE 26 [0001237] A solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (498.02 mg, 1.23 mmol, 1 eq), 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (400 mg, 1.59 mmol, 1.3 eq), 1 -methylimidazole (301.83 mg, 3.68 mmol, 293.04 uL, 3 eq) in ACN (5 mL) was added TCFH (687.68 mg, 2.45 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was poured into H2O (5 mL) and then extracted with EtOAc (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75 * 30 mm * 3 um; mobile phase: [water(FA)-ACN]; B%: 40%-80%, 8 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 445.69 umol, 36.37% yield, 95% purity) as a yellow solid. MS (ESI) m/z 639.0 [M+H]+.
[0001238] Step 3: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001239] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5 -a] pyri din-3 -yl)-cyano-methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 469.14 umol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK AD (250mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 16%-16%, 6 min) to give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]- 6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (80.47 mg, 125.84 umol, 26.82% yield) as a white solid. MS (ESI) m/z 639.0 [M+H]+.
[0001240] 'H NMR (400 MHz, DMSO-de) 5 = 9.85 (s, 1H), 9.33 (d, J = 7.0 Hz, 1H), 8.84 (d, J = 7.0 Hz, 1H), 8.32 (s, 1H), 7.70 (d, J = 7.2 Hz, 1H), 6.95 (t, J = 7.1 Hz, 1H), 6.59 (d, J = 6.8 Hz, 1H), 4.41 - 4.30 (m, 1H), 4.22 (s, 1H), 3.98 - 3.88 (m, 2H), 3.67 (d, J = 10.3 Hz, 1H), 1.54 (dd, J = 5.7, 7.5 Hz, 1H), 1.35 - 1.31 (m, 4H), 1.20 - 1.13 (m, 3H), 0.97 (s, 3H), 0.82 - 0.79 (m, 3H), 0.77 - 0.75 (m, 2H), 0.39 - 0.31 (m, 2H)
553
SUBSTITUTE SHEET RULE 26 [0001241] To give (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (81.49 mg, 127.44 umol, 27.16% yield) as a white solid. MS (ESI) m/z 639.0 [M+H]+.
[0001242] 'H NMR (400 MHz, DMSO-de) 5 = 9.80 (br s, 1H), 9.35 (d, J = 6.5 Hz, 1H), 8.84 (d, J = 7.0 Hz, 1H), 8.30 (s, 1H), 7.69 (d, J = 7.3 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 6.57 (d, J = 6.5 Hz, 1H), 4.35 (br d, J = 7.1 Hz, 1H), 4.27 (s, 1H), 3.95 - 3.82 (m, 2H), 3.68 (br d, J = 10.5 Hz, 1H), 1.57 (br dd, J = 5.4, 7.2 Hz, 1H), 1.38 (d, J = 7.6 Hz, 1H), 1.30 (s, 3H), 1.15 (d, J = 6.1 Hz, 3H), 1.03 (s, 3H), 0.82 (s, 3H), 0.75 (br d, J = 11.1 Hz, 1H), 0.64 - 0.57 (m, 1H), 0.35 - 0.27 (m, 2H)
Example 150: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 212A)
Figure imgf000555_0001
[0001243] Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001244] A mixture of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (0.25 g, 1.17 mmol, 1 eq) and (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (472.39 mg, 1.29 mmol, 1.1 eq) in ACN (10 mL) was added 1 -methylimidazole (288.73 mg, 3.52 mmol, 280.32 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (657.84 mg, 2.34 mmol, 2 eq), the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with
554
SUBSTITUTE SHEET RULE 26 EA 45 mL (15 mL * 3). The combined organic layers were washed with brine 100 m (20 mL * 5), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Waters Xbridge BEH C18 250*70mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-60%,20min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide as a yellow solid. MS (ESI) m/z 562.3 [M+H]+
[0001245] Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001246] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (500 mg) was separated by SFC(condition: column: REGIS (s,s) WHELK-01 (250mm*50mm,10um);mobile phase: [Hexane-IPA];B%: 50%-50%,10min) to give (lR,2S,5S)- N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 2 (170 mg, 302.72 umol, 25.82% yield, 100% purity) as a white solid. MS (ESI) m/z 562.3 [M+H]+
[0001247] 'H NMR (400 MHz, DMSO-de) 5 = 9.84 (br s, 1H), 9.69 (br d, J = 5.5 Hz, 1H), 9.41 (s, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.13 (br s, 1H), 4.46 (s, 1H), 4.39 - 4.27 (m, 2H), 3.95 - 3.77 (m, 2H), 3.62 - 3.53 (m, 1H), 3.19 (s, 3H), 1.58 (dd, J = 5.3, 7.3 Hz, 1H), 1.47 (d, J = 7.6 Hz, 1H), 1.12 - 1.02 (m, 6H), 0.88 (s, 3H)
Example 151: Synthesis of (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano- methyl]-3-[(2S,3R)-3-methoxy-2-[[l-
555
SUBSTITUTE SHEET RULE 26 (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 213A)
Figure imgf000557_0001
[0001248] Step 1: (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol
[0001249] The 4-bromopyrazolo[l,5-a]pyridine-3-carboxylic acid (1.3 g, 5.39 mmol, 1 eq) in THF (15 mL) was added CDI (1.31 g, 8.09 mmol, 1.5 eq). The mixture was stirred at 20 °C for 17.5 h under N2 atmosphere. Then the mixture was added to a solution of NaBFLi (1.02 g, 26.97 mmol, 5 eq) in H2O (15 mL) and was stirred for 30 min at 20 °C. Upon completion, the mixture was diluted with IM HC1 (20 mL) and the solution was neutralised with saturated aqueous NaHCCh, then extracted with EA (40 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by prep-TLC (SiCh, Petroleum ether:Ethyl acetate = 0:1) to get product (4-bromopyrazolo[l,5-a]pyridin-3- yl)methanol (2 g, 7.13 mmol, 81% purity) as white solid. MS (ESI) m/z 227.0 [M+H]+
556
SUBSTITUTE SHEET RULE 26 [0001250] Step 2: 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde
[0001251] The (4-bromopyrazolo[l,5-a]pyridin-3-yl)methanol (0.5 g, 2.20 mmol, 1 eq) in DCM (25 mb) was added MnCh (3.83 g, 44.04 mmol, 20 eq). The mixture was stirred at 20 °C for 48 h. Upon completion, the mixture was filtered and concentrated in vacuum to get crude product 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (1.8 g, crude) as white solid. MS (ESI) m/z 225.0 [M+H]+
[0001252] Step 3: 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[0001253] A solution of 4-bromopyrazolo[l,5-a]pyridine-3-carbaldehyde (0.6 g, 2.67 mmol, 1 eq), Ti(i-PrO)4 (1.14 g, 4.00 mmol, 1.18 mL, 1.5 eq), NH3/MeOH (7 M, 1.52 mL, 4 eq) in DCM (6 mL) was stirred at 20 °C for 1 h, then TMSCN (793.53 mg, 8.00 mmol, 1.00 mL, 3 eq) was added at 0 °C. The mixture was stirred at 20 °C for 2 h. Upon completion, the mixture was poured into H2O (50 mL) at 20 °C, and then extracted with DCM (50 mL * 5). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressur to get crude product 2-amino-2-(4-bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.63 g, crude) as yellow solid. MS (ESI) m/z 251.0 [M+H]+
[0001254] Step 4: methyl (2S,3R)-2-amino-3-methoxy-butanoate
[0001255] The (3R)-2-(tert-butoxycarbonylamino)-3-methoxy-butanoic acid (2 g, 8.57 mmol, 1 eq) in HCl/MeOH (4 M, 60 mL, 27.99 eq) was stirred at 60 °C for 6 h. Upon completion, the mixture was concentrated in vacuum to get crude product methyl (2S,3R)-2-amino-3-methoxy- butanoate (1.6 g, crude) as white solid. MS (ESI) m/z 148.2 [M+H]+
[0001256] Step 5: methyl (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoate
[0001257] To a solution of methyl (2S,3R)-2-amino-3-methoxy-butanoate (1.6 g, 8.71 mmol, 1 eq, HC1) in DCM (30 mL) was added l-(trifluoromethyl)cyclopropanecarboxylic acid (1.34 g, 8.71 mmol, 1 eq), HATU (6.63 g, 17.43 mmol, 2 eq) and DIEA (3.38 g, 26.14 mmol, 4.55 mL, 3 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition water (30 mL) and extracted with DCM (25 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 9: 1) to get product
557
SUBSTITUTE SHEET RULE 26 methyl (2S,3R)-3-methoxy-2-[[l -(trifluoromethyl)cyclopropanecarbonyl]amino]butanoate (2.29 g, 7.60 mmol, 87.22% yield, 94% purity) as yellow oil. MS (ESI) m/z 284.1 [M+H]+
[0001258] Step 6: (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoic acid
[0001259] The methyl (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoate 3.43 g, 12.11 mmol, 1 eq) in THF (15 mL) and H2O (15 m ) was added LiOH.HzO (1.52 g, 36.33 mmol, 3 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted by addition H2O (100 mL), and then added HC1 (I M, aq.) to adjust the pH = 3 - 4, and extracted with EA (100 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoic acid (3.3 g, crude) as yellow oil. MS (ESI) m/z 270.1 [M+H]+
[0001260] Step 7: methyl (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2- carboxylate
[0001261] To a solution of (2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoic acid (2 g, 7.43 mmol, 1 eq) in DCM (10 mL) was added methyl (lR,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate (1.53 g, 7.43 mmol, 1 eq, HC1), HATU (5.65 g, 14.86 mmol, 2 eq) and DIEA (2.88 g, 22.29 mmol, 291.14 uL, 3 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition water (100 mL) and extracted with DCM (80 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 9:1 to 17:3) to give the product methyl (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (1.7 g, 3.68 mmol, 35.86% yield) as white solid. MS (ESI) m/z 421.2 [M+H]+
558
SUBSTITUTE SHEET RULE 26 [0001262] Step 8: (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxylic acid
[0001263] To a solution of methyl (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylate (1.7 g, 4.04 mmol, 1 eq) in THF (9 mL) and H2O (9 mL) was added LiOH.FEO (509.00 mg, 12.13 mmol, 3 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was adjusted pH~l with HC1 (I M, aq.) and then was extracted with EA (80 mL * 3), then the organic phase was dried with Na2SO4, filtered and concentrated in vacuum to give the crude product (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.6 g, crude) as yellow oil. MS (ESI) m/z 407.2 [M+H]+
[0001264] Step 9: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001265] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.02 g, 2.51 mmol, 1 eq), 2-amino-2-(4- bromopyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.63 g, 2.51 mmol, 1 eq) in ACN (10 mL) was added TCFH (1.41 g, 5.02 mmol, 2 eq) and 1 -methylimidazole (618.03 mg, 7.53 mmol, 600.03 uL, 3 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was quenched by addition water (80 mL) and extracted with DCM (50 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250 * 70 mm, 15 um); mobile phase: [water (FA)- ACN]; B%: 40%-75%, 20 min) to get product (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3- yl)-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.88 g, 1.38 mmol, 54.85% yield, 100% purity) as white solid. MS (ESI) m/z 639.1 [M+H]+
559
SUBSTITUTE SHEET RULE 26 [0001266] Step 10: (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3- [(2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001267] The ( 1 R,2S, 5 S)-N- [(4-bromopyrazolo [ 1 , 5-a] pyri din-3 -yl)-cyano-methyl]-3- [(2S,3R)-3-methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (0.88 g, 1.38 mmol, 1 eq) was separation by SFC (column: REGIS (S,S) WHELK-01 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 6 min) to get product (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)- cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[[l- (trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (0.3 g, 467.74 umol, 33.99% yield, 99.7% purity) as white solid. MS (ESI) m/z 639.1 [M+H]+
[0001268] 'H NMR (400 MHz, DMSO-de) 5 = 9.31 (d, J = 7.0 Hz, 1H), 8.84 (d, J = 6.9 Hz, 1H), 8.31 (s, 1H), 7.70 (d, J = 7.3 Hz, 2H), 6.94 (t, J = 7.2 Hz, 1H), 6.57 (d, J = 7.0 Hz, 1H), 4.38 (t, J = 7.3 Hz, 1H), 4.23 (s, 1H), 3.93 - 3.78 (m, 2H), 3.68 - 3.53 (m, 1H), 3.23 (s, 3H), 1.53 (dd, J = 4.7, 7.3 Hz, 1H), 1.33 - 1.20 (m, 5H), 1.10 (d, J = 6.3 Hz, 3H), 1.00 - 0.95 (m, 3H), 0.84 (s, 3H).
[0001269] (lR,2S,5S)-N-[(4-bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-3-[(2S,3R)-3- methoxy-2-[[l-(trifluoromethyl)cyclopropanecarbonyl]amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (0.31 g, 483.33 umol, 35.12% yield, 99.7% purity) was obtained as white solid. MS (ESI) m/z 639.1 [M+H]+
[0001270] 'H NMR (400 MHz, DMSO-de) 5 = 9.54 - 9.05 (m, 1H), 8.83 (d, J = 6.9 Hz, 1H), 8.30 (s, 1H), 7.90 - 7.61 (m, 2H), 6.94 (t, J = 7.2 Hz, 1H), 6.56 (d, J = 6.4 Hz, 1H), 4.36 (t, J = 7.4 Hz, 1H), 4.27 (s, 1H), 3.86 (br d, J = 2.6 Hz, 2H), 3.60 - 3.47 (m, 1H), 3.23 - 3.09 (m, 3H), 1.56 (br d, J = 7.4 Hz, 1H), 1.39 - 1.16 (m, 5H), 1.10 - 0.82 (m, 9H).
560
SUBSTITUTE SHEET RULE 26 Example 152: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 214A)
Figure imgf000562_0001
[0001271] Step 1: 3-(2-trimethylsilylethynyl)thieno[2,3-c]pyridine-4-carbaldehyde
[0001272] A solution of 3-bromothieno[2,3-c]pyridine-4-carbaldehyde (4.5 g, 18.59 mmol, 1 eq) and N-isopropylpropan-2-amine (5.64 g, 55.76 mmol, 7.88 mL, 3 eq) in DMSO (45 mL) was added Cui (1.42 g, 7.44 mmol, 0.4 eq), palladium;triphenylphosphane (4.30 g, 3.72 mmol, 0.2 eq) and ethynyl(trimethyl)silane (3.65 g, 37.18 mmol, 5.15 mL, 2 eq) and stirred at 25 °C for 16 h. Upon completion, the reaction mixture was quenched by H2O (20 mL), and then extracted with EtOAc (20 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, PE:EA = 100:1 to 40: 1) to give 3-(2- trimethylsilylethynyl)thieno[2,3-c]pyridine-4-carbaldehyde (2 g, 7.71 mmol, 41.48% yield) as a brown oil. MS (ESI) m/z 260.1 [M+H]+.
[0001273] Step 2: 3-ethynylthieno[2,3-c]pyridine-4-carbaldehyde
[0001274] A solution of 3-(2-trimethylsilylethynyl)thieno[2,3-c]pyridine-4-carbaldehyde (2 g, 7.71 mmol, 1 eq) in MeOH (10 mL) was added K2CO3 (2.66 g, 19.28 mmol, 2.5 eq) and stirred at 25 °C for 1 h. Upon completion, the reaction mixture was give 3-ethynylthieno[2,3-c]pyridine-
561
SUBSTITUTE SHEET RULE 26 4-carbaldehyde (1.5 g, crude) as a yellow liquid and used to next step directly. MS (ESI) m/z 188.2 [M+H]+.
[0001275] Step 3: 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile
[0001276] A solution of 3-ethynylthieno[2,3-c]pyridine-4-carbaldehyde (1.5 g, 8.01 mmol, 1 eq) in DCM (5 mL) was added Ti(i-PrO)4 (2.28 g, 8.01 mmol, 2.36 mL, 1 eq) and NH MeOH (7 M, 42 mL, 36.69 eq) and stirred at 25 °C for 1 h, then added TMSCN (2.38 g, 24.04 mmol, 3.01 mL, 3 eq) and stirred at 25 °C for 15 h. Upon completion, the solution was quenched with H2O (20 mL), and then extracted with EA (15 mL * 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (1.5 g, crude) as brown oil. MS (ESI) m/z 214.0 [M+H]+.
[0001277] Step 4:(lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001278] A mixture of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (500 mg, 2.34 mmol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (957.55 mg, 2.34 mmol, 1 eq), TCEH (986.76 mg, 3.52 mmol, 1.5 eq) and 1 -methylimidazole (577.50 mg, 7.03 mmol, 560.68 uL, 3 eq), then stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by water (10 mL), and then extracted with EA (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC(column: Waters Xbridge BEH Cl 8 250 * 70 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 20 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (800 mg, 1.33 mmol, 56.52% yield) as yellow solid. MS (ESI) m/z 604.2 [M+H]+.
[0001279] Step 5: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
562
SUBSTITUTE SHEET RULE 26 [0001280] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (800 mg, 1.33 mmol, 1 eq) was purified by SFC (column: (s,s) WHELK-01 (250 mm * 30 mm, 5 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 10 min) to give (lR,2S,5S)-3- [(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-ethynylthieno[2,3- c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (110 mg, 182.22 umol, 13.75% yield, 100% purity) as a white solid. MS (ESI) m/z 604.1 [M+H]+.
[0001281] 'H NMR (400 MHz, DMSO-de) 5 = 9.61 (br s, 1H), 9.43 (s, 1H), 9.37 (br d, J = 6.6 Hz, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.04 (br d, J = 6.6 Hz, 1H), 4.85 - 4.48 (m, 1H), 4.44 - 4.36 (m, 1H), 4.25 (s, 1H), 4.00 - 3.74 (m, 2H), 3.69 - 3.56 (m, 1H), 1.58 - 1.50 (m, 1H), 1.38 (d, J = 7.6 Hz, 1H), 1.20 - 1.06 (m, 12H), 0.99 - 0.94 (m, 3H), 0.80 (s, 3H)
[0001282] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (87 mg, 144.12 umol, 10.88% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 604.1 [M+H]+.
[0001283] 'H NMR (400 MHz, DMSO-de) 5 = 9.61 (br s, 1H), 9.56 - 9.46 (m, 1H), 9.44 - 9.34 (m, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.14 (br s, 1H), 4.51 (s, 1H), 4.38 (br d, J = 7.4 Hz, 1H), 4.34 - 4.23 (m, 1H), 3.95 (dd, J = 5.4, 10.5 Hz, 1H), 3.90 - 3.81 (m, 1H), 3.64 (d, J = 10.6 Hz, 1H), 1.61 - 1.53 (m, 1H), 1.48 (d, J = 7.5 Hz, 1H), 1.10 - 0.97 (m, 15H), 0.85 (s, 3H)
563
SUBSTITUTE SHEET RULE 26 Example 153: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino] butanoyl] -3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 215A)
Figure imgf000565_0001
[0001284] Step 1 : 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde
[0001285] To a solution of 5-bromoisoquinoline-4-carbaldehyde (2 g, 8.47 mmol, 1 eq) in DMSO (30 mb) was added i-PnNH (2.57 g, 25.42 mmol, 3.59 mL, 3 eq), and then was added Cui (645.42 mg, 3.39 mmol, 0.4 eq) and Pd(PPhs)4 (979.02 mg, 847.23 umol, 0.1 eq). After the addition, ethynyl(trimethyl)silane (1.66 g, 16.94 mmol, 2.35 mL, 2 eq) was added to slowly under N2, the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction mixture was diluted with H2O (60 mL) and celite filtered then extracted with EtOAc (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 96:4 to 91:9) to give 5-(2-trimethylsilylethynyl)isoquinoline-4- carbaldehyde (1.69 g, 5.78 mmol, 68.25% yield, 86.69% purity) as a yellow solid. MS (ESI) m/z 309.2 [M+H]+.
[0001286] Step 2: 5-ethynylisoquinoline-4-carbaldehyde
[0001287] To a solution of 5-(2-trimethylsilylethynyl)isoquinoline-4-carbaldehyde (1.69 g, 6.67 mmol, 1 eq) in MeOH (8 mL) was added K2CO3 (2.30 g, 16.68 mmol, 2.5 eq). The mixture was stirred at 20 °C for 40 min. Upon completion, the mixture was filtered to give 5-
564
SUBSTITUTE SHEET RULE 26 ethynylisoquinoline-4-carbaldehyde (1.21 g, crude) as a yellow liquid, which was used for next step directly.
[0001288] Step 3: 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile
[0001289] To a solution of 5-ethynylisoquinoline-4-carbaldehyde (1.21 g, 6.68 mmol, 1 eq) in DCM (10 mL) was added Ti(i-PrO)4 (2.85 g, 10.02 mmol, 2.96 mL, 1.5 eq) and NH3/MeOH (7 M, 10 mL, 10.48 eq) stirred 1 h at 20 °C, then TMSCN (1.99 g, 20.03 mmol, 2.51 mL, 3 eq) was added at 20°C. The mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with DCM (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO-i, filtered and concentrated under reduced pressure to give 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (740 mg, crude) as a yellow oil. MS (ESI) m/z 208.1 [M+H]+.
[0001290] Step 4: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001291] A mixture of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3. L0]hexane-2-carboxylic acid (330 mg, 812.02 umol, 1 eq), 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (185.10 mg, 893.22 umol, 1.1 eq) and 1 -methylimidazole (200.01 mg, 2.44 mmol, 194.18 uL, 3 eq) in ACN (5 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphate (455.67 mg, 1.62 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C 18 150 * 40 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 8 mm) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4- isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2-carboxamide (400 mg, 671.58 umol, 82.70% yield) as a yellow oil. MS (ESI) m/z 596.3 [M+H]+.
565
SUBSTITUTE SHEET RULE 26 [0001292] Step 5: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001293] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3- (l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (400 mg) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 6 min) to give (lR,2S,5S)-N-[cyano-(5- ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (114.45 mg, 192.16 umol, 28.61% yield, 100% purity) as a white solid. MS (ESI) m/z 596.3 [M+H]+.
[0001294] 'H NMR (400 MHz, DMSO-de) 8 = 9.82 (s, 1H), 9.52 - 9.41 (m, 2H), 8.81 (s, 1H),
8.34 (d, J = 8.1 Hz, 1H), 8.14 (d, J = 6.5 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 4.70 (s, 1H), 4.37 (d, J = 7.3 Hz, 1H), 4.25 (s, 1H), 3.98 - 3.88 (m, 2H), 3.67 (d, J = 10.5 Hz, 1H), 1.53 (dd, J = 5.4, 7.4 Hz, 1H), 1.38 - 1.32 (m, 4H), 1.20 (d, J = 6.4 Hz, 3H), 0.97 (s, 3H), 0.82 - 0.75 (m, 4H), 0.70 - 0.62 (m, 1H), 0.42 - 0.30 (m, 2H).
[0001295] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3- (l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (105.74 mg, 177.53 umol, 26.44% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 596.3 [M+H]+.
[0001296] 'H NMR (400 MHz, DMSO-de) 8 = 9.88 - 9.76 (m, 1H), 9.72 (d, J = 7.1 Hz, 1H),
9.46 (s, 1H), 8.82 (s, 1H), 8.33 (d, J = 7.4 Hz, 1H), 8.14 (d, J = 7.1 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.68 (d, J = 7.0 Hz, 1H), 4.66 (s, 1H), 4.37 - 4.29 (m, 2H), 3.94 - 3.83 (m, 2H), 3.71 - 3.63 (m, 1H), 1.58 (dd, J = 5.5, 7.3 Hz, 1H), 1.47 (d, J = 7.5 Hz, 1H), 1.27 (s, 3H), 1.09 (d, J = 6.1 Hz, 3H), 1.04 (s, 3H), 0.85 (s, 3H), 0.75 - 0.69 (m, 1H), 0.62 - 0.52 (m, 1H), 0.32 - 0.25 (m, 2H).
566
SUBSTITUTE SHEET RULE 26 Example 154: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 216A)
Figure imgf000568_0001
[0001297] Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001298] A mixture of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (0.25 g, 1.17 mmol, 1 eq) and (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (472.39 mg, 1.29 mmol, 1.1 eq) in ACN (10 mL) was added 1 -methylimidazole (288.73 mg, 3.52 mmol, 280.32 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (657.84 mg, 2.34 mmol, 2 eq), the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O 20 mL and extracted with EA 45 mL (15 mL * 3). The combined organic layers were washed with BRINE 100 mL (20 mL * 5), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude was purified by prep-HPLC (neutral condition; column: Waters Xbridge BEH C18 250*70mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-60%,20min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide as a yellow solid. MS (ESI) m/z 562.3 [M+H]+
[0001299] Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
567
SUBSTITUTE SHEET RULE 26 [0001300] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (500 mg) was separated by SFC(condition: column: REGIS (s,s) WHELK-01 (250mm*50mm,10um);mobile phase: [Hexane-IPA];B%: 50%-50%,10min) to give (lR,2S,5S)- N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 2 (170 mg, 302.72 umol, 25.82% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 562.3 [M+H]+
[0001301] 'H NMR (400 MHz, DMSO-de) 5 = 9.84 (br s, 1H), 9.69 (br d, J = 5.5 Hz, 1H), 9.41 (s, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.13 (br s, 1H), 4.46 (s, 1H), 4.39 - 4.27 (m, 2H), 3.95 - 3.77 (m, 2H), 3.62 - 3.53 (m, 1H), 3.19 (s, 3H), 1.58 (dd, J = 5.3, 7.3 Hz, 1H), 1.47 (d, J = 7.6 Hz, 1H), 1.12 - 1.02 (m, 6H), 0.88 (s, 3H)
Example 155: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]he ane-2-carbo amide (Compound 218A)
Figure imgf000569_0001
[0001302] Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001303] A solution of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (310.58 mg, 1.02 mmol, 70% purity, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (500 mg, 1.02 mmol, 80% purity, 1 eq) and NMI (251.10 mg, 3.06 mmol, 243.79 uL, 3 eq) then followed by TCFH (572.07 mg, 2.04 mmol, 2 eq).
568
SUBSTITUTE SHEET RULE 26 The mixture was stirred at 20 °C for 2 h. Upon completion, the mixture was diluted with H2O (5 mL) and extracted with EA (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-60%, 10 min) to give (lR,2S,5S)-N-[cyano- (3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (240 mg, 383.93 umol, 37.66% yield, 94% purity) as a white solid. MS (ESI) m/z 588.2 [M+H]+.
[0001304] Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001305] The (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)- 3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (230 mg, 391.41 umol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-O1 (250mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 33%-33%, 6 min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (63.65 mg, 102.58 umol, 26.21% yield, 94.7% purity) as a white solid. MS (ESI) m/z 588.2 [M+H]+.
[0001306] 'H NMR (400 MHz, DMSO-de) 5 = 9.92 - 9.82 (m, 1H), 9.46 (br d, J = 6.1 Hz, 1H), 9.44 (s, 1H), 8.70 (s, 1H), 8.63 (s, 1H), 7.02 (br d, J = 6.4 Hz, 1H), 4.52 (s, 1H), 4.44 - 4.35 (m, 1H), 4.23 (s, 1H), 4.08 - 3.81 (m, 2H), 3.77 (br d, J = 10.3 Hz, 1H), 3.47 - 3.35 (m, 1H), 1.59 - 1.49 (m, 1H), 1.41 - 1.33 (m, 1H), 1.23 (br d, J = 6.1 Hz, 3H), 0.98 (s, 3H), 0.82 (s, 3H), 0.64 - 0.54 (m, 1H), 0.48 - 0.30 (m, 3H)
[0001307] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (67.54 mg, 112.76 umol, 28.81% yield, 98.1% purity) was obtained as a white solid. MS (ESI) m/z 588.2 [M+H]+.
[0001308] 'H NMR (400 MHz, DMSO-de) 5 = 9.92 - 9.82 (m, 1H), 9.73 (br s, 1H), 9.42 (s, 1H), 8.71 (s, 1H), 8.63 (s, 1H), 7.21 - 7.06 (m, 1H), 4.49 (s, 1H), 4.41 - 4.33 (m, 1H), 4.31 (s,
569
SUBSTITUTE SHEET RULE 26 1H), 3.90 (br dd, J = 5.0, 10.3 Hz, 1H), 3.85 - 3.69 (m, 2H), 3.30 (br d, J = 2.9 Hz, 1H), 1.63 -
1.54 (m, 1H), 1.48 (br d, J = 7.5 Hz, 1H), 1.13 (br d, J = 6.1 Hz, 3H), 1.04 (s, 3H), 0.86 (s, 3H), 0.52 - 0.26 (m, 4H)
Example 156: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2- trifluoroacetyl)amino] butanoyl] -N- [cyano-(3-prop- 1-ynylthieno [2,3-c] pyridin-4-yl)methyl] - 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 219A)
Figure imgf000571_0001
[0001309] Step 1: tert-butyl (2S,3R)-2-(benzyloxycarbonylamino)-3-hydroxy-butanoate
[0001310] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (645.09 mg, 1.58 mmol, 1 eq) in ACN (12 mL) was added NMI (389.05 mg, 4.74 mmol, 377.72 uL, 3 eq) and TCFH (886.36 mg, 3.16 mmol, 2 eq), and then 2-amino-2-(3-prop-l- ynylthieno[2,3-c]pyridin-4-yl)acetonitrile (359 mg, 1.58 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction was quenched by addition H2O (20 mL) at 25 °C, and then extracted with DCM (20 mL * 2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80 * 40 mm * 3 um; mobile phase: [water (NH4HCCh)-ACN]; B%: 50%-70%, 8 min). Compound (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(3- prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (540 mg, 874.24 umol, 55.34% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 618.3 [M+H]+.
570
SUBSTITUTE SHEET RULE 26 [0001311] Step 2: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001312] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (540 mg, 874.24 umol, 1 eq) was separation by SFC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 23%-23%,5 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (261 mg, 422.55 umol, 26.75% yield, 100% purity) as a white solid. MS (ESI) m/z 618.2 [M+H]+.
[0001313] 'H NMR (400 MHz, DMSO-d6) 5 = 9.60 (br s, 1H), 9.41 (s, 1H), 9.21 (d, J = 6.4 Hz, 1H), 8.70 (s, 1H), 8.40 (s, 1H), 7.07 (br d, J = 6.3 Hz, 1H), 4.40 (br s, 1H), 4.25 (s, 1H), 4.04 - 3.85 (m, 2H), 3.67 (d, J = 10.6 Hz, 1H), 2.07 (s, 3H), 1.56 (dd, J = 5.3, 7.5 Hz, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.11 (m, 9H), 1.06 (d, J = 6.1 Hz, 3H), 1.00 - 0.95 (m, 3H), 0.80 (s, 3H).
[0001314] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (265 mg, 429.02 umol, 27.16% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 618.2 [M+H]+.
[0001315] 'H NMR (400 MHz, DMSO-d6) 5 = 9.62 (br s, 1H), 9.39 (s, 1H), 9.34 (d, J = 6.8 Hz, 1H), 8.73 - 8.66 (m, 1H), 8.40 (s, 1H), 7.21 (br d, J = 6.1 Hz, 1H), 4.38 (br s, 1H), 4.29 (s, 1H), 3.95 (dd, J = 5.3, 10.6 Hz, 1H), 3.85 (dd, J = 6.2, 7.9 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 2.08 (s, 3H), 1.59 (dd, J = 5.5, 7.4 Hz, 1H), 1.44 (d, J = 7.6 Hz, 1H), 1.12 - 1.03 (m, 15H), 0.83 (s, 3H).
571
SUBSTITUTE SHEET RULE 26 Example 157: Synthesis of (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl (methyl |-6.6-dimethyl-3-|(2S.3R)-3-( 1 -methylcyclopropoxy )-2-|( 2.2.2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 220A)
Figure imgf000573_0001
[0001316] Step 1: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001317] A solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (700 mg, 1.72 mmol, 9.79e-l eq) in DCM (5 mL) was added 2-amino-2-(3-prop-l-ynylthieno[2,3- c]pyridin-4-yl)acetonitrile (400 mg, 1.76 mmol, 1 eq), DMAP (645.02 mg, 5.28 mmol, 3 eq) and EDCI (674.75 mg, 3.52 mmol, 2 eq), then stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by H2O (5 mL) and extracted with EA (5 mL* 3). The organic layers were washed with HC1 (IM, 5 mL), then wash with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC((column: Welch Xtimate C 18 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)- ACN]; B%: 35%-70%, 23 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3- c]pyridin-4-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (800 mg, 1.30 mmol, 73.83% yield) as a yellow solid.MS (ESI) m/z 616.3 [M+H]+.
[0001318] Step 2: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
572
SUBSTITUTE SHEET RULE 26 [0001319] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (800 mg, 1.30 mmol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 27%-27%, 6 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (111.96 mg, 180.76 umol, 13.91% yield, 99.4% purity) as a white solid. MS (ESI) m/z 616.3 [M+H]+.
[0001320] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.82 (d, J= 8.2 Hz, 1H), 9.41 (s, 1H), 9.29 (d, J= 6.6 Hz, 1H), 8.70 (s, 1H), 8.41 (s, 1H), 7.06 (br d, J= 6.3 Hz, 1H), 4.37 (t, J= 8.3 Hz, 1H), 4.24 (s, 1H), 4.01 - 3.79 (m, 2H), 3.69 (d, J= 10.5 Hz, 1H), 2.07 (s, 3H), 1.56 (dd, J= 5.3, 7.5 Hz, 1H), 1.36 (s, 1H), 1.35 - 1.31 (m, 3H), 1.21 - 1.06 (m, 3H), 1.01 - 0.93 (m, 3H), 0.83 - 0.74 (m, 4H), 0.71 - 0.62 (m, 1H), 0.41 - 0.29 (m, 2H)
[0001321] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (143.24 mg, 232.66 umol, 17.90% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 616.3 [M+H]+.
[0001322] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.82 (br d, J= 7.7 Hz, 1H), 9.43 (br d, J= 6.7 Hz, 1H), 9.39 (s, 1H), 8.76 - 8.64 (m, 1H), 8.40 (s, 1H), 7.20 (br d, J= 5.5 Hz, 1H), 4.35 (br t, J = 8.2 Hz, 1H), 4.28 (s, 1H), 3.94 - 3.83 (m, 2H), 3.69 (d, J= 10.6 Hz, 1H), 2.07 (s, 3H), 1.58 (dd, J= 5.4, 7.5 Hz, 1H), 1.44 (d, J= 7.6 Hz, 1H), 1.30 (s, 3H), 1.15 (d, J= 6.1 Hz, 3H), 1.08 - 1.01 (m, 3H), 0.82 (s, 3H), 0.77 - 0.70 (m, 1H), 0.65 - 0.57 (m, 1H), 0.37 - 0.25 (m, 2H).
573
SUBSTITUTE SHEET RULE 26 Example 158: Synthesis of (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 221A)
Figure imgf000575_0001
[0001323] Step 1: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-
[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001324] To a solution of 2-amino-2-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)acetonitrile (360 mg, 1.58 mmol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (580.24 mg, 1.58 mmol, 1 eq), TCFH (666.62 mg, 2.38 mmol, 1.5 eq) and NMI (390.14 mg, 4.75 mmol, 378.77 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (0.5 mL) at 25 °C, and then diluted with H2O (10 mL) and extracted with ethyl acetate (5 mL * 2). The combined organic layers were washed with sat. NaCl (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)- ACN] ; B%: 35%-60%, 10 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide (300 mg, 521.19 umol, 1 eq) as white solid. MS (ESI) m/z 576.4 [M+H]+
[0001325] Step 2: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
574
SUBSTITUTE SHEET RULE 26 [0001326] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)- 3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (300 mg, 521.19 umol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 8 min) to give the product (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (84 mg, 145.93 umol, 9.21% yield). MS (ESI) m/z 576.2 [M+H]+
[0001327] 'H NMR (400 MHz, DMSO-de) 5 = 9.85 (br s, 1H), 9.52 - 9.30 (m, 2H), 8.72 (s, 1H), 8.39 (s, 1H), 7.19 (br d, I = 4.5 Hz, 1H), 4.35 (br d, I = 9.0 Hz, 1H), 4.28 (s, 1H), 3.96 - 3.82 (m, 2H), 3.58 (dd, I = 6.3, 8.9 Hz, 1H), 3.21 (s, 3H), 2.05 (s, 3H), 1.60 (dd, I = 5.2, 7.3 Hz, 1H), 1.43 (d, I = 7.6 Hz, 1H), 1.16 (d, I = 6.1 Hz, 3H), 1.05 (s, 3H), 0.86 (s, 3H)
[0001328] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)- 3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (89 mg, 154.62 umol, 9.76% yield) was obtained as a white solid. MS (ESI) m/z 576.2 [M+H]+
[0001329] 'H NMR (400 MHz, DMSO-de) 5 = 9.85 (br s, 1H), 9.50 - 9.30 (m, 2H), 8.72 (s, 1H), 8.39 (s, 1H), 7.27 - 7.12 (m, 1H), 4.40 - 4.15 (m, 2H), 3.97 - 3.76 (m, 2H), 3.58 (dd, 1 = 6.3, 8.9 Hz, 1H), 3.21 (s, 3H), 2.05 (s, 3H), 1.60 (dd, I = 5.3, 7.3 Hz, 1H), 1.43 (d, I = 7.5 Hz, 1H), 1.16 (d, I = 6.1 Hz, 3H), 1.05 (s, 3H), 0.86 (s, 3H)
Example 159: Synthesis of (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 222A)
575
SUBSTITUTE SHEET RULE 26
Figure imgf000577_0001
[0001330] Step 1: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001331] A solution of 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4-yl)acetonitrile (310.58 mg, 1.02 mmol, 70% purity, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3- (cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (500 mg, 1.02 mmol, 80% purity, 1 eq) and NMI (251.10 mg, 3.06 mmol, 243.79 uL, 3 eq) then followed by TCFH (572.07 mg, 2.04 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (5 mL) and extracted with EA (5 mL * 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex C 18 80 * 40 mm * 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 8 min) to give (lR,2S,5S)-N-[cyano-(3-prop- l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 452.60 umol, 47.57% yield, 95% purity) as a white solid. MS (ESI) m/z 630.4 [M+H]+.
[0001332] Step 2: (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001333] The (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 476.42 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 28%-28%, 7 min) to give (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-
576
SUBSTITUTE SHEET RULE 26 yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (62.73 mg, 96.03 umol, 20.16% yield, 96.4% purity) as a white solid. MS (ESI) m/z 630.2 [M+H]+.
[0001334] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.73 (br d, J= 7.3 Hz, 1H), 9.41 (s, 1H), 9.24 (d, J= 6.6 Hz, 1H), 8.69 (s, 1H), 8.40 (s, 1H), 7.06 (br d, J= 6.3 Hz, 1H), 4.43 (br t, J = 7.5 Hz, 1H), 4.25 (s, 1H), 3.97 (dd, J= 5.4, 10.5 Hz, 1H), 3.93 - 3.84 (m, 1H), 3.69 (d, J= 10.5 Hz, 1H), 2.15 - 2.05 (m, 5H), 1.79 - 1.70 (m, 2H), 1.62 - 1.54 (m, 2H), 1.53 - 1.45 (m, 1H), 1.36 (d, J = 7.5 Hz, 1H), 1.28 (s, 3H), 1.09 (d, J= 6.2 Hz, 3H), 0.98 (s, 3H), 0.80 (s, 3H).
[0001335] (lR,2S,5S)-N-[cyano-(3-prop-l-ynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (70.13 mg, 111.37 umol, 23.38% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 630.2 [M+H]+.
[0001336] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.72 (br s, 1H), 9.42 - 9.30 (m, 2H), 8.71 (s, 1H), 8.40 (s, 1H), 7.21 (br d, J= 4.9 Hz, 1H), 4.41 (br d, J= 6.3 Hz, 1H), 4.30 (s, 1H), 3.94 (dd, J= 5.3, 10.4 Hz, 1H), 3.88 - 3.77 (m, 1H), 3.68 (d, J= 10.6 Hz, 1H), 2.11 - 2.06 (m, 3H), 2.06 - 1.96 (m, 2H), 1.75 - 1.62 (m, 2H), 1.61 - 1.50 (m, 2H), 1.49 - 1.40 (m, 2H), 1.23 (s, 3H), 1.11 - 0.97 (m, 6H), 0.84 (s, 3H).
Example 160: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide. (Compound 223A)
Figure imgf000578_0001
577
SUBSTITUTE SHEET RULE 26 [0001337] Step 1: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001338] A solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (700 mg, 1.69 mmol, 50% purity, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (758.76 mg, 1.86 mmol, 1.1 eq) in DCM (30 mL) was added DMAP (619.00 mg, 5.07 mmol, 3 eq), EDCI (647.55 mg, 3.38 mmol, 2 eq), the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (60 mL), and then extracted with DCM (40 mL * 3). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 70 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 20 min) to give desired compound as a white solid, which was further separated by SFC (column: REGIS(S,S)WHELK-Ol(250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 40%-40%, 6 min) to give a (lR,2S,5S)-3-[(2S,3R)-3- tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-ethynyl-4- isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (128 mg, 210.97 umol, 12.49% yield, 98.5% purity) was obtained as a white solid. MS (ESI) m/z 598.3 [M+H]+.
[0001339] 'H NMR (400 MHz, DMSO-t/r,) 8 = 9.62 (br d, J= 8.6 Hz, 1H), 9.49 - 9.45 (m, 1H), 9.41 (br d, J= 6.7 Hz, 1H), 8.81 (s, 1H), 8.34 (d, J = 8.2 Hz, 1H), 8.14 (d, J = 7.3 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.58 (d, J= 6.6 Hz, 1H), 4.70 (s, 1H), 4.41 (br t, J= 8.1 Hz, 1H), 4.26 (s, 1H), 4.00 - 3.88 (m, 2H), 3.65 (br d, J= 10.6 Hz, 1H), 1.57 - 1.51 (m, 1H), 1.36 (d, J= 7.6 Hz, 1H), 1.15 - 1.07 (m, 12H), 0.98 (s, 3H), 0.80 (s, 3H).
[0001340] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (152 mg, 253.32 umol, 15.00% yield, 99.6% purity) was obtained as a white solid. MS (ESI) m/z 598.3 [M+H]+.
578
SUBSTITUTE SHEET RULE 26 [0001341] 'H NMR (400 MHz, DMSO-t/r,) 8 = 9.61 (br t, J= 8.8 Hz, 2H), 9.46 (s, 1H), 8.81
(s, 1H), 8.33 (dd, J= 1.0, 8.2 Hz, 1H), 8.14 (dd, J= 1.0, 7.3 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.68 (d, J= Hz, 1H), 4.68 (s, 1H), 4.43 - 4.31 (m, 2H), 3.96 (dd, J= 5.3, 10.6 Hz, 1H), 3.89 - 3.80 (m, 1H), 3.65 (d, J= 10.7 Hz, 1H), 1.63 - 1.54 (m, 1H), 1.48 (d, J= 7.5 Hz, 1H), 1.06 - 0.98 (m, 15H), 0.85 (s, 3H).
Example 161: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 224A)
Figure imgf000580_0001
[0001342] Step 1: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001343] A solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (406.65 mg, 1.18 mmol, 60% purity, 1.1 eq) in ACN (5 mL) was added (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (500 mg, 1.07 mmol, 90% purity, 1 eq) and NMI (263.63 mg, 3.21 mmol, 255.95 uL, 3 eq) then followed by TCFH (600.64 mg, 2.14 mmol, 2 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (5 mL) and extracted with EA (10 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 100:1 to 40: 1) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-
579
SUBSTITUTE SHEET RULE 26 carboxamide (400 mg, 459.29 umol, 38.62% yield, 70% purity) as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001344] Step 2: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001345] The (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (400 mg, 459.29 umol, 70% purity, 1 eq was separated by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu- IPA]; B%: 40%-40%, 7 min) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (62.23 mg, 98.10 umol, 21.36% yield, 96.1% purity) as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001346] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.86 - 9.72 (m, 1H), 9.53 - 9.40 (m, 2H), 8.81 (s, 1H), 8.37 - 8.29 (m, 1H), 8.14 (dd, J= 1.1, 7.2 Hz, 1H), 7.79 (t, J= 7.7 Hz, 1H), 7.57 (d, J = 6.8 Hz, 1H), 4.71 (s, 1H), 4.43 (br t, J= 7.6 Hz, 1H), 4.25 (s, 1H), 3.96 (dd, J= 5.4, 10.6 Hz, 1H), 3.89 (dd, J= 6.4, 8.1 Hz, 1H), 3.66 (d, J= 10.5 Hz, 1H), 2.14 - 2.04 (m, 2H), 1.79 - 1.70 (m, 2H), 1.60 - 1.46 (m, 3H), 1.36 (d, J= 7.5 Hz, 1H), 1.28 (s, 3H), 1.12 (d, J= 6.1 Hz, 3H), 1.04 - 0.96 (m, 3H), 0.81 (s, 3H).
[0001347] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3- (1 -methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (70.78 mg, 110.30 umol, 24.01% yield, 95% purity) was obtained as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001348] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.86 - 9.70 (m, 1H), 9.66 (d, J= 7.5 Hz, 1H), 9.46 (s, 1H), 8.81 (s, 1H), 8.33 (dd, J= 0.9, 8.1 Hz, 1H), 8.18 - 8.08 (m, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.68 (d, J= 7.0 Hz, 1H), 4.69 (s, 1H), 4.40 (br d, J= 8.1 Hz, 1H), 4.33 (s, 1H), 4.01 - 3.88 (m, 1H), 3.86 - 3.76 (m, 1H), 3.72 - 3.62 (m, 1H), 2.09 - 1.92 (m, 2H), 1.74 - 1.65 (m, 1H), 1.64 - 1.54 (m, 2H), 1.52 - 1.31 (m, 3H), 1.25 - 1.18 (m, 3H), 1.12 - 0.97 (m, 6H), 0.96 - 0.72 (m, 3H).
580
SUBSTITUTE SHEET RULE 26 Example 162: Synthesis of (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3- [(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 225A)
Figure imgf000582_0001
[0001350] To a solution of 5-bromoisoquinoline-4-carbaldehyde (1 g, 4.24 mmol, 1 eq) in NHVMeOH (7 M) (10 mL) was added NH4CI (679.77 mg, 12.71 mmol, 3 eq) stirred at 25 °C for 1 h, then added TMSCN (1.26 g, 12.71 mmol, 1.59 mL, 3 eq) at 0 °C, the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was concentrated under reduced pressure to remove NHa/MeOH. The reaction mixture was quenched by addition H2O (30 mL), and then extracted with EA (10 mL * 3). The combined organic layers weredried over Na2SO4, filtered and concentrated in vacuum and was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 0: 1) to give the product 2-amino-2-(5-bromo-4- isoquinolyl)acetonitrile (730 mg, 2.59 mmol, 61.15% yield, 93% purity) as yellow oil. MS (ESI) m/z 262.2, 264.2 [M+H]+
[0001351] Step 2: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
581
SUBSTITUTE SHEET RULE 26 [0001352] To a solution of 2-amino-2-(5-bromo-4-isoquinolyl)acetonitrile (700 mg, 2.48 mmol, 93% purity, 1 eq) in ACN (10 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.29 g, 2.98 mmol, 1.2 eq) and NMI (693.31 mg, 8.44 mmol, 673.12 uL, 3 eq) and then was added TCFH (1.67 g, 5.96 mmol, 2 eq), the mixture was stirred at 25 °C for 1 h. Upon completion, the mixture was diluted with H2O (50 mL) and filtered then was extracted with EA (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuum and was purified by prep-HPLC (column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3) - ACN]; B%: 40% - 70%, 10 min) to give the product (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.43 mmol, 57.73% yield, 97% purity) as yellow solid. MS (ESI) m/z 676.2, 678.2 [M+H]+
[0001353] Step 3: (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001354] The (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (1 g, 1.48 mmol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm * 30 mm, 10 um); mobile phase: [Neu - IP A]; B%: 26% - 26%, 6 min) to give product (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)- 3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (112.74 mg, 166.65 umol, 11.27% yield, 100% purity) as white solid. MS (ESI) m/z 676.1, 678.1 [M+H]+
[0001355] 'H NMR (400 MHz, DMSO-t/r,) 5 = 10.01 - 9.69 (m, 1H), 9.65 - 9.43 (m, 2H), 8.89 (s, 1H), 8.31 (ddd, J= 1.2, 7.8, 18.8 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.51 (br d, J= 6.8 Hz, 1H), 4.40 (br d, J= 8.4 Hz, 1H), 4.24 (s, 1H), 4.12 - 3.99 (m, 1H), 3.94 (dd, J= 5.4, 10.4 Hz, 1H), 3.66 (d, J= 10.6 Hz, 1H), 1.56 (dd, J= 5.4, 7.4 Hz, 1H), 1.48 - 1.32 (m, 2H), 1.26 (d, J = 6.0 Hz, 3H), 1.06 - 0.95 (m, 3H), 0.80 (s, 3H), 0.72 - 0.60 (m, 1H), 0.56 - 0.39 (m, 3H), 0.38 - 0.17 (m, 2H), 0.15 - -0.03 (m, 2H).
582
SUBSTITUTE SHEET RULE 26 [0001356] (lR,2S,5S)-N-[(5-bromo-4-isoquinolyl)-cyano-methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (165.93 mg, 244.59 umol, 16.55% yield,
99.723% purity) was obtained as a white solid. MS (ESI) m/z 676.1, 678.1 [M+H]+
[0001357] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.78 (s, 1H), 9.69 - 9.45 (m, 2H), 8.89 (s, 1H), 8.30 (dd, J= 7.8, 18.8 Hz, 2H), 7.68 (t, J= 7.8 Hz, 1H), 7.55 - 7.41 (m, 1H), 4.45 - 4.33 (m, 1H), 4.32 - 4.23 (m, 1H), 4.07 - 3.97 (m, 1H), 3.96 - 3.87 (m, 1H), 3.73 - 3.64 (m, 1H), 1.63 - 1.53 (m, 1H), 1.48 - 1.33 (m, 2H), 1.28 - 1.16 (m, 3H), 1.05 - 1.01 (m, 3H), 0.90 - 0.77 (m, 3H), 0.71 - 0.57 (m, 1H), 0.51 (br s, 3H), 0.31 - 0.13 (m, 2H), 0.12 -0.07 (m, 2H).
Example 163: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-
[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 226A)
Figure imgf000584_0001
[0001358] Step 1: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001359] To a solution of 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (900 mg, 2.17 mmol, 50% purity, 1 eq) in ACN (10 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (1.17 g, 2.17 mmol, 80% purity, 1 eq), TCFH (913.91 mg, 3.26 mmol, 1.5 eq) and NMI (534.86 mg, 6.51 mmol, 519.28 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O (0.5 mL) at 25 °C, and then diluted with H2O (10 mL) and extracted with ethyl acetate (5 mL * 2). The combined organic layers were washed with sat. NaCl (5 mL), dried over Na2SO4, filtered
583
SUBSTITUTE SHEET RULE 26 and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (column: Waters Xbridge BEH C 18 250 * 70 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 20 mm) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-4- isoquinolyl)methyl]-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (400 mg, 643.45 umol, 29.63% yield) as white solid. MS (ESI) m/z 622.3 [M+H]+
[0001360] Step 2: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxamide
[0001361] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 521.19 umol, 1 eq) was purified by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 9 min) to give the product (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3- [(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (65 mg, 104.56 umol, 16.25% yield) as white solid. MS (ESI) m/z 622.2 [M+H]+
[0001362] 'H NMR (400 MHz, DMSO-de) 5 = 9.89 - 9.69 (m, 1H), 9.53 - 9.36 (m, 2H), 8.81 (s, 1H), 8.34 (dd, J = 1.1, 8.2 Hz, 1H), 8.14 (dd, J = 1.1, 7.3 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.58 (d, J = 6.6 Hz, 1H), 4.71 (s, 1H), 4.41 (br d, J = 8.2 Hz, 1H), 4.25 (s, 1H), 4.12 - 3.99 (m, 1H), 3.93 (dd, J = 5.3, 10.3 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 1.54 (dd, J = 5.4, 7.4 Hz, 1H), 1.46 - 1.38 (m, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.28 (d, J = 6.1 Hz, 3H), 0.97 (s, 3H), 0.80 (s, 3H), 0.67 (br dd, J = 5.2, 11.1 Hz, 1H), 0.55 - 0.40 (m, 3H), 0.38 - 0.29 (m, 1H), 0.26 - 0.18 (m, 1H), 0.14 - 0.07 (m, 1H), 0.05 - 0.02 (m, 1H).
[0001363] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 as white solid (62 mg, 99.73 umol, 15.50% yield) was obtained as white solid. MS (ESI) m/z 622.2 [M+H]+
[0001364] 'H NMR (400 MHz, DMSO-de) 5 = 9.72 (m, 2H), 9.46 (s, 1H), 8.82 (s, 1H), 8.38 - 8.29 (m, 1H), 8.14 (dd, J = 0.8, 7.2 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.69 (d, J = 7.2 Hz, 1H),
584
SUBSTITUTE SHEET RULE 26 4.66 (s, 1H), 4.40 - 4.30 (m, 2H), 4.02 - 3.87 (m, 2H), 3.67 (d, J = 10.6 Hz, 1H), 1.62 - 1.54 (m, 1H), 1.47 (d, J = 7.6 Hz, 1H), 1.38 - 1.30 (m, 1H), 1.17 (d, J = 6.2 Hz, 3H), 1.05 (s, 3H), 0.85 (s, 3H), 0.61 (td, J = 5.5, 11.0 Hz, 1H), 0.46 - 0.34 (m, 3H), 0.24 (br dd, J = 4.5, 10.4 Hz, 1H), 0.19 - 0.11 (m, 1H), 0.08 - 0.01 (m, 1H), 0.00 - 0.08 (m, 1H).
Example 164: Synthesis of (lR,2S,5S)-N-((3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7- yl)(cyano)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclopropoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 227A)
Figure imgf000586_0001
[0001365] Step 1: 7-bromo-3-chloro-lH-pyrrolo[3,2-c]pyridine
[0001366] To a solution of 7-bromo-lH-pyrrolo[3,2-c]pyridine (24.5 g, 124.35 mmol, 1 eq) in AcOH (250 mL) was added NCS (19.93 g, 149.21 mmol, 1.2 eq), the mixture was stirred at 25 °C for 12 h. Upon completion, the reaction mixture was poured into NaHCCh (500 mL) and filtered and concentrated under reduced pressure to give a 7-bromo-3-chloro-lH-pyrrolo[3,2- c]pyridine (34 g, crude) as yellow solid. MS (ESI) m/z 231.1 [M+H]+.
585
SUBSTITUTE SHEET RULE 26 [0001367] Step 2: 7-bromo-3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridine
[0001368] To a solution of 7-bromo-3-chloro-lH-pyrrolo[3,2-c]pyridine (34 g, 146.88 mmol, 1 eq)in THF (340 mL) at 0 °C under N2, LiHMDS (1 M, 440.65 mL, 3 eq) was added and the mixture was stirred for 1 h at 0 °C. Then iodoethane (91.63 g, 587.53 mmol, 46.99 mL, 4 eq) was added and the mixture was warmed to 25 °C slowly and stirred for 11 h. The reaction mixture was poured into water (500 mL) and extracted with Ethyl acetate (300 mL * 3). The combined organics dried over Na2SO4, filtered, concentrated in vacuo. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 9/1) to give 7-bromo-3- chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridine (8 g, 30.82 mmol, 20.99% yield) as yellow solid. MS (ESI) m/z 259.1 [M+H]+.
[0001369] Step 3: 3-chloro-l-ethyl-7-vinyl-lH-pyrrolo[3,2-c]pyridine
[0001370] To a solution of 7-bromo-3 -chloro- l-ethyl-pyrrolo[3,2-c]pyridine (8 g, 30.82 mmol, 1 eq) in Tol. (224 mL) and H2O (56 mL) was added K3PO4 (13.09 g, 61.65 mmol, 2 eq) and potassium;trifluoro(vinyl)boranuide (4.95 g, 36.99 mmol, 1.2 eq), then the mixture was added ditert-butyl(cyclopentyl)phosphane;dichloropalladium;iron (2.01 g, 3.08 mmol, 0.1 eq) under the atmosphere of N2. Then the mixture was stirred at 80 °C for 16 h. Upon completion, the reaction was pour into H2O (500 mL) and then extracted with Ethyl acetate (300 mL * 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 9/1) to give 3 -chloro- l-ethyl-7-vinyl- 1H- pyrrolo[3,2-c]pyridine (3.9 g, 18.87 mmol, 61.22% yield) as yellow solid. MS (ESI) m/z 207.2 [M+H]+.
[0001371] Step 4: 3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridine-7-carbaldehyde
[0001372] To a solution of 3-chloro-l-ethyl-7-vinyl-lH-pyrrolo[3,2-c]pyridine (3.9 g, 18.87 mmol, 1 eq) and K2OSO4.2H2O (695.31 mg, 1.89 mmol, 0.1 eq) in THF (40 mL), H2O (8 mL) was stirred for 1 h. Then NalCh (113.22 mmol, 6.27 mL, 6 eq) was added and the mixture was stirred at 25°C for 11 h. Upon completion, the mixture was pour into H2O (100 mL) and then extracted with EA (150 mL * 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 9/1) to give 3 -chloro- 1 -ethyl- 1H-
586
SUBSTITUTE SHEET RULE 26 pyrrolo[3,2-c]pyridine-7-carbaldehyde (1.6 g, 7.67 mmol, 40.64% yield, N/A purity) as yellow solid. MS (ESI) m/z 209.2 [M+H]+.
[0001373] 'H NMR (400 MHz, DMSO-de) 5 = 10.20 (s, 1H), 9.02 (s, 1H), 8.86 (s, 1H), 7.88 (s, 1H), 4.66 - 4.56 (m, 2H), 1.29 (t, 3H).
[0001374] Step 5: 2-amino-2-(3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7-yl)acetonitrile
[0001375] A mixture of 3 -chi oro-1 -ethyl- lH-pyrrolo[3,2-c]pyridine-7-carbaldehy de (400 mg, 1.92 mmol, 1 eq) and NH3/MeOH (7 M, 3.09 mL, 11.3 eq), Ti(i-PrO)4 (2.88 mmol, 848.72 uL, 1.5 eq) in DCM (15 mL) was stirred at 25 °C for 1 h. Then TMSCN (5.75 mmol, 719.52 uL, 3 eq) was added at 0 °C, the mixture was stirred at 25 °C for 2 h. Upon completion, the reaction was pour into H2O (20 mL) and then extracted with EA (40 mL * 3). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO-i, filtered and concentrated in vacuo to give 2-amino-2-(3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7-yl)acetonitrile (400 mg, crude) as yellow solid. MS (ESI) m/z 235.2 [M+H]+.
[0001376] Step 6: (lR,2S,5S)-N-((3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7- yl)(cyano)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclopropoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001377] A mixture of 2-amino-2-(3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7- yl)acetonitrile (400 mg, 1.70 mmol, 1 eq), (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (692.67 mg, 1.70 mmol, 1 eq) and TCEH (621.69 mg, 2.22 mmol, 1.3 eq), NMI (3.41 mmol, 271.71 uL, 2 eq) in ACN (15 mL) was stirred at 25 °C for 1 h. Upon completion, the reaction was pour into H2O (30 mL) and then extracted with EA (15 mL * 3). The combined organic phase was washed with brine (30 mL), dried over anhydrous Na2SO-i, filtered and concentrated in vacuo. The residue was purified by prep-HPLC (column: Welch Xtimate Cl 8 250 * 70 mm # 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-70%, 20 min) to give (lR,2S,5S)-N-((3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7-yl)(cyano)methyl)-6,6-dimethyl-3- ((2S,3R)-3-(l-methylcyclopropoxy)-2-(2,2,2-trifluoroacetamido)butanoyl)-3- azabicyclo[3.1.0]hexane-2-carboxamide (350 mg, 100% purity) as white solid, which was further separated by SEC (condition: column: DAICEL CHIRALPAK IC (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 23%-23%, 7 min) to give (lR,2S,5S)-N-((3-chloro-l-ethyl-lH-
587
SUBSTITUTE SHEET RULE 26 pyrrolo[3,2-c]pyridin-7-yl)(cyano)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l -methylcyclopropoxy)- 2-(2,2,2-trifluoroacetamido)butanoyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (181.8 mg, 291.78 umol, 17.12% yield, 100% purity) as white solid. MS (ESI) m/z 623.4 [M+H]+.
[0001378] 'H NMR (400 MHz, DMSO-de) 5 = 9.82 (d, 1H), 9.60 (d, 1H), 8.88 (s, 1H), 8.52 (s, 1H), 7.85 (s, 1H), 6.61 (d, 1H), 4.36 (t, 1H), 4.23 - 4.05 (m, 3H), 4.00 - 3.86 (m, 2H), 3.69 (d, 1H), 1.66 - 1.56 (m, 1H), 1.46 - 1.36 (m, 3H), 1.35 - 1.28 (m, 4H), 1.19 (d, 3H), 1.02 (s, 3H), 0.85 - 0.71 (m, 4H), 0.69 - 0.61 (m, 1H), 0.41 - 0.29 (m, 2H).
[0001379] (lR,2S,5S)-N-((3-chloro-l-ethyl-lH-pyrrolo[3,2-c]pyridin-7-yl)(cyano)methyl)- 6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclopropoxy)-2-(2,2,2-trifluoroacetamido)butanoyl)-3- azabicyclo[3.1.0]hexane-2-carboxamide (142.5 mg, 226.89 umol, 13.31% yield, 99.2% purity) was obtained as white solid. MS (ESI) m/z 623.4 [M+H]+.
[0001380] 'H NMR (400 MHz, DMSO-de) 5 = 9.83 (d, 1H), 9.39 (d, 1H), 8.86 (s, 1H), 8.52 (s, 1H), 7.83 (s, 1H), 6.57 (d, 1H), 4.35 - 4.09 (m, 4H), 3.98 - 3.91 (m, 1H), 3.91 - 3.81 (m, 1H), 3.59 (d, 1H), 1.56 (s, 1H), 1.41 (t, 3H), 1.30 (s, 3H), 1.24 (d, 1H), 1.05 - 0.91 (m, 6H), 0.84 - 0.70 (m, 4H), 0.66 - 0.58 (m, 1H), 0.34 (t, 2H).
Example 165: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 228A)
Figure imgf000589_0001
[0001381] Step 1: 3-bromo-5-methyl-pyrazolo[l,5-a]pyrazin-4-one
588
SUBSTITUTE SHEET RULE 26 [0001382] To a solution of 3-bromo-5H-pyrazolo[l,5-a]pyrazin-4-one (2 g, 9.34 mmol, 1 eq) in THF (25 mL) was added NaH (448.51 mg, 11.21 mmol, 60% purity, 1.2 eq) at 0 °C. The mixture was stirred at 0 °C for 30 min, then Mel (3.98 g, 28.03 mmol, 1.75 mL, 3 eq) was added. The mixture was stirred at 25 °C for 16 h under N2 atmosphere. Upon completion, the reaction was quenched by addition NH4CI 20 mL at 25 °C, and then extracted with EA 50mL (25 mL *
2). The combined organic layers were washed with brine 50 mL (50 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10:1 to 3:1). Compound 3- bromo-5-methyl-pyrazolo[l,5-a]pyrazin-4-one (1.5 g, 4.70 mmol, 50.26% yield, 71.4% purity) was obtained as a white solid. MS (ESI) m/z 228.1 [M+H]+.
[0001383] Step 2: 5-methyl-3-vinyl-pyrazolo[l,5-a]pyrazin-4-one
[0001384] To a solution of 3-bromo-5-methyl-pyrazolo[l,5-a]pyrazin-4-one (1.5 g, 6.58 mmol, 1 eq) in H2O (20.0 mL) and dioxane (100.0 mL) was added potassium;trifluoro(vinyl)boranuide (1.06 g, 7.89 mmol, 1.2 eq) and CS2CO3 (6.43 g, 19.73 mmol, 3 eq). Then ditert-butyl(cyclopentyl)phosphane; dichloropalladium; iron (428.69 mg, 657.76 umol, 0.1 eq) was added under N2. The mixture was stirred at 80 °C for 16 h. Upon completion, the reaction was poured into H2O 100 mL, and then extracted with DCM (100 mL *
3). The combined organic layers were washed with brine (100 mL * 2), dried over [Na2SO4], filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10:1 to 3: 1). Compound 5- methyl-3-vinyl-pyrazolo[l,5-a]pyrazin-4-one (930 mg, 4.41 mmol, 66.99% yield, 83% purity) was obtained as a yellow solid. MS (ESI) m/z 176.2 [M+H]+.
[0001385] Step 3: 5-methyl-4-oxo-pyrazolo[l,5-a]pyrazine-3-carbaldehyde
[0001386] To a mixture of 5-methyl-3-vinyl-pyrazolo[l,5-a]pyrazin-4-one (900 mg, 5.14 mmol, 1 eq) in dioxane (90 mL) and H2O (18 mL) was added 2,6-dimethylpyridine (1.10 g, 10.27 mmol, 1.20 mL, 2 eq) and dipotassium; di oxi do(dioxo)osmium; dihydrate (378.58 mg, 1.03 mmol, 0.2 eq). The mixture was stirred at 25 °C for 30 min, then NalCh (5.49 g, 25.69 mmol, 1.42 mL, 5 eq) was added at 0 °C, the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction was filtered and then diluted with H2O 50 mL and extracted with EA 100 mL (50 mL * 2). The combined organic layers were washed with BRINE 50 mL (50 mL * 1), dried over
589
SUBSTITUTE SHEET RULE 26 Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 0:1).
[0001387] Compound 5-methyl-4-oxo-pyrazolo[l,5-a]pyrazine-3-carbaldehyde (550 mg, 2.37 mmol, 46.11% yield, 76.3% purity) was obtained as a yellow solid. MS (ESI) m/z 178.3 [M+H]+.
[0001388] Step 4: 2-amino-2-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)acetonitrile
[0001389] To a solution of 5-methyl-4-oxo-pyrazolo[l,5-a]pyrazine-3-carbaldehyde (350 mg, 1.98 mmol, 1 eq) in NH3/MeOH (7 M, 5 mL, 17.72 eq) was added NH4C1 (317.04 mg, 5.93 mmol, 3 eq) , after 1 h, TMSCN (783.98 mg, 7.90 mmol, 988.62 uL, 4 eq) was added drp-wise, the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was concentrated under reduced pressure to give a residue and used directly next step. Compound 2-amino-2-(5-methyl- 4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)acetonitrile (350 mg, 1.72 mmol, 87.18% yield) was obtained as a yellow solid. MS (ESI) m/z 204.3 [M+H]+.
[0001390] Step 5: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001391] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (703.46 mg, 1.72 mmol, 1 eq) in ACN (10 mL) was added TCFH (966.56 mg, 3.44 mmol, 2 eq) and NMI (424.24 mg, 5.17 mmol, 411.88 uL, 3 eq). Then 2-amino-2-(5-methyl-4-oxo- pyrazolo[l,5-a]pyrazin-3-yl)acetonitrile (350 mg, 1.72 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction was quenched by addition H2O 50 mL at 25 °C, and then extracted with DCM 100 mL (50 mL * 2). The combined organic layers were washed with BRINE 50 mL (50 mL * 1), dried over Na2SO-i, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250 * 70 mm * 10 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 35%- 65%, 20 min). Compound (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 503.87 umol, 27.94% yield, 95.0% purity) was obtained as a white solid. MS (ESI) m/z 538.2 [M-55+H]+
590
SUBSTITUTE SHEET RULE 26 [0001392] Step 6: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001393] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 503.87 umol, 1 eq) was separation by SFC (column: REGIS(S,S)WHELK-O1 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 55%-55%,10min) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (70 mg, 117.57 umol, 6.83% yield, 99.7% purity) as a white solid. MS (ESI) m/z 538.2 [M-55+H]+.
[0001394] 1H NMR (400 MHz, DMSO-d6) 5 = 9.71 - 9.45 (m, 1H), 9.37 - 9.23 (m, 1H), 8.12 - 8.02 (m, 1H), 7.87 - 7.80 (m, 1H), 7.26 - 7.21 (m, 1H), 6.49 - 6.43 (m, 1H), 4.40 (br t, J = 8.1 Hz, 1H), 4.26 - 4.21 (m, 1H), 4.05 - 3.78 (m, 2H), 3.69 - 3.62 (m, 1H), 3.48 - 3.45 (m, 3H), 1.57 (dd, J = 5.4, 7.4 Hz, 1H), 1.40 - 1.32 (m, 1H), 1.16 - 1.07 (m, 9H), 1.07 - 0.91 (m, 6H), 0.85 - 0.80 (m, 3H).
[0001395] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (70 mg, 117.81 umol, 6.84% yield, 99.9% purity) was obtained as a white solid. MS (ESI) m/z 538.2 [M-55+H]+.
[0001396] 'H NMR (400 MHz, DMSO-d6) 5 = 9.72 - 9.54 (m, 1H), 9.27 (d, J = 7.6 Hz, 1H), 8.14 - 7.99 (m, 1H), 7.89 - 7.79 (m, 1H), 7.30 - 7.18 (m, 1H), 6.50 - 6.43 (m, 1H), 4.39 (br t, J = 7.8 Hz, 1H), 4.23 (s, 1H), 4.03 - 3.83 (m, 2H), 3.70 - 3.59 (m, 1H), 3.51 - 3.42 (m, 3H), 1.55 (dd, J = 5.4, 7.4 Hz, 1H), 1.34 (d, J = 7.5 Hz, 1H), 1.20 - 1.07 (m, 9H), 1.06 - 0.93 (m, 6H), 0.82 (s, 3H).
591
SUBSTITUTE SHEET RULE 26 Example 166: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 229 A)
Figure imgf000593_0001
[0001397] Step 1: 4-bromo-lH-pyrazole-3 -carboxylic acid
[0001398] A solution of lH-pyrazole-3 -carboxylic acid (100 g, 892.17 mmol, 1 eq) in ACN (1000 mL) was added NBS (158.79 g, 892.17 mmol, 1 eq) and the mixture was stirred at 20 °C for 15 h. Upon completion, the reaction mixture was diluted with water (500 mL) and extracted with EtOAc (500 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 4-bromo-lH-pyrazole-3 -carboxylic acid (200 g, crude) as yellow solid. MS (ESI) m/z 190.9 [M-H]'.
[0001399] Step 2: 4-bromo-N-(2,2-dimethoxyethyl)-lH-pyrazole-3-carboxamide
[0001400] A solution of 4-bromo-lH-pyrazole-3 -carboxylic acid (200 g, 1.05 mol, 1 eq) in thionyl chloride (675.29 g, 5.68 mol, 411.76 mL, 5.42 eq) was stirred at 100 °C for 3 h. Upon completion, the reaction was concentrated in the vacuum to give 4-bromo-lH-pyrazole-3-
592
SUBSTITUTE SHEET RULE 26 carbonyl chloride (200 g, crude) as black brown oil, then a solution of 4-bromo-lH-pyrazole-3- carbonyl chloride (200 g, 954.98 mmol, 1 eq) in DCM (1 L) was added TEA (676.43 g, 6.68 mol, 930.45 mL, 7 eq), then 2,2-dimethoxyethanamine (120.48 g, 1.15 mol, 124.85 mL, 1.2 eq) in DCM (500 mL) was added and the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction was quenched by H2O (1000 mL), and was extracted with DCM (600 mL * 3). The combined organic phase was washed with brine (1500 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum and purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate=l:O to 1: 1) to give 4-bromo-N-(2,2-dimethoxyethyl)-lH-pyrazole-3- carboxamide (160 g, 402.73 mmol, 42.17% yield, 70% purity) as yellow oil. MS (ESI) m/z 278.1 [M-H]-
[0001401] Step 3: 3-bromo-5H-pyrazolo[l,5-a]pyrazin-4-one
[0001402] A mixture of 4-bromo-N-(2,2-dimethoxyethyl)-lH-pyrazole-3-carboxamide (80 g, 287.66 mmol, 1 eq) in DCM (150 mL) was added TLA (126.36 g, 1.11 mol, 82.05 mL, 3.85 eq) and was stirred at 20 °C for 16 h, and then concentrated in vacuo. The residue was suspended in PPA (287.66 mmol, 200 mL, 1 eq) and heated to 145 °C for 4.5 h. Upon completion, the mixture was treated with ice water, and the pH was adjusted to 10 by (NaHCOy aq,). The mixture was extracted with DCM (1 L * 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated in the vacuum and purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:9 to 0: 1) to give 3-bromo-5H-pyrazolo[l,5-a]pyrazin-4-one (8.75 g, 40.88 mmol, 14.21% yield) as yellow solid. MS (ESI) m/z 214.1 [M+H]+
[0001403] Step 4: 3-bromo-4-methoxy-pyrazolo[l,5-a]pyrazine
[0001404] A solution of 3-bromo-5H-pyrazolo[l,5-a]pyrazin-4-one (6.6 g, 30.84 mmol, 1 eq) in dioxane (60 mL) was added Ag2O (14.29 g, 61.68 mmol, 2 eq) then Mel (8.75 g, 61.68 mmol, 3.84 mL, 2 eq) and the mixture was stirred at 40 °C for 12 h. Upon completion, the reaction mixture was filtered and quenched by water (200 mL) and extracted with DCM (200 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure and purified by column chromatography (SiCh, Petroleum ether :Ethyl acetate = 1:0 to 7:3) to give 3-bromo-4-methoxy-pyrazolo[l,5-a]pyrazine (900 mg, 3.95 mmol, 12.80% yield) as yellow solid. MS (ESI) m/z 228.1 [M+H]+.
[0001405] Step 5: 4-methoxy-3-vinyl-pyrazolo[l,5-a]pyrazine
593
SUBSTITUTE SHEET RULE 26 [0001406] A solution of 3-bromo-4-methoxy-pyrazolo[l,5-a]pyrazine (900 mg, 3.95 mmol, 1 eq) in dioxane (20 mL), H2O (5 mL) was added potassium;trifluoro(vinyl)boranuide (634.37 mg,
4.74 mmol, 1.2 eq) then CS2CO3 (2.57 g, 7.89 mmol, 2 eq) and followed by Pd(dppf)Ch (288.77 mg, 394.66 umol, 0.1 eq) and the mixture was stirred at 80 °C for 4 h. Upon completion, the reaction was quenched by H2O (30 mL) and was extracted with ethyl acetate (30 mL * 3). The combined organic phase was washed with brine (10 mL * 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuum and purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate=l:O to 1 :1) to give 4-methoxy-3-vinyl-pyrazolo[l,5-a]pyrazine (460 mg, 1.84 mmol, 46.57% yield, 70% purity) as yellow solid. MS (ESI) m/z 176.1 [M+H]+.
[0001407] Step 6: 4-methoxypyrazolo[l,5-a]pyrazine-3-carbaldehyde
[0001408] A solution of 4-methoxy-3-vinyl-pyrazolo[l,5-a]pyrazine (460 mg, 2.63 mmol, 1 eq) in H2O (1 mL), dioxane (5 mL) was added K2OSO4.2H2O (193.50 mg, 525.15 umol, 0.2 eq) and the mixture was stirred at 20 °C for 0.5 h, then the mixture was added NalCh (2.81 g, 13.13 mmol, 727.49 uL, 5 eq) and stirred at 20 °C for 13.5 h. Uppon completion, the reaction was quenched by H2O (30 mL) and was extracted with ethyl acetate (30 mL * 3). The combined organic phase was washed with brine (10 mL * 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum and purified by prep-TLC(SiO2, PE:EA = 1 : 1) to give 4- methoxypyrazolo[l,5-a]pyrazine-3-carbaldehyde (230 mg, 1.25 mmol, 47.47% yield, 96% purity) as yellow solid. MS (ESI) m/z 178.1 [M+H]+.
[0001409] Step 7: 2-amino-2-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)acetonitrile
[0001410] A solution of 4-methoxypyrazolo[l,5-a]pyrazine-3-carbaldehyde (230 mg, 1.25 mmol, 96% purity, 1 eq) in NHVMeOH (7 M, 4.80 mL, 26.96 eq) was added NH4CI (200.00 mg,
3.74 mmol, 3 eq) and the mixture was stirred at 20 °C for 0.5 h, then was cooled at 0 °C and added TMSCN (494.58 mg, 4.99 mmol, 623.68 uL, 4 eq) and the mixture was stirred at 20 °C for 0.5 h. Upon completion, the reaction was quenched by H2O (10 mL), and was extracted with DCM (10 mL * 3). The combined organic phase was washed with brine (10 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 2-amino-2-(4- methoxypyrazolo[l,5-a]pyrazin-3-yl)acetonitrile (240 mg, crude) as yellow solid. MS (ESI) m/z 204.1 [M+H]+.
594
SUBSTITUTE SHEET RULE 26 [0001411] Step 8: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001412] A solution of 2-amino-2-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)acetonitrile (240 mg, 1.18 mmol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (530.12 mg, 1.30 mmol, 1.1 eq) then NMI (290.92 mg, 3.54 mmol, 282.44 uL, 3 eq) and followed by TCFH (662.78 mg, 2.36 mmol, 2 eq) and the mixture was stirred at 20 °C for 2 h. Upon completion, the reaction was quenched by H2O (30 mL), and was extracted with DCM (30 mL * 3). The combined organic phase was washed with brine (50 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum and purified by column chromatography ( S 1O2, Petroleum ether:Ethyl acetate=l :O to 3:7) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (480 mg, 727.77 umol, 61.68% yield, 90% purity) as white solid. MS (ESI) m/z 594.2 [M+H]+.
[0001413] Step 9: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001414] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (480 mg, 808.63 umol, 1 eq) was separated by SFC (column: DAI CEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 20%-20%, 7 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-[cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (66.88 mg, 112.67 umol, 13.93% yield, 100% purity) as white solid. MS (ESI) m/z 538.2 [M+H-56]+.
[0001415] 'H NMR (400 MHz, DMSO-de) 8 = 9.67 - 9.50 (m, 1H), 9.11 (d, J = 7.2 Hz, 1H), 8.44 - 8.39 (m, 1H), 8.25 - 8.14 (m, 1H), 7.54 - 7.48 (m, 1H), 6.46 - 6.22 (m, 1H), 4.39 (br s,
595
SUBSTITUTE SHEET RULE 26 1H), 4.24 (s, 1H), 4.06 - 4.00 (m, 3H), 4.00 - 3.84 (m, 2H), 3.66 - 3.42 (m, 1H), 1.60 - 1.40 (m, 1H), 1.27 (d, J = 7.5 Hz, 1H), 1.16 - 1.08 (m, 9H), 1.01 - 0.95 (m, 6H), 0.81 (s, 3H)
[0001416] (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N- [cyano-(4-methoxypyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2(82.6 mg, 139.15 umol, 17.21% yield, 100% purity) was obtained as white solid. MS (ESI) m/z 538.2 [M+H-56]+.
[0001417] 'H NMR (400 MHz, DMSO-de) 8 = 9.66 - 9.40 (m, 1H), 9.19 (d, J = 6.7 Hz, 1H),
8.48 - 8.41 (m, 1H), 8.24 - 8.20 (m, 1H), 7.53 (d, J = 4.9 Hz, 1H), 6.39 - 6.17 (m, 1H), 4.39 (t, J = 8.0 Hz, 1H), 4.27 (s, 1H), 4.06 (s, 3H), 3.99 - 3.82 (m, 2H), 3.71 - 3.43 (m, 1H), 1.62 - 1.56 (m, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.13 - 0.96 (m, 15H), 0.82 (s, 3H)
Example 167: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 230A)
Figure imgf000597_0001
[0001418] Step 1: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxamide
[0001419] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (0.54 g, 1.47 mmol, 1 eq), 2-amino-2-(5-ethynyl-4-isoquinolyl)acetonitrile (507.71 mg, 1.47 mmol, 60% purity, 1 eq) in ACN (15 mL) was added TCFH (824.91 mg, 2.94 mmol, 2 eq) and NMI (362.06 mg, 4.41 mmol, 351.52 uL, 3 eq). The mixture was stirred at 20 °C for 1 h. Upon completion, the mixture was diluted with H2O (100 mL) and then extracted with DCM (80 mL * 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under
596
SUBSTITUTE SHEET RULE 26 reduced pressure and was purified by prep-HPLC (column: Cl 8 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCCh)-ACN]; B%: 35%-60%, 10 min) to get product (3S,3aS,6aR)-N-[(4- bromopyrazolo[l,5-a]pyridin-3-yl)-cyano-methyl]-2-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrole-3- carboxamide (0.65 g, 1.17 mmol, 79.59% yield) as yellow solid. MS (ESI) m/z 556.3 [M+H]+
[0001420] Step 2: (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001421] The (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-methoxy- 2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (0.65 g, 1.17 mmol, 1 eq) was separation by SFC (column: REGIS (S, S) WHELK- 01 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 30%-30%, 8 min) to get product (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 1 (0.21 g, 374.23 umol, 31.98% yield, 99% purity) as white solid. MS (ESI) m/z 556.3 [M+H]+
[0001422] 'H NMR (400 MHz, DMSO-de) 5 = 9.86 (br s, 1H), 9.50 - 9.43 (m, 2H), 8.81 (s, 1H), 8.34 (dd, J = 1.1, 8.2 Hz, 1H), 8.14 (dd, J = 1.1, 7.3 Hz, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 6.7 Hz, 1H), 4.70 (s, 1H), 4.41 - 4.23 (m, 2H), 3.95 - 3.79 (m, 2H), 3.72 - 3.64 (m, 1H), 3.26 (s, 3H), 1.54 (dd, J = 5.2, 7.5 Hz, 1H), 1.35 (d, J = 7.6 Hz, 1H), 1.19 (d, J = 6.2 Hz, 3H), 0.98 (s, 3H), 0.84 (s, 3H)
[0001423] (lR,2S,5S)-N-[cyano-(5-ethynyl-4-isoquinolyl)methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (0.21 g, 374.23 umol, 31.98% yield, 99% purity) was obtained as white solid. MS (ESI) m/z 556.3 [M+H]+
[0001424] 'H NMR (400 MHz, DMSO-de) 5 = 9.91 - 9.70 (m, 2H), 9.46 (s, 1H), 8.82 (s, 1H), 8.33 (dd, J = 1.0, 8.2 Hz, 1H), 8.18 - 8.09 (m, 1H), 7.79 (t, J = 7.7 Hz, 1H), 7.67 (d, J = 7.0 Hz, 1H), 4.63 (s, 1H), 4.34 (s, 2H), 3.95 - 3.79 (m, 2H), 3.57 (dd, J = 6.2, 8.6 Hz, 1H), 3.18 (s, 3H), 1.59 (dd, J = 5.3, 7.3 Hz, 1H), 1.46 (d, J = 7.6 Hz, 1H), 1.10 - 1.02 (m, 6H), 0.89 (s, 3H).
597
SUBSTITUTE SHEET RULE 26 Example 168: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl (methyl |-6.6-dimethyl-3-|(2S.3R)-3-( 1 -methylcyclopropoxy )-2-|( 2.2.2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound
231 A)
Figure imgf000599_0001
[0001425] Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001426] To a solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (476.41 mg, 1.17 mmol, 1 eq) in ACN (5 mL) was added NMI (288.75 mg, 3.52 mmol, 280.34 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (657.84 mg, 2.34 mmol, 2 eq). And then 2-amino-2-(3-ethynylthieno [2,3-c]pyridin-4-yl)acetonitrile (250 mg, 1.17 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was blow-dried with N2 to give a residue. The residue was purified by prep- HPLC ( column: Waters Xbridge BEH C18 250*70mm*10um;mobile phase: [water(NH4HCO3)- ACN];B%: 40%-70%,20min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (450 mg, 725.52 umol, 61.89% yield, 97% purity) as a white solid. MS (ESI) m/z 602.3 [M+H]+
[0001427] Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
598
SUBSTITUTE SHEET RULE 26 [0001428] The (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide was separated by SFC (column: REGIS(S,S)WHELK- Ol(250mm * 25mm,10um);mobile phase: [Neu-IPA];B%: 50%-50%,6min) to give (1R,2S,5S)- N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl) amino] butanoyl] -3 -azabicyclo [3.1 ,0]hexane-2- carboxamide (130 mg, 216.08 umol, 28.89% yield) as a white solid. MS (ESI) m/z 602.3 [M+H]+
[0001429] 'H NMR (400 MHz, MeOD-d4) 5 = 9.30 (s, 1H), 8.79 (s, 1H), 8.44 (s, 1H), 7.14 (s, 1H), 4.54 - 4.44 (m, 1H), 4.32 (s, 1H), 4.10 - 3.96 (m, 2H), 3.94 - 3.82 (m, 2H), 1.60 (dd, J = 5.3, 7.5 Hz, 1H), 1.49 (d, J = 7.6 Hz, 1H), 1.39 (s, 3H), 1.26 (d, J = 6.2 Hz, 3H), 1.05 - 1.02 (m, 3H), 0.89 (s, 3H), 0.82 - 0.77 (m, 2H), 0.42 (dt, J = 1.7, 3.1 Hz, 2H)
[0001430] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (120 mg, 199.46 umol, 26.67% yield) was obtained as a white solid. MS (ESI) m/z 602.3 [M+H]+
[0001431] 'H NMR (400 MHz, MeOD-d4) 5 = 9.42 - 9.26 (m, 1H), 8.82 - 8.75 (m, 1H), 8.43 (s, 1H), 7.25 (s, 1H), 4.51 - 4.34 (m, 2H), 4.00 (t, J = 6.6 Hz, 1H), 3.97 - 3.91 (m, 2H), 3.85 - 3.80 (m, 1H), 1.62 (dd, J = 5.1, 7.5 Hz, 1H), 1.51 (d, J = 7.6 Hz, 1H), 1.31 (s, 3H), 1.16 (d, J = 6.2 Hz, 3H), 1.11 - 1.07 (m, 3H), 0.93 (s, 3H), 0.75 - 0.67 (m, 1H), 0.63 - 0.56 (m, 1H), 0.35 - 0.26 (m, 2H)
599
SUBSTITUTE SHEET RULE 26 Example 169: Synthesis of (lR,2S,5S)-N-[[3-chloro-l-(2- triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7-yl]-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-
[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Compound 232A)
Figure imgf000601_0001
[0001432] Step 1: 7-vinyl-lH-pyrrolo[3,2-c]pyridine
[0001433] A solution of 7-bromo-lH-pyrrolo[3,2-c]pyridine (5 g, 25.38 mmol, 1 eq) and potassium;trifluoro(vinyl)boranuide (4.08 g, 30.45 mmol, 1.2 eq) in H2O (15 mL) and Tol. (60 mL) was added ditert-butyl (cyclopentyl)phosphane;dichloropalladium;iron (1.65 g, 2.54 mmol, 0.1 eq) and CS2CO3 (24.80 g, 76.13 mmol, 3 eq) under N2 atmosphere. The mixture was stirred at 80 °C for 16 h. Upon completion, the reaction was filtered to give the filtrate, then the filtrate was diluted with H2O (30 mL) and extracted with EA (25 mL * 3). The combined organic layers were washed with brine 40 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, Petroleum
600
SUBSTITUTE SHEET RULE 26 ether:Ethyl acetate = 1:0 to 0:1). To give the product 7-vinyl-lH-pyrrolo[3,2-c]pyridine (2.7 g, 16.29 mmol, 64.20% yield, 87% purity) as a yellow solid. MS (ESI) m/z 145.2 [M+H]+.
[0001434] Step 2: 3-chloro-7-vinyl-lH-pyrrolo[3,2-c]pyridine
[0001435] A solution of 7-vinyl-lH-pyrrolo[3,2-c]pyridine (2.7 g, 18.73 mmol, 1 eq) in DMF (40 mL) was added NCS (3.00 g, 22.47 mmol, 1.2 eq) and was stirred at 25 °C for 5 h. Upon completion, the reaction was diluted with H2O (80 mL) and extracted with EA (50 mL * 3). The combined organic layers were washed with brine 80 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:0 to 0:1). To give the product 3-chloro- 7-vinyl-lH-pyrrolo[3,2-c]pyridine (2 g, 9.85 mmol, 52.61% yield, 88% purity) as a yellow solid. MS (ESI) m/z 179.1 [M+H]+.
[0001436] Step 3: 2-(3-chloro-7-vinyl-pyrrolo[3,2-c]pyridin-l-yl)ethynyl-triisopropyl-silane
[0001437] A solution of 3-chloro-7-vinyl-lH-pyrrolo[3,2-c]pyridine (500 mg, 2.80 mmol, 1 eq), K3PO4 (1.19 g, 5.60 mmol, 2 eq), Nl,N2-dimethylcyclohexane-l,2-diamine (199.09 mg, 1.40 mmol, 0.5 eq), 2-bromoethynyl(triisopropyl)silane (1.46 g, 5.60 mmol, 2 eq) in DMSO (10 mL) was added Cui (53.31 mg, 279.93 umol, 0.1 eq) under N2 atmosphere and the mixture was stirred at 130 °C for 16 h. Upon completion, the reaction was diluted with H2O (20 mL) and extracted with EA (10 mL * 3). The combined organic layers were washed with brine 15 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 50: 1 to 20: 1). Compound 2-(3-chloro-7-vinyl-pyrrolo[3,2-c]pyridin-l-yl)ethynyl-triisopropyl-silane (310 mg, 725.39 umol, 25.91% yield, 84% purity) was obtained as a yellow oil. MS (ESI) m/z 359.3 [M+H]+.
[0001438] Step 4: 3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2-c]pyridine-7- carbaldehyde
[0001439] To a mixture of 2-(3-chloro-7-vinyl-pyrrolo[3,2-c]pyridin-l-yl)ethynyl-triisopropyl- silane (300 mg, 835.70 umol, 1 eq) in dioxane (25 mL) and H2O (5 mL) was added dipotassium; dioxido(dioxo)osmium;dihydrate (61.58 mg, 167.14 umol, 0.2 eq) and 2,6- dimethylpyridine (179.10 mg, 1.67 mmol, 194.67 uL, 2 eq). The mixture was stirred at 25 °C for
601
SUBSTITUTE SHEET RULE 26 30 min, then NalCh (893.75 mg, 4.18 mmol, 231.54 uL, 5 eq) was added at 0 °C, the mixture was stirred at 25 °C for 16 h. Upon completion, the reaction was filtered and then diluted with H2O 50 mL and extracted with EA 100 mL (50 mL * 2). The combined organic layers were washed with brine 50 mL (50 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, Petroleum ether:Ethyl acetate = 10:1 to 3:1). Compound 3-chloro-l-(2- triisopropylsilylethynyl)pyrrolo[3,2-c]pyridine-7-carbaldehyde (200 mg, 434.96 umol, 52.05% yield, 78.5% purity) was obtained as a yellow oil. MS (ESI) m/z 361.2 [M+H]+.
[0001440] Step 5: 2-amino-2-[3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7- yl]acetonitrile
[0001441] To a solution of 3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2-c]pyridine-7- carbaldehyde (90 mg, 249.34 umol, 1 eq) in NEE/MeOH (7 M, 3 mL, 84.22 eq) was added NH4CI (40.01 mg, 748.02 umol, 3 eq), after 1 h, TMSCN (98.94 mg, 997.36 umol, 124.77 uL, 4 eq) was added drop-wise, the mixture was stirred at 25 °C for 1 h. Upon completion, the reaction was quenched by addition H2O 20 mL and stirred for 5 min, then filtered and extracted with DCM 40 mL (20 mL * 2). The combined organic layers were washed with brine 30 mL (30 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-[3- chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7-yl]acetonitrile (70 mg, 180.88 umol, 72.54% yield) as a yellow solid. MS (ESI) m/z 387.3 [M+H]+.
[0001442] Step 6: (lR,2S,5S)-N-[[3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2- c]pyridin-7-yl]-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001443] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (66.26 mg, 180.88 umol, 1 eq) in ACN (4 mL) was added TCFH (101.50 mg, 361.76 umol, 2 eq) and NMI (44.55 mg, 542.65 umol, 43.25 uL, 3 eq). Then 2-amino-2-[3-chloro-l-(2- triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7-yl]acetonitrile (70 mg, 180.88 umol, 1 eq) was added. The mixture was stirred at 25 °C for 16 h. Upon completion, the reaction was quenched by addition H2O 20 mL at 25 °C, and then extracted with DCM 40 mL (20 mL * 2). The combined organic layers were washed with BRINE 40 mL (40 mL * 1), dried over Na2SO4,
602
SUBSTITUTE SHEET RULE 26 filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, Petroleum ether:Ethyl acetate = 1: 1) to give (lR,2S,5S)-N-[[3-chloro-l-(2- triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7-yl]-cyano-methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (30 mg, 85.7% purity ) as a white solid. MS (ESI) m/z 735.5 [M+H]+.
[0001444] Step 7: (lR,2S,5S)-N-[[3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2- c]pyridin-7-yl]-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001445] (lR,2S,5S)-N-[[3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7-yl]- cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (30 mg, 85.7% purity) was separation by SFC (column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 urn); mobile phase: [Neu-IPA]; B%: 11%- 11%, 21 min) to give (lR,2S,5S)-N-[[3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2- c]pyridin-7-yl]-cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (4.0 mg, 5.18 umol, 2.86% yield, 95.2% purity) was obtained as a white solid. MS (ESI) m/z 735.2 [M+H]+.
[0001446] 'H NMR (400 MHz, MeOD-d4) 5 = 8.93 (s, 1H), 8.63 (s, 1H), 7.74 (s, 1H), 7.36 (s, 1H), 4.59 (br s, 1H), 4.50 (d, J = 8.0 Hz, 1H), 4.41 (s, 1H), 3.99 - 3.94 (m, 1H), 3.76 - 3.70 (m, 1H), 3.36 (s, 3H), 1.59 - 1.53 (m, 1H), 1.34 - 1.23 (m, 7H), 1.23 - 1.15 (m, 18H), 1.05 (s, 3H), 0.98 (s, 3H).
[0001447] (lR,2S,5S)-N-[[3-chloro-l-(2-triisopropylsilylethynyl)pyrrolo[3,2-c]pyridin-7-yl]- cyano-methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (4.0 mg, 5.06 umol, 2.80% yield, 93.1% purity) was obtained as a white solid. MS (ESI) m/z 735.2 [M+H]+.
[0001448] 'H NMR (400 MHz, MeOD-d4) 5 = 8.80 (s, 1H), 8.57 (s, 1H), 7.63 (s, 1H), 7.27 (s, 1H), 4.38 - 4.28 (m, 2H), 3.90 - 3.80 (m, 2H), 3.53 - 3.43 (m, 1H), 3.08 (s, 3H), 1.56 (br dd, J = 4.5, 6.9 Hz, 1H), 1.30 (d, J = 7.6 Hz, 1H), 1.22 - 1.13 (m, 6H), 1.12 - 1.08 (m, 18H), 1.01 (s, 3H), 0.91 - 0.85 (m, 3H).
603
SUBSTITUTE SHEET RULE 26 Example 170: Synthesis of (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino] butanoyl] -3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 233A)
Figure imgf000605_0001
[0001449] Step l:5-prop-l-ynylisoquinoline-4-carbaldehyde
[0001450] A mixture of 5-bromoisoquinoline-4-carbaldehyde (2 g, 8.47 mmol, 1 eq), 4, 4, 5, 5- tetramethyl-2-prop-l-ynyl-l,3,2-dioxaborolane (4.22 g, 25.42 mmol, 3 eq), Cs2CO3 (5.52 g, 16.94 mmol, 2 eq), ditert-butyl(cyclopentyl)phosphane; di chloropalladium; iron (276.09 mg, 423.61 umol, 0.05 eq) in dioxane (20 mb) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80 °C for 3 hr under N2 atmosphere. Upon completion, The residue was poured into water (15 mL) and filtered. The aqueous phase was extracted with ethyl acetate (15 mL*3). The combined organic phase was washed with brine (15 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=20/l, 0/1) to give 5-prop-l-ynylisoquinoline-4-carbaldehyde (800 mg, 3.69 mmol, 43.53% yield, 90% purity) as a brown solid. MS (ESI) m/z 196.3 [M+H]+.
[0001451] Step 2: 2-amino-2-(5-prop-l-ynyl-4-isoquinolyl)acetonitrile
[0001452] To a solution of 5-prop-l-ynylisoquinoline-4-carbaldehyde (800 mg, 4.10 mmol, 1 eq) in DCM (20 mL) was added NH3/MeOH (7 M, 4.10 mL, 7 eq) and Ti(i-PrO)4 (1.75 g, 6.15 mmol, 1.81 mL, 1.5 eq), the solution was stirred at 20 °C for 1 h, then TMSCN (1.22 g, 12.29
604
SUBSTITUTE SHEET RULE 26 mmol, 1.54 mL, 3 eq) was added at 0 °C, the solution was sitrred at 20°C for 15 h. Upon completion, The residue was poured into water(15 mL) and filtered. The aqueous phase was extracted with ethyl acetate (15 mL*3). The combined organic phase was washed with brine (15 mL*3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=20/l, 0/1) to give 2-amino-2-(5-prop-l-ynyl-4- isoquinolyl)acetonitrile (600 mg, 2.18 mmol, 52.93% yield, 80% purity) as a brown oil. MS (ESI) m/z 222.3 [M+H]+.
[0001453] Step 3:(lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001454] To a mixture of 2-amino-2-(5-prop-l-ynyl-4-isoquinolyl)acetonitrile (600 mg, 2.71 mmol, 1 eq) and (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1.10 g, 2.71 mmol, 1 eq) in ACN (10 mL) was added TCFH (1.52 g, 5.42 mmol, 2 eq) and NMI (779.26 mg, 9.49 mmol, 756.57 uL, 3.5 eq) in one portion at 25°C. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was quenched by addition water 20 mL, and then extracted with DCM (10 mL * 3). The combined organic layers were washed with brine (10 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC(column: Waters Xbridge BEH Cl 8 250*70mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 55%-80%,15min) to give a (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (300 mg, 492.10 umol, 18.15% yield) as a rown solid. MS (ESI) m/z 610.3[M+H]+.
[0001455] Step 4: (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 and 2
[0001456] (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (300 mg, 492.10 umol, 1 eq) was separated by SFC
605
SUBSTITUTE SHEET RULE 26 (column: DAI CEL CHIRALPAK AD(250mm*30mm,10um);mobile phase: [Neu-IPA];B%: 33%-33%,7min) to give (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (62 mg, 96.61 umol, 19.63% yield, 95% purity) as white solid. MS (ESI) m/z 610.3 [M+H]+
[0001457] 'H NMR (400 MHz, DMSO-de) 5 = 9.82 (br d, J = 8.3 Hz, 1H), 9.53 - 9.38 (m, 1H), 9.31 (br d, J = 6.4 Hz, 1H), 8.85 - 8.69 (m, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.08 - 7.94 (m, 1H), 7.81 - 7.54 (m, 2H), 4.35 (t, J = 8.4 Hz, 1H), 4.29 (s, 1H), 3.94 - 3.83 (m, 2H), 3.70 (d, J = 10.5 Hz, 1H), 2.13 (s, 3H), 1.66 - 1.50 (m, 1H), 1.44 (d, J = 7.5 Hz, 1H), 1.29 (s, 3H), 1.19 - 1.11 (m, 3H), 1.04 (s, 3H), 0.82 (s, 3H), 0.77 - 0.71 (m, 1H), 0.64 - 0.56 (m, 1H), 0.36 - 0.24 (m, 2H)
[0001458] (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (62 mg, 101.70 umol, 20.67% yield, 100% purity) Isomer 2 was obtained as a white solid. MS (ESI) m/z 610.3 [M+H]+
[0001459] 'H NMR (400 MHz, DMSO-de) 5 = 9.82 (d, J = 8.2 Hz, 1H), 9.55 - 9.37 (m, 2H), 8.82 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H), 7.81 - 7.71 (m, 2H), 4.35 (t, J = 8.4 Hz, 1H), 4.29 (s, 1H), 3.97 - 3.82 (m, 2H), 3.70 (br d, J = 10.4 Hz, 1H), 2.89 (s, 1H), 2.13 (s, 3H), 1.62 - 1.56 (m, 1H), 1.44 (d, J = 7.5 Hz, 1H), 1.29 (s, 3H), 1.19 - 1.11 (m, 3H), 1.04 (s, 3H), 0.82 (s, 3H), 0.77 - 0.71 (m, 1H), 0.65 - 0.56 (m, 1H), 0.35 - 0.26 (m, 2H)
Example 171: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-(l-cyclopropyl cyclopropoxy)-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
(Compound 234A)
Figure imgf000607_0001
SUBSTITUTE SHEET RULE 26 [0001460] Step 1: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(l -cyclopropyl cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001461] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino] butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (506.94 mg, 1.17 mmol, 1 eq) in ACN (10 mL) was added NMI (288.73 mg, 3.52 mmol, 280.32 uL, 3 eq) and [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (657.84 mg, 2.34 mmol, 2 eq). And then 2-amino-2-(3-ethynylthieno[2,3-c]pyridin-4- yl)acetonitrile (250 mg, 1.17 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O (20 mL) and extracted with Ethyl acetate 45 mL (15 mL * 3). The combined organic layers were washed with BRINE 100 mL (20 mL * 5), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition; column: Waters Xbridge BEH Cl 8 250*70mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-70%,20min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (364 mg, 538.74 umol, 45.96% yield, 92.9% purity) was obtained as a light yellow solid. MS (ESI) m/z 628.4 [M+H]+.
[0001462] Step 2: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(l -cyclopropyl cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001463] The residue was purified by separated by SEC (column:column:REGIS(S,S)WHELK-Ol (250mm*25mm,10um);mobile phase: [Neu-IPA];B%: 33%-33%,8min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3- [(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (88.05 mg, 140.28 umol, 25.45% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 628.4 [M+H]+.
[0001464] 'H NMR (400 MHz, DMSO-t/r,) 8 = 9.80 (br d, J= 8.3 Hz, 1H), 9.43 (s, 2H), 8.79 - 8.53 (m, 2H), 7.04 (br d, J= 6.6 Hz, 1H), 4.52 (s, 1H), 4.41 (t, J= 8.3 Hz, 1H), 4.24 (s, 1H), 4.11
607
SUBSTITUTE SHEET RULE 26 - 4.01 (m, 1H), 3.93 (dd, J= 5.4, 10.4 Hz, 1H), 3.67 (d, J= 10.4 Hz, 1H), 1.61 - 1.50 (m, 1H), 1.48 - 1.34 (m, 2H), 1.28 (d, J= 6.1 Hz, 3H), 0.97 (s, 3H), 0.80 (s, 3H), 0.73 - 0.61 (m, 1H), 0.56 - 0.39 (m, 3H), 0.37 - 0.28 (m, 1H), 0.26 - 0.17 (m, 1H), 0.15 - 0.04 (m, 2H)
[0001465] (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxamide (169.28 mg, 269.69 umol, 48.92% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 628.4 [M+H]+.
[0001466] 'H NMR (400 MHz, DMSO-t/r,) 8 = 9.78 (br d, J= 7.7 Hz, 1H), 9.65 (br d, J= 5.7
Hz, 1H), 9.42 (s, 1H), 8.71 (s, 1H), 8.63 (s, 1H), 7.15 (br s, 1H), 4.50 (s, 1H), 4.37 (br t, J= 8.0 Hz, 1H), 4.31 (s, 1H), 4.05 - 3.86 (m, 2H), 3.66 (d, J= 10.5 Hz, 1H), 1.62 - 1.54 (m, 1H), 1.48 (d, J= 7.5 Hz, 1H), 1.40 - 1.28 (m, 1H), 1.18 (d, J = 6.1 Hz, 3H), 1.06 - 1.01 (m, 3H), 0.84 (s, 3H), 0.61 (td, J= 5.5, 11.0 Hz, 1H), 0.46 - 0.36 (m, 3H), 0.30 - 0.21 (m, 1H), 0.20 - 0.11 (m, 1H), 0.10 - 0.06 (m, 2H).
Example 172: Synthesis of (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 235A)
Figure imgf000609_0001
[0001467] Step 1: (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3-
[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001468] To a solution of 2-amino-2-(5-prop-l-ynyl-4-isoquinolyl)acetonitrile (400 mg, 1.45 mmol, 80% purity, 1 eq) in ACN (4 mL) was added (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-
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SUBSTITUTE SHEET RULE 26 carboxylic acid (760.06 mg, 1.45 mmol, 80% purity, 1 eq), TCFH (608.70 mg, 2.17 mmol, 1.5 eq) and NMI (356.24 mg, 4.34 mmol, 345.86 uL, 3 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was quenched by addition H2O (0.5 mL) at 25 °C, and then diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL * 2). The combined organic layers were washed with sat. NaCl (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Cl 8 80*40mm*3um;mobile phase: [water (NH4HCO3)-ACN];B%: 47%- 67%,8min) to give (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (250 mg, 400.86 umol, 27.72% yield, 100% purity) as white solid. MS (ESI) m/z 624.5 [M+H]+
[0001469] Step 2: (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001470] (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (240 mg, 384.82 umol, 1 eq) was purified by SFC (column: DAI CEL CHIRALPAK AD (250mm*30mm,10um);mobile phase: [Neu-IPA];B%: 27%-27%,9min) to give the product (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]- 6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (63 mg, 101.02 umol, 26.25% yield, 100% purity). MS (ESI) m/z 624.2 [M+H]+
[0001471] 'H NMR (400 MHz, DMSO-de) 5 = 9.73 (m, J = 6.1 Hz, 1H), 9.49 - 9.42 (m, 1H), 9.27 (d, J = 6.2 Hz, 1H), 8.80 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 6.6 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.62 (d, J = 6.3 Hz, 1H), 4.44 (m, J = 6.0 Hz, 1H), 4.27 (s, 1H), 4.01 - 3.85 (m, 2H), 3.68 (d, J = 10.5 Hz, 1H), 2.14 - 2.04 (m, 5H), 1.80 - 1.69 (m, 2H), 1.61 - 1.45 (m, 3H), 1.37 - 1.33 (m, 1H), 1.27 (s, 3H), 1.09 (d, J = 6.2 Hz, 3H), 1.00 - 0.95 (m, 3H), 0.80 (s, 3H)
[0001472] (lR,2S,5S)-N-[cyano-(5-prop-l-ynyl-4-isoquinolyl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-
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SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (67 mg, 107.43 umol, 27.92% yield, 100% purity) was obtained as white solid. MS (ESI) m/z 624.3 [M+H]+
[0001473] 'H NMR (400 MHz, DMSO-de) 5 = 9.73 (br d, J = 6.5 Hz, 1H), 9.54 - 9.33 (m, 2H), 8.81 (s, 1H), 8.24 (dd, J = 1.0, 8.2 Hz, 1H), 7.99 (d, J = 6.7 Hz, 1H), 7.83 - 7.67 (m, 2H), 4.45 - 4.38 (m, 1H), 4.30 (s, 1H), 3.94 (dd, J = 5.4, 10.6 Hz, 1H), 3.87 - 3.78 (m, 1H), 3.69 (d, J = 10.5 Hz, 1H), 2.13 (s, 3H), 2.07 - 1.97 (m, 2H), 1.76 - 1.56 (m, 3H), 1.55 - 1.41 (m, 3H), 1.22 (s, 3H), 1.10 - 0.96 (m, 6H), 0.84 (s, 3H)
Example 173: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 236A)
Figure imgf000611_0001
[0001474] Step 1: (lR,2S,5S)-N-[cyano-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7- naphthyridin-4-yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxamide
[0001475] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (536.41 mg, 1.46 mmol, 1 eq) in ACN (3 mb) was added NMI (360.65 mg, 4.39 mmol, 350.14 uL, 3 eq) and 2-amino-2-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4- yl]acetonitrile (500 mg, 1.61 mmol, 1.1 eq). And then TCFH (821.69 mg, 2.93 mmol, 2 eq) was
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SUBSTITUTE SHEET RULE 26 added at 0 °C. The mixture was stirred at 25 °C for 1 h. Upon completion, the combined was diluted with water (20 mL) and extracted with Ethyl acetate (20 mL). And then filtered to give a solution was extracted with Ethyl acetate (20 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 10/1 to 1/1) to give (lR,2S,5S)-N-[cyano-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4-yl]methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (905 mg, 1.37 mmol, 93.82% yield) as a light yellow solid. MS (ESI) m/z 659.5 [M+H]+
[0001476] Step 2: (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]- 3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001477] To a solution of (lR,2S,5S)-N-[cyano-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7- naphthyridin-4-yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (900 mg, 1.37 mmol, 1 eq) in THF (8 mL) was added 18-CROWN-6 (361.12 mg, 1.37 mmol, 1 eq) and KF (238.12 mg, 4.10 mmol, 96.02 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the combined was diluted with water (20 mL) and extracted with DCM (20 mL). And then filtered to give a solution was extracted with DCM (20 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (column: Waters Xbridge BEH C18 250*70mm*10um;mobile phase: [water( NH4HCO3)- ACN];B%: 35%-65%,20min) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7- naphthyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (450 mg, 636.76 umol, 46.61% yield, 83% purity) as a white solid. MS (ESI) m/z 587.4 [M+H]+
[0001478] Step 3: (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]- 3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001479] The (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-
611
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxamide was separated by SFC (column: DAICEL CHIRALPAK AD(250mm*30mm,10 um);mobile phase: [Neu-IPA];B%: 26%-26%,5min) to give (1R,2S,5S)- N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino] butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 170.49 umol, 22.22% yield, 100% purity) as a white solid. MS (ESI) m/z 587.3 [M+H]+
[0001480] 'H NMR (400 MHz, DMSO-de) 5 = 9.85 (br s, 1H), 9.52 (s, 1H), 9.45 - 9.38 (m, 2H), 8.81 (s, 1H), 7.38 (d, J = 6.6 Hz, 1H), 4.81 (s, 1H), 4.37 (br d, J = 8.3 Hz, 1H), 4.24 (s, 1H), 4.12 (s, 3H), 3.97 - 3.88 (m, 1H), 3.86 - 3.78 (m, 1H), 3.73 - 3.62 (m, 1H), 3.26 (s, 3H), 1.55 (dd, J = 5.4, 7.4 Hz, 1H), 1.35 (d, J = 7.6 Hz, 1H), 1.18 (d, J = 6.1 Hz, 3H), 0.98 (s, 3H), 0.84 (s, 3H)
[0001481] (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (100 mg, 170.14 umol, 22.18% yield, 99.8% purity) was obtained as a white solid. MS (ESI) m/z 587.3 [M+H]+
[0001482] 'H NMR (400 MHz, DMSO-de) 5 = 9.83 (br s, 1H), 9.72 (br d, J = 7.0 Hz, 1H), 9.50 (s, 1H), 9.41 (s, 1H), 8.82 (s, 1H), 7.51 (d, J = 7.0 Hz, 1H), 4.73 (s, 1H), 4.39 - 4.28 (m, 2H), 4.12 (s, 3H), 3.95 - 3.86 (m, 1H), 3.86 - 3.79 (m, 1H), 3.62 - 3.53 (m, 1H), 3.19 (s, 3H), 1.59 (dd, J = 5.4, 7.4 Hz, 1H), 1.45 (d, J = 7.5 Hz, 1H), 1.08 - 1.04 (m, 6H), 0.89 (s, 3H)
612
SUBSTITUTE SHEET RULE 26 Example 174: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4- yl (methyl |-6.6-dimethyl-3-|(2S.3R)-3-( 1 -methylcyclopropoxy )-2-|( 2.2.2- trifluoroacetyl)amino] butanoyl] -3-azabicyclo [3.1.0]hexane-2-carboxamide (Compound 237A)
Figure imgf000614_0001
[0001483] Step 1: 3-methoxy-5-vinyl-2,7-naphthyridine
[0001484] To a mixture of potassium;trifluoro(vinyl)boranuide (4.84 g, 36.14 mmol, 1.2 eq) and 5-bromo-3-methoxy-2,7-naphthyridine (7.2 g, 30.12 mmol, 1 eq) in dioxane (120 mL) and H2O (30 mL) was added CS2CO3 (29.44 g, 90.35 mmol, 3 eq). The mixture was added Pd(dppf)Ch (2.20 g, 3.01 mmol, 0.1 eq) under N2 and stirred at 70 °C for 16 h. Upon completion, the reaction mixture was concentrated and then diluted with H2O (200 mL) and extracted with DCM 400 mL (200 mL * 2). The combined organic layers were washed with brine 200 mL (200 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate =
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SUBSTITUTE SHEET RULE 26 20:1 to 3: 1) to give 3-methoxy-5-vinyl-2,7-naphthyridine (4.8 g, 24.49 mmol, 81.31% yield, 95% purity) as a yellow solid. MS (ESI) m/z 187.2 [M+H]+.
[0001485] Step 2: 6-methoxy-2,7-naphthyridine-4-carbaldehyde
[0001486] To a mixture of 3-methoxy-5-vinyl-2,7-naphthyridine (4.8 g, 25.78 mmol, 1 eq) in dioxane (150 mL) and H2O (30 mL) added 2,6-dimethylpyridine (5.52 g, 51.55 mmol, 6.00 mL, 2 eq), K2OSO4.2H2O (1.90 g, 5.16 mmol, 0.2 eq). The mixture was stirred at 25 °C for 30 min, then NaIO4 (27.57 g, 128.89 mmol, 7.14 mL, 5 eq) was added at 0 °C, the mixture was stirred at 25 °C for 16 h. The reaction mixture was filtered and then diluted with H2O (50 mL) and extracted with Ethyl acetate 200 mL (100 mL * 2). The combined organic layers were washed with brine 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, Petroleum ether:Ethyl acetate = 20: 1 to 1 :1) to give 6-methoxy-2,7-naphthyridine-4-carbaldehyde (3 g, 14.35 mmol, 55.66% yield, 90% purity) as a yellow solid. MS (ESI) m/z 189.2 [M+H]+
[0001487] Step 3: 5-bromo-6-methoxy-2,7-naphthyridine-4-carbaldehyde
[0001488] To a mixture of 6-methoxy-2,7-naphthyridine-4-carbaldehyde (1.6 g, 8.50 mmol, 1 eq) in AcOH (10 mL) was added NBS (1.59 g, 8.93 mmol, 16.44 uL, 1.05 eq) at 25 °C. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was added drop-wise into Sat. aq. NaHCCh (50 mL, pH~8) and extracted with Ethyl acetate (50 mL * 3). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated. Then crude product was triturated with Ethyl acetate 50 mL at 25 °C to afford 5- bromo-6-methoxy-2,7-naphthyridine-4-carbaldehyde (2.2 g, 7.83 mmol, 92.04% yield, 95% purity) as a yellow solid. MS (ESI) m/z 267/269 [M+H]+.
[0001489] Step 4: 6-methoxy-5-(2-trimethylsilylethynyl)-2,7-naphthyridine-4-carbaldehyde
[0001490] To a mixture of 5-bromo-6-methoxy-2,7-naphthyridine-4-carbaldehyde (1.9 g, 7.11 mmol, 1 eq) and 3-trimethylsilylprop-2-ynoic acid (2.02 g, 14.23 mmol, 10.37 uL, 2 eq) in Tol (190 mL) was added CS2CO3 (6.95 g, 21.34 mmol, 3 eq) and XPhos (678.27 mg, 1.42 mmol, 0.2 eq), then XPhos Pd G3 (602.16 mg, 711.40 umol, 0.1 eq) was added to the above mixture under N2. The mixture was stirred at 100 °C for 3 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to remove solvent. The residue was diluted with H2O
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SUBSTITUTE SHEET RULE 26 (100 mL) and extracted with DCM 400 mL (200 mL * 2). The combined organic layers were washed with brine 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 3:1) to give 6-methoxy-5-(2-trimethylsilylethynyl)-2,7- naphthyridine-4-carbaldehyde (1.40 g, 4.66 mmol, 65.55% yield, 95% purity) as a yellow solid. MS (ESI) m/z 285.3 [M+H]+
[0001491] Step 5: 2-amino-2-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4- yl]acetonitrile
[0001492] To a mixture of 6-methoxy-5-(2-trimethylsilylethynyl)-2,7-naphthyridine-4- carbaldehyde (1.2 g, 4.22 mmol, 1 eq) in DCM (20 mL) was added tetraisopropoxytitanium (1.80 g, 6.33 mmol, 1.87 mL, 1.5 eq) and NH3/MeOH (7 M, 2.41 mL, 4 eq). The mixture was stirred at 25 °C for 30 min, then added TMSCN (837.23 mg, 8.44 mmol, 1.06 mL, 2 eq) at 0 °C, and stirred for 2.5 h. Upon completion, the reaction mixture was diluted with H2O (50 mL) and extracted with DCM 100 mL (50 mL * 2). The combined organic layers were washed with brine 50 mL (50 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue to give 2-amino-2-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4- yl]acetonitrile (1.16 g, crude) was obtained as a brown oil. MS (ESI) m/z 311.3 [M+H]+
[0001493] Step 6: (lR,2S,5S)-N-[cyano-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7- naphthyridin-4-yl]methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2-carboxamide
[0001494] To a solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (654.58 mg, 1.61 mmol, 1 eq) in ACN (10 mL) was added TCFH (903.85 mg, 3.22 mmol, 2 eq) and NMI (396.73 mg, 4.83 mmol, 385.18 uL, 3 eq). Then 2-amino-2-[6-methoxy-5-(2- trimethylsilylethynyl)-2,7-naphthyridin-4-yl]acetonitrile (500 mg, 1.61 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition H2O 20 mL, and then extracted with DCM 40 mL (20 mL * 2). The combined organic layers were washed with brine 40 mL (40 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250 * 70 mm * 10 um; mobile phase: [water( NH4HCO3)-
615
SUBSTITUTE SHEET RULE 26 ACN]; B%: 50%-85%, 20 min). Compound (lR,2S,5S)-N-[cyano-[6-methoxy-5-(2- trimethylsilylethynyl)-2,7-naphthyridin-4-yl]methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l -methyl cyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide (600 mg, 858.61 umol, 53.31% yield) was obtained as a yellow oil. MS (ESI) m/z 699.5 [M+H]+.
[0001495] Step 7: (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-
6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001496] To a solution of (lR,2S,5S)-N-[cyano-[6-methoxy-5-(2-trimethylsilylethynyl)-2,7- naphthyridin-4-yl]methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino] butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (600 mg, 858.61 umol, 1 eq) in THF (50 mL) was added KF (149.65 mg, 2.58 mmol, 60.34 uL, 3 eq) and 18- CROWN-6 (226.94 mg, 858.61 umol, 1 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with FEO 50 mL and extracted with EA 150 mL (50 mL * 3). The combined organic layers were washed with brine 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 10: 1 to 3:1) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (500 mg) as a white solid. MS (ESI) m/z 627.4 [M+H]+
[0001497] Step 8: (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-
6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0] hexane-2-carboxamide
[0001498] (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0] hexane-2-carboxamide (500 mg) was separated by SFC (column: REGIS(S,S)WHELK-O1( 250 mm * 25 mm, 10 urn); mobile phase: [Neu-IPA]; B%: 30%- 30%, 7 min) to give (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-
6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-
616
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (72 mg, 114.10 umol, 13.29% yield, 99.3% purity) as a white solid. MS (ESI) m/z 627.3 [M+H]+
[0001499] 'H NMR (400 MHz, DMSO-de) 5 = 9.83 (br d, J = 7.1 Hz, 1H), 9.52 (s, 1H), 9.45 - 9.40 (m, 2H), 8.81 (s, 1H), 7.40 (d, J = 6.6 Hz, 1H), 4.82 (s, 1H), 4.36 (br t, J = 7.8 Hz, 1H), 4.22 (s, 1H), 4.12 (s, 3H), 3.97 - 3.89 (m, 2H), 3.66 (d, J = 10.5 Hz, 1H), 1.54 (dd, J = 5.4, 7.4 Hz, 1H), 1.38 - 1.32 (m, 4H), 1.19 (d, J = 6.1 Hz, 3H), 0.98 (s, 4H), 0.80 (s, 3H), 0.67 (br dd, J = 5.7, 10.6 Hz, 1H), 0.41 - 0.31 (m, 2H).
[0001500] (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0] hexane-2-carboxamide Isomer 2 (75 mg, 119.69 umol, 13.94% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 627.3 [M+H]+
[0001501] 'H NMR (400 MHz, DMSO-d6) 5 = 9.83 (br d, J = 6.8 Hz, 1H), 9.69 (br d, J = 7.1 Hz, 1H), 9.51 (s, 1H), 9.42 (s, 1H), 8.83 (s, 1H), 7.52 (d, J = 7.1 Hz, 1H), 4.78 (s, 1H), 4.38 - 4.29 (m, 2H), 4.13 (s, 3H), 3.93 - 3.84 (m, 2H), 3.67 (d, J = 10.5 Hz, 1H), 1.59 (dd, J = 5.4, 7.3 Hz, 1H), 1.47 (d, J = 7.6 Hz, 1H), 1.29 (s, 3H), 1.13 - 1.02 (m, 6H), 0.85 (s, 3H), 0.75 - 0.70 (m, 1H), 0.61 - 0.56 (m, 1H), 0.32 - 0.28 (m, 2H).
617
SUBSTITUTE SHEET RULE 26 Example 175: Synthesis of (lR,2S,5S)-N-[cyano-(5-ethynyl-6-methoxy-2,7-naphthyridin-4- yl (methyl |-6.6-dimethyl-3-|(2S.3R)-3-( 1 -methylcyclopropoxy )-2-|( 2.2.2- trifluoroacetyl)amino] butanoyl] -3-azabicyclo [3.1.0]hexane-2-carboxamide (Compound 238A)
Figure imgf000619_0001
[0001502] Step 1: 3-(2-methoxyethoxy)-5-vinyl-2,7-naphthyridine
[0001503] To a mixture of potassium;trifluoro(vinyl)boranuide (965.16 mg, 7.21 mmol, 1.2 eq) and 5-bromo-3-(2-methoxyethoxy)-2,7-naphthyridine (1.7 g, 6.00 mmol, 1 eq) in dioxane (20 mL) and H2O (5 mL) was added CS2CO3 (5.87 g, 18.01 mmol, 3 eq). Then Pd(dppf)Ch (439.36 mg, 600.45 umol, 0.1 eq) was added under N2 and the the mixture was stirred at 70 °C for 18 h. Upon completion, the reaction mixture was concentrated to remove most dioxane before diluted with H2O 200 mL. The mixture was extracted with DCM 400 mL (200 mL * 2). The combined organic layer was washed with brine 200 mL (200 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether: Ethyl acetate = 20: 1 to 1: 1) to afford 3-(2-
618
SUBSTITUTE SHEET RULE 26 methoxyethoxy)-5-vinyl-2,7-naphthyridine (1.1 g, 4.54 mmol, 75.58% yield, 95% purity) as a yellow solid. MS (ESI) m/z 231.3[M+H]+.
[0001504] Step 2: 6-(2-methoxyethoxy)-2,7-naphthyridine-4-carbaldehyde
[0001505] To a mixture of 3-(2-methoxyethoxy)-5-vinyl-2,7-naphthyridine (1.1 g, 4.78 mmol, 1 eq) in dioxane (30 mL) and H2O (6 mL) added 2,6-dimethylpyridine (1.02 g, 9.55 mmol, 1.11 mL, 2 eq) and K2OSO4.2H2O (352.04 mg, 955.43 umol, 0.2 eq). The mixture was stirred at 25 °C for 30 min, then NalCh (5.11 g, 23.89 mmol, 1.32 mL, 5 eq) was added at 0 °C, the mixture was stirred at 25 °C for 15.5 h. Upon completion, the reaction mixture was filtered, diluted with H2O 50 mL and then extracted with EA 200 mL (100 mL * 2). The combined organic layer was washed with BRINE 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography ( S 1O2, Petroleum ether: Ethyl acetate = 20: 1 to 1: 1) to afford 6-(2-methoxyethoxy)-2,7-naphthyridine- 4-carbaldehyde (0.8 g, 3.27 mmol, 68.50% yield, 95% purity) as a yellow solid. MS (ESI) m/z 233.2[M+H]+.
[0001506] Step 3: 5-bromo-6-(2-methoxyethoxy)-2,7-naphthyridine-4-carbaldehyde
[0001507] To a mixture of 6-(2-methoxyethoxy)-2,7-naphthyridine-4-carbaldehyde (800 mg, 3.44 mmol, 1 eq) in AcOH (20 mL) was added NBS (643.76 mg, 3.62 mmol, 16.44 uL, 1.05 eq) at 25 °C. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was added drop-wise into sat. aq. NaHCCh (50 mL, pH~8) and extracted with EA (3*50 mL). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, concentrated. The crude product was triturated with EA 10 mL at 25 °C to afford 5-bromo-6-(2- methoxyethoxy)-2,7-naphthyridine-4-carbaldehyde (0.72 g, 2.29 mmol, 66.51% yield, 99% purity) as a white solid. MS (ESI) m/z 311/313 [M+H]+.
[0001508] Step 4: 6-(2-methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7-naphthyridine-4- carbaldehyde
[0001509] To a mixture of 3-trimethylsilylprop-2-ynoic acid (715.41 mg, 5.03 mmol, 10.37 uL, 2.5 eq) and 5-bromo-6-(2-methoxyethoxy)-2,7-naphthyridine-4-carbaldehyde (626 mg, 2.01 mmol, 1 eq) in Xylene (62 mL) was added CS2CO3 (1.97 g, 6.04 mmol, 3 eq) and XPhos (191.83 mg, 402.40 umol, 0.2 eq). Then XPhos Pd G3 (170.31 mg, 201.20 umol, 0.1 eq) was added to the
619
SUBSTITUTE SHEET RULE 26 above mixture under N2. The mixture was stirred at 125 °C for 4 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to remove solvent. The residue was diluted with H2O 100 mL and extracted with DCM 400 mL (200 mL * 2). The combined organic layers were washed with brine 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether: Ethyl acetate = 5: 1 to 3: 1) to afford 6-(2- methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7-naphthyridine-4-carbaldehyde (158 mg, 457.01 umol, 22.71% yield, 95% purity) as a yellow solid. MS (ESI) m/z 329.2 [M+H]+
[0001510] Step 5: 2 -amino-2-[6-(2-methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7- naphthyridin-4-yl]acetonitrile
[0001511] To a mixture of 6-(2-methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7- naphthyridine-4-carbaldehyde (250 mg, 761.18 umol, 1 eq) in DCM (10 mL) was added tetraisopropoxytitanium (324.50 mg, 1.14 mmol, 336.97 uL, 1.5 eq) and NHVMeOH (7 M, 434.96 uL, 4 eq). The mixture was stirred at 25 °C for 30 min before TMSCN (151.03 mg, 1.52 mmol, 190.46 uL, 2 eq) was added at 0 °C. The mixture was stirred for 2 h at 25 °C. Upon completion, the reaction mixture was diluted with H2O 50 mL and extracted with DCM 100 mL (50 mL * 2). The combined organic layer was washed with brine 50 mL (50 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a 2-amino-2-[6-(2- methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4-yl]acetonitrile (230 mg, crude) as a yellow solid, which was usded for next without further purification. MS (ESI) m/z 355.3 [M+H]+.
[0001512] Step 6: (lR,2S,5S)-N-[cyano-[6-(2-methoxyethoxy)-5-(2-trimethylsilylethynyl)- 2,7-naphthyridin-4-yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- 6,6-dimethyl-3-azabicyclo [3.1 ,0]hexane-2-carboxamide
[0001513] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (227.36 mg, 620.63 umol, 1.1 eq) in ACN (10 mL) was added 1 -METHYLIMIDAZOLE (138.97 mg, 1.69 mmol, 134.92 uL, 3 eq) and TCFH (316.61 mg, 1.13 mmol, 2 eq). 2-amino-2-[6-(2- methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4-yl]acetonitrile (200 mg, 564.21 umol, 1 eq) was added. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction
620
SUBSTITUTE SHEET RULE 26 mixture was diluted with H2O 100 mL and extracted with EA 200 mL (100 mL * 2). The combined organic layer was washed with brine 100 mL (100 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether: Ethyl acetate = 10: 1 to 1 : 1) to afford (lR,2S,5S)-N-[cyano-[6-(2-methoxyethoxy)-5-(2-trimethylsilylethynyl)-2,7-naphthyridin-4- yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (328 mg, 406.04 umol, 71.97% yield, 87% purity) as a yellow solid. MS (ESI) m/z 703.3 [M+H]+.
[0001514] Step 7: (lR,2S,5S)-N-[cyano-[5-ethynyl-6-(2-methoxyethoxy)-2,7-naphthyridin-4- yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxamide
[0001515] To a solution of (lR,2S,5S)-N-[cyano-[6-(2-methoxyethoxy)-5-(2- trimethylsilylethynyl)-2,7-naphthyridin-4-yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (308 mg, 438.25 umol, 1 eq) in THF (10 mL) was added KF (127.31 mg, 2.19 mmol, 51.34 uL, 5 eq) and 18-CROWN-6 (231.68 mg, 876.50 umol, 2 eq). The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O 50 mL and extracted with EA 100 mL (50 mL * 2). The combined organic layers were washed with brine 50 mL (50 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue.
[0001516] The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=10/l to 0/1) to get 280 mg (lR,2S,5S)-N-[cyano-[5-ethynyl-6-(2-methoxyethoxy)-2,7- naphthyridin-4-yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (280 mg, 109.96 umol, 90% yield, 90% purity) as yellow oil. MS (ESI) m/z 631.2 [M+H]+
[0001517] Step 8: (lR,2S,5S)-N-[cyano-[5-ethynyl-6-(2-methoxyethoxy)-2,7-naphthyridin-4- yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxamide
[0001518] (lR,2S,5S)-N-[cyano-[5-ethynyl-6-(2-methoxyethoxy)-2,7-naphthyridin-4- yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide was seperated by SFC (column: REGIS(S,S)WHELK-
621
SUBSTITUTE SHEET RULE 26 01 (250mm*25mm,10um); mobile phase: [Neu-IPA];B%: 30%-30%,9min) to get the (lR,2S,5S)-N-[cyano-[5-ethynyl-6-(2-methoxyethoxy)-2,7-naphthyridin-4-yl]methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-
3azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (65 mg, 102.54 umol, 23.40% yield, 99.48% purity) as white solid. MS (ESI) m/z 631.3 [M+H]+
[0001519] 'H NMR (400 MHz, DMSO+D2O-de) 8 = 9.50 (s, 1H), 9.38 (s, 1H), 8.79 (s, 1H),
7.37 (s, 1H), 4.78 (s, 1H), 4.67 (q, J = 4.0 Hz, 2H), 4.36 (d, J = 8.5 Hz, 1H), 4.22 (s, 1H), 3.97 - 3.87 (m, 1H), 3.81 (d, J = 10.4 Hz, 1H), 3.76 - 3.62 (m, 3H), 3.31 (s, 3H), 3.25 (s, 3H), 1.55 (dd, J = 5.5, 7.4 Hz, 1H), 1.35 (d, J = 7.6 Hz, 1H), 1.17 (d, J = 6.1 Hz, 3H), 0.97 (s, 3H), 0.83 (s, 3H)
[0001520] 'H NMR (400 MHz, DMSO-de) 8 = 9.87 (br d, J = 6.8 Hz, 1H), 9.51 (s, 1H), 9.43
(d, J = 6.6 Hz, 1H), 9.39 (s, 1H), 8.81 (s, 1H), 7.39 (d, J = 6.6 Hz, 1H), 4.81 (s, 1H), 4.70 - 4.65 (m, 2H), 4.37 (t, J = 7.6 Hz, 1H), 4.23 (s, 1H), 3.96 - 3.87 (m, 1H), 3.86 - 3.79 (m, 1H), 3.76 - 3.62 (m, 3H), 3.32 (br s, 3H), 3.26 (s, 3H), 1.55 (dd, J = 5.3, 7.6 Hz, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.18 (d, J = 6.3 Hz, 3H), 0.98 (s, 3H), 0.84 (s, 3H)
[0001521] (lR,2S,5S)-N-[cyano-[5-ethynyl-6-(2-methoxyethoxy)-2,7-naphthyridin-4- yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0] hexane-2-carboxamide Isomer 2 (70 mg, 109.96 umol, 25.09% yield, 99.06% purity) was obtained as a white solid. MS (ESI) m/z 631.3 [M+H]+
[0001522] 'H NMR (400 MHz, DMSO+D2O-de) 8 = 9.48 (s, 1H), 9.37 (s, 1H), 8.80 (s, 1H),
7.50 (s, 1H), 4.76 - 4.58 (m, 3H), 4.39 - 4.25 (m, 2H), 3.95 - 3.85 (m, 1H), 3.84 - 3.78 (m, 1H), 3.77 - 3.70 (m, 2H), 3.61 - 3.53 (m, 1H), 3.32 (s, 3H), 3.18 (s, 3H), 1.62 - 1.56 (m, 1H), 1.44 (d, J = 7.7 Hz, 1H), 1.10 - 1.00 (m, 6H), 0.88 (s, 3H)
[0001523] 'H NMR (400 MHz, DMSO-de) 8 = 9.94 - 9.81 (m, 1H), 9.75 (br d, J = 7.0 Hz,
1H), 9.49 (s, 1H), 9.38 (s, 1H), 8.82 (s, 1H), 7.54 - 7.47 (m, 1H), 4.74 (s, 1H), 4.71 - 4.63 (m, 2H), 4.38 - 4.29 (m, 2H), 3.96 - 3.87 (m, 1H), 3.86 - 3.80 (m, 1H), 3.78 - 3.70 (m, 2H), 3.63 - 3.52 (m, 1H), 3.33 - 3.30 (m, 3H), 3.19 (s, 3H), 1.64 - 1.54 (m, 1H), 1.46 (d, J = 7.7 Hz, 1H), 1.10 - 1.01 (m, 6H), 0.89 (s, 3H)
622
SUBSTITUTE SHEET RULE 26 Example 176: Synthesis of (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-[(2S,3R) -3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound
239A)
Figure imgf000624_0001
[0001524] Step 1: (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0] hexane-2-carboxamide
[0001525] To a solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (200.00 mg, 492.13 umol, 1 eq) in ACN (10 mL) was added NMI (121.22 mg, 1.48 mmol), TCFH (276.16 mg, 984.25 umol, 2 eq), and 2-amino-2-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)acetonitrile (100 mg, 492.13 umol, 1 eq) in turn. The mixture was stirred at 25 °C for 2 hr. Upon completion, the reaction mixture was quenched by addition of H2O 20 mL at 25 °C, and then extracted with DCM 40 mL (20 mL * 2). The combined organic layers were washed with BRINE 40 mL (40 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiCh, PE:EA = 0:1) to give (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (160 mg) as a white solid. MS (ESI) m/z 592.3 [M+H]+.
[0001526] Step 2: (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1. 0]hexane-2-carboxamide
623
SUBSTITUTE SHEET RULE 26 [0001527] The mixture was purified by separated by SFC (column: DAICEL CHIRALPAK IC(250 mm*30 mm, 10 um);mobile phase: [Neu-IPA];B%: 33%-33%,8 min) to give (1R,2S,5S)- N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 1 (75 mg, 125.70 umol, 25.54% yield, 99.15% purity) as a white solid. MS (ESI) m/z 592.3 [M+H]+.
[0001528] 'H NMR (400 MHz, DMSO-d6) 5 = 9.84 (br d, J = 7.7 Hz, 1H), 9.38 (d, J = 7.9 Hz, 1H), 8.08 (s, 1H), 7.84 (d, J = 5.9 Hz, 1H), 7.23 (d, J = 5.9 Hz, 1H), 6.44 (d, J = 7.5 Hz, 1H),
4.36 - 4.31 (m, 1H), 4.22 (s, 1H), 3.92 - 3.83 (m, 2H), 3.66 - 3.61 (m, 1H), 3.45 (s, 3H), 1.57 - 1.52 (m, 1H), 1.36 (d, J = 7.5 Hz, 1H), 1.28 (s, 3H), 1.11 (d, J = 5.9 Hz, 3H), 1.01 (s, 3H), 0.81 (s, 3H), 0.75 - 0.70 (m, 1H), 0.70 - 0.70 (m, 1H), 0.34 - 0.27 (m, 2H)
[0001529] (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0] hexane-2-carboxamide Isomer 2 (62 mg, 104.80 umol, 21.30% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 592.3 [M+H]+.
[0001530] 'H NMR (400 MHz, DMSO-d6) 5 = 9.85 (br d, J = 7.2 Hz, 1H), 9.33 - 9.28 (m, 1H), 8.06 (s, 1H), 7.83 (d, J = 5.9 Hz, 1H), 7.22 (d, J = 5.9 Hz, 1H), 6.45 (d, J = 7.5 Hz, 1H),
4.37 - 4.29 (m, 1H), 4.20 (s, 1H), 3.94 - 3.86 (m, 2H), 3.67 - 3.61 (m, 1H), 3.44 (s, 3H), 1.55 - 1.50 (m, 1H), 1.30 (s, 4H), 1.11 (d, J = 6.4 Hz, 3H), 0.96 (s, 3H), 0.81 - 0.74 (m, 4H), 0.65 - 0.60 (m, 1H), 0.36 - 0.29 (m, 2H)
624
SUBSTITUTE SHEET RULE 26 Example 177: Synthesis of (lR,2S,5S)-N-(cyano(5-methyl-4-oxo-4,5-dihydropyrazolo[l,5- a|pyraziii-3-yl)niethyl)-6.6-dimethyl-3-((2S.3R)-3-( l-methylcyclobutoxy )-2-(2.2.2- trifluoroacetamido)butanoyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 240A)
Figure imgf000626_0001
[0001531] Step 1: 2-amino-2-(5-methyl-4-oxo-4,5-dihydropyrazolo[l,5-a]pyrazin-3- yl)acetonitrile
[0001532] To a solution of 5-methyl-4-oxo-pyrazolo[l,5-a]pyrazine-3-carbaldehyde (300 mg, 1.69 mmol, 1 eq) in NH3/MeOH (7 M, 6.00 mL, 24.80 eq) was added NH4C1 (271.74 mg, 5.08 mmol, 3 eq). The mixture was stirred at 20 °C for 0.5 h before TMSCN (671.98 mg, 6.77 mmol, 847.39 uL, 4 eq) was added. The mixture was stirred at 20 °C for 1 h. Upon completion, the reaction mixture was diluted with water (3 mL) and extracted with DCM (5 mL * 10). The combined organic layers were dried over Na2SO-i, filtered and concentrated under reduced pressure to give a 2-amino-2-(5-methyl-4-oxo-pyrazolo[l,5-a] pyrazin-3-yl)acetonitrile (347 mg, crude) as a white solid. MS (ESI) m/z 204.1 [M+H]+
[0001533] Step 2: (lR,2S,5S)-N-(cyano(5-methyl-4-oxo-4,5-dihydropyrazolo[l,5-a]pyrazin-3- yl)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclobutoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-3 -azabicyclo[3.1.0]hexane-2- carboxamide
625
SUBSTITUTE SHEET RULE 26 [0001534] To a solution of (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclobutoxy)-2- [(2,2,2-trifluoroacetyl) amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (717.94 mg, 1.71 mmol, 1 eq) in ACN (10 mb) was added NMI (420.62 mg, 5.12 mmol, 408.37 uL, 3 eq) and TCFH (958.28 mg, 3.42 mmol, 2 eq). Then 2-amino-2-(5-methyl-4-oxo-pyrazolo[l,5- a]pyrazin-3-yl)acetonitrile (347 mg, 1.71 mmol, 1 eq) was added. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (15 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250*70mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-70%,20min ) to give (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3- [(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (350 mg, 549.04 umol, 32.15% yield, 95% purity) as a light yellow solid. MS (ESI) m/z 606.3 [M+H]+
[0001535] Step 3: (lR,2S,5S)-N-(cyano(5-methyl-4-oxo-4,5-dihydropyrazolo[l,5-a]pyrazin-3- yl)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclobutoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-3 -azabicyclo[3.1.0]hexane-2- carboxamide
[0001536] The residue was separated by SFC (column: REGIS(S,S)WHELK- 01(250mm*25mm,10um);mobile phase: [Neu-IPA];B%: 35%-35%,7min) to give (1R,2S,5S)-N- [cyano-(5-methyl-4-oxo-pyrazolo [l,5-a]pyrazin-3-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2- carboxamide (68.01 mg, 111.65 umol, 19.32% yield, 99.42% purity) as a white solid. MS (ESI) m/z 606.3 [M+H]+
[0001537] 'H NMR (400 MHz, DMSO-t/e) 5 = 9.77 (br d, J= 1.1 Hz, 1H), 9.38 (d, J = 7.7 Hz, 1H), 8.10 (s, 1H), 7.85 (d, J= 5.9 Hz, 1H), 7.25 (d, J= 6.1 Hz, 1H), 6.46 (d, J= 7.5 Hz, 1H), 4.42 (br t, J= 7.5 Hz, 1H), 4.25 (s, 1H), 3.96 (dd, J= 5.3, 10.5 Hz, 1H), 3.83 (dd, J= 6.2, 8.0 Hz, 1H), 3.65 (br d, J= 10.7 Hz, 1H), 3.48 - 3.44 (m, 3H), 2.11 - 2.00 (m, 2H), 1.76 - 1.65 (m, 2H), 1.59 - 1.45 (m, 3H), 1.26 - 1.23 (m, 3H), 1.07 - 0.97 (m, 7H), 0.84 (s, 3H)
[0001538] (lR,2S,5S)-N-[cyano-(5-methyl-4-oxo-pyrazolo[l,5-a]pyrazin-3-yl)methyl]-6,6- dimethyl-3 -[(2S,3R)-3-(l-methylcyclobutoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-
626
SUBSTITUTE SHEET RULE 26 azabicyclo[3.1.0]hexane-2-carboxamide (66.39 mg, 109.63 umol, 18.97% yield, 100% purity) was obtained as a white solid.
[0001539] 'H NMR (400 MHz, DMSO-t/e) 8 = 9.76 (br d, J= 8.4 Hz, 1H), 9.29 (d, J= 7.5 Hz,
1H), 8.08 (s, 1H), 7.84 (d, J= 6.0 Hz, 1H), 7.24 (d, J= 6.0 Hz, 1H), 6.47 (d, J= 7.5 Hz, 1H),
4.41 (t, J= 8.3 Hz, 1H), 4.23 (s, 1H), 3.97 (dd, J = 5.3, 10.4 Hz, 1H), 3.87 (dd, J= 6.1, 8.0 Hz, 1H), 3.66 (br d, J= 10.5 Hz, 1H), 3.46 (s, 3H), 2.14 - 2.03 (m, 2H), 1.81 - 1.67 (m, 2H), 1.62 - 1.45 (m, 3H), 1.30 - 1.24 (m, 3H), 1.09 - 0.94 (m, 7H), 0.83 (s, 3H)
Example 178: Synthesis of (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-d]pyridazin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 249A)
Figure imgf000628_0001
627
SUBSTITUTE SHEET RULE 26 [0001540] Step 1: 4-bromo-2-formyl-thiophene-3-carboxylic acid
[0001541] To a mixture of N-isopropylpropan-2-amine (61.09 g, 603.73 mmol, 85.32 mL, 2.5 eq) in THF (800 mL) was added n-BuLi (2.5 M, 217.34 mL, 2.25 eq) at -30 °C and the mixture was stirred for Ih at -30 °C. The reaction mixture was allowed cool to -60 °C, added slowly a mixture of 4-bromothiophene-3 -carboxylic acid (50 g, 241.49 mmol, 1 eq) and HMPA (4.16 g, 23.22 mmol, 4.08 mL, 9.62e-2 eq) in THF (400 mL) and stirred for 1 h at -60 °C. After DMF (30.02 g, 410.73 mmol, 31.60 mL, 1.70 eq) was added slowly to above reaction mixture at - 60 °C, the reaction mixture was stirred at -60 °C for 1 h. Upon completion, the reaction mixture was added the 2 N HC1 to adjust pH=2 and extected with ethyl acetate (2000 mL * 2). The organic phase was washed with brine (1000 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Give 4-bromo-2-formyl-thiophene-3 -carboxylic acid (110 g, crude) as a yellow solid. MS (ESI) m/z 234.9 [M+H]+.
[0001542] Step 2: methyl 4-bromo-2-formyl-thiophene-3-carboxylate
[0001543] To a solution of 4-bromo-2-formyl-thiophene-3-carboxylic acid (55 g, 233.99 mmol, 1 eq) in DMF (500 mL) was added K2CO3 (97.02 g, 701.96 mmol, 3 eq) and iodomethane (45.50 g, 320.56 mmol, 19.96 mL, 1.37 eq). The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was added water (1000 mL) and extected with ethyl acetate (500 mL * 3). The organic phase was washed with brine (300 mL), dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate=100/l to 1/1) to give methyl 4- bromo-2-formyl-thiophene-3-carboxylate (105 g, 358.32 mmol, 38.28% yield, 85% purity) as a light white solid. MS (ESI) m/z 248.9 [M+H]+.
[0001544] Step 3: 3-bromothieno[2,3-d]pyridazin-4-ol
[0001545] To a solution of methyl 4-bromo-2-formyl-thiophene-3-carboxylate (47.5 g, 190.70 mmol, 1 eq) in EtOH (600 mL) was dropwise N2H4.H2O (35.80 g, 572.10 mmol, 34.76 mL, 80% purity, 3 eq) at 80 °C, the reaction mixture was stirred at 80 °C for 2 h. Upon completion, the reaction mixture was cooled to 25 °C and filtered to give 3-bromothieno[2,3-d]pyridazin-4-ol (22 g, crude) as a light yellow solid. MS (ESI) m/z 230.9 [M+H]+.
[0001546] Step 4: 3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4-ol
628
SUBSTITUTE SHEET RULE 26 [0001547] To a solution of 3-bromothieno[2,3-d]pyridazin-4-ol (15 g, 64.92 mmol, 1 eq) and ethynyl(trimethyl)silane (63.76 g, 649.15 mmol, 89.93 mL, 10 eq) in DMF (150 mL) was added iodocopper (2.47 g, 12.98 mmol, 0.2 eq), dichloropalladium;triphenylphosphane (4.56 g, 6.49 mmol, 0.1 eq) and TEA (131.38 g, 1.30 mol, 180.71 mL, 20 eq), the mixture was stirred at 50 °C for 12 h. Upon completion, the reaction mixture was diluted with H2O (300 mL) and extracted with DCM (100 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, Petroleum ether/Ethyl acetate = 50/1 to 1/1) to give 3-(2-trimethylsilylethynyl)thieno[2,3- d]pyridazin-4-ol (8 g, 28.99 mmol, 44.66% yield, 90% purity) as a yellow solid. MS (ESI) m/z 249.0 [M+H]+.
[0001548] Step 5: [3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4-yl] trifluoromethanesulfonate
[0001549] To a solution of 3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4-ol (7 g, 28.18 mmol, 1 eq) in DCM (70 mL) was added Tf20 (7.67 g, 27.18 mmol, 4.48 mL, 1.5 eq), then pyridine (3.34 g, 42.27 mmol, 3.41 mL, 1.5 eq) was added. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with H2O (300 mL) and extracted with DCM (100 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, Petroleum ether/Ethyl acetate = 40/1 to 1/1) to give [3-(2-trimethylsilylethynyl)thieno[2,3- d]pyridazin-4-yl] trifluoromethanesulf onate (5 g, 11.83 mmol, 41.97% yield, 90% purity) as a brown solid. MS (ESI) m/z 381.0 [M+H]+.
[0001550] Step 6: trimethyl-[2-(4-vinylthieno[2,3-d]pyridazin-3-yl)ethynyl]silane
[0001551] To a solution of [3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4-yl] trifluoromethanesulfonate (4 g, 10.51 mmol, 1 eq) in dry Tol. (40 mL) was added tributyl(vinyl)tin (20.00 g, 63.08 mmol, 18.35 mL, 6.00 eq) and the reaction was mixture was purged with argon for 0.5 h. Pd(PPhs)4 (1.21 g, 1.05 mmol, 0.1 eq) was added and the mixture heated at 110 °C for 12 hr. Upon completion, the residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 30/0 to 1/1) to give trimethyl- [2-(4- vinylthieno[2,3-d]pyridazin-3-yl)ethynyl]silane (780 mg, 2.72 mmol, 25.84% yield, 90% purity) as a yellow solid. MS (ESI) m/z 259.1 [M+H]+.
629
SUBSTITUTE SHEET RULE 26 [0001552] Step 7: 3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazine-4-carbaldehyde
[0001553] To a solution of trimethyl-[2-(4-vinylthieno[2,3-d]pyridazin-3-yl)ethynyl]silane (800 mg, 3.09 mmol, 1 eq) inTHF (20 mL) and H2O (4 mL) was added K2OSO4.2H2O (227.99 mg, 618.77 umol, 0.2 eq), after stirred 0.5 h at 25 °C, NalCh (2.64 g, 12.37 mmol, 685.75 uL, 4 eq) was added, the solution was stirred at 25 °C for 4.5 h. Upon completion, The reaction mixture was quenched by addition H2O (100 mL), and then extracted with EA (30 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC(SiO2, Petroleum ether/Ethyl acetate = 1/1) to give 3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazine-4-carbaldehyde (300 mg, 921.71 umol, 29.77% yield, 80% purity) as a yellow solid. MS (ESI) m/z 261.0 [M+H]+.
[0001554] Step 8: 2-amino-2-[3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4- yl]acetonitrile
[0001555] To a solution of 3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazine-4-carbaldehyde (300 mg, 1.15 mmol, 1 eq) in DCM (10 mL) was added T i(i-PrO)4 (491.17 mg, 1.73 mmol, 510.04 uL, 1.5 eq) and NEh/MeOH (7 M, 3.29 mL, 20 eq), after stirred 1 h at 25 °C, then TMSCN (514.36 mg, 5.18 mmol, 648.63 uL, 4.5 eq) was added, the solution was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was quenched by addition H2O (30 mL), and then extracted with EA (10 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-[3-(2- trimethylsilylethynyl)thieno[2,3-d]pyridazin-4-yl]acetonitrile (220 mg, crude) as a yellow solid. MS (ESI) m/z 287.1 [M+H]+.
[0001556] Step 9: (lR,2S,5S)-N-[cyano-[3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4- yl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001557] To a solution of 2-amino-2-[3-(2-trimethylsilylethynyl)thieno[2,3-d]pyridazin-4- yl]acetonitrile (220 mg, 768.08 umol, 1 eq) and (lR,2S,5S)-3-[(2S)-3,3-dimethyl-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (279.86 mg, 768.08 umol, 1 eq) in ACN (20 mL) was added NMI (189.18 mg, 2.30 mmol, 183.67 uL, 3 eq), then TCEH (646.53 mg, 2.30 mmol, 3 eq) was added. The mixture was stirred at 20 °C for 2 h. Upon completion, the reaction mixture was poured into H2O (60 mL) and
630
SUBSTITUTE SHEET RULE 26 extracted with DCM (20mL * 3). The combined organic layers was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC(SiO2, Petroleum ether/Ethyl acetate = 1/1) to give (lR,2S,5S)-N-[cyano-[3-(2-trimethylsilylethynyl)thieno[2,3- d]pyridazin-4-yl]methyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (250 mg, 326.64 umol, 43.59% yield, 85% purity) as a brown solid. MS (ESI) m/z 635.2 [M+H]+.
[0001558] Step 10: (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-d]pyridazin-4-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001559] To a solution of (lR,2S,5S)-N-[cyano-[3-(2-trimethylsilylethynyl)thieno[2,3- d]pyridazin-4-yl]methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (200 mg, 315.09 umol, 1 eq) in THF (10 mL) was added 1,4,7,10,13,16-hexaoxacyclooctadecane (166.57 mg, 630.18 umol, 2 eq), then KF (109.84 mg, 1.89 mmol, 44.29 uL, 6 eq) was added. The mixture was stirred at 25 °C for 2 h. The reaction mixture was poured into H2O (60 mL) and extracted with DCM (20 mL * 3). The combined organic layers was filtered and concentrated under reduced pressure to give a residue. The crude was purified by prep-HPLC(neutral) (column: Waters Xbridge BEH Cl 8 100*30mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-55%,8min) to give (lR,2S,5S)-N-[cyano-(3-ethynylthieno[2,3-d]pyridazin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2- [(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2-carboxamide (75 mg, 126.65 umol, 40.20% yield, 95% purity) as a white solid. MS (ESI) m/z 563.2 [M+H]+.
[0001560] 'H NMR (400 MHz, DMSO-de) 8 = 10.18 - 9.70 (m, 2H), 9.67 - 9.43 (m, 1H), 9.54 (br d, J = 7.0 Hz, 1H), 8.90 - 8.54 (m, 1H), 7.07 (d, J = 7.0 Hz, 1H), 4.64 - 4.26 (m, 3H), 3.98 - 3.79 (m, 2H), 3.71 - 3.55 (m, 1H), 3.28 - 3.20 (m, 3H), 1.60 - 1.49 (m, 1H), 1.43 - 1.33 (m, 1H), 1.25 - 1.11 (m, 3H), 1.05 - 0.94 (m, 3H), 0.89 - 0.78 (m, 3H)
631
SUBSTITUTE SHEET RULE 26 Example 179: Synthesis of (lR,2S,5S)-N-(cyano(3-ethynyl-l-methyl-lH-pyrazolo[3,4- c| pyridin-4-yl (methyl )-6.6-dimethyl-3-(( 2S.3R)-3-( 1 -methylcyclopropoxy )-2-( 2.2.2- trifluoroacetamido)butanoyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 250A)
Figure imgf000633_0001
[0001561] Step 1: l-methyl-3-((trimethylsilyl)ethynyl)-lH-pyrazolo[3,4-c]pyridine-4- carbaldehyde
[0001562] To a solution of trimethyl-[2-(l-methyl-4-vinyl-pyrazolo[3,4-c]pyridin-3- yl)ethynyl] silane (2.5 g, 9.79 mmol, 1 eq) in THF (80 mL) and H2O (25 mL) was added dipotassium; di oxi do(dioxo)osmium; dihydrate (360.68 mg, 978.90 umol, 0.1 eq) and stirred for 0.5 h, then added sodium;periodate (2.09 g, 9.79 mmol, 542.43 uL, 1 eq). The mixture was stirred at 25 °C for 11.5 h. Upon completion, the reaction mixture was pour into H2O (50 mL) and then extracted with EA (50 mL * 3). The combined organic phase was washed with brine (50 mL * 1), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 1:0 to 5: 1) to givel-methyl-3-(2-trimethylsilylethynyl)pyrazolo[3,4-c]pyridine-4-carbaldehyde (1.5 g, 5.83 mmol, 59.54% yield) as a yellow solid. MS (ESI) m/z 258.2 [M+H]+.
[0001563] Step2: 2-amino-2-(3-ethynyl-l-methyl-lH-pyrazolo[3,4-c]pyridin-4-yl)acetonitrile
632
SUBSTITUTE SHEET RULE 26 [0001564] A soluton of l-methyl-3-(2-trimethylsilylethynyl)pyrazolo[3,4-c]pyridine-4- carbaldehyde (1.5 g, 5.83 mmol, 1 eq), Ti(i-PrO)4 (2.48 g, 8.74 mmol, 2.58 mL, 1.5 eq) and NH3/MeOH (7 M, 8.33 mL, 10.01 eq) in DCM (20 mL) was stirred for 1 h, then TMSCN (1.73 g, 17.49 mmol, 2.19 mL, 3 eq) was added. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was diluted with H2O (20 mL), filtered and then extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (20 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(3-ethynyl-l- methyl-pyrazolo[3,4-c]pyridin-4-yl)acetonitrile (1.1 g, crude) as a black brown solid. MS (ESI) m/z 212.1 [M+H]+.
[0001565] Step 3: (lR,2S,5S)-N-(cyano(3-ethynyl-l-methyl-lH-pyrazolo[3,4-c]pyridin-4- yl)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclopropoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-3-azabicyclo[3.L0]hexane-2-carboxamide
[0001566] A solution of 2-amino-2-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4- yl)acetonitrile (250 mg, 1.18 mmol, 1 eq) in ACN (5 mL) was added (lR,2S,5S)-6,6-dimethyl- 3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (529.11 mg, 1.30 mmol, 1.1 eq) and 1- methylimidazole (388.69 mg, 4.73 mmol, 377.37 uL, 4 eq). Then TCFH (398.51 mg, 1.42 mmol, 1.2 eq) was added to the mixture and the mixture was sitrred at 25 °C for 1 h. Upon completion, the reaction mixture was quenched by addition of H2O 5 mL at 0°C, and extracted with DCM (5 mL * 3). The combined organic layers were washed with brine (5 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 10: 1 to 0:1) to give (1R,2S,5S)- N-[cyano-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4-yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3- (l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2- carboxamide (760 mg, 1.17 mmol, 98.52% yield, 92% purity) as yellow oil. MS (ESI) m/z 600.3 [M+H]+.
[0001567] Step 4: (lR,2S,5S)-N-(cyano(3-ethynyl-l-methyl-lH-pyrazolo[3,4-c]pyridin-4- yl)methyl)-6,6-dimethyl-3-((2S,3R)-3-(l-methylcyclopropoxy)-2-(2,2,2- trifluoroacetamido)butanoyl)-3-azabicyclo[3.L0]hexane-2-carboxamide
633
SUBSTITUTE SHEET RULE 26 [0001568] (lR,2S,5S)-N-[cyano-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4-yl)methyl]-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (760 mg, 1.27 mmol, 1 eq) was separated by SFC (column: REGIS(S,S)WHELK-01 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 36%-36%, 7 min) to give (lR,2S,5S)-N-[cyano-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (84.89 mg, 137.90 umol, 10.88% yield, 97.4% purity) as white solid. MS (ESI) m/z 600.3 [M+H]+.
[0001569] 'H NMR (400 MHz, ACETONITRILE-ds) 6 = 9.16 (s, 1H), 8.55 (s, 1H), 7.49 (br d, J = 6.8 Hz, 1H), 7.43 (br d, J = 6.7 Hz, 1H), 6.56 (d, J = 6.8 Hz, 1H), 4.47 (dd, J = 5.7, 7.5 Hz, 1H), 4.27 (s, 1H), 4.20 (s, 3H), 3.98 - 3.90 (m, 2H), 3.77 (s, 1H), 3.71 (d, J = 10.6 Hz, 1H), 1.59 - 1.53 (m, 1H), 1.51 - 1.48 (m, 1H), 1.29 (s, 3H), 1.01 - 0.99 (m, 6H), 0.86 (s, 3H), 0.75 - 0.70 (m, 2H), 0.40 - 0.36 (m, 2H)
[0001570] (lR,2S,5S)-N-[cyano-(3-ethynyl-l-methyl-pyrazolo[3,4-c]pyridin-4-yl)methyl]- 6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (73.91 mg, 123.26 umol, 9.73% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 600.3 [M+H]+.
[0001571] 'H NMR (400 MHz, ACETONITRILE-ds) 6 = 9.16 (s, 1H), 8.55 (s, 1H), 7.48 (br d, J = 6.6 Hz, 2H), 6.57 (d, J = 6.9 Hz, 1H), 4.49 - 4.43 (m, 1H), 4.40 (s, 1H), 4.20 (s, 3H), 3.97 (s, 1H), 3.79 (s, 1H), 3.75 - 3.64 (m, 2H), 1.57 - 1.53 (m, 1H), 1.51 (d, J = 4.8 Hz, 1H), 1.14 (s, 3H), 1.10 (d, J = 6.4 Hz, 3H), 1.04 (s, 3H), 0.88 (s, 3H), 0.55 - 0.47 (m, 1H), 0.20 (s, 2H), 0.04 - 0.00 (m, 1H)
634
SUBSTITUTE SHEET RULE 26 Example 180: Synthesis of (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4- c] pyridin-4-yl)methyl] -3- 1 ( 2 S.3R )-3-met hoxy-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 251A)
Figure imgf000636_0001
[0001572] Step 1: l-methyl-3-prop-l-ynyl-4-vinyl-pyrazolo[3,4-c]pyridine
[0001573] 3-iodo-l-methyl-4-vinyl-pyrazolo[3,4-c]pyridine (4 g, 14.03 mmol, 1 eq), but-2- ynoic acid (1.77 g, 21.05 mmol, 1.5 eq) and Cui (133.61 mg, 701.55 umol, 0.05 eq), Pd(0Ac)2 (157.50 mg, 701.55 umol, 0.05 eq), TEA (2.84 g, 28.06 mmol, 3.91 mL, 2 eq), PPh3 (368.01 mg, 1.40 mmol, 0.1 eq) in DMF (80 mL) in a sealed tube was heated at 50 °C and stirred for 16 h. Four batches were set up in parallel. Upon completion, the reaction mixture was poured into H2O (150 mL) at 25 °C, and then extracted with EtOAc (150 mL * 3). The combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 1/0 to 2/1) to give l-methyl-3-prop-l-ynyl-4-vinyl-pyrazolo[3,4-c]pyridine (6.9 g, 31.49 mmol, 74.80% yield, 90% purity) as a yellow solid. MS (ESI) m/z 198.2 [M+H]+.
[0001574] Step 2: l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridine-4-carbaldehyde
[0001575] To a solution of l-methyl-3-prop-l-ynyl-4-vinyl-pyrazolo[3,4-c]pyridine (3.2 g, 16.22 mmol, 1 eq) in THF (60 mL) and H2O (12 mL), was added K2OSO4.2H2O (1.20 g, 3.24 mmol, 0.2 eq), stirred for 30 min, then NalCh (10.41 g, 48.67 mmol, 2.70 mL, 3 eq) was added. The mixture was stirred at 25 °C for 15.5 h. Upon completion, the reaction mixture was poured
635
SUBSTITUTE SHEET RULE 26 into H2O (100 mL) at 25 °C, and then extracted with EtOAc (110 mL * 3). The combined organic layers were washed with brine (100 mL), dried over NaiSCh, filtered and concentrated under reduced pressure to give l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridine-4-carbaldehyde (2.2 g, crude) as a yellow solid. MS (ESI) m/z 200.0 [M+H]+.
[0001576] Step 3: 2-amino-2-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4-yl)acetonitrile
[0001577] To a solution of l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridine-4-carbaldehyde (2.1 g, 10.54 mmol, 1 eq) in DCM (50 mL) was added Ti(i-PrO)4 (4.49 g, 15.81 mmol, 4.67 mL, 1.5 eq) and NEh/MeOH (7 M, 6.02 mL, 4 eq), stirred for 1 h, then TMSCN (3.14 g, 31.63 mmol, 3.96 mL, 3 eq) was added. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was poured into H2O (40 mL) at 25 °C, and then extracted with DCM (60 mL * 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-( 1-methy 1-3 -prop- 1-ynyl- pyrazolo[3,4-c]pyridin-4-yl)acetonitrile (1.55 g, crude) as a red solid. MS (ESI) m/z 226.1 [M+H]+.
[0001578] Step 4: (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001579] To a solution of 2-amino-2-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4- yl)acetonitrile (300 mg, 1.33 mmol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (585.48 mg, 1.60 mmol, 1.2 eq), NMI (328.05 mg, 4.00 mmol, 318.50 uL, 3 eq) in ACN (6 mL) was added TCFH (747.38 mg, 2.66 mmol, 2 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was poured into H2O (20 mL) at 25 °C, and then extracted with DCM (25 mL * 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 30%-60%,8min) to give (lR,2S,5S)-N-[cyano-(l-methyl- 3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4-yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (415 mg, 687.37 umol, 51.61% yield, 95% purity) as a yellow solid. MS (ESI) m/z 574.4 [M+H]+.
636
SUBSTITUTE SHEET RULE 26 [0001580] Step 5: (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
[0001581] (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (415 mg, 723.54 umol, 1 eq) was separated by (column: DAICEL CHIRALPAK AD(250mm*30mm,10um);mobile phase: [Neu-IPA];B%: 30%- 30%,8min) to give (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (170 mg, 295.50 umol, 40.84% yield, 99.7% purity) as a yellow solid. MS (ESI) m/z 574.2 [M+H]+.
[0001582] 'H NMR (400 MHz, DMSO-de) 8 = 9.88 (d, J = 6.4 Hz, 1H), 9.42 - 9.21 (m, 2H),
8.45 (s, 1H), 6.63 (d, J = 7.0 Hz, 1H), 4.45 - 4.31 (m, 1H), 4.26 - 4.15 (m, 4H), 3.98 - 3.81 (m, 2H), 3.68 (dd, J = 6.3, 8.5 Hz, 1H), 3.26 (s, 3H), 2.10 (s, 3H), 1.58 (dd, J = 5.2, 7.4 Hz, 1H), 1.37 (d, J = 7.6 Hz, 1H), 1.20 (d, J = 6.2 Hz, 3H), 0.97 (s, 3H), 0.83 (s, 3H).
[0001583] (lR,2S,5S)-N-[cyano-(l-methyl-3-prop-l-ynyl-pyrazolo[3,4-c]pyridin-4- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (101.6 mg, 176.25 umol, 24.36% yield, 99.5% purity) was obtained as a yellow solid. MS (ESI) m/z 574.2 [M+H]+.
[0001584] 'H NMR (400 MHz, DMSO-de) 8 = 9.86 (d, J = 4.6 Hz, 1H), 9.45 (d, J = 6.6 Hz, 1H), 9.30 (s, 1H), 8.47 (s, 1H), 6.65 (d, J = 6.7 Hz, 1H), 4.44 - 4.33 (m, 1H), 4.30 (s, 1H), 4.20 (s, 3H), 3.96 - 3.80 (m, 2H), 3.59 (dd, J = 6.2, 8.8 Hz, 1H), 3.21 (s, 3H), 2.10 (s, 3H), 1.60 (dd, J = 5.2, 7.4 Hz, 1H), 1.44 (d, J = 7.6 Hz, 1H), 1.22 - 1.11 (m, 3H), 1.05 (s, 3H), 0.87 (s, 3H).
637
SUBSTITUTE SHEET RULE 26 Example 181: Synthesis of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2- [(2,2,2- trifluoroacetyl)amino] butanoyl] -N-(3-cyano-6-azatricyclo [6.3.1.04,12] dodeca- l(ll),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 301A)
Figure imgf000639_0001
Step 1: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-(3- cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001585] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (300 mg, 734.55 umol, 1 eq) in ACN (5 mL) was added 1 -methylimidazole (180.93 mg, 2.20 mmol, 175.66 uL, 3 eq), TCFH (412.20 mg, 1.47 mmol, 2 eq), and then added 3-amino-6- azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaene-3-carbonitrile (143.40 mg, 734.55 umol, 1 eq). The mixture was stirred at 20 °C for 16 h. Upon completion, the reaction mixture was diluted by addition H2O (30 mL), and then extracted with DCM (20 mL * 3). The combined organic layers were washed with brine (30 mL), dried over, filtered and concentrated under reduced pressure to give a residue. The residue was purifed by pre-HPLC (column: Waters Xbridge Prep OBD C18 150 * 40 mm * 10 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 45%-75%, 8 min) to give the product (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifhjoroacetyl)amino]butanoyl]-N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l (11), 4(12), 5,7,9- pentaen-3-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 306.45 umol, 41.72% yield, 99.7% purity) as a brown solid. MS (ESI) m/z 586.2 [M+H]+.
Step 2: (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-N-(3- cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
638
SUBSTITUTE SHEET RULE 26 [0001586] The (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]- N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l 1), 4(12), 5,7, 9-pentaen-3-yl)-6, 6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (180 mg, 307.37 umol, 1 eq) was separated by SFC (column: DAICEL CHIRALPAK IG (250mm * 30mm, 10 um); mobile phase: [Neu-ETOH]; B%: 12%-12%, 15 mm) to give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (53.33 mg, 90.52 umol, 29.45% yield, 99.4% purity) as a white solid. MS (ESI) m/z 586.2 [M+H]+.
[0001587] 'H NMR (400 MHz, DMSO-de) 5 = 9.57 (br d, J = 8.3 Hz, 1H), 9.48 - 9.39 (m, 2H), 8.96 - 8.87 (m, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.83 - 7.69 (m, 2H), 4.40 (t, J = 7.9 Hz, 1H), 4.28 (d, J = 18.2 Hz, 1H), 4.21 (s, 1H), 4.01 (dd, J = 5.3, 10.4 Hz, 1H), 3.92 (dd, J = 6.3, 7.3 Hz, 1H), 3.78 (d, J = 18.4 Hz, 1H), 3.65 (br d, J = 10.4 Hz, 1H), 1.60 (dd, J = 5.4, 7.5 Hz, 1H), 1.37 (d, J = 7.5 Hz, 1H), 1.17 - 1.09 (m, 11H), 1.03 - 0.98 (m, 4H), 0.81 (s, 3H).
[0001588] To give (lR,2S,5S)-3-[(2S,3R)-3-tert-butoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-N-(3-cyano-6-azatricyclo[6.3.1.04 12]dodeca-l (11), 4(12), 5,7,9- pentaen-3-yl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (55.86 mg, 85.66 umol, 27.87% yield, 89.8% purity) as a white solid. MS (ESI) m/z 586.2 [M+H]+.
[0001589] 'H NMR (400 MHz, DMSO-de) 5 = 9.57 (br d, J = 8.2 Hz, 1H), 9.45 (s, 1H), 9.38 (s, 1H), 8.82 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.82 - 7.69 (m, 2H), 4.39 (t, J = 7.9 Hz, 1H), 4.31 - 4.20 (m, 2H), 4.04 - 3.86 (m, 3H), 3.66 (d, J = 10.5 Hz, 1H), 1.58 (dd, J = 5.4, 7.4 Hz, 1H), 1.42 (d, J = 7.6 Hz, 1H), 1.16 - 1.11 (m, 12H), 1.01 - 0.98 (m, 3H), 0.81 (s, 3H).
639
SUBSTITUTE SHEET RULE 26 Example 182: Synthesis of (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-
1(11), 4(12), 5,7, 9-pentaen-3-yl)-6,6-dimethyl- 3-[(2S, 3R)-3-(l-methylcyclopropoxy)-2-[(2, 2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound
302A)
Figure imgf000641_0001
Step 1: methyl isoquinoline-4-carboxylate
[0001590] To a solution of 4-bromoisoquinoline (55 g, 264.35 mmol, 1 eq) in MeOH (600 mL) was added TEA (267.50 g, 2.64 mol, 367.95 mL, 10 eq), Xantphos (30.59 g, 52.87 mmol, 0.2 eq) and Pd(OAc)2 (5.93 g, 26.44 mmol, 0.1 eq) in turn. After the addition, the resulting mixture was stirred at 75 °C under CO (264.35 mmol, 1 eq) (30 psi) for 48 h. Two batches were set up in parallel. Upon completion, the combined reaction mixture was filtered. The filtrate was concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 10: 1 to 2:1) to afford methyl isoquinoline-4-carboxylate (81 g, 393.76 mmol, 74.48% yield, 91% purity) as white solid and other curde product (55 g) which need further purification.
Step 2: methyl 5-bromoisoquinoline-4-carboxylate
640
SUBSTITUTE SHEET RULE 26 [0001591] To a mixture of methyl isoquinoline-4-carboxylate (81 g, 432.71 mmol, 1 eq) in H2SO4 (560 mL) at 0 °C was added NBS (110.90 g, 623.10 mmol, 1.44 eq) slowly in portions. After the addition, the resulting mixture was stirred at 25 °C for 12 h. To the reaction mixture was added ice (300 g) and EtOAc (1 L). Upon completion, the resulting mixture was adjusted pH to 7-8 with sat.aqueous NaHCCh. The organic layer was separated, dried over Na2SO4, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 10: 1 to 6:1) to afford methyl 5- bromoisoquinoline-4-carboxylate (80 g, 269.68 mmol, 62.32% yield, 89.7% purity) as yellow solid. MS (ESI) m/z 266.0 [M+H]+.
Step 3: methyl 5-allylisoquinoline-4-carboxylate
[0001592] To a solution of methyl 5-bromoisoquinoline-4-carboxylate (20 g, 75.16 mmol, 1 eq), PPhs (1.97 g, 7.52 mmol, 0.1 eq) and allyl(tributyl)stannane (37.33 g, 112.74 mmol, 34.57 mL, 1.5 eq) in Tol. (200 mL) under N2 was added Pd2(dba)3 (6.88 g, 7.52 mmol, 0.1 eq). After the addition, the mixture was stirred at 110 °C for 12 h. Upon completion, the reaction mixture cooled to 25 °C, diluted with EtOAc (1000 mL) and water (1000 mL). The mixture was extracted with EtOAc (1000 mL * 3). The combined organic layer was dried over Na2SO4, filtered, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 20: 1 to 3:1) to afford methyl 5- allylisoquinoline-4-carboxylate (26 g, 93.81 mmol, 62.41% yield, 82% purity) as yellow oil. MS (ESI) m/z 228.1 [M+H]+.
Step 4: methyl 5-(2-methoxy-2-oxo-ethyl)isoquinoline-4-carboxylate
[0001593] Methyl 5-allylisoquinoline-4-carboxylate (28 g, 123.21 mmol, 1 eq) in anhydrous DCM (1 L) was cooled to -60 °C. And then 250 mL 2.5 M NaOH/MeOH was added. After the addition, ozone was bubbled pass through the reaction solution at 15 psi for 6 h. The reaction mixture was quenched by (500 mL) 10% aqueous citric acid, extracted with DCM (1 L * 2). Upon completion, the combined organic layer was dried over Na2SO4, fitlered, conentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiCh, Petroleum ether:Ethyl acetate = 20: 1 to 8:1) to afford methyl 5-(2-methoxy-2-oxo- ethyl)isoquinoline-4-carboxylate (6.8 g, 16.58 mmol, 13.45% yield, 63.2% purity). MS (ESI) m/z 260.1 [M+H]+.
641
SUBSTITUTE SHEET RULE 26 Step 5: methyl 3-oxo-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4,6,8(12),9-pentaene-2-carboxylate
[0001594] A mixture of methyl 5-(2-methoxy-2-oxo-ethyl)isoquinoline-4-carboxylate (3.4 g, 13.11 mmol, 1 eq) and t-BuOK (4.41 g, 39.34 mmol, 3 eq) in toluene (40 mL) was stirred at 50 °C for 2 h. Two batches were set up in parallel. Upon completion, the reaction mixture was concentrated in a vacuum to afford methyl 3-oxo-6-azatricyclo[6.3.1.04,12]dodeca- 1(1 l),4,6,8(12),9-pentaene-2-carboxylate (6 g, crude) as red solid, which was used for next step without further purification. MS (ESI) m/z 228.0 [M+H]+.
Step 6: 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one
[0001595] A solution of methyl 3-oxo-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4, 6, 8(12), 9- pentaene-2-carboxylate (3 g, 13.20 mmol, Crude purity, 1 eq) in 6 M HC1 (60 mL) was strried at 110 °C for 1 h. Two batches were set up in parallel. Upon completion, the reaction mixture was cooled to 25 °C, poured into sat.aq.NaHCCh (1.5 L), diluted with EtOAc (1 L). The resulting mixture was filtered and the filter cake was wahsed with EtOAc (500 mL). The combined filtrate was extracted with EtOAc (1.5 L * 2). The organic layer was separated, dried over Na2SO4, filtered, concentrated in a vacuum to afford a residue. The residue was purified by column chromatography (SiO2, Petroleum ether :Ethyl acetate = 3: 1 to 5:1) to give 6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one (2.36 g, 13.95 mmol, 47.20% yield) as a yellow oil. MS (ESI) m/z 170.0 [M+H]+.
Step 7: 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one
[0001596] To a solution of 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one (1.5 g, 8.87 mmol, 1 eq) and Ti(i-PrO)4 (3.78 g, 13.30 mmol, 3.93 mL, 1.5 eq) in DCM (15 mL) was added NH3/MeOH (7 M, 5.07 mL, 4 eq) stirred 1 h at 25 °C, then TMSCN (2.64 g, 26.60 mmol, 3.33 mL, 3 eq) was added at 25 °C. The mixture was stirred at 25 °C for 15 h. The reaction mixture was poured into H2O (30 mL) at 25 °C, and then extracted with EtOAc (30 mL * 3). Upon completion, the combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 5: 1 to 0:1) to give 3- amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaene-3-carbonitrile (600 mg, 2.70 mmol, 30.50% yield, 88% purity) as a black brown oil. MS (ESI) m/z 196.2 [M+H]+.
642
SUBSTITUTE SHEET RULE 26 Step 8: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001597] A mixture of 3-amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4(12), 5,7, 9-pentaene- 3 -carbonitrile (300 mg, 1.54 mmol, 1 eq), (lR,2S,5S)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (749.43 mg, 1.84 mmol, 1.2 eq) and 1 -methylimidazole (378.51 mg, 4.61 mmol, 367.49 uL, 3 eq) in ACN (10 mL) was added[chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (862.35 mg, 3.07 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was poured into H2O 30 mL at 20 °C, and then extracted with EtOAC (30 mL * 3). Upon completion, the combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 10 min) to give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6- dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3- azabicyclo[3.1.0]hexane-2-carboxamide (410 mg, 636.78 umol, 41.44% yield, 90.64% purity) as a yellow solid. MS (ESI) m/z 584.3 [M+H]+.
Step 9: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3. L0]hexane-2-carboxamide
[0001598] (lR,2S,5S)-N-(3-cyano-6-azatncyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-6,6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3. L0]hexane-2-carboxamide (600 mg) was separated by SFC (column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 35%-35%, 7 min) to give (lR,2S,5S)-N-(3-cyano-6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-6,6-dimethyl-3-[(2S,3R)-3-(l- methylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.L0]hexane-2- carboxamide Isomer 1 (121.74 mg, 208.60 umol, 29.69% yield, 100% purity) as a white solid. MS (ESI) m/z 584.3 [M+H]+.
643
SUBSTITUTE SHEET RULE 26 [0001599] 'H NMR (400 MHz, DMSO-de) 8 = 9.77 (s, 1H), 9.50 (s, 1H), 9.41 (s, 1H), 8.88 (s,
1H), 8.05 (d, J = 8.1 Hz, 1H), 7.82 - 7.76 (m, 1H), 7.74 - 7.70 (m, 1H), 4.36 (d, J = 8.2 Hz, 1H), 4.28 (d, J = 18.3 Hz, 1H), 4.19 (s, 1H), 3.99 - 3.91 (m, 2H), 3.78 (d, J = 18.3 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 1.60 (dd, J = 5.3, 7.4 Hz, 1H), 1.38 (s, 1H), 1.36 - 1.34 (m, 3H), 1.22 (d, J = 6.2 Hz, 3H), 1.01 (s, 3H), 0.80 (s, 3H), 0.77 (br d, J = 4.6 Hz, 1H), 0.71 - 0.64 (m, 1H), 0.42 - 0.30 (m, 2H).
[0001600] To give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-
1(11), 4(12), 5,7, 9-pentaen-3-yl)-6, 6-dimethyl-3-[(2S,3R)-3-(l-methylcyclopropoxy)-2-[(2, 2,2- trifluoroacetyl)amino]butanoyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (141.72 mg, 241.74 umol, 34.41% yield, 99.55% purity) as a white solid. MS (ESI) m/z 584.3 [M+H]+.
[0001601] 'H NMR (400 MHz, DMSO-de) 8 = 9.78 (d, J = 7.0 Hz, 1H), 9.50 (s, 1H), 9.38 (s,
1H), 8.82 (s, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.75 - 7.70 (m, 1H), 4.36 (s, 1H), 4.26 (d, J = 18.2 Hz, 1H), 4.22 (s, 1H), 3.97 - 3.89 (m, 3H), 3.69 (d, J = 10.4 Hz, 1H), 1.57 (dd, J = 5.3, 7.4 Hz, 1H), 1.42 (d, J = 7.6 Hz, 1H), 1.35 (s, 3H), 1.22 (d, J = 6.1 Hz, 3H), 1.01 - 0.97 (m, 3H), 0.81 (s, 3H), 0.77 (d, J = 4.8 Hz, 1H), 0.69 (d, J = 5.1 Hz, 1H), 0.40 - 0.32 (m, 2H).
Example 183: Synthesis of (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- l(ll),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 303A)
Figure imgf000645_0001
Step 1: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1 ,0]hexane-2-carboxamide
644
SUBSTITUTE SHEET RULE 26 [0001602] A mixture of 3-amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l 1), 4(12), 5,7, 9-pentaene- 3 -carbonitrile (300 mg, 1.54 mmol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (675.54 mg, 1.84 mmol, 1.2 eq) and 1 -methylimidazole (378.50 mg, 4.61 mmol, 367.47 uL, 3 eq) in ACN (1 mL) was added [chloro(dimethylamino)methylene]-dimethyl- ammonium;hexafluorophosphate (862.35 mg, 3.07 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C for 1 h. The reaction mixture was poured into H2O (30 mL) at 20 °C, and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C 18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 10 min) to give (lR,2S,5S)-N-(3-cyano-6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (404 mg, 709.54 umol, 46.17% yield, 95.46% purity) as a yellow solid. MS (ESI) m/z 544.2 [M+H]+.
Step 2: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide
[0001603] (lR,2S,5S)-N-(3-cyano-6-azatncyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9- pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (600 mg) was separated by SFC (column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA]; B%: 35%-35%, 11 min) to give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (109.08 mg, 200.69 umol, 27.00% yield, 100% purity) as a white solid. MS (ESI) m/z 544.2 [M+H]+.
[0001604] 'H NMR (400 MHz, DMSO-de) 8 = 9.82 (br s, 1H), 9.51 (s, 1H), 9.41 (s, 1H), 8.88 (s, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.74 - 7.69 (m, 1H), 4.36 (br d, J = 8.2 Hz, 1H), 4.27 (d, J = 18.3 Hz, 1H), 4.19 (s, 1H), 3.96 (dd, J = 5.4, 10.3 Hz, 1H), 3.85 - 3.74 (m, 2H), 3.70 - 3.63 (m, 1H), 3.28 (s, 3H), 1.62 (dd, J = 5.4, 7.5 Hz, 1H), 1.36 (d, J = 7.6 Hz, 1H), 1.22 (d, J = 6.2 Hz, 3H), 1.02 (s, 3H), 0.84 (s, 3H).
645
SUBSTITUTE SHEET RULE 26 [0001605] To give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- 1(1 l),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer
2 (138.4 mg, 245.21 umol, 32.99% yield, 96.3% purity) as a white solid. MS (ESI) m/z 544.2 [M+H]+.
[0001606] 'H NMR (400 MHz, DMSO-de) 8 = 9.81 (s, 1H), 9.51 (s, 1H), 9.37 (s, 1H), 8.81 (s,
1H), 8.03 (d, J = 8.1 Hz, 1H), 7.83 - 7.75 (m, 1H), 7.75 - 7.70 (m, 1H), 4.36 (d, J = 8.2 Hz, 1H), 4.31 - 4.21 (m, 2H), 3.99 - 3.82 (m, 3H), 3.70 - 3.60 (m, 1H), 3.27 (s, 3H), 1.59 (dd, J = 5.4, 7.4 Hz, 1H), 1.42 (d, J = 7.6 Hz, 1H), 1.22 (d, J = 6.1 Hz, 3H), 1.00 (s, 3H), 0.85 (s, 3H).
Example 184: Synthesis of (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- l(ll),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Compound 304A)
Figure imgf000647_0001
Step 1: 3-amino-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaene-3-carbonitrile
[0001607] To a solution of 6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-one (860 mg, 5.08 mmol, 1 eq) and Ti(i-PrO)4 (2.17 g, 7.63 mmol, 2.25 mL, 1.5 eq) in DCM (8 mL)
646
SUBSTITUTE SHEET RULE 26 was added NH3/MeOH (7 M, 2.90 mL, 4 eq) stirred 1 h at 25 °C, then TMSCN (2.77 g, 27.96 mmol, 3.50 mL, 5.5 eq) was added at 25 °C. The mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether:Ethyl acetate = 5: 1 to 1:3) to give 3-amino-6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaene-3-carbonitrile (220 mg, 991.70 umol, 19.51% yield, 88% purity) as a black brown oil. MS (ESI) m/z 196.1 [M+H]+.
Step 2: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide
[0001608] To a solution of 3-amino-6-azatricyclo[6.3.1.04, 12] dodeca- 1(11), 4(12), 5,7,9- pentaene-3-carbonitrile (220 mg, 1.13 mmol, 1 eq), (lR,2S,5S)-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (584.79 mg, 1.35 mmol, 1.2 eq) and 1- methylimidazole (277.58 mg, 3.38 mmol, 269.49 uL, 3 eq) in ACN (5 mL) was added [chloro(dimethylamino)methylene]-dimethyl-ammonium;hexafluorophosphate (632.39 mg, 2.25 mmol, 2 eq) at 0 °C. The mixture was stirred at 25 °C for 1 h. After the addition, the reaction mixture was poured into H2O (30 mL), and then extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: C 18 (250 * 50 mm * 10 um); mobile phase: [water (NH4HCO3)-ACN]; B%: 40%- 70%, 10 min) to give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- 1(1 l),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide (313 mg, 488.64 umol, 43.36% yield, 95.173% purity) as a yellow oil. MS (ESI) m/z 610.4 [M+H]+.
Step 3: (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3- yl)-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6- dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide
647
SUBSTITUTE SHEET RULE 26 [0001609] (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04, 12] dodeca- 1 (11), 4(12), 5,7,9- pentaen-3 -y 1) - 3 - [(2S,3R)-3-(l -cyclopropylcyclopropoxy)-2- [(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (310 mg) was separated by SFC (column: DAICEL CHIRALPAK IG (250 mm * 30 mm, 10 um); mobile phase: [Neu-IPA];B%: 45%-45%, 5 min) to give (lR,2S,5S)-N-(3-cyano-6- azatricyclo[6.3.1.04,12]dodeca-l(l l),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-(l- cyclopropylcyclopropoxy)-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (73.14 mg, 119.97 umol, 23.59% yield, 100% purity) as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001610] 'H NMR (400 MHz, DMSO-de) 8 = 9.73 (d, J = 8.2 Hz, 1H), 9.49 (s, 1H), 9.41 (s,
1H), 8.89 (s, 1H), 8.08 - 7.99 (m, 1H), 7.83 - 7.76 (m, 1H), 7.75 - 7.69 (m, 1H), 4.40 (t, J = 8.2 Hz, 1H), 4.28 (d, J = 18.4 Hz, 1H), 4.20 (s, 1H), 4.08 - 4.02 (m, 1H), 3.97 (dd, J = 5.5, 10.4 Hz, 1H), 3.79 (d, J = 18.4 Hz, 1H), 3.67 (d, J = 10.4 Hz, 1H), 1.61 (dd, J = 5.4, 7.4 Hz, 1H), 1.46 -
1.39 (m, 1H), 1.37 (d, J = 7.5 Hz, 1H), 1.30 (d, J = 6.2 Hz, 3H), 1.01 (s, 3H), 0.80 (s, 3H), 0.73 - 0.63 (m, 1H), 0.56 - 0.43 (m, 3H), 0.38 - 0.30 (m, 1H), 0.23 (br dd, J = 4.7, 10.7 Hz, 1H), 0.16 - 0.08 (m, 1H), 0.06 - 0.00 (m, 1H).
[0001611] To give (lR,2S,5S)-N-(3-cyano-6-azatricyclo[6.3.1.04,12]dodeca- 1(1 l),4(12),5,7,9-pentaen-3-yl)-3-[(2S,3R)-3-(l-cyclopropylcyclopropoxy)-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxamide Isomer 2 (74.36 mg, 121.49 umol, 23.89% yield, 99.6% purity) as a white solid. MS (ESI) m/z 610.4 [M+H]+.
[0001612] 1 H NMR (400 MHz, DMSO-de) 8 = 9.74 (br d, J = 8.1 Hz, 1H), 9.49 (s, 1H), 9.42 - 9.34 (m, 1H), 8.83 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.82 - 7.76 (m, 1H), 7.75 - 7.69 (m, 1H),
4.40 (t, J = 8.2 Hz, 1H), 4.30 - 4.20 (m, 2H), 4.09 - 4.01 (m, 1H), 3.99 - 3.88 (m, 2H), 3.68 (d, J = 10.4 Hz, 1H), 1.62 - 1.55 (m, 1H), 1.42 (br d, J = 7.6 Hz, 2H), 1.30 (d, J = 6.2 Hz, 3H), 0.99 (s, 3H), 0.81 (s, 3H), 0.73 - 0.63 (m, 1H), 0.56 - 0.43 (m, 3H), 0.38 - 0.30 (m, 1H), 0.27 - 0.19 (m, 1H), 0.14 - 0.07 (m, 1H), 0.07 - 0.00 (m, 1H).
648
SUBSTITUTE SHEET RULE 26 Example 185: Synthesis of (lR,2S,5S)-N-[cyano-(7-fhioro-4-methyl-pyrazolo[l,5-a]pyridin- 3-yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 499)
Figure imgf000650_0001
Step 1: methyl 4-bromo-7-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate
[0001613] To a mixture of pre-dried CsF (1.18 g, 7.77 mmol, 286.54 uL, 10 eq) and 4A MOLECULAR SIEVE (2.1 g, 15.54 mmol, 20 eq) under N2 was added DMSO (11 mL). The resulting mixture was stirred at 25 °C for 0.5 h. And then methyl 4-bromo-7-chloro- pyrazolo[l,5-a]pyridine-3-carboxylate (225 mg, 777.17 umol, 1 eq) was added. The resulting mixture was stirred at 40 °C for 15.5 h. Upon completion, the reaction mixture was diluted with saturated NH-iCl aqueous solution (150 mL) and was extracted with EA (150 mL*3), dried with Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by prep- HPLC (column: Welch Ultimate XB-CN 250*70*10um;mobile phase: [water(HCl)-ACN];B%: 20%-50%,20min) to give methyl 4-bromo-7-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate (800 mg, 2.78 mmol, 29.84% yield, 95% purity) as a yellow solid. MS (ESI) m/z 273.1 [M+H]+
649
SUBSTITUTE SHEET RULE 26 Step 2: methyl 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate
[0001614] To a solution of methyl 4-bromo-7-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate (700 mg, 2.56 mmol, 1 eq) in dioxane (35 mL) was added K2CO3 (708.60 mg, 5.13 mmol, 2 eq) and 2,4,6-trimethyl-l,3,5,2,4,6-trioxatriborinane (965.44 mg, 3.85 mmol, 1.08 mL, 50% purity, 1.5 eq). And then cyclopentyl(diphenyl)phosphane;dichloropalladium;iron (187.58 mg, 256.36 umol, 0.1 eq) was added. The mixture was stirred at 90 °C for 16 h. Upon completion, the reaction mixture was diluted with saturated NH4CI aqueous solution (80 mL) and was extracted with EA (70 mL*3), dried with Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 20/1 to 3/1) to give methyl 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate (560 mg, 2.56 mmol, 99.68% yield, 95% purity) as a white solid. MS (ESI) m/z 209.2 [M+H]+
Step 3: (7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methanol
[0001615] To a solution of methyl 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate (560 mg, 2.69 mmol, 1 eq) in THF (63 mL) was cooled to -65 °C. And then DIBAL-H (I M, 9.41 mL, 3.5 eq) was added. The mixture was stirred at 25 °C for 2 h. And then DIBAL-H (I M, 9.41 mL, 3.5 eq) was added at -65 °C. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixtrure was quenched by 20% aqueous Potassium Sodium Tartrate (80 mL) and diluted with EtOAc (70 mL*3). Then organic phase was separated and dried over Na2SO4, filtered and concentrated in a vacuum to afford (7-fluoro-4-methyl-pyrazolo[l,5- a]pyridin-3-yl)methanol (370 mg, 1.79 mmol, 66.42% yield, 87% purity) as a white solid. MS (ESI) m/z 181.2 [M+H]+
Step 4: 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde
[0001616] To a solution of (7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methanol (370 mg, 2.05 mmol, 1 eq) in DCM (30 mL) was added MnCh (3.57 g, 41.07 mmol, 20 eq). The mixture was stirred at 30 °C for 4 h. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure to give 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde (310 mg, crude) as a white solid. MS (ESI) m/z 179.2 [M+H]+
Step 5: 2-amino-2-(7-fluoro-4-methyl-pyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
650
SUBSTITUTE SHEET RULE 26 [0001617] To a solution of 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde (290 mg, 1.63 mmol, 1 eq) in DCM (5 mL) was added NHa/MeOH (7 M, 2 mL, 8.60 eq) and Ti(i- PrO)4 (693.94 mg, 2.44 mmol, 720.60 uL, 1.5 eq). The mixture was stirred at 25 °C for 0.5 h. And then TMSCN (484.44 mg, 4.88 mmol, 610.90 uL, 3 eq) was added. The mixture was stirred at 25 °C for 15.5 h. Upon completion, the combined was diluted with water (30 mL) and extracted with DCM (30 mL). And then filtered to give a solution was extracted with DCM (30 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)acetonitrile (320 mg, crude) as a yellow solid.
Step 6: (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-
3.3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane- 2-carboxamide
[0001618] To a solution of 2-amino-2-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3- yl)acetonitrile (240 mg, 1.18 mmol, 1 eq) in ACN (10 mL) was added (lR,2S,5S)-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0]hexane-2- carboxylic acid (428.23 mg, 1.18 mmol, 1 eq) and NMI (289.49 mg, 3.53 mmol, 281.06 uL, 3 eq). And then TCFH (659.53 mg, 2.35 mmol, 2 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the combined was diluted with water (20 mL) and extracted with EA (15 mL). And then filtered to give a solution was extracted with EA (15 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-70%,8min) to give (lR,2S,5S)-N-[cyano-(7-fhroro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0] hexane-2- carboxamide (100 mg, 181.64 umol, 15.45% yield, 100% purity) as a yellow solid. MS (ESI) m/z 551.2 [M+H]+
Step 7: (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-
3.3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.L0] hexane-2-carboxamide
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SUBSTITUTE SHEET RULE 26 [0001619] The (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]- 3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 181.64 umol, 100% purity) was separated by SFC (column: DAI CEL CHIRALPAK IG (250mm*30mm,10um);mobile phase: [Neu-IPA];B%: 20%-20%,5min) to give (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3- yl)methyl]-3-[(2S)-3,3-dimethyl -2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (25 mg, 45.41 umol, 25.00% yield, 100% purity) as a white solid. MS (ESI) m/z 551.3 [M+H]+
[0001620] 'H NMR (400 MHz, DMSO-de) 5 = 9.42 - 9.29 (m, 2H), 8.29 (s, 1H), 7.13 (t, I = 6.4 Hz, 1H), 6.85 (dd, I = 5.6, 7.6 Hz, 1H), 6.55 (d, I = 6.8 Hz, 1H), 4.40 (br s, 1H), 4.22 (s, 1H), 3.93 (dd, I = 5.4, 10.3 Hz, 1H), 3.72 (d, I = 10.4 Hz, 1H), 2.54 (s, 3H), 1.60 (dd, I = 5.5, 7.4 Hz, 1H), 1.29 (d, I = 7.6 Hz, 1H), 1.04 - 0.97 (m, 12H), 0.81 (s, 3H)
[0001621] (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (27 mg, 49.04 umol, 27.00% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 551.3 [M+H]+
[0001622] 'H NMR (400 MHz, DMSO-de) 5 = 9.44 - 9.31 (m, 1H), 9.22 (d, I = 7.3 Hz, 1H), 8.36 - 8.22 (m, 1H), 7.13 (t, I = 6.6 Hz, 1H), 6.85 (dd, I = 5.8, 7.5 Hz, 1H), 6.48 (d, I = 7.3 Hz, 1H), 4.36 (d, I = 8.4 Hz, 1H), 4.22 (s, 1H), 3.93 (dd, 1 = 5.6, 10.3 Hz, 1H), 3.65 (d, I = 11.0 Hz, 1H), 2.54 (s, 3H), 1.56 (dd, 1 = 5.5, 7.4 Hz, 1H), 1.22 (d, I = 7.6 Hz, 1H), 1.00 - 0.98 (m, 3H), 0.91 (s, 9H), 0.81 (s, 3H)
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SUBSTITUTE SHEET RULE 26 Example 186: Synthesis of (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3- yl)methyl]-3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 500)
Figure imgf000654_0001
Step 1: 2-amino-2-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile
[0001623] A solution of 4-ethynylpyrazolo[l,5-a]pyridine-3-carbaldehyde (0.6 g, 3.53 mmol,
1 eq) in NHa/MeOH (8 mL, 7 M) was added TMSCN (1.05 g, 10.58 mmol, 1.32 mL, 3 eq) was stirred at 25 °C for 1 h, then added NH4CI (565.81 mg, 10.58 mmol, 3 eq) at 0 °C, the mixture was stirred at 25 °C for 15 h. Upon completion, the reaction mixture was quenched by addition H2O (50 mL), and then extracted with EA (30 mL * 3). The combined organic layers were washed with brine (20 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.6 g, crude) as yellow solid.
Step 2: (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001624] To a solution of (lR,2S,5S)-3-[(2S,3R)-3-methoxy-2-[(2,2,2- trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1 ,0]hexane-2-carboxylic acid (448.09 mg, 1.22 mmol, 1 eq), 2-amino-2-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)acetonitrile (0.6
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SUBSTITUTE SHEET RULE 26 g, 1.22 mmol, 40% purity, 1 eq), in ACN (10 mL) was added TCFH (686.41 mg, 2.45 mmol, 2 eq) and NMI (301.27 mg, 3.67 mmol, 292.50 uL, 3 eq). The mixture was stirred at 25 °C for 1 h. Upon completion, the reaction mixture was diluted with H2O 80mL and then extracted with EA (50 mL * 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18 250 * 50 mm * 10 um; mobile phase: [water (NH4HCO3)- ACN] ; B%: 35%-55%, 10 min) to give (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl]- 3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.22 g, 404.02 umol, 33.03% yield) as white solid. MS (ESI) m/z 545.1[M+H]+
Step 3: (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide
[0001625] The (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3- [(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (0.22 g, 404.02 umol, 1 eq) was separation by SFC (column: REGIS (S, S) WHELK-01 (250 mm * 25 mm, 10 um); mobile phase: [Neu-IPA]; B%: 33%-33%, 7 min) to give (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl]- 3-[(2S,3R)-3-methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (0.073 g, 134.06 umol, 33.18% yield, 100% purity) as a white solid. MS (ESI) m/z 545.2[M+H]+
[0001626] 'H NMR (400 MHz, DMSO-t/r,) 5 = 9.87 (br d, J= 6.6 Hz, 1H), 9.29 (d, J= 6.8 Hz, 1H), 8.84 (d, J= 7.0 Hz, 1H), 8.30 (s, 1H), 7.59 (d, J= 7.2 Hz, 1H), 7.01 (t, J= 7.1 Hz, 1H), 6.52 (d, J= 6.8 Hz, 1H), 4.74 (s, 1H), 4.37 (br s, 1H), 4.23 (s, 1H), 3.91 (br d, J= 5.2 Hz, 1H), 3.86 - 3.80 (m, 1H), 3.70 - 3.63 (m, 1H), 3.26 (s, 3H), 1.54 (dd, J= 5.4, 7.3 Hz, 1H), 1.33 (d, J = 7.6 Hz, 1H), 1.18 (d, J= 6.2 Hz, 3H), 1.00 - 0.95 (m, 3H), 0.85 (s, 3H)
[0001627] (lR,2S,5S)-N-[cyano-(4-ethynylpyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S,3R)-3- methoxy-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide Isomer 2 (0.072 g, 132.23 umol, 32.73% yield, 100% purity) was obtained as a white solid. MS (ESI) m/z 545.2[M+H]+.
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SUBSTITUTE SHEET RULE 26 [0001628] 'H NMR (400 MHz, DMSO-t/e) 5 = 9.84 (br d, J = 6.1 Hz, 1H), 9.38 (d, J = 6.6 Hz, 1H), 8.83 (d, J= l.Q Hz, 1H), 8.29 (s, 1H), 7.58 (d, J= l.Q Hz, 1H), 7.01 (t, J= Hz, 1H), 6.55 (d, J= 6.6 Hz, 1H), 4.62 (s, 1H), 4.35 (br s, 1H), 4.28 (s, 1H), 3.90 (br d, J= 5.1 Hz, 1H), 3.87 - 3.81 (m, 1H), 3.60 (br dd, J = 6.3, 8.5 Hz, 1H), 3.21 (s, 3H), 1.62 - 1.53 (m, 1H), 1.41 (d, J = 7.5 Hz, 1H), 1.14 (d, J = 6.2 Hz, 3H), 1.06 - 1.01 (m, 3H), 0.87 (s, 3H)
Example 187: Synthesis of (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin- 3-yl)methyl] -3- [(2S)-3,3-dimethyl-2- [(2,2,2-trifluoroacetyl)amino] butanoyl] -6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Compound 499)
Figure imgf000656_0001
Step 1: methyl 4-bromo-7-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate
[0001629] To a mixture of pre-dried CsF (1.18 g, 7.77 mmol, 286.54 uL, 10 eq) and 4A MOLECULAR SIEVE (2.1 g, 15.54 mmol, 20 eq) under N2 was added DMSO (11 mL). The resulting mixture was stirred at 25 °C for 0.5 h. And then methyl 4-bromo-7-chloro- pyrazolo[l,5-a]pyridine-3-carboxylate (225 mg, 777.17 umol, 1 eq) was added. The resulting mixture was stirred at 40 °C for 15.5 h. Upon completion, the reaction mixture was diluted with saturated NH4CI aqueous solution (150 mL) and was extracted with EA (150 mL*3), dried with
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SUBSTITUTE SHEET RULE 26 Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by prep- HPLC (column: Welch Ultimate XB-CN 250*70*10um;mobile phase: [water(HCl)-ACN];B%: 20%-50%,20min) to give methyl 4-bromo-7-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate (800 mg, 2.78 mmol, 29.84% yield, 95% purity) as a yellow solid. MS (ESI) m/z 273.1 [M+H]+
Step 2: methyl 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate
[0001630] To a solution of methyl 4-bromo-7-fluoro-pyrazolo[l,5-a]pyridine-3-carboxylate (700 mg, 2.56 mmol, 1 eq) in dioxane (35 mL) was added K2CO3 (708.60 mg, 5.13 mmol, 2 eq) and 2,4,6-trimethyl-l,3,5,2,4,6-trioxatriborinane (965.44 mg, 3.85 mmol, 1.08 mL, 50% purity, 1.5 eq). And then cyclopentyl(diphenyl)phosphane;dichloropalladium;iron (187.58 mg, 256.36 umol, 0.1 eq) was added. The mixture was stirred at 90 °C for 16 h. Upon completion, the reaction mixture was diluted with saturated NH4CI aqueous solution (80 mL) and was extracted with EA (70 mL*3), dried with Na2SO4, filtered and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether/Ethyl acetate = 20/1 to 3/1) to give methyl 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate (560 mg, 2.56 mmol, 99.68% yield, 95% purity) as a white solid. MS (ESI) m/z 209.2 [M+H]+
Step 3: (7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methanol
[0001631] To a solution of methyl 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carboxylate (560 mg, 2.69 mmol, 1 eq) in THF (63 mL) was cooled to -65 °C. And then DIBAL-H (I M, 9.41 mL, 3.5 eq) was added. The mixture was stirred at 25 °C for 2 h. And then DIBAL-H (I M, 9.41 mL, 3.5 eq) was added at -65 °C. The mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixtrure was quenched by 20% aqueous Potassium Sodium Tartrate (80 mL) and diluted with EtOAc (70 mL*3). Then organic phase was separated and dried over Na2SO4, filtered and concentrated in a vacuum to afford (7-fluoro-4-methyl-pyrazolo[l,5- a]pyridin-3-yl)methanol (370 mg, 1.79 mmol, 66.42% yield, 87% purity) as a white solid. MS (ESI) m/z 181.2 [M+H]+
Step 4: 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde
[0001632] To a solution of (7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methanol (370 mg, 2.05 mmol, 1 eq) in DCM (30 mL) was added MnCh (3.57 g, 41.07 mmol, 20 eq). The mixture was stirred at 30 °C for 4 h. Upon completion, the reaction mixture was filtered and concentrated
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SUBSTITUTE SHEET RULE 26 under reduced pressure to give 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde (310 mg, crude) as a white solid. MS (ESI) m/z 179.2 [M+H]+
Step 5: 2-amino-2-(7-fluoro-4-methyl-pyrazolo[l ,5-a]pyridin-3-yl)acetonitrile
[0001633] To a solution of 7-fluoro-4-methyl-pyrazolo[l,5-a]pyridine-3-carbaldehyde (290 mg, 1.63 mmol, 1 eq) in DCM (5 mL) was added NEb/MeOH (7 M, 2 mL, 8.60 eq) and Ti(i- PrO)4 (693.94 mg, 2.44 mmol, 720.60 uL, 1.5 eq). The mixture was stirred at 25 °C for 0.5 h. And then TMSCN (484.44 mg, 4.88 mmol, 610.90 uL, 3 eq) was added. The mixture was stirred at 25 °C for 15.5 h. Upon completion, the combined was diluted with water (30 mL) and extracted with DCM (30 mL). And then filtered to give a solution was extracted with DCM (30 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 2-amino-2-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)acetonitrile (320 mg, crude) as a yellow solid.
Step 6: (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide
[0001634] To a solution of 2-amino-2-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3- yl)acetonitrile (240 mg, 1.18 mmol, 1 eq) in ACN (10 mL) was added (lR,2S,5S)-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxylic acid (428.23 mg, 1.18 mmol, 1 eq) and NMI (289.49 mg, 3.53 mmol, 281.06 uL, 3 eq). And then TCFH (659.53 mg, 2.35 mmol, 2 eq) was added. The mixture was stirred at 25 °C for 1 h. Upon completion, the combined was diluted with water (20 mL) and extracted with EA (15 mL). And then filtered to give a solution was extracted with EA (15 mL * 2). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150*40mm*10um;mobile phase: [water( NH4HCO3)-ACN];B%: 40%-70%,8min) to give (lR,2S,5S)-N-[cyano-(7-fhroro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)-3,3- dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2- carboxamide (100 mg, 181.64 umol, 15.45% yield, 100% purity) as a yellow solid. MS (ESI) m/z 551.2 [M+H]+
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SUBSTITUTE SHEET RULE 26 Step 7: (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3-[(2S)- 3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxamide
[0001635] The (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]- 3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (100 mg, 181.64 umol, 100% purity) was separated by SFC (column: DAI CEL CHIRALPAK IG (250mm*30mm,10um);mobile phase: [Neu-IPA];B%: 20%-20%,5min) to give (lR,2S,5S)-N-[cyano-(7-fhioro-4-methyl-pyrazolo[l,5-a]pyridin-3- yl)methyl]-3-[(2S)-3,3-dimethyl -2-[(2,2,2-trifhioroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 1 (25 mg, 45.41 umol, 25.00% yield, 100% purity) as a white solid. MS (ESI) m/z 551.3 [M+H]+
[0001636] 'H NMR (400 MHz, DMSO-de) 5 = 9.42 - 9.29 (m, 2H), 8.29 (s, 1H), 7.13 (t, I = 6.4 Hz, 1H), 6.85 (dd, I = 5.6, 7.6 Hz, 1H), 6.55 (d, I = 6.8 Hz, 1H), 4.40 (br s, 1H), 4.22 (s, 1H), 3.93 (dd, I = 5.4, 10.3 Hz, 1H), 3.72 (d, I = 10.4 Hz, 1H), 2.54 (s, 3H), 1.60 (dd, I = 5.5, 7.4 Hz, 1H), 1.29 (d, I = 7.6 Hz, 1H), 1.04 - 0.97 (m, 12H), 0.81 (s, 3H)
[0001637] (lR,2S,5S)-N-[cyano-(7-fluoro-4-methyl-pyrazolo[l,5-a]pyridin-3-yl)methyl]-3- [(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide Isomer 2 (27 mg, 49.04 umol, 27.00% yield, 100% purity) as a white solid. MS (ESI) m/z 551.3 [M+H]+
[0001638] 'H NMR (400 MHz, DMSO-de) 5 = 9.44 - 9.31 (m, 1H), 9.22 (d, I = 7.3 Hz, 1H), 8.36 - 8.22 (m, 1H), 7.13 (t, I = 6.6 Hz, 1H), 6.85 (dd, I = 5.8, 7.5 Hz, 1H), 6.48 (d, I = 7.3 Hz, 1H), 4.36 (d, I = 8.4 Hz, 1H), 4.22 (s, 1H), 3.93 (dd, 1 = 5.6, 10.3 Hz, 1H), 3.65 (d, I = 11.0 Hz, 1H), 2.54 (s, 3H), 1.56 (dd, 1 = 5.5, 7.4 Hz, 1H), 1.22 (d, I = 7.6 Hz, 1H), 1.00 - 0.98 (m, 3H), 0.91 (s, 9H), 0.81 (s, 3H).
Example 188: Evaluation of broad-spectrum coronaviral 3CL pro Inhibitors
[0001639] CoV 3CLPro's representing members from each of the a, 0, y phylogenetic groups and subgroups can be expressed and purified to high purity to be assayed for inhibition by the designed library: FIPV-, PEDV-, and NL63-3CL pro from the oc-CoV lineage, HKU1-, OC43-,
658
SUBSTITUTE SHEET RULE 26 SARS-, HKU4-, HKU5-, and HKU9-3CLpro from the -CoV lineage, and IBV-3CLpro from the y CoV lineage (St. John, et al., Bioorg Med Chem Lett 2015, 25(22):5072-5077; Grum-Tokars, et al., Virus Res 2008, 133(1 ) :63-73). The compounds can be tested against all ten 3CLpro's individually to determine inhibition. For example, the enzymatic activity of a given 3CLpro in the presence of a library member at a concentration of 100 pM is measured using a synthetic FRET peptide substrate containing the consensus nsp4-nsp5 cleavage site known for 3CLpro's: HilyteFluor.TM.-488-ESATLQSGLRKAK-(QXL 520)-NH2 (AnaSpec, Inc ). IC50 values are then determined for compounds that produced greater than 50% inhibition of a given 3CLpro at 100 pM.
Example 189: Evaluation of antiviral activity of compounds against COVID-19 (nCoV- 2019, SARS-CoV2) Mpro in the enzymatic assay
[0001640] Compounds were assayed using standard methods to assess compound activity and IC50. As an exemplary for assessment of the SARS-COV2 Mpro, the C-His6-tagged Mpro (NC_045512) was cloned, expressed in E. coli and purified. The assay buffer contained 20 mM of Tns-HCl (pH 7.3), 100 mM of NaCl, 1 mM of EDTA, 5mM of TCEP and 0.1%BSA. The final concentrations of the Mpro protein and substrate were 25 nM and 25 pM, respectively, in the Mpro enzymatic assay. The Km of the Mpro substrate for the protease was 13.5 pM.
[0001641] The compounds were added to an assay plate. For 100% inhibition control (HPE, hundred percent effect), 1 pM GC376 was added. For no inhibition control (ZPE, zero percent effect), no compound was added. Each activity testing point had a relevant background control to normalize the fluorescence interference of compound.
[0001642] IC50 values of compounds were calculated with the GraphPad Prism software using the nonlinear regression model of log(inhibitor) vs. response — Variable slope (four parameters). The inhibition activity was calculated using the formula below, IC50 values is calculated using the Inhibition% data.
Inhibition%> =[ (Sample- Average ZPE )/(Average HPE-Average ZPE)] * 100% # # HEP: Hundred percent effect controls. Containing substrate + enzyme + 1 pM GC376. ZPE: Zero percent effective controls. Containing enzyme + substrate, no compound.
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SUBSTITUTE SHEET RULE 26 Sample: Compound activity testing wells. Containing compound + enzyme + substrate. BG: Compound background control wells. Containing compound + substrate, no enzyme.
Example 190: Evaluation of antiviral activity of compounds against human coronavirus (HCov) 229E and OC43 in the cytopathic effect (CPE) assays
[0001643] Compounds are assayed using standard methods against multiple coronaviral strains, including HCoV 229E and OC43 strains. The antiviral activity of compounds is calculated based on the protection of the virus-induced CPE at each concentration normalized by the virus control.
[0001644] Reagents and instruments for use in this assay include luminescent cell viability assay kit CellTiter Gio (Promega) and Microplate Reader Synergy2 (BioTek).
Virus - HCoV 229E
[0001645] Cytopathic effect (CPE) is measured by CellTiter Gio following the manufacturer’s manual. The antiviral activity of compounds is calculated based on the protection of the virus- induced CPE at each concentration normalized by the virus control.
Virus - HCov OC43
[0001646] Reference compound used is remdesivir; detection reagent: CellTiter Gio.) The CPE is measured by CellTiter Gio following the manufacturer’s manual. The antiviral activity of compounds is calculated based on the protection of the virus-induced CPE at each concentration normalized by the virus control.
[0001647] The cytotoxicity of compounds is assessed under the same conditions, but without virus infection, in parallel. Cell viability is measured with CellTiter Gio. The antiviral activity and cytotoxicity of compounds are expressed as % Inhibition and % Viability, respectively, and calculated with formulas.
[0001648] Table 4, Table 5 and Table 6 show activity data.
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SUBSTITUTE SHEET RULE 26 Table 4. Activity data for compounds.
Figure imgf000662_0001
661
SUBSTITUTE SHEET RULE 26
Figure imgf000663_0001
A > 30 pM, B > 10 pM and <30 pM, C >2 pM and <10 pM, D <2 pM.
Table 5. Activity data for compounds.
Figure imgf000663_0002
Figure imgf000663_0003
662
SUBSTITUTE SHEET RULE 26
Figure imgf000664_0001
Figure imgf000664_0002
663
SUBSTITUTE SHEET RULE 26
Figure imgf000665_0001
Figure imgf000665_0002
664
SUBSTITUTE SHEET RULE 26
Figure imgf000666_0001
Figure imgf000666_0002
665
SUBSTITUTE SHEET RULE 26
Figure imgf000667_0001
Figure imgf000667_0002
A > 30 pM, B > 10 pM and <30 pM, C >2 pM and <10 pM, D <2 pM.
Table 6. Activity data for compounds.
Figure imgf000667_0003
666
SUBSTITUTE SHEET RULE 26
Figure imgf000668_0001
667
SUBSTITUTE SHEET RULE 26
Figure imgf000669_0001
668
SUBSTITUTE SHEET RULE 26
Figure imgf000670_0001
669
SUBSTITUTE SHEET RULE 26
Figure imgf000671_0001
670
SUBSTITUTE SHEET RULE 26
Figure imgf000672_0001
671
SUBSTITUTE SHEET RULE 26
Figure imgf000673_0001
672
SUBSTITUTE SHEET RULE 26
Figure imgf000674_0001
673
SUBSTITUTE SHEET RULE 26
Figure imgf000675_0001
674
SUBSTITUTE SHEET RULE 26
Figure imgf000676_0001
675
SUBSTITUTE SHEET RULE 26
Figure imgf000677_0001
A > 30 pM, B > 10 pM and <30 pM, C >2 pM and <10 pM, D <2 pM.
Example 188. Evaluation the in vitro antiviral activity of compounds against SARS-CoV-2 in the cell-based replicon assay
[0001649] Compounds are assayed at 8 concentrations, 3 -fold dilution, in duplicate wells. The highest test concentrations of test compounds and reference compound is 10 pM and 1 pM, respectively. The main reagents used in this assay was luminescent cell viability assay kit CellTiter Gio (Promega). The main instrument used in this assay was Acumen Cellista (TTP LabTech).
[0001650] The antiviral assay is summarized in Table 7.
Table 7. Method of the SARS-CoV-2 replicon assay
Figure imgf000677_0002
[0001651] The replicon RNA was generated by the mMACHINE T7 Ultra Kit. Huh7 cells were transfected with purified replicon RNAs and seeded in plates containing serially diluted compounds, then cultured at 37°C and 5% CO2 for 1 day. The final volume of the cell culture was 60pl per well, and the final concentrations of DMSO in the test plates are 0.5%.
Fluorescence intensity were determined using Acumen Cellista (TTP LabTech), and the antiviral activity of compounds were calculated based on the inhibition of expression of GFP. Cell viability were measured with CellTiter Gio following the manufacturer’s manual.
[0001652] The antiviral activity and cytotoxicity of compounds were expressed as % Inhibition and % Viability, respectively, and calculated with the formulas below:
Inhibition (%) = (Raw data CPD -Average ZPE) / (Average HPE - Average ZPE) *100
676
SUBSTITUTE SHEET RULE 26 Viability (%) = (Raw data CPD -Average HPE) / (Average ZPE- Average HPE) *100
Raw data CPD: values of the sample-treated wells
Average ZPE: average value of no-compound treatment control
Average HPE: average value of medium control (medium only) wells.
[0001653] ECso and CC50 values were calculated using the GraphPad Prism software using the nonlinear regression model of log(inhibitor) vs. response — Variable slope (four parameters).
[0001654] Table 8 shows activity data.
Table 8. Activity data for compounds.
Figure imgf000678_0001
Figure imgf000678_0002
677
SUBSTITUTE SHEET RULE 26
Figure imgf000679_0002
Figure imgf000679_0001
A > 30 pM, B > 10 pM and <30 pM, C >2 pM and <10 pM, D <2 pM.
INCORPORATION BY REFERENCE
[0001655] All publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent was specifically and individually incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
EQUIVALENTS AND SCOPE
[0002] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
678
SUBSTITUTE SHEET RULE 26 [0003] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0004] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[0005] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art
679
SUBSTITUTE SHEET RULE 26 will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
680
SUBSTITUTE SHEET RULE 26

Claims

What is claimed is:
1. A protease inhibitory compound represented by Formula I:
Figure imgf000682_0001
Formula I or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R1 is selected from the group consisting of -CH(Rn)-NHR12, a 9-10 membered bicyclic heteroaryl having a ring nitrogen, and a 5-6 membered monocyclic heteroaryl having a ring nitrogen; wherein R1 may optionally be substituted by one, two, or three substituents each independently selected from R5;
R11 is selected from the group consisting of branched Ci-Cealkyl, -CFL-phenyl, -CH2- naphthyl, and -CH2-(8-10 membered bicyclic heteroaryl);
R12 is selected from the group consisting of -C(O)RB, -C(O)ORB, -C(O)NHRB, -RB, - NHC(O)RB, -NHC(O)ORB, -NHC(O)NHRB, and -NHRB; wherein R12 or RB may optionally be substituted by one or more substituents each independently selected from R13;
RB is selected from the group consisting of Ci-Cealkyl, C3-C4cycloalkyl, phenyl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl;
R2 is hydrogen or Ci-Csalkyl;
R3a is hydrogen or Ci-Csalkyl;
R3 is selected from the group consisting of Ci-Cealkyl, Cs-Cecycloalkyl, phenyl, 4-6 heterocyclyl and 5-6 membered heteroaryl; wherein R3 may optionally be substituted by one, two, or three substituents each independently selected from R5; or R3 is -Si(CH3)3; or R2 and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6; or R3a and R3, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered
681
SUBSTITUTE SHEET RULE 26 spirocyclic carbocyclyl which may optionally be substituted by one, two or three substituents each independently selected from R6;
R4a is hydrogen or Ci-Csalkyl optionally substituted by one or more halogens;
R4 is selected from the group consisting of a 9-10 membered bicyclic heteroaryl having at least one ring nitrogen, a 5-6 membered monocyclic heteroaryl having at least one ring nitrogen, a 4-10 membered heterocyclyl, and a 12-membered tricyclic heteroaryl having at least one nitrogen; wherein R4 may optionally be substituted by one, two, or three substituents each independently selected from R7; or
R4a and R4, together with the carbon to which they are attached, may be joined together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from R8;
R5, R6, R7, R8 and R13 are independently selected for each occurrence from the group consisting of deuterium, oxo, halogen, hydroxyl, cyano, -N(R56)2, -N(R56)C(O)R56, -C(O)N(R56)2, -OPO(OH)2, -CO2H, -SO2CH3, -CF3, C9H21S1-, Ci-Cealkyl, C2-C6alkynyl, Ci-C6alkoxy, C3- Cecycloalkyl, C3-Cecycloalkoxy, phenyl, phenoxy, and 5-6 membered heteroaryl; wherein Ci- Cealkyl, Ci-Cealkoxy, Cs-Cecycloalkyl, Cs-Cecycloalkoxy, phenyl, phenoxy and heteroaryl may optionally be substituted by one or more substituents each independently selected from the group consisting of halogen, hydroxyl, Ci-Csalkyl, C2-Cealkynyl, Ci-Cealkoxy, and -CF3;
R56 is hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens; and Rais hydrogen or Ci-Csalkyl optionally substituted by one, two or three halogens.
2. The compound of claim 1, wherein R2 and R3, together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic, 5-10 membered fused or bridged bicyclic, or 5-10 membered spirocyclic heterocyclyl.
3. The compound of claim 1 or 2, wherein the compound of Formula I is represented by:
Figure imgf000683_0001
682
SUBSTITUTE SHEET RULE 26 wherein m is selected from 0, 1 and 2; and n is selected from 0, 1, 2 and 3.
4. The compound of any one of claims 1-3, wherein the compound of Formula I is represented by:
Figure imgf000684_0001
wherein m is 0, 1 or 2; n is 0, 1, 2, or 3; and t is 0, 1 or 2.
5. The compound of any one of claims 1-4, wherein R6 when present is independently selected for each occurrence from the group consisting of fluoro, chloro and -CH3.
6. The compound of claim 1 or 2, wherein R2 and R3, together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
Figure imgf000684_0002
683
SUBSTITUTE SHEET RULE 26
Figure imgf000685_0001
7. The compound of claim 1, wherein R3a and R3, together with the atoms to which they are attached, join together to form a 4-6 membered monocyclic or 5-10 membered spirocyclic carbocyclyl selected from the group consisting of:
Figure imgf000685_0002
8. The compound of claim 1, wherein R2 is hydrogen.
9. The compound of claim 1 or 8, wherein the compound of Formula I is represented by:
684
SUBSTITUTE SHEET RULE 26
Figure imgf000686_0001
wherein t is 0, 1 or 2.
10. The compound of any one of claims 1 and 8-9, wherein R3 is selected from the group consisting of:
Figure imgf000686_0002
11. The compound of any one of claims 1-10, wherein R5 when present is independently selected for each occurrence from the group consisting of fluoro, chloro, bromo, -CH3, -CF3, isopropyl, tert- butyl, cyclopropyl and -OCH3.
12. The compound of any one of claims 1-11, wherein R11 is selected from the group consisting of:
Figure imgf000686_0003
685
SUBSTITUTE SHEET RULE 26
Figure imgf000687_0001
13. The compound of any one of claim 1, wherein the compound of Formula I is represented by:
Figure imgf000687_0002
wherein m is 0, 1 or 2; and n is 0, 1, 2 or 3, and R6 when present is independently selected for each occurrence from the group consisting of fluoro, chloro and -CH3.
14. The compound of any one of claims 1-13, wherein R12 is -C(O)CF3.
15. The compound of any one of claims 1-14, wherein R4a is hydrogen.
16. The compound of any one of claims 1-15, wherein R4 is selected from the group consisting of:
686
SUBSTITUTE SHEET RULE 26
Figure imgf000688_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000689_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000690_0001
wherein:
X is O, S, or NRa;
R7 is independently selected for each occurrence from the group consisting of chloro, fluoro, bromo, -CN, -CCH, -CH3, -CH2F, -CHF2, -CF3, cyclopropyl, -OH, -OCH3 and -OCF3;
R77 and R78 are independently selected, for each occurrence, from the group consisting of hydrogen, chloro, fluoro, bromo, -CN, -CCH, -CH3, -CH2F, -CHF2, CF3, -OCH3, -OCF3 and cyclopropyl; and
Ra is selected from the group consisting of hydrogen, Ci-C3alkyl, C3-Cecycloalkyl, and phenyl, wherein Ci-C3alkyl, C3-Cecycloalkyl, and phenyl may optionally be substituted by one or more halogens.
17. The compound of claim 16, wherein R4 is selected from the group consisting of:
Figure imgf000690_0002
689
SUBSTITUTE SHEET RULE 26
Figure imgf000691_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000692_0001
691
SUBSTITUTE SHEET RULE 26 D
Figure imgf000693_0001
692
SUBSTITUTE SHEET RULE 26
Figure imgf000694_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000695_0001
694
SUBSTITUTE SHEET RULE 26
Figure imgf000696_0001
The compound of any one of claims 1-15, wherein
Figure imgf000696_0002
wherein two
R80 may be joined, together with the atoms to which they are attached, to form a 5-6 membered heterocyclyl or 5-6 membered heteroaryl.
19. The compound of any one of claims 1-15, wherein R4 is selected from the group consisting
Figure imgf000696_0003
20. The compound of any one of claims 1-14, wherein R4a and R4, together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of:
Figure imgf000696_0004
695
SUBSTITUTE SHEET RULE 26
Figure imgf000697_0001
wherein R8 and R9 are independently selected for each occurrence from the group consisting of chloro, fluoro, -CH3, -CH2F, -CHF2, -CF3, -OH and -OCH3; or two geminal R9 form oxo.
21. The compound of any one of claims 1-12 wherein R4a and R4, together with the carbon to which they are attached, join together to form:
Figure imgf000697_0002
696
SUBSTITUTE SHEET RULE 26
Figure imgf000698_0001
22. A compound represented by Formula II:
Figure imgf000698_0002
or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R10 is selected from the group consisting of -C(O)-Ci-Cealkyl, -C(O)-NRa-Ci-Cealkyl, - C(O)-O-Ci-Cealkyl, -C(O)-O-CH2-phenyl, and Ci-Cealkyl, wherein R10 may optionally be substituted on an available carbon by one or more substituents each independently selected from the group of halogen, hydroxy, -CF3, -NRaRb, -C(O)NRaRb, and Ci-Csalkoxy;
R15 is selected from the group consisting of:
Figure imgf000698_0003
697
SUBSTITUTE SHEET RULE 26
Figure imgf000699_0001
R20 is hydrogen;
R30 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R30 may optionally be substituted by one, two or three substituents each independently selected from R33; or R30 -Si(CH3)3;
R33 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R20 and R30, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
Figure imgf000699_0002
R40a is hydrogen Ci-Csalkyl; and
698
SUBSTITUTE SHEET RULE 26 R40 is selected from the group consisting of:
Figure imgf000700_0001
699
SUBSTITUTE SHEET RULE 26
Figure imgf000701_0001
700
SUBSTITUTE SHEET RULE 26
Figure imgf000702_0001
701
SUBSTITUTE SHEET (RULE 26) WO 2023/044171
Figure imgf000703_0001
702
SUBSTITUTE SHEET (RULE 26)
Figure imgf000704_0001
703
SUBSTITUTE SHEET RULE 26
Figure imgf000705_0001
or R40a and R40, together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of:
Figure imgf000705_0002
704
SUBSTITUTE SHEET RULE 26
Figure imgf000706_0001
23. A compound represented by Formula III:
Figure imgf000706_0002
Formula III or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof, wherein:
R100 is independently selected for each occurrence from the group consisting of halogen and Ci-Csalkoxy;
R200 is hydrogen or Ci-Csalkyl;
R300 is selected from the group consisting of isopropyl, /-butyl, cyclopropyl and cyclohexyl, wherein R300 may optionally be substituted by one, two or three substituents each independently selected from R330; or R300 -Si(CH3)3;
R330 is independently selected from each occurrence from the group consisting of fluoro, chloro, -CH3 and -CF3; or R200 and R300, together with the atoms to which they are attached, may be joined together to form a 4-6 membered monocyclic, 6-10 membered fused bicyclic, or 6-10 membered spirocyclic heterocyclyl selected from the group consisting of:
705
SUBSTITUTE SHEET RULE 26
Figure imgf000707_0001
706
SUBSTITUTE SHEET RULE 26
Figure imgf000708_0001
707
SUBSTITUTE SHEET RULE 26
Figure imgf000709_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000710_0001
709
SUBSTITUTE SHEET RULE 26
Figure imgf000711_0001
710
SUBSTITUTE SHEET RULE 26
Figure imgf000712_0001
SUBSTITUTE SHEET RULE 26 or R400a and R400, together with the carbon to which they are attached, join together to form an 8-14 membered partially unsaturated bicyclic or tricyclic heterocyclyl selected from the group consisting of:
Figure imgf000713_0001
712
SUBSTITUTE SHEET RULE 26
24. A compound selected from the group consisting of:
Figure imgf000714_0001
713
SUBSTITUTE SHEET RULE 26
Figure imgf000715_0001
714
SUBSTITUTE SHEET RULE 26
Figure imgf000716_0001
715
SUBSTITUTE SHEET RULE 26 XVO 2023/044171
Figure imgf000717_0001
716
SUBSTITUTE SHEET (RULE 26)
Figure imgf000718_0001
717
SUBSTITUTE SHEET RULE 26
Figure imgf000719_0001
718
SUBSTITUTE SHEET RULE 26
Figure imgf000720_0001
719
SUBSTITUTE SHEET RULE 26
Figure imgf000721_0001
720
SUBSTITUTE SHEET RULE 26
Figure imgf000722_0001
or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof .
25. A compound selected from the group consisting of:
Figure imgf000722_0002
Figure imgf000722_0003
721
SUBSTITUTE SHEET RULE 26
Figure imgf000723_0002
Figure imgf000723_0001
722
SUBSTITUTE SHEET RULE 26
Figure imgf000724_0001
Figure imgf000724_0002
723
SUBSTITUTE SHEET RULE 26
Figure imgf000725_0001
724
SUBSTITUTE SHEET RULE 26
Figure imgf000726_0002
Figure imgf000726_0001
725
SUBSTITUTE SHEET RULE 26
Figure imgf000727_0001
Figure imgf000727_0002
726
SUBSTITUTE SHEET RULE 26
Figure imgf000728_0001
Figure imgf000728_0002
727
SUBSTITUTE SHEET RULE 26
Figure imgf000729_0003
Figure imgf000729_0001
_ _ or a pharmaceutically acceptable salt, a stereoisomer, and/or an N-oxide thereof.
26. A compound selected from the group consisting of:
Figure imgf000729_0002
Figure imgf000729_0004
728
SUBSTITUTE SHEET RULE 26
Figure imgf000730_0002
Figure imgf000730_0001
729
SUBSTITUTE SHEET RULE 26
Figure imgf000731_0002
Figure imgf000731_0001
730
SUBSTITUTE SHEET RULE 26
Figure imgf000732_0001
Figure imgf000732_0002
731
SUBSTITUTE SHEET RULE 26
Figure imgf000733_0001
Figure imgf000733_0002
732
SUBSTITUTE SHEET RULE 26
Figure imgf000734_0002
Figure imgf000734_0001
733
SUBSTITUTE SHEET RULE 26
Figure imgf000735_0001
Figure imgf000735_0002
734
SUBSTITUTE SHEET RULE 26
Figure imgf000736_0002
Figure imgf000736_0001
735
SUBSTITUTE SHEET RULE 26
Figure imgf000737_0002
Figure imgf000737_0001
736
SUBSTITUTE SHEET RULE 26
Figure imgf000738_0002
Figure imgf000738_0001
737
SUBSTITUTE SHEET RULE 26
Figure imgf000739_0001
Figure imgf000739_0002
738
SUBSTITUTE SHEET RULE 26
Figure imgf000740_0002
Figure imgf000740_0001
739
SUBSTITUTE SHEET RULE 26
Figure imgf000741_0001
Figure imgf000741_0002
740
SUBSTITUTE SHEET RULE 26
Figure imgf000742_0002
Figure imgf000742_0001
741
SUBSTITUTE SHEET RULE 26
Figure imgf000743_0001
Figure imgf000743_0002
742
SUBSTITUTE SHEET RULE 26
Figure imgf000744_0002
Figure imgf000744_0001
743
SUBSTITUTE SHEET RULE 26
Figure imgf000745_0002
Figure imgf000745_0001
744
SUBSTITUTE SHEET RULE 26
Figure imgf000746_0002
Figure imgf000746_0001
745
SUBSTITUTE SHEET RULE 26 Docket No. PARB-015WO
Figure imgf000747_0001
Figure imgf000747_0002
746
SUBSTITUTE SHEET RULE 26
Figure imgf000748_0002
Figure imgf000748_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000749_0001
Figure imgf000749_0002
748
SUBSTITUTE SHEET RULE 26
Figure imgf000750_0001
Figure imgf000750_0002
749
SUBSTITUTE SHEET RULE 26
Figure imgf000751_0002
Figure imgf000751_0001
750
SUBSTITUTE SHEET RULE 26
Figure imgf000752_0002
Figure imgf000752_0001
751
SUBSTITUTE SHEET RULE 26
Figure imgf000753_0001
Figure imgf000753_0002
752
SUBSTITUTE SHEET RULE 26
Figure imgf000754_0001
Figure imgf000754_0002
753
SUBSTITUTE SHEET RULE 26
Figure imgf000755_0002
Figure imgf000755_0001
754
SUBSTITUTE SHEET RULE 26
Figure imgf000756_0002
Figure imgf000756_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000757_0002
Figure imgf000757_0001
SUBSTITUTE SHEET RULE 26
Figure imgf000758_0002
Figure imgf000758_0001
757
SUBSTITUTE SHEET RULE 26
Figure imgf000759_0001
Figure imgf000759_0002
758
SUBSTITUTE SHEET RULE 26
Figure imgf000760_0001
Figure imgf000760_0002
759
SUBSTITUTE SHEET RULE 26
Figure imgf000761_0002
Figure imgf000761_0001
760
SUBSTITUTE SHEET RULE 26
Figure imgf000762_0001
Figure imgf000762_0002
761
SUBSTITUTE SHEET RULE 26
Figure imgf000763_0001
Figure imgf000763_0002
762
SUBSTITUTE SHEET RULE 26
Figure imgf000764_0002
Figure imgf000764_0003
Figure imgf000764_0001
763
SUBSTITUTE SHEET RULE 26
Figure imgf000765_0002
Figure imgf000765_0001
764
SUBSTITUTE SHEET RULE 26
Figure imgf000766_0001
Figure imgf000766_0002
765
SUBSTITUTE SHEET RULE 26
Figure imgf000767_0002
Figure imgf000767_0001
or a pharmaceutically acceptable salt thereof.
27. A protease inhibitory compound represented by Formula X:
766
SUBSTITUTE SHEET RULE 26
Figure imgf000768_0001
Formula X or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
Figure imgf000768_0002
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3;
R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD;
RD is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
R102 is -NRm(C=O)Rm;
Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C3- Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or
-CF3;
R103 is 9- 10 membered bicyclic heteroaryl having at least one ring nitrogen, wherein R103 may optionally be substituted by one, two, or three substituents each independently selected from RE; and
RE is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2- Cealkynyl, Ci-Cealkoxy, and Ci-Cealkoxy-OCFF.
28. The compound of claim 27, wherein Formula X is represented by:
767
SUBSTITUTE SHEET RULE 26
Figure imgf000769_0001
29. The compound of claim 27, wherein
Figure imgf000769_0002
Cealkyl or C3-
Cecycloalkyl, and R105 is Ci-Cealkyl, wherein the R101 is optionally substituted by one -CH3 or cyclopropyl.
Figure imgf000769_0003
30. The compound of claim 29, wherein the R101 is selected from the group consisting
Figure imgf000769_0004
31. The compound of claim 27, wherein
Figure imgf000769_0005
768
SUBSTITUTE SHEET RULE 26
Figure imgf000770_0001
32. The compound of claim 31 , wherein the
Figure imgf000770_0002
selected from
Figure imgf000770_0003
33. The compound of claim 27, wherein R101 is selected from the group consisting of:
Figure imgf000770_0004
34. The compound of claim 27, wherein R105 is -CH3.
35. The compound of any one of claims 27-34, wherein R102 is -NH(C=O)CF3.
36. The compound of any one of claims 27-35, wherein R103 is selected from the group consisting of:
Figure imgf000770_0005
37. A compound selected from the group consisting of:
769
SUBSTITUTE SHEET RULE 26
Figure imgf000771_0002
Figure imgf000771_0003
Figure imgf000771_0001
770
SUBSTITUTE SHEET RULE 26
Figure imgf000772_0002
Figure imgf000772_0001
Figure imgf000772_0003
771
SUBSTITUTE SHEET RULE 26
Figure imgf000773_0003
Figure imgf000773_0002
Figure imgf000773_0001
772
SUBSTITUTE SHEET RULE 26
Figure imgf000774_0002
Figure imgf000774_0001
Figure imgf000774_0003
773
SUBSTITUTE SHEET RULE 26
Figure imgf000775_0001
Figure imgf000775_0005
Figure imgf000775_0003
or pharmaceutically acceptable salts thereof.
38. A compound selected from the group consisting of:
Figure imgf000775_0002
Figure imgf000775_0004
774
SUBSTITUTE SHEET RULE 26
Figure imgf000776_0002
Figure imgf000776_0001
775
SUBSTITUTE SHEET RULE 26
Figure imgf000777_0002
Figure imgf000777_0001
776
SUBSTITUTE SHEET RULE 26
Figure imgf000778_0002
Figure imgf000778_0001
777
SUBSTITUTE SHEET RULE 26
Figure imgf000779_0002
Figure imgf000779_0001
778
SUBSTITUTE SHEET RULE 26
Figure imgf000780_0001
Figure imgf000780_0002
779
SUBSTITUTE SHEET RULE 26
39. A protease inhibitory compound represented by Formula XI:
Figure imgf000781_0001
Formula XI or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
Figure imgf000781_0002
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3;
R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RF;
RF is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, cyano, and
C3 -Cecycloalkyl, wherein the alkyl may optionally be substituted by one or more halo;
R102 is -NRm(C=O)Rm;
Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C3-
Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3;
R106 and R106a, together with the carbon to which they are attached, are joined together to form an 10-14 membered partially unsaturated tricyclic heterocyclyl optionally substituted by one, two, or three substituents each independently selected from RH; and
RH is, for each occurrence, selected from the group consisting of halo, Ci-Cealkyl, C2- Cealkynyl, and Ci-Cealkoxy.
40. The compound of claim 39, wherein Formula XI is represented by:
780
SUBSTITUTE SHEET RULE 26
Figure imgf000782_0001
41. The compound of claim 39, wherein
Figure imgf000782_0002
Cealkyl or C3-
Cecycloalkyl, and R105 is Ci-Cealkyl, wherein the R101 is optionally substituted by one -CH3 or cyclopropyl.
781
SUBSTITUTE SHEET RULE 26
Figure imgf000783_0001
42. The compound of claim 41, wherein the R101 is selected from the group consisting
Figure imgf000783_0002
43. The compound of claim 39, wherein R101 is selected from the group consisting of:
Figure imgf000783_0003
44. The compound of claim 39, wherein R105 is -CH3.
45. The compound of any one of claims 39-43, wherein R102 is -NH(C=O)CF3.
46. The compound of any one of claims 39-44, wherein R106 and R106a, together with the carbon to which they are attached, are joined together to form a 12 membered partially unsaturated tricyclic heterocyclyl ring.
47. The compound of claim 46, wherein the heterocyclyl ring is represented
Figure imgf000783_0004
48. A compound selected from the group consisting of:
782
SUBSTITUTE SHEET RULE 26
Figure imgf000784_0003
Figure imgf000784_0004
or pharmaceutically acceptable salts thereof.
49. A protease inhibitory compound represented by Formula X-Ia or Formula X-Ib:
Figure imgf000784_0001
or a pharmaceutically acceptable salt, and/or a stereoisomer thereof, wherein:
R104 is selected from the group consisting of Ci-Cealkyl,
Figure imgf000784_0002
wherein R105 is Ci-Cealkyl, and n is 1, 2, or 3;
R101 is selected from the group consisting of Ci-Cealkyl, C2-Cealkenyl, Cs-Cecycloalkyl, and Cs-Cecycloalkenyl, wherein R101 may optionally be substituted by one, two, three, or four substituents each independently selected from RD;
783
SUBSTITUTE SHEET RULE 26 RD is selected from the group consisting of halogen, Ci-Cealkyl, Ci-Cealkoxy, C3- Cecycloalkyl, and cyano, wherein the alkyl may optionally be substituted by one or more halo;
R102 is -NRm(C=O)Rm; and
Rm is, for each occurrence, selected from the group consisting of H, Ci-ealkyl and C3- Cecycloalkyl, wherein the alkyl or cycloalkyl is optionally substituted by one, two or three halo or -CF3.
50. A pharmaceutical composition comprising a compound of any one of claims 1-49 and a pharmaceutically acceptable excipient.
51. A method of ameliorating or treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of any one of claims 1-49.
52. The method of claim 51 , wherein the viral infection is from a virus selected from the group consisting of an RNA virus, a DNA virus, a coronavirus, a papillomavirus, a pneumovirus, a picornavirus, an influenza virus, an adenovirus, a cytomegalovirus, a polyomavirus, a poxvirus, a flavivirus, an alphavirus, an ebola virus, a morbillivirus, an enterovirus, an orthopneumovirus, a lentivirus, an arenavirus, a herpes virus, and a hepatovirus.
53. The method of claim 51 , wherein the viral infection is from a virus selected from the group consisting of Norwalk virus, feline calicivirus, MD145, murine norovirus, an astrovirus, vesicular exanthema of swine virus, rabbit hemorrhagic disease virus, enterovirus (EV)-68 virus, EV-71 virus, poliovirus, coxsackievirus, foot-and-mouth disease virus, hepatitis A, porcine teschovirus, rhinovirus, human coronavirus, transmissible gastroenteritis virus, murine hepatitis virus, bovine coronavirus, feline infectious peritonitis virus, and severe acute respiratory syndrome coronavirus.
54. The method of any one of claims 51-53, wherein the viral infection is a coronavirus infection.
784
SUBSTITUTE SHEET RULE 26
55. The method of any one of claims 51-54, wherein the viral infection is a coronavirus selected from the group consisting of: 229E alpha coronavirus, NL63 alpha coronavirus, OC43 beta coronavirus, HKU1 beta coronavirus, Middle East Respiratory Syndrome (MERS) coronavirus (MERS -Co V), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and SARS-CoV-2 (COVID-19).
56. The method of any one of claims 51-55, wherein the viral infection is SARS-CoV-2.
57. The method of any one of claims 51-53, wherein the viral infection is an arenavirus infection.
58. The method of claim 57, wherein the arenavirus is selected from the group consisting of: Junin virus, Lassa virus, Lujo virus, Machupo virus, and Sabia virus.
59. The method of any one of claims 51-53, wherein the viral infection is an influenza infection.
60. The method of claim 59, wherein the influenza is influenza H1N1, H3N2 or H5N1.
61. A method of inhibiting transmission of a virus, a method of inhibiting viral replication, a method of minimizing expression of viral proteins, or a method of inhibiting virus release, comprising administering a therapeutically effective amount of a compound of any one of claims 1-49 to a patient suffering from the virus, and/or contacting an effective amount of a compound of any one of claims 1-49 with a virally infected cell.
62. The method of any one of claims 51-61, further comprising administering another therapeutic.
63. The method of any one of claims 51-62, further comprising administering an additional anti-viral therapeutic.
785
SUBSTITUTE SHEET RULE 26
64. The method of claim 63, wherein the additional anti-viral therapeutic is selected from the group consisting of ribavirin, favipiravir, ST-193, oseltamivir, zanamivir, peramivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir, grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX-355, valacyclovir, VIR- 576, and zalcitabine.
65. The method of claim 62, wherein the another therapeutic is selected from the group consisting of protease inhibitors, fusion inhibitors, M2 proton channel blockers, polymerase inhibitors, 6- endonuclease inhibitors, neuraminidase inhibitors, reverse transcriptase inhibitor, aciclovir, acyclovir, protease inhibitors, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, docosanol, edoxudine, entry inhibitors, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, inosine, integrase inhibitor, interferons, lopinavir, loviride, moroxydine, nexavir, nucleoside analogues, penciclovir, pleconaril, podophyllotoxin, ribavirin, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zodovudine.
66. The method of claim 62, wherein the additional anti-viral therapeutic is selected from the group consisting of lamivudine, an interferon alpha, a VAP anti-idiotypic antibody, enfuvirtide, amantadine, rimantadine, pleconaril, aciclovir, zidovudine, fomivirsen, a morpholino, a protease inhibitor, double-stranded RNA activated caspase oligomerizer (DRACO), rifampicin, zanamivir, oseltamivir, danoprevir, ritonavir, remdesivir, cobicistat, elvitegravir, emtricitabine, tenofovir, tenofovir disoproxil, tenofovir alafenamide hemifumarate, abacavir, dolutegravir, efavirenz, elbasvir, ledipasvir, glecaprevir, sofosbuvir, bictegravir, dasabuvir, lamivudine, atazanavir, ombitasvir, lamivudine, stavudine, nevirapine, rilpivirine, paritaprevir, simeprevir, daclatasvir,
786
SUBSTITUTE SHEET RULE 26 grazoprevir, pibrentasvir, adefovir, amprenavir, ampligen, aplaviroc, anti-caprine antibody, balavir, cabotegravir, cytarabine, ecoliever, epigallocatechin gallate, etravirine, fostemsavir, gemcitabine, griffithsin, imunovir, indinavir, maraviroc, methisazone, MK-2048, nelfmavir, nevirapine, nitazoxanide, norvir, plerixafor, PRO 140, raltegravir, pyramidine, saquinavir, telbivudine, TNX-355, valacyclovir, VIR- 576, and zalcitabine.
67. A method of prophylactically treating a patient at risk of viral infection, comprising administering to the patient an effective amount of a compound of any one of claims 1-49.
68. The method of claim 67, wherein the compound is administered before viral exposure.
69. The method of claim 67 or 68, wherein the compound is administered after viral exposure.
787
SUBSTITUTE SHEET RULE 26
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11963967B2 (en) 2020-10-16 2024-04-23 Gilead Sciences, Inc. Phospholipid compounds and uses thereof
WO2024086111A1 (en) * 2022-10-17 2024-04-25 Aligos Therapeutics, Inc. Anti-viral compounds
US12030904B2 (en) 2023-04-14 2024-07-09 Gilead Sciences, Inc. Phospholipid compounds and uses thereof

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049008A1 (en) * 1999-02-20 2000-08-24 Astrazeneca Ab Di- and tripeptide nitrile derivatives as inhibitors of cathepsin l and cathepsin s
WO2001019816A1 (en) * 1999-09-13 2001-03-22 Boehringer Ingelheim Pharmaceuticals, Inc. Novel spiroheterocyclic compounds useful as reversible inhibitors of cysteine proteases
WO2005113580A1 (en) * 2004-05-21 2005-12-01 Pfizer Inc. Anticoronviral compounds and compositions, their pharmaceutical uses and materials for their synthesis
WO2013049382A2 (en) * 2011-09-27 2013-04-04 Kansas State University Research Foundation Broad-spectrum antivirals against 3c or 3c-like proteases of picornavirus-like supercluster: picornaviruses, caliciviruses and coronaviruses
WO2017114509A1 (en) * 2015-12-31 2017-07-06 中国科学院上海药物研究所 Aldehyde and preparation and application thereof
WO2018042343A2 (en) * 2016-08-30 2018-03-08 Glaxosmithkline Intellectual Property (No.2) Limited Compounds that inhibit 3c and 3cl proteases and methods of use thereof
EP3835296A1 (en) * 2018-08-09 2021-06-16 Shanghai Institute of Materia Medica, Chinese Academy of Sciences Ketoamide compound and preparation method, pharmaceutical composition, and use thereof
WO2021176369A1 (en) * 2020-03-06 2021-09-10 Pfizer Inc. Methods of inhibiting sars-cov-2 replication and treating coronavirus disease 2019
US11124497B1 (en) * 2020-04-17 2021-09-21 Pardes Biosciences, Inc. Inhibitors of cysteine proteases and methods of use thereof
WO2021252644A1 (en) * 2020-06-09 2021-12-16 Pardes Biosciences, Inc. Inhibitors of cysteine proteases and methods of use thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049008A1 (en) * 1999-02-20 2000-08-24 Astrazeneca Ab Di- and tripeptide nitrile derivatives as inhibitors of cathepsin l and cathepsin s
WO2001019816A1 (en) * 1999-09-13 2001-03-22 Boehringer Ingelheim Pharmaceuticals, Inc. Novel spiroheterocyclic compounds useful as reversible inhibitors of cysteine proteases
WO2005113580A1 (en) * 2004-05-21 2005-12-01 Pfizer Inc. Anticoronviral compounds and compositions, their pharmaceutical uses and materials for their synthesis
WO2013049382A2 (en) * 2011-09-27 2013-04-04 Kansas State University Research Foundation Broad-spectrum antivirals against 3c or 3c-like proteases of picornavirus-like supercluster: picornaviruses, caliciviruses and coronaviruses
WO2017114509A1 (en) * 2015-12-31 2017-07-06 中国科学院上海药物研究所 Aldehyde and preparation and application thereof
WO2018042343A2 (en) * 2016-08-30 2018-03-08 Glaxosmithkline Intellectual Property (No.2) Limited Compounds that inhibit 3c and 3cl proteases and methods of use thereof
EP3835296A1 (en) * 2018-08-09 2021-06-16 Shanghai Institute of Materia Medica, Chinese Academy of Sciences Ketoamide compound and preparation method, pharmaceutical composition, and use thereof
WO2021176369A1 (en) * 2020-03-06 2021-09-10 Pfizer Inc. Methods of inhibiting sars-cov-2 replication and treating coronavirus disease 2019
US11124497B1 (en) * 2020-04-17 2021-09-21 Pardes Biosciences, Inc. Inhibitors of cysteine proteases and methods of use thereof
WO2021252644A1 (en) * 2020-06-09 2021-12-16 Pardes Biosciences, Inc. Inhibitors of cysteine proteases and methods of use thereof

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BAI BING ET AL: "Peptidomimetic nitrile warheads as SARS-CoV-2 3CL protease inhibitors", RSC MEDICINAL CHEMISTRY, vol. 12, no. 10, 20 August 2021 (2021-08-20), pages 1722 - 1730, XP055965007, Retrieved from the Internet <URL:https://pubs.rsc.org/en/content/articlepdf/2021/md/d1md00247c> DOI: 10.1039/D1MD00247C *
BANERJEE ABHISEK ET AL: "Development of potent and selective Cathepsin C inhibitors free of aortic binding liability by application of a conformational restriction strategy", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 47, 15 June 2021 (2021-06-15), Amsterdam NL, pages 128202, XP093001054, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2021.128202 *
CARREIRAKVAERNO: "Classics in Stereoselective Synthesis", 2009, WILEY-VCH
GREENEWUTS: "Protective Groups in Organic Synthesis", 1999
HALFORD BETHANY: "To conquer COVID-19, create the perfect pill", CHEMICAL & ENGINEERING NEWS (2021), 99(19), 20 May 2021 (2021-05-20), pages 28 - 31, XP055847761, Retrieved from the Internet <URL:https://cen.acs.org/pharmaceuticals/drug-discovery/covid-19-oral-antiviral-pill-candidates/99/i19> [retrieved on 20211005] *
HOFFMAN ROBERT ET AL: "The Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19", CHEMRXIVTM, 22 July 2020 (2020-07-22), pages 1 - 106, XP055803261, Retrieved from the Internet <URL:https://s3-eu-west-1.amazonaws.com/itempdf74155353254prod/12631496/The_Discovery_of_Ketone-Based_Covalent_Inhibitors_of_Coronavirus_3CL_Proteases_for_the_Potential_Therapeutic_Treatment_o_v1.pdf> [retrieved on 20210511], DOI: 10.26434/chemrxiv.12631496.v1 *
LIU CHENG ET AL: "Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development", ANTIVIRAL RESEARCH, vol. 187, 27 January 2021 (2021-01-27), NL, pages 105020, XP055810028, ISSN: 0166-3542, DOI: 10.1016/j.antiviral.2021.105020 *
RAUTIOKUMPULAINEN, NATURE REVIEWS DRUG DISCOVERY, vol. 7, 2008, pages 255
STEINHAUER DAHOLLAND JJ: "Rapid evolution qf RNA viruses", ANNU. REV. MICROBIOL., vol. 41, 1987, pages 409 - 33
WENHAO DAI ET AL: "Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease", SCIENCE (AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE), 22 April 2020 (2020-04-22), United States, pages eabb4489, XP055707767, Retrieved from the Internet <URL:https://science.sciencemag.org/content/sci/368/6497/1331.full.pdf> DOI: 10.1126/science.abb4489 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11963967B2 (en) 2020-10-16 2024-04-23 Gilead Sciences, Inc. Phospholipid compounds and uses thereof
WO2024086111A1 (en) * 2022-10-17 2024-04-25 Aligos Therapeutics, Inc. Anti-viral compounds
US12030904B2 (en) 2023-04-14 2024-07-09 Gilead Sciences, Inc. Phospholipid compounds and uses thereof

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