WO2022129047A2 - Novel modulators of the nmda receptor - Google Patents

Novel modulators of the nmda receptor Download PDF

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Publication number
WO2022129047A2
WO2022129047A2 PCT/EP2021/085688 EP2021085688W WO2022129047A2 WO 2022129047 A2 WO2022129047 A2 WO 2022129047A2 EP 2021085688 W EP2021085688 W EP 2021085688W WO 2022129047 A2 WO2022129047 A2 WO 2022129047A2
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Prior art keywords
amino
propanoic acid
fluorobenzamido
fluoro
ethyl
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PCT/EP2021/085688
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French (fr)
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WO2022129047A3 (en
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John Paul Kilburn
Erhad ASCIC
Mauro Marigo
Laurent David
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H. Lundbeck A/S
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Publication of WO2022129047A2 publication Critical patent/WO2022129047A2/en
Publication of WO2022129047A3 publication Critical patent/WO2022129047A3/en

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Definitions

  • the present invention is directed to compounds which are modulators of the NMDA receptor, pharmaceutical compositions comprising the compounds, and their use in treatment of neurological disorders or neuropsychiatric disorders such as depression, in particular major depressive disorder (MDD) and treatment-resistant depression (TRD).
  • MDD major depressive disorder
  • TRD treatment-resistant depression
  • a rough working estimate of prevalence is that depression affects l/5th of the population at some point, affecting women in a higher proportion than men (5-9% and 2-3% incidence respectively in the US, representing an overall incidence of 6.6%).
  • the North-American Center for Disease Control has reported that from 2005-2008, 8.9% of the US population was prescribed an antidepressant during any given month, antidepressants being also prescribed for anxiety, pain, and other non-mood disorders [Murray et al., Global Burden of Disease Study. Lancet. May 17, 1997; 349(9063): 1436-1442],
  • Antidepressants are marketed and thus known to the skilled person. Some examples of different types of antidepressant are selective serotonin reuptake inhibitors (SSRIs), Serotonin-norepinephrine reuptake inhibitors (SNRIs), Monoamine oxidase inhibitors (MAOIs), and Tricyclic antidepressants. Typical limitation of known antidepressants are delayed onset of efficacy and low remission rates after multiple courses of pharmacotherapy, and for some antidepressants severe side-effects [Jick et al., Antidepressants and the risk of suicidal behaviors. Jama. Jul. 21 2004; 292(3 ) :338-343] .
  • NMDA N-Methyl-D-Aspartate
  • TRD treatment resistant depression
  • ketamine a pore-blocking antagonist of the NMDA receptor
  • MDD treatment with ketamine has the drawbacks of sedation, psychotomimetic side effects and requirement of intravenous or intranasal administration.
  • NMDA receptors are tetrameric ligand-gated ion channels which are also involved in essential physiological processes such as synaptic plasticity and development.
  • NMDA receptors are heterotetramers comprising two GluNl subunits and two GluN2/GluN3 subunits. This means that they assemble as either diheteromeric or triheteromeric receptors.
  • the majority of native NMDA receptors consist of two GluNl subunits and two GluN2 subunits.
  • Activation of the NMDA receptors requires simultaneous binding at two different binding sites. Glutamate, the major excitatory neurotransmitter in the central nervous system, binds to the GluN2 subunits and glycine binds to the GluNl and GluN3 subunits.
  • D-cycloserine Another known modulator of the NMDA receptor is D-cycloserine, which is a partial glycine site agonist.
  • D-cycloserine has been intensively studied due to its neuroactive properties and potential utility in treatment of depression and depression disorders such as MDD [Heresco-Levy, U., Javitt, D.C., Gelfin, Y., Gorelik, E., Bar, M., Blanaru, M., Kremer, I., 2006. Controlled trial of d-cycloserine adjuvant therapy for treatmentresistant major depressive disorder. J. Affect. Disord.
  • a compound which is a modulator of the NMDA receptor with affinity for the glycine site may fulfil such unmet needs.
  • R 1 is selected from the group consisting of hydrogen and halogen
  • R 2 and R 4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C1- C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1- C
  • a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or diluents.
  • a method for the treatment of depression comprising the administration of a therapeutically effective amount of a compound of formula I, or acceptable salt thereof, or a pharmaceutical composition to a patient in need thereof.
  • a compound of formula I, or a pharmaceutically acceptable salt thereof for use as a medicament comprising a compound of formula I, or a pharmaceutically acceptable salt thereof.
  • a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of depression.
  • Y-axis Baseline-normalized power (dB);
  • X-axis bars from left to right: 10% HPbetaCD (vehicle), 3 mg/kg compound 3f, 10 mg/kg compound 3f, and 30 mg/kg compound 3f.
  • Figure 2 Effects of compound 3f in rat maximal electroshock threshold model (MEST) according to Example 4.
  • Y-axis Estimated seizure threshold (CC50) current (mA);
  • X-axis bar to the left: 10% HPbetaCD (vehicle); bar to the right: 10 mg/kg compound 3f.
  • the inventors have identified new compounds that has affinity for the glycine site of the NMDA receptor as seen in Table 2.
  • (C x -C y ) alkyl refers to a hydrocarbyl radical obtained when one hydrogen atom is removed from a linear (i.e. unbranched) or branched saturated hydrocarbon.
  • the number of carbon atoms in the hydrocarbyl substituent (i.e. alkyl) is indicated by the prefix “(Cx-Cy)”, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent.
  • Said ”(Cx-Cy) alkyl” comprises 1-6, preferably 1-4, more preferably 1-3, such as 1-2 or such as 2-3 carbon atoms.
  • (C 1 -C 6 ) alkyl refers to an alkyl substituent containing from 1 to 6 carbon atoms.
  • alkyl groups include the subclasses normal alkyl (n-alkyl), secondary and tertiary alkyl.
  • “(C 1 -C 6 ) alkyl” includes groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl, tert. -butyl, n-pentyl, isopentyl, neopentyl, n- hexyl and isohexyl.
  • alkylene refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms, for example, of 1 to 4 carbon atoms.
  • Representative examples of alkylene include, but are not limited to, -CH 2 -, - CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, and - CH 2 CH 2 CH 2 CH 2 CH 2 -.
  • (C x -C y ) alkoxy refers to a moiety of the formula –OR’, wherein R’ indicates (C x -C y )alkyl as defined above.
  • R’ indicates (C x -C y )alkyl as defined above.
  • (C 1 -C 4 )alkoxy refers to such moiety wherein the alkyl contains from one to four carbon atoms.
  • Examples of “(C 1 - C 4 )alkoxy” include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy and tert- butoxy.
  • phenoxy refers refers to a moiety of the formula –O-phenyl, i.e.
  • halogen indicates a substituent selected from the 7 th main group of the periodic table, such as fluoro, chloro, bromo and iodo (F, Cl, Br and I).
  • cyano refers to a CN group appended to the parent molecule through the carbon atom of the CN group.
  • halo(C x -C y )alkyl as used herein, means a (C x -C y )a I ky I group as defined herein, which is substituted with one or more halogen as defined herein, e.g. fluoro, chloro or bromo. Examples include, but are not limited to, trifluormethyl and difluoromethyl.
  • hydroxyl or "hydroxy,” as used herein, means an -OH group.
  • (C z -C w )alkoxy(C x -C y )alkyl refers to an (C z -C w )alkoxy group, as defined herein, appended to the parent molecular moiety through an (C x -C y )a I ky I group, as defined herein. Examples include, but are not limited to, methoxymethyl and ethoxymethyl.
  • phenyl is intended to mean a benzene radical, with one H removed at the attachment point.
  • hydroxy(C x -C y )alkyl or "hydroxyl(C x -C y )alkyl” as used herein, means a (C x - C y )alkyl group as defined herein, which is substituted with one or more -OH group(s). Examples include, but are not limited to hydroxymethyl, hydroxyethyl, hydroxypropyl
  • heteroaryl refers to a radical of a heteroaromatic ring.
  • heteroaryl refers to a radical of a heteroaromatic ring.
  • the term “5- or 6-membered heteroaryl” means a radical of a five or six membered heteroaromatic monocyclic ring containing at least one heteroatom independently selected from the group consisting of N, O and S, such as containing 1 to 4 heteroatoms independently selected from O, S, and N, such as 1 to 3 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O and N.
  • the five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds.
  • the heteroaryl radical may be connected to the parent moiety through a carbon atom or a nitrogen atom contained anywhere within the heteroaryl group.
  • Representative examples of 5- or 6-membered heteroaryl include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl.
  • the term '5- or 6-membered heteroaromatic rings comprising 1 or 2 nitrogen' comprises, but is not limited to, pyrrol, pyrazol, imidazole, pyridine, pyrimidin, pyridazine and pyrazine.
  • heterocyclyl as used herein, means a radical of an aliphatic heterocycle.
  • '4 - 6 membered heterocyclyl' as used herein, means a radical of a four-, five- or six-membered monocyclic ring containing at least one heteroatom independently selected from the group consisting of O, N, and S.
  • the four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S.
  • the five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S.
  • monocyclic heterocycles include, but are not limited to, azetidinyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3- dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetra hydropyranyl.
  • the term '5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen' comprises, but is not limited to, pyrrolidine, pyrazoline, piperidine, tetrahydropyrimidine dihydropyridine, imidazoline, imidazolidine.
  • (C x -C y )cycloalkyl refers to a saturated cycloalkane hydrocarbon radical comprising from x to y carbon atoms, such as from 3-6 carbon atoms, such as 3-5 or such as 3-4 carbon atoms.
  • the cycloalkyl may be monocyclic or bicyclic, wherein the two rings are bridged, fused, or spirocyclic.
  • Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
  • each substituent is selected independent of the other. Each substituent may therefore be identical or different from the other substituent(s).
  • optionally substituted means "unsubstituted or substituted", and therefore the general formulas described herein encompasses compounds containing the specified optional substituent(s) as well as compounds that do not contain the optional substituent(s).
  • groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • a modulator of the NMDA receptor with affinity to the glycine site is a compound which modulates through binding the orthosteric glycine binding site on the NMDA receptor so as to increase or decrease the flux of Ca + + through the ligand-gated channel.
  • the term "therapeutically effective amount" of a compound is intended to indicate an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease (e.g. depression) and its complications in a therapeutic intervention comprising the administration of said compound.
  • An amount adequate to accomplish this is defined as “therapeutically effective amount”.
  • Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, e.g. by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician.
  • treatment means the management and care of a patient for the purpose of combating a disease.
  • the term is intended to include the full spectrum of treatments for a given disease (e.g. depression) from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the depression, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the depression.
  • a given disease e.g. depression
  • the patient to be treated is preferably a mammal, in particular a human being.
  • disease can be used synonymous with disorder, condition, malfunction, dysfunction and the like.
  • the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof wherein R 1 is selected from the group consisting of hydrogen and halogen; R 2 and R 4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 - C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkyl, (C 1 -C 4
  • the invention provides a compound according to the above embodiment, or a pharmaceutically acceptable salt thereof, wherein R 2 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 - C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C 4 )
  • the invention provides a compound according to the above embodiment, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 - C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 1 is selected from the group consisting of hydrogen and fluoro.
  • R 3 is selected from the group consisting of hydrogen, fluoro and (C 1 -C 3 )alkyl.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 are hydrogen.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 2 is selected from the group consisting of hydrogen, halogen and (C 1 -C 6 )alkyl and wherein R 4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6- membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 - C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 2 and R 4 are not hydrogen.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 are hydrogen;
  • R 2 is selected from the group consisting of halogen and (C 1 -C 6 )alkyl;
  • R 4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of 5- or 6-membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 - C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C 4 )alkyl, halo(C 1 -C 4 )alkyl, phenyl, phenoxy and -C(O)NH 2 .
  • R 4 is selected from the group consisting of 5- or 6-membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 - C 4 )alkoxy, (
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is pyrazolyl, wherein said pyrazolyl is optionally substituted with one or more substituents independently selected from the group consisting of fluor, chloro, (C 1 - C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C 4 )alkyl, and halo(C 1 -C 2 )alkyl.
  • R 2 is fluoro.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 2 and R 4 each independently are selected from the group consisting of halogen, -NRaRb, 5- or 6- membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 - C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alk
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1- C4)alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C 4 )
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 - C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C 4
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of 5- or 6-membered heteroaryl, phenyl, 4 - 6 membered heterocyclyl and (C 3 -C 6 )cycloalkyl, wherein said 5- or 6-membered heteroaryl, phenyl, 4-6 membered heterocyclyl and (C 3 -C 6 )cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )alkoxy(C 1 -C 4 )alkyl, halo(C1- C4)alkyl, hydroxy(C 1 -C 4 )alkyl, phenyl, phenoxy and -C(O)NH2; and R 2 is selected from the group consisting of 5-
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 - C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of, fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo(C 1 -C 2 )
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 - C 4 )alkyl, (C 1 -C 4 )alkoxy, halo(C 1 -C 2 )alkyl, (
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 5- or 6-membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy halo(C 1 - C 2 )alkyl, (C 1 -C 2 )alkoxy(C 1 -C 2 )alkyl, hydroxy(C 1 -C 2
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 5- or 6-membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 2 )alkoxy(C 1 -C 2 )alkyl, halo(C 1 -C 2 )alkyl, phenyl, phenoxy and -C(O)NH 2 .
  • the 5- or 6-membered heteroaryl is selected from the group
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 5- membered heteroaryl is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 - C 2 )alkoxy(C 1 -C 2 )alkyl, and halo(C 1 -C 2 )alkyl.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 2 )alkoxy(C 1 -C 2 )alkyl and halo(C 1 -C 2 )alkyl; and wherein R 2 is selected from the group consisting of halogen and (C 1 -C 6 )alkyl; and wherein R 1 and R 3 both represent hydrogen.
  • R 4 is selected from the group consisting of pyrazolyl, isoxazolyl, ox
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 - C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 2 )alkoxy(C 1 -C 2 )alkyl and halo(C 1 -C 2 )alkyl; and wherein R 2 is selected from the group consisting of halogen and (C 1 -C 6 )alkyl; and wherein R 1 and R 3 both represent hydrogen.
  • R 4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 - C 4 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 4 - 6 membered heterocyclyl is selected from the group consisting of morpholinyl, piperidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, wherein said 4 - 6 membered heterocyclyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy halo(C 1 - C2)alkyl, (C 1 -C 2 )alkoxy(C 1 -C 2 )alkyl, hydroxy(C 1 -C 2 )alkyl, phenyl, phenoxy and -C(O)NH2.
  • the 4 - 6 membered heterocyclyl is selected from the group consisting of morpholinyl, pipe
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 2 is selected from the group consisting of morpholinyl, piperidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, wherein said 4 - 6 membered heterocyclyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy halo(C 1 -C 2 )alkyl, (C 1 - C 2 )alkoxy(C 1 -C 2 )alkyl, hydroxy(C 1 -C 2 )alkyl, phenyl, phenoxy and -C(O)NH 2 ; and wherein wherein R 2 is selected from the group consisting of halogen and (C 1 - C 6 )alkyl; and
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of (C 3 -C 6 )cycloalkyl, wherein said (C 3 -C 6 )cycloalkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, halo(C 1 -C 4 )alkyl, hydroxy(C1- C4)alkyl, phenyl, phenoxy and -C(O)NH2.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein at least one of R 2 or R 4 does not represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 2 and R 4 do not represent hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 1 represents hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 3 is selected from the group consisting of hydrogen, halogen and (C 1 -C 6 )alkyl.
  • the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 3 is selected from the group consisting of hydrogen, fluoro, chloro and (C 1 -C 3 )alkyl.
  • R 3 is selected from the group consisting of hydrogen, fluoro, methyl and ethyl.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 3 is hydrogen.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 1 is selected from the group consisting of hydrogen and fluoro. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 1 is hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 both represent hydrogen.
  • the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 1 is selected from the group consisting of hydrogen and fluoro; R 2 is selected from the group consisiting of hydrogen, fluoro, chloro and (C 1 -C 4 )alkyl; R 3 is selected from the group consisting of hydrogen, fluoro, (C 1 -C 2 )alkyl and (C 1 - C 2 )alkoxy; R 4 is selected from the group consisiting of hydrogen, fluoro, -NRaRb, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl, thienyl, phenyl, (C 1 -C 4 )alkyl,
  • the invention provides a compound according to formula I of the embodiment above, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaryl is optionally substituted with one or more substituents independently selected from (C 1 - C 4 )alkyl, and wherein said 5- or 6-membered heterocycle is optionally substituted with one or more substituents independently selected from (C 1 -C 4 )alkyl and oxo; and wherein R 3 and R 4 each independently are selected from the group consisting of hydrogen, halogen, (C 1 -C 6 )alkyl and (C 1 -C 4 )alkoxy; or R 2 and R 3 , together with the carbon atoms to which they are attached form a 5- or 6 membere
  • the invention provides a compound according to the embodiment above, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 , or R 2 and R 3 , together with the carbon atoms to which they are attached form a ring selected from the group consisting of pyridine, imidazole, dihydropyridine and tetrahydropyridine, wherein said pyridine and imidazole are optionally substituted with one or more substituents independently selected from (C 1 -C 4 )alkyl, and wherein said dihydropyridine and tetrahydropyridine are optionally substituted with one or more substituents independently selected from (C 1 -C 4 )alkyl and oxo.
  • the invention provides a compound according to any one of the embodiments above, selected from the list consisting of (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido
  • the invention provides a compound according to any one of the embodiments above, selected from the list consisting of (R)-2-amino-3-((3-(4-chloro-2-ethyl-pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 3f below (R)-2-amino-3-((3-(2-ethylphenyl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 4l below (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid with the formula 3b below or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above, selected from the list consisting of (R)-2-amino-3-((3-(4-chloro-2-ethyl-pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 3f below (R)-2-amino-3-((3-(2-ethylphenyl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 4l below or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above as a salt of hydrochloric acid.
  • the invention provides a compound according to any one of the embodiments above as a salt of hydrobromic acid.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid, or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid hydrochloride.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid hydrobromide.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid, or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid hydrochloride. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid hydrobromide.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid.
  • Reference to compounds encompassed by the invention includes the free substance (zwitter ion) of compounds of the invention, pharmaceutically acceptable salts of compounds of the invention, such as acid addition salts or base addition salts, and polymorphic and amorphic forms of compounds of the invention and of pharmaceutically acceptable salts thereof.
  • the compounds of the invention and pharmaceutically acceptable salts thereof may potentially exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like.
  • optically active acids suitable for this purpose may include, but are not limited to, d- or l- tartaric, mandelic or camphorsulfonic acids.
  • Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix.
  • the compounds of the present invention may also be resolved by the formation and chromatographic separation of diastereomeric derivatives from chiral derivatizing reagents, such as, chiral alkylating or acylating reagents, followed by cleavage of the chiral auxiliary.
  • any of the above methods may be applied either to resolve the optical antipodes of the compounds of the invention per se or to resolve the optical antipodes of synthetic intermediates, which can then be converted by methods described herein into the optically resolved final products which are the compounds of the invention. Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S. Wilen in Enantiomers, Racemates, and Resolutions, John Wiley and Sons, New York, 1981. Optically active compounds can also be prepared from optically active starting materials.
  • isotopically labelled compounds which are similar to those claimed in formula I, wherein one or more atoms are represented by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., 2 H, 3 H, 11 C, 13 C, 15 N, 18 F and the like). Particular mention is made of 2 H substituted compounds i.e. compounds wherein one or more H atoms are represented by deuterium. In one embodiment of the invention one or more of the hydrogen atoms of the compound of formula I are represented by deuterium. It is recognized that elements are present in natural isotopic abundances in most synthetic compounds and result in inherent incorporation of deuterium.
  • the natural isotopic abundance of hydrogen isotopes such as deuterium is immaterial (about 0.015%) relative to the degree of stable isotopic substitution of compounds indicated herein.
  • designation of an atom as deuterium at a position indicates that the abundance of deuterium is significantly greater than the natural abundance of deuterium.
  • Any atom not designated as a particular isotope is intended to represent any stable isotope of that atom, as will be apparent to the ordinarily skilled artisan.
  • Pharmaceutically acceptable salts The compounds of this invention are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof.
  • salts may be prepared in a conventional manner by treating a solution or suspension of a free base of formula I with a molar equivalent of a pharmaceutically acceptable acid.
  • suitable organic and inorganic acids are described below.
  • Pharmaceutically acceptable salts in the present context is intended to indicate non- toxic, i.e. physiologically acceptable salts.
  • pharmaceutically acceptable salts include salts formed with inorganic and/or organic acids on the nitrogen atoms in the parent molecule.
  • Said acids may be selected from for example hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fumaric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid, saccharin, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid.
  • the pharmaceutically acceptable salt is a hydrogen chloride salt.
  • the pharmaceutically acceptable salt is a hydrogen bromide salt.
  • pharmaceutically acceptable salts also include salts formed with inorganic and/or organic bases on the acidic groups of compounds of formula I.
  • Said bases may be selected from for example alkali metal bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, alkaline earth bases, such as calcium hydroxide and magnesium hydroxide, and organic bases, such as trimethylamine and trimethylamine. Additional examples of useful acids and bases to form pharmaceutically acceptable salts can be found e.g. in Stahl and Wermuth (Eds) “Handbook of Pharmaceutical salts. Properties, selection, and use”, Wiley-VCH, 2008.
  • the invention encompasses compounds of the invention for use as a medicament, as well as for use in treatment of all diseases and disorders listed below.
  • compounds of the present invention may be useful in the treatment of depression and depressive disorders.
  • a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of depression.
  • the diagnosis of depression usually follows a clinical evaluation by a psychiatrist or other mental health professionals.
  • DSM IV Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV) published by the American psychiatric association and the ICD (ICD-10: International Statistical Classification of Diseases and Related Health Problems – 10 th Revision, published periodically by the World Health Organization) or any other psychiatric classification system.
  • Signs and symptoms of depression are for example depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach.
  • compounds of the invention are for use to prevent, alter, reduce or alleviate one or more signs or symptoms of depression selected from the group consisting of depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach.
  • the skilled person is familiar with various test for measuring the improvement of depressive symptoms. Examples of tests for measuring the improvements are, but not limited to, the Hamilton Rating Scale for Depression (HAM-D) or Montgomery– ⁇ sberg Depression Rating Scale (MADRS) scale.
  • the depression is major depressive disorder.
  • the depression is treatment-resistant depression.
  • the depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • Clinically used NMDA antagonist such as ketamine and dextromethorphan are generally effective in patients with neurophatic pain [Hy et al.,Expert Rev Clin Pharmacol. 2011 May 1; 4(3): 379–388].
  • compound of formula I or a pharmaceutically acceptable salt thereof is used in the treatment of pain.
  • the pain is neuropathic pain.
  • a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of suicidal ideation.
  • compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of a neurological disorder or neuropsychiatric disorder.
  • the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof to a patient (e.g. a human patient) in need thereof.
  • a patient e.g. a human patient
  • the invention provides a method for the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof.
  • a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof.
  • the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof.
  • depression such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression
  • the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • depression such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • depression such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • the compounds of formula I are for use as stand- alone treatment as the sole active compound.
  • the compounds of formula I may be used in combination with other agents useful in the treatment of disorders such as depression.
  • combined use in combination with” and “a combination of” and the like as used herein in the context of the method of the invention comprising the combined administration of therapeutically effective amounts of a compound of formula I, and another compound, which compound is useful in the treatment a neurodegenerative disease or disorder, is intended to mean the administration of a compound of formula I simultaneously or sequentially, in any order, together with said other compound.
  • the two compounds may be administered simultaneously or sequentially with a time gap between the administrations of the two compounds.
  • the two compounds may be administered either as part of the same pharmaceutical formulation or composition, or in separate pharmaceutical formulations or compositions.
  • the two compounds may be administered on the same day or on different days.
  • the patient to be treated may already be in treatment with one or more other compounds useful in the treatment of depression when treatment with a compound of the invention initiated.
  • the patient may already be in treatment with a compound of the invention when treatment with one or more other compounds useful in the treatment of a depression or psychosis is initiated.
  • therapeutically active compounds which may advantageously be combined with compounds of the invention include sedatives or hypnotics, such as benzodiazepines; anticonvulsants, such as lamotrigine, valproic acid, topiramate, gabapentin, carbamazepine; mood stabilizers such as lithium; dopaminergic drugs, such as dopamine agonists and L-Dopa; drugs to treat ADHD, such as atomoxetine; psychostimulants, such as modafinil, ketamine, methylphenidate and amphetamine; other antidepressants, such as mirtazapine, mianserin, vortioxetine, cipralex, and buproprion; hormones, such as T3, estrogen, DHEA and testosterone; atypical antipsychotics, such as olanzapine,
  • compositions comprising a compound of formula I may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, buccal, sublingual, pulmonal, transdermal and parenteral (e.g. subcutaneous, intramuscular, and intravenous) route. It will be appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient. Doses In one embodiment, the compound of the present invention is administered in an amount from about 0.5 mg/kg body weight to about 50 mg/kg body weight per day. In particular, daily dosages may be in the range of 1 mg/kg body weight to about 30 mg/kg body weight per day.
  • the exact dosages will depend upon the frequency and mode of administration, the sex, the age, the weight, and the general condition of the subject to be treated, the nature and the severity of the condition to be treated, any concomitant diseases to be treated, the desired effect of the treatment and other factors known to those skilled in the art.
  • the frequency of administration is 1, 2, 3, 4, or 5 times per day
  • the frequency of administration is once weekly.
  • the frequency of administration is twice weekly.
  • a typical oral dosage for adults will be in the range of 1-3000 mg/day of a compound of the present invention, such as 700-2800 mg/day, such as 1000-2000 mg/day or 1200- 1700mg/day.
  • the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 100 to 1000 mg, such as 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 750 mg or up to 1000 mg of a compound of the present invention.
  • the frequency of administration is 1, 2, 3, 4, or 5 times per day
  • the frequency of administration is once weekly.
  • the frequency of administration is twice weekly.
  • a typical IV dosage for adults will be in the range of 20-300 mg/day of a compound of the present invention, such as 50-200 mg/day, such as 70-150 mg/day or 75-125 mg/day.
  • the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 10 to 300 mg, such as 10 mg, 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg of a compound of the present invention.
  • the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly.
  • excipient or “pharmaceutically acceptable excipient” refers to pharmaceutical excipients including, but not limited to, fillers, antiadherents, binders, coatings, colours, disintegrants, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, solvents, vehicles and adjuvants.
  • the present invention also provides a pharmaceutical composition comprising a compound of formula I, such as one of the compounds disclosed in the Experimental Section herein, and one or more pharmaceutically acceptable carriers or diluents.
  • the present invention also provides a process for making a pharmaceutical composition comprising a compound of formula I.
  • compositions according to the invention may be formulated with pharmaceutically acceptable excipients in accordance with conventional techniques such as those disclosed in Remington, “The Science and Practice of Pharmacy”, 22 th edition (2013), Edited by Allen, Loyd V., Jr.
  • Pharmaceutical compositions for oral administration include solid oral dosage forms such as tablets, capsules, powders and granules; and liquid oral dosage forms such as solutions, emulsions, suspensions and syrups as well as powders and granules to be dissolved or suspended in an appropriate liquid.
  • Solid oral dosage forms may be presented as discrete units (e.g. tablets or hard or soft capsules), each containing a predetermined amount of the active ingredient, and preferably one or more suitable excipients.
  • the solid dosage forms may be prepared with coatings such as enteric coatings or they may be formulated so as to provide modified release of the active ingredient such as delayed or extended release according to methods well known in the art.
  • the solid dosage form may be a dosage form disintegrating in the saliva, such as for example an oral-dispersible tablet.
  • excipients suitable for solid oral formulation include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, povidone, croscarmellose sodium, sucrose, cyclodextrin, talcum, gelatin, pectin, magnesium stearate, stearic acid and lower alkyl ethers of cellulose.
  • the solid formulation may include excipients for delayed or extended release formulations known in the art, such as glyceryl monostearate or hypromellose.
  • the formulation may for example be prepared by mixing the active ingredient with solid excipients and subsequently compressing the mixture in a conventional tableting machine; or the formulation may for example be placed in a hard capsule e.g. in powder, pellet or mini tablet form.
  • the amount of solid excipient will vary widely but will typically range from about 25 mg to about 1 g per dosage unit.
  • Liquid oral dosage forms may be presented as for example elixirs, syrups, oral drops or a liquid filled capsule.
  • Liquid oral dosage forms may also be presented as powders for a solution or suspension in an aqueous or non-aqueous liquid.
  • excipients suitable for liquid oral formulation include, but are not limited to, ethanol, propylene glycol, glycerol, polyethylenglycols, poloxamers, sorbitol, poly-sorbate, mono and di- glycerides, cyclodextrins, coconut oil, palm oil, and water.
  • Liquid oral dosage forms may for example be prepared by dissolving or suspending the active ingredient in an aqueous or non-aqueous liquid, or by incorporating the active ingredient into an oil-in-water or water-in-oil liquid emulsion.
  • compositions for parenteral administration include sterile aqueous and nonaqueous solutions, dispersions, suspensions or emulsions for injection or infusion, concentrates for injection or infusion as well as sterile powders to be reconstituted in sterile solutions or dispersions for injection or infusion prior to use.
  • excipients suitable for parenteral formulation include, but are not limited to water, coconut oil, palm oil and solutions of cyclodextrins.
  • Aqueous formulations should be suitably buffered if necessary and rendered isotonic with sufficient saline or glucose.
  • compositions include suppositories, inhalants, creams, gels, dermal patches, implants and formulations for buccal or sublingual administration. It is requisite that the excipients used for any pharmaceutical formulation comply with the intended route of administration and are compatible with the active ingredients.
  • Scheme 1 Compounds of general formula I (Scheme 1) may be prepared from compounds with general formula IV by standard de-protection procedures.
  • compounds of general formula I may be prepared from compounds with general formula IV where Pg 1 is Cbz (carboxybenzyl) and Pg 2 is benzyl using conditions such as HBr in acetic acid.
  • Compounds with general formula IV may be prepared by reacting protected amines of general formula III with carboxylic acids (or salt thereof) of general formula II by standard peptide coupling such as using O-(Benzotriazol-1-yl)-N,N,N ⁇ ,N ⁇ - tetramethyluronium tetrafluoroborate in the presence of a base such as N,N- diisopropylethylamine in a solvent such as N,N-dimethylformamide.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R 2 can be hetaryl, aryl or alkenyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VI can be obtained from compounds of general formula V, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as potassium triphosphate.
  • a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II)
  • a base such as potassium triphosphate.
  • similar reaction conditions may be applied for introducing R 4 substituents.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 3) where R 2 is hetaryl or heterocycle as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VII can be obtained from compounds of general formula V, under reaction conditions such as in the presence of a catalyst such as copper (II) acetate and a base such as pyridine.
  • reaction conditions may be applied for introducing R 4 substituents.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 4) where R 2 is NR a R b as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VI can be obtained by reacting compounds of general formula VIII together with amines of general formula R a R b NH.
  • similar reaction conditions may be applied for introducing R 4 substituents.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 5) where R 2 can be alkyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VI can be obtained by reacting compounds of general formula IX together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ -Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2-dicyclohexylphosphino-2 ⁇ ,6 ⁇ -bis(N,N-dimethylamino)biphenyl (Cphos). Likewise, similar reaction conditions may be applied for introducing R 4 substituents.
  • a catalyst such as [1,1 ⁇ -Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • a base such as caesium acetate
  • Benzoic acids of general formula II (or salt thereof), (Scheme 6) where R 3 can be alkyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VI can be obtained by reacting compounds of general formula X together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ -Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2-dicyclohexylphosphino-2 ⁇ ,6 ⁇ -bis(N,N-dimethylamino)biphenyl (Cphos).
  • a catalyst such as [1,1 ⁇ -Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • a base such as caesium acetate
  • alkyl Zinc bromides under reaction conditions such as in the presence of
  • reaction conditions may be applied for introducing R 1 substituents.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 7) where R 2 can be alkyloxy or cycloalkyoxy as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VI can be obtained by reacting compounds of general formula XI together with aldehydes or ketones, under reaction conditions such as in the presence of a base such as isopropylmagnesium chloride complex with lithium chloride.
  • similar reaction conditions may be applied for introducing R 4 substituents.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 8) where R 2 can be hetaryl, aryl or alkenyly as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VI can be obtained from compounds of general formula XII, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ -Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as cesium carbonate.
  • a catalyst such as [1,1 ⁇ -Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II)
  • a base such as cesium carbonate.
  • Preparative HPLC Preparative-HPLC (Method AA): Instrument: Gilson GX-281 Liquid Handler, SHIMADZU LC-8A LCMS2010; Column: YMC-Actus Triart C18150*305 ⁇ m; Mobile Phase A: water (0.05% HCl v/v); Mobile phase B: MeCN; Gradient: B from 5% to 35% in 10 minutes then hold at 100% for 3 minutes; FlowRate(ml/min): 25; Column temperature: 35°C and Wavelength: 220nm 254nm.
  • Preparative HPLC (Method BB): Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Xtimate C18150*25mm*5 ⁇ m; Mobile Phase A: water (0.05% ammonia hydroxide v/v); Mobile phase B: MeCN; Gradient: from 42% to 72% in 10 minutes then hold at 100% for 2.5 minutes; Flow Rate (ml/min): 25; Column temperature: 25°C and Wavelength: 220nm 254nm
  • Preparative HPLC (Method CC): Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Agela Durashell C18150 mm ⁇ 25mm ⁇ 5 ⁇ m; Mobile Phase A: water (0.225%TFA, v/v); Mobile phase B: MeCN; Gradient: B from 32% to 62% in 10 minutes, hold 100% B for 2 minutes; Flow Rate (ml
  • NMR 1 H NMR spectra were recorded at 300, 400, 500 or 600 MHz on Bruker Avance instruments. TMS was used as internal reference standard. Chemical shift values are expressed in ppm.
  • Abbreviations are in accordance with to the ACS Style Guide: "The ACS Style guide – A manual for authors and editors" Janet S.
  • Step 1 methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
  • Step 2 3-(4-Ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate 230 mg, 0.88 mmol
  • THF 2 mL
  • H2O 2 mL
  • LiOH.H2O 110 mg, 2.63 mmol
  • the mixture was stirred at 20 °C for 16 hours.
  • Step 3 (R)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate
  • 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid 200 mg, 0.81 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (353 mg, 0.97 mmol, HCl salt)
  • TBTU 388 mg, 1.21 mmol
  • DIPEA 312 mg, 2.42 mmol
  • Step 4 (R)-2-Amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate 270 mg, 0.48 mmol
  • 30% HBr in AcOH 5 mL
  • Step 1 methyl 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate
  • 3-bromo-4-ethyl-1-methyl-1H-pyrazole 200 mg, 1.06 mmol
  • (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid 251 mg, 1.27 mmol
  • Pd(dtbpf)Cl 2 69 mg, 0.11 mmol
  • K 3 PO 4 (674 mg, 3.17 mmol) in the mixture of dioxane (4 mL) and H 2 O (0.5 mL) was degassed and purged with N 2 (3 times), and then the mixture was stirred at 110 °C for 16 hours under N 2 atmosphere.
  • Step 2 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid
  • methyl 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate 130 mg, 0.5 mmol
  • THF 2 mL
  • H2O 2 mL
  • LiOH.H2O 62 mg, 1.49 mmol
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was concentrated to remove MeOH and THF.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3- yl)-5-fluorobenzamido)propanoate
  • 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid 100 mg, 402 ⁇ mol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 159 mg, 0.44 mmol, HCl salt)
  • TBTU (194 mg, 604 ⁇ mol) and DIPEA 156 mg, 1.21 mmol
  • Step 4 (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5- fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H- pyrazol-3-yl)-5-fluorobenzamido)propanoate 150 mg, 0.27 mmol
  • 30% HBr in AcOH (4 mL) was stirred at 50°C for 16 hours. The mixture was concentrated.
  • Step 1 methyl 3-(3-ethylpyridin-2-yl)-5-fluorobenzoate
  • 2-chloro-3-ethylpyridine 300 mg, 2.12 mmol
  • (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid 503 mg, 2.54 mmol
  • Pd(dtpbf) Cl 2 138 mg, 0.21 mmol
  • K 3 PO 4 1.35 g, 6.36 mmol
  • dioxane 3 mL
  • H 2 O 0.5 mL
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-2-yl)-5- fluorobenzamido)propanoate
  • 3-(3-ethylpyridin-2-yl)-5-fluorobenzoic acid 320 mg, 1.30 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 524 mg, 1.44 mmol, HCl salt
  • TBTU (628 mg, 1.96 mmol
  • DIPEA 506 mg, 3.91 mmol
  • Step 1 Methyl 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoate and methyl 3-(3-ethyl-1H- pyrazol-1-yl)-5-fluorobenzoate
  • 3- ethyl-1H-pyrazole (0.25 g, 2.6 mmol)
  • Cu(OAc)2 945 mg, 5.2 mmol
  • Py 1,2-dichloroethane (25 mL) was added 4A MS (0.2 g) under O2 (15 psi).
  • Step 2 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid
  • the mixture was extracted with MTBE (20 mL ⁇ 2).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (654 mg, 1.79 mmol, HCl salt), 3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid (0.42 g, 1.79 mmol), TBTU (864 mg, 2.69 mmol) and DIPEA (1.16 g, 8.97 mmol) in DMF (20 mL) was stirred at 25°C for 16 hours.
  • Step 4 (R)-2-amino-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate (0.1 g, 0.18 mmol) and HBr in AcOH (2 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was diluted with AcOH (10 mL). The part of solvent was removed.
  • Step 1 3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid and 3-(4-ethyl-1H-imidazol-1- yl)-5-fluorobenzoic acid
  • 5-ethyl-1H-imidazole 500 mg, 5.20 mmol
  • methyl 3-bromo-5- fluorobenzoate (1.21 g, 5.20 mmol) in DMF (10 mL)
  • Cs2CO3 6.78 g, 20.81 mmol
  • CuI 99 mg, 0.52 mmol
  • L-Proline 120 mg, 1.04 mmol
  • the reaction mixture was quenched with water (30 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • the combined organic layers were washed with brine (30 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • the sample was purified by preparation HPLC (Method CC).
  • Step 2 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoate
  • 3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid 70 mg, 0.30 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 120 mg, 0.33 mmol, HCl salt
  • TBTU 144 mg, 0.45 mmol
  • DIPEA 116 mg, 0.90 mmol
  • Step 1 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoate
  • 3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid 700 mg, 2.99 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.20 g, 3.29 mmol, HCl salt)
  • TBTU (1.44 g, 4.48 mmol
  • DIPEA (1.16 g, 8.97 mmol
  • Step 2 methyl 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoate
  • 2-bromo-1-ethyl-1H-imidazole 300 mg, 1.71 mmol
  • (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid 509 mg, 2.57 mmol
  • Pd(dppf)Cl2 251 mg, 0.34 mmol
  • K3PO4 (728 mg, 3.43 mmol) in 1,4-dioxane (10 mL) and H2O (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 110°C for 16 hours under N2 atmosphere.
  • the reaction mixture was concentrated.
  • Step 3 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoate (270 mg, 1.09 mmol) in a mixture of MeOH (8 mL) and H2O (4 mL) was added LiOH.H2O (137 mg, 3.26 mmol). The mixture was stirred at 20°C for 16 hours.
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-2-yl)-5- fluorobenzamido)propanoate
  • 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoic acid 600 mg, crude
  • DMF 8 mL
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (841 mg, 2.56 mmol, HCl salt), HATU (974 mg, 2.56 mmol) and DIPEA (331 mg, 2.56 mmol).
  • the mixture was stirred at 20°C for 16 hours.
  • the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL ⁇ 2).
  • the combined organic layers were dried over MgSO4, filtered and concentrated.
  • the residue was purified by column chromatography (SiO2, eluent of 0 ⁇ 80% ethyl acetate/Petroleum ether) to give a yellow oil.
  • the yellow oil was diluted with ethyl acetate (50 mL) and washed with brine (25 mL ⁇ 2).
  • the combined organic layers were dried over MgSO4, filtered and concentrated.
  • Step 5 (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-2-yl)- 5-fluorobenzamido)propanoate 190 mg, 0.35 mmol
  • 30% HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated.
  • Step 2 methyl 3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzoate
  • MeCN MeCN
  • iodosylbenzene 768 mg, 3.49 mmol
  • 1,1,1- trifluoro-N-(trifluoromethylsulfonyl)methanesulfonamide 818 mg, 2.91 mmol
  • Step 3 3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzoic acid
  • MeOH MeOH
  • LiOH.H 2 O 48 mg, 1.14 mmol
  • H 2 O H 2 O
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was concentrated.
  • the residue was dissolved in water (10 mL) and acidified with aqueous 2N HCl to adjust pH to 4 and extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 5 (R)-2-amino-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-2-methyloxazol-4- yl)-5-fluorobenzamido)propanoate 140 mg, 0.25 mmol
  • 30% HBr in AcOH 5 mL
  • the mixture was concentrated.
  • the residue was dissolved in water (6 mL) and washed with MTBE (6 mL ⁇ 2).
  • Step 1 methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate
  • 3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 877 mg, 4.43 mmol
  • 4-bromo-5-ethyl-isoxazole 0.6 g, 3.41 mmol
  • Na2CO3 7.23 mg, 6.82 mmol
  • H2O 4 mL
  • dioxane 16 mL
  • Pd(dppf)Cl2.CH2Cl2 557 mg, 0.68 mmol
  • the mixture was purged with N2, sealed and heated at 120°C for 40 minutes in the microwave.
  • the mixture was concentrated.
  • Step 4 (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate 60 mg, 0.11 mmol
  • HBr in AcOH 2 mL, 30% in AcOH
  • Step 2 methyl 3-butyryl-5-fluorobenzoate To a mixture of 3-butanoyl-5-fluoro-benzoic acid (4.5 g, crude) in MeOH (50 mL) was added SOCl 2 (5.09 g, 42.8 mmol, 3.1 mL) at 0°C. The mixture was stirred at 70°C for 16 hours.
  • Step 3 (E)-methyl 3-(2-((dimethylamino)methylene)butanoyl)-5-fluorobenzoate
  • a mixture of methyl 3-butyryl-5-fluorobenzoate (0.5 g, 2.23 mmol) and DMF-DMA (17.9 g, 150 mmol) were stirred at 110°C for 16 hours.
  • the mixture was concentrated to give (E)-methyl 3-(2-((dimethylamino)methylene)butanoyl)-5-fluorobenzoate (0.6 g, crude).
  • Step 5 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoic acid
  • a mixture of methyl 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoate (50 mg, crude) and LiOH.H2O (17 mg, 0.4 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was stirred at 25°C for 0.5 hour. The mixture was adjusted to pH 3 ⁇ 4 with aq. KHSO4. The mixture was concentrated to give 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoic acid (47 mg, crude).
  • Step 7 (R)-2-amino-3-(3-(4-ethylisoxazol-5-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylisoxazol-5-yl)-5- fluorobenzamido)propanoate 50 mg, 0.09 mmol
  • HBr in AcOH 2 mL, 30% in AcOH
  • Step 2 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
  • methyl 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate 130 mg, 0.50 mmol
  • MeOH 10 mL
  • H2O 2 mL
  • LiOH.H2O 62 mg, 1.49 mmol
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
  • 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid 100 mg, 0.40 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 176 mg, 0.48 mmol, HCl salt
  • T3P 0.4 mL, 50% in DMF
  • DIPEA 0.2 mL,1.15 mmol
  • Step 1 methyl 3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzoate
  • 5-bromo-4-ethyl-2-methyloxazole 120 mg, 0.63 mmol
  • (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid 125 mg, 0.63 mmol
  • H 2 O 0.5 mL
  • Pd(dppf)Cl 2 .CH 2 Cl 2 52 mg, 0.063 mmol
  • Na 2 CO 3 201 mg, 1.90 mmol
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5- fluorobenzamido)propanoate
  • TBTU 68 mg, 0.21 mmol
  • DIPEA 54 mg, 0.42 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (51 mg, 0.14 mmol, HCl salt).
  • Step 4 (R)-2-amino-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-2-methyloxazol-5- yl)-5-fluorobenzamido)propanoate 60 mg, 0.11 mmol
  • 30% HBr in AcOH 5 mL
  • Step 1 4-Bromo-5-ethyl-1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H- pyrazole
  • NaH 82 mg, 2.06 mmol, 60% in mineral oil
  • CH 3 I 268 mg, 1.89 mmol
  • Step 2 Methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate and methyl 3-(3- ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate
  • 3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 300 mg, 1.52 mmol
  • dioxane 10 mL
  • 4-bromo-5-ethyl-1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H-pyrazole 300 mg, crude. The reaction mixture was stirred at 100°C for 16 hours.
  • reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL ⁇ 3). The combined organic layers were washed with brine (10 mL ⁇ 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give the mixture of two isomers (200 mg), which were seperated by SFC separation to give methyl 3-(3-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro- benzoate (70 mg), and methyl 3-(5-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate (90 mg).
  • Step 3 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid
  • methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate 90 mg, 0.34 mmol
  • MeOH 3 mL
  • H 2 O 2 mL
  • LiOH.H 2 O 43 mg, 1.03 mmol
  • the reaction mixture was stirred at 25°C for 1 hour.
  • the MeOH was removed.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,4-diethyl-1H-pyrazol-3-yl)-5- fluorobenzamido)propanoate
  • TEA 43 mg, 0.43 mmol
  • R 4-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate
  • Step 1 3-ethyl-5-methylisoxazole and 5-ethyl-3-methylisoxazole To a solution of NH 2 OH.HCl (2.5 g, 36.0 mmol) in a mixture of MeOH (10 mL) and H 2 O (20 mL) were added Na 2 CO 3 (1.91 g, 17.99 mmol). When bubbles were absent, hexane- 2,4-dione (4.93 g, 43.2 mmol, 1.2 eq) was added dropwise. The mixture was heated at 100°C for 16 hours. The reaction mixture was extracted with DCM (40 mL ⁇ 3).
  • Step 2 4-bromo-3-ethyl-5-methylisoxazole and 4-bromo-5-ethyl-3-methylisoxazole A mixture of 3-ethyl-5-methylisoxazole and 5-ethyl-3-methylisoxazole (4.0 g, crude) dissolved in DMF (20 mL) was added NBS (7.05 g, 39.59 mmol) and the reaction mixture was stirred at 25°C for 16 hours.
  • reaction mixture was quenched with water (20 mL), and extracted with ethyl acetate (30 mL ⁇ 3). The combined organic layers were washed with brine (30 mL ⁇ 3), dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give a mixture of 4-bromo-3-ethyl-5-methylisoxazole and 4-bromo-5- ethyl-3-methylisoxazole (2.5 g, crude).
  • the reaction mixture was stirred at 120°C for 40 minutes under microwave.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3).
  • the combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 10%) to give methyl 3-(3-ethyl-5-methylisoxazol-4-yl)-5- fluorobenzoate and methyl 3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzoate (1.1 g).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-3-methylisoxazol-4-yl)- 5-fluorobenzamido)propanoate and (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-((3-(3- ethyl-5-methylisoxazol-4-yl)-5-fluorobenzyl)amino)propanoate
  • Step 1 1-ethyl-4-methyl-1H-pyrazole To a solution of 4-methyl-1H-pyrazole (2.0 g, 24.4 mmol) in DMF (10 mL) was added NaH (1.27 g, 31.7 mmol, 60% in mineral oil) at 0°C and stirred at 0°C for 0.5 hour. Then iodoethane (4.56 g, 29.2 mmol) was added at 0°C and stirred at 30°C for 3 hours. The reaction mixture was quenched with water (20 mL), and then extracted with ethyl acetate (30 mL ⁇ 3). The combined organic layers were washed with brine (30 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • Step 2 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H- pyrazole
  • NBS NBS
  • the reaction was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100°C for 16 hours under N 2 atmosphere.
  • the reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 15%).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate
  • 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid 90 mg, 0.36 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 145 mg, 0.40 mmol, HCl salt
  • TBTU (175 mg, 0.54 mmol
  • DIPEA 141 mg, 1.09 mmol
  • Step 2 methyl 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate
  • methyl 2'-bromo-6'-chloro-5-fluoro-[1,1'-biphenyl]-3-carboxylate 240 mg, 0.70 mmol
  • triethylborane 1 M in hexane, 0.84 mL
  • Pd(dppf)Cl2 51 mg, 0.07 mmol
  • K2CO3 290 mg, 2.10 mmol
  • Step 3 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid
  • methyl 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate 100 mg, 0.34 mmol
  • MeOH 4 mL
  • H2O 2 mL
  • LiOH.H2O 29 mg, 0.68 mmol
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-6'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
  • TBTU 156 mg, 0.48 mmol
  • DIPEA 125 mg, 0.97 mmol
  • reaction mixture was stirred at 30°C for 16 hours.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • Step 1 1,4-diethyl-1H-pyrazole To a solution of 4-ethyl-1H-pyrazole (1.0 g, 10.4 mmol) in DMF (10 mL) were added NaH (624 mg, 15.6 mmol, 60% in mineral oil) at 0°C. The reaction mixture was stirred at 0°C for 30 min. CH3CH2I (1.95 g, 12.5 mmol) was added to the reaction mixture and stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL ⁇ 3). The combined organic layers were washed with brine (10 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • Step 2 5-bromo-1,4-diethyl-1H-pyrazole and 3-bromo-1,4-diethyl-1H-pyrazole
  • NBS 860 mg, 4.83 mmol
  • the reaction mixture was stirred at 20°C for 16 hours.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • the combined organic layers were washed with brine (10 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • reaction mixture was stirred at 100°C for 16 hours under N2.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • the combined organic layers were washed with brine (10 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 10%).
  • Step 4 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • methyl 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoate 150 mg, 0.54 mmol
  • MeOH MeOH
  • H 2 O 3 mL
  • LiOH.H 2 O 39 mg, 0.93 mmol
  • the reaction mixture was stirred at 20°C for 2 hours.
  • the MeOH was removed.
  • Step 6 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,4-diethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
  • TEA 108 mg, 1.07 mmol
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate 143 mg, 0.39 mmol, HCl salt).
  • Step 1 3-fluoro-5-(2-hydroxypropan-2-yl)benzoic acid
  • n-BuLi 2.5 M in hexane, 8.04 mL
  • acetone (1.06 g, 18.26 mmol) was added and the mixture was allowed to warm to 25 °C for 1 hour.
  • Step 2 methyl 3-fluoro-5-(2-hydroxypropan-2-yl)benzoate To a solution of 3-fluoro-5-(2-hydroxypropan-2-yl)benzoic acid (300 mg, 1.11 mmol) in a mixture of DCM (10 mL) and MeOH (2 mL) was added TMSCHN 2 (2 M in hexane, 1.67 mL). The mixture was stirred at 25 °C for 1 hour.
  • Step 3 methyl 3-fluoro-5-(2-methoxypropan-2-yl)benzoate
  • DMF dimethyl methoxypropan-2-yl
  • Step 4 3-fluoro-5-(2-methoxypropan-2-yl)benzoic acid
  • a solution of methyl 3-fluoro-5-(2-methoxypropan-2-yl)benzoate (90 mg, 0.40 mmol) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (51 mg, 1.19 mmol).
  • the mixture was stirred at 25 °C for 2 hours.
  • the mixture was concentrated.
  • the residue was added water (8 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(2-methoxypropan-2- yl)benzamido)propanoate
  • TBTU 182 mg, 0.57 mmol
  • DIPEA 146 mg, 1.13 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
  • the mixture was stirred at 25 °C for 16 hours.
  • the mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 2), dried over Na2SO4, filtered and concentrated.
  • Step 6 (R)-2-amino-3-(3-fluoro-5-(2-methoxypropan-2-yl)benzamido)propanoic acid
  • Pd/C 50 mg, 10% Pd, 50% water
  • Step 2 3-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole
  • THF 8 mL
  • NaH 121 mg, 3.04 mmol, 60% in mineral oil
  • MeI 431 mg, 3.04 mmol
  • the reaction was quenched with water (10 mL) and extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were concentrated.
  • Step 3 methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoate
  • 3-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole 140 mg, 0.68 mmol
  • (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 135 mg, 0.68 mmol
  • Pd(dppf)Cl2 50 mg, 0.068 mmol
  • K2CO3 283 mg, 2.05 mmol
  • Step 4 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoic acid
  • methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzoate 130 mg, 0.47 mmol
  • MeOH MeOH
  • H 2 O 1 mL
  • LiOH.H2O 59 mg, 1.40 mmol
  • the mixture was stirred at 25 °C for 16 hours.
  • the mixture was concentrated.
  • the residue was added water (8 mL) and adjusted pH to 5 with 2N HCl and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-(methoxymethyl)-1- methyl-1H-pyrazol-3-yl)benzamido)propanoate
  • TBTU 201 mg, 0.62 mmol
  • DIPEA 161 mg, 1.25 mmol
  • R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 152 mg, 0.42 mmol, HCl salt).
  • Step 6 (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4- (methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzamido)propanoate 80 mg, 0.14 mmol
  • Pd/C 40 mg, 10% Pd, 50% water
  • the mixture was stirred under nitrogen atmosphere at 0°C for 0.5 hour, then the reaction mixture was added iodomethane (778 mg, 5.48 mmol) at 0°C, and the mixture was stirred at 30°C for 1 hour.
  • the reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL ⁇ 3). The combined organic layers were washed with brine (30 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • Step 2 (5-bromo-1-methyl-1H-pyrazol-4-yl)methanol
  • THF 10 mL
  • DIBAL-H 1 M, 5.15 mL
  • the mixture was stirred at 30°C for 16 hours.
  • the reaction mixture was quenched with saturated aqueous sodium potassium tartrate (10 mL), extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 3 5-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole
  • THF tetrahydrofuran
  • NaH 126 mg, 3.14 mmol, 60% NaH in mineral oil
  • MeI 446 mg, 3.14 mmol
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3).
  • Step 4 methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzoate
  • Step 5 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzoic acid
  • methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5- yl)benzoate 240 mg, 0.86 mmol
  • LiOH.H2O 72 mg, 1.72 mmol
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 6 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-(methoxymethyl)-1- methyl-1H-pyrazol-5-yl)benzamido)propanoate
  • 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzoic acid 100 mg, 0.38 mmol
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate 138 mg, 0.38 mmol, HCl salt)
  • TBTU (182 mg, 0.57 mmol
  • DIPEA 147 mg, 1.14 mmol
  • reaction mixture was stirred at 30°C for 2 hours.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • Step 2 methyl 3-(2-amino-4-oxopent-2-en-3-yl)-5-fluorobenzoate
  • MeOH MeOH
  • Raney-Ni 250 mg
  • H23 times The suspension was degassed and purged with H23 times.
  • the mixture was stirred under H2 (1 atm) at 25°C for 6 hours.
  • the mixture was filtered and concentrated to give methyl 3- (2-amino-4-oxopent-2-en-3-yl)-5-fluorobenzoate (530 mg).
  • Step 3 methyl 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoate
  • P2S5 (1.41 g, 6.33 mmol
  • chloranil 519 mg, 2.11 mmol,
  • Step 4 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoic acid To a solution of methyl 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoate (150 mg, 0.57 mmol) in the mixture of THF (8 mL) and H 2 O (4 mL) was added LiOH.H 2 O (36 mg, 0.85 mmol), the mixture was stirred at 25°C for 1 hour.
  • Step 6 (R)-2-amino-3-(3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethylisothiazol-4- yl)-5-fluorobenzamido)propanoate (170 mg, 0.30 mmol) in 30% HBr/HOAc (6 mL) was stirred at 50°C for 15 hours. The mixture was concentrated.
  • Step 2 methyl 3-fluoro-5-(1-methylcyclopropyl)benzoate To a solution of ZnEt2 (1 M in Hexane, 15.45 mL) in DCM (20 mL) was added TFA (0.2 mL) in DCM (5 mL). The mixture was stirred at 0°C for 20 minutes.
  • Step 3 3-fluoro-5-(1-methylcyclopropyl)benzoic acid To a solution of methyl 3-fluoro-5-(1-methylcyclopropyl)benzoate (550 mg, 2.64 mmol) in a mixture of THF (15 mL) and H2O (10 mL) was added LiOH.H2O (554 mg, 13.21 mmol).
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methylcyclopropyl)benzamido)propanoate
  • 3-fluoro-5-(1-methylcyclopropyl)benzoic acid 150 mg, 0.772 mmol
  • DMF 20 mL
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (338 mg, 0.927 mmol, HCl salt)
  • TBTU (372 mg, 1.16 mmol)
  • DIPEA 299 mg, 2.32 mmol
  • Step 2 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid To a solution of methyl 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoate (330 mg, 1.33 mmol) in a mixture of THF (5 mL) and H2O (2.5 mL) was added LiOH.H2O (112 mg, 2.66 mmol) and the mixture was stirred at 30°C for 16 hours.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)- 5-fluorobenzamido)propanoate
  • 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid 300 mg, 1.28 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (514 mg, 1.41 mmol, HCl salt)
  • TBTU (617 mg, 1.92 mmol
  • DIPEA 497 mg, 3.84 mmol
  • Step 1 3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzoic acid
  • xylene xylene
  • 3-amino-5-fluorobenzoic acid 474 mg, 3.06 mmol
  • 4-methylbenzenesulfonic acid 132 mg, 0.76 mmol
  • the reaction mixture was concentrated and the residue was washed thoroughly twice with 4 ml of a 1:1 (v/v) ethyl acetate : ethanol mixture.
  • Step 2 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-4H-1,2,4-triazol-4- yl)-5-fluorobenzamido)propanoate
  • 3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzoic acid 100 mg, 0.43 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 17.1 mg, 0.47 mmol, HCl salt
  • TBTU 205 mg, 0.64 mmol
  • DIPEA 165 mg, 1.28 mmol
  • reaction mixture was stirred at 30°C for 16 hours.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na2SO4 and concentrated.
  • Step 2 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoic acid
  • methyl 3-(3,5-dimethyl-1,2,4-triazol-1-yl)-5-fluoro-benzoate 90 mg, 0.36 mmol
  • LiOH.H 2 O 30 mg, 0.72 mmol
  • the reaction mixture was diluted with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-1,2,4-triazol- 1-yl)-5-fluorobenzamido)propanoate
  • 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoic acid 80 mg, 0.34 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 136 mg, 0.37 mmol, HCl salt
  • TBTU 164 mg, 0.51 mmol
  • DIPEA 132 mg, 1.02 mmol
  • Step 1 methyl 3-(but-1-en-2-yl)-5-fluorobenzoate and (E)-methyl 3-(but-2-en-2-yl)-5- fluorobenzoate
  • 3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 1.0 g, 5.05 mmol
  • 2-bromobut-1-ene 1.02 g, 7.58 mmol
  • K2CO3 2.09 g, 15.15 mmol
  • Pd(dppf)Cl2 554 mg, 0.76 mmol
  • Step 5 (R)-2-amino-3-(3-(1-ethylcyclopropyl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethylcyclopropyl)-5- fluorobenzamido)propanoate 140 mg, 0.27 mmol
  • Pd/C 40 mg, 10% Pd, 50% water
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(diethylamino)-5- fluorobenzamido)propanoate
  • To a mixture of 3-(diethylamino)-5-fluorobenzoic acid (130 mg, 0.62 mmol) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (269 mg, 0.74 mmol) in DMF (8 mL) was added T 3 P (587 mg, 0.92 mmol, 50% in DMF) and DIPEA (239 mg, 1.85 mmol), the mixture was stirred at 30°C for 16 hours.
  • Step 4 (R)-2-amino-3-(3-(diethylamino)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(diethylamino)-5- fluorobenzamido)propanoate (170 mg, 0.33 mmol) in HBr/ HOAc (5 mL, 30% purity)
  • the mixture was stirred at 50°C for 16 hours.
  • the mixture was concentrated.
  • the residue was added MTBE (20 mL) and stirred for 5 hours and filtered to give a yellow solid.
  • Step 1 methyl 3-(3-ethylpyridin-4-yl)-5-fluorobenzoate
  • Step 2 3-(3-ethylpyridin-4-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(3-ethylpyridin-4-yl)-5-fluorobenzoate (210 mg, 0.81 mmol) in a mixture of THF (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (68 mg, 1.62 mmol) and the mixture was stirred at 25°C for 3 hours.
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 1 methyl 3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzoate
  • 2,3-dichloro-5-methylpyridine 200 mg, 1.23 mmol
  • (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid 244 mg, 1.23 mmol
  • Pd(dppf)Cl 2 90 mg, 0.12 mmol
  • K 2 CO 3 512 mg, 3.70 mmol
  • Step 2 3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzoic acid
  • methyl 3-(3-chloropyridin-2-yl)-5-fluorobenzoate 250 mg, 0.89 mmol
  • LiOH.H 2 O 75 mg, 1.79 mmol
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 1 methyl 3-(3-ethylmorpholino)-5-fluorobenzoate
  • a mixture of methyl 3-bromo-5-fluorobenzoate (200 mg, 0.86 mmol), 3- ethylmorpholine (143 mg, 0.94 mmol, HCl salt) ,CPhos-Pd-G3 (69 mg, 0.08 mmol) and Cs2CO3 (839 mg, 2.57 mmol) in dioxane (4 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 90°C for 16 hours under N2 atmosphere.
  • the mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL ⁇ 3).
  • Step 2 3-(3-ethylmorpholino)-5-fluorobenzoic acid
  • a solution of methyl 3-(3-ethylmorpholino)-5-fluorobenzoate (200 mg, 0.75 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (63 mg, 1.50 mmol) and the mixture was stirred at 15°C for 16 hours.
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 4 (2R)-2-amino-3-(3-(3-ethylmorpholino)-5-fluorobenzamido)propanoic acid
  • (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylmorpholino)-5- fluorobenzamido)propanoate 250 mg, 0.44 mmol
  • 30% HBr/HOAc 10 mL
  • Step 1 methyl 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoate To a mixture of methyl 3-bromo-5-fluorobenzoate (200 mg, 0.86 mmol) and 2- ethylpiperidine (117 mg, 1.03 mmol) in dioxane (4 mL) was added Cs 2 CO 3 (839 mg, 2.57 mmol) and CPhos-Pd-G3 (69 mg, 0.086 mmol) in glovebox, the mixture was stirred at 90°C for 16 hours in glovebox. The mixture was concentrated.
  • Step 4 (2R)-2-amino-3-(3-(2-ethylpiperidin-1-yl)-5-fluorobenzamido)propanoic acid
  • (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpiperidin-1-yl)-5- fluorobenzamido)propanoate 100 mg, 0.18 mmol
  • 30% HBr/HOAc 3 mL
  • Step 1 methyl 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-carboxylate
  • 2-bromo-1-chloro-4-methylbenzene 300 mg, 1.46 mmol
  • (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid 289 mg, 1.46 mmol
  • Pd(dppf)Cl2 107 mg, 0.15 mmol
  • K 2 CO 3 (605 mg, 4.38 mmol) in a mixture of dioxane (4 mL) and H 2 O (1 mL) was degassed and purged with N 2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N 2 atmosphere.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-5'-methyl-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
  • TBTU (182 mg, 0.57 mmol
  • DIPEA 146 mg, 1.13 mmol
  • Step 2 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-carboxylic acid
  • a solution of methyl 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-carboxylate (200 mg, 0.72 mmol) in a mixture of THF (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (60 mg, 1.44 mmol) and the mixture was stirred at 25°C for 16 hours.
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-4'-methyl-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
  • TBTU 328 mg, 1.02 mmol
  • DIPEA 264 mg, 2.04 mmol
  • Step 4 (R)-2-amino-3-(2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-4'-methyl- [1,1'-biphenyl]-3-ylcarboxamido)propanoate 200 mg, 0.35 mmol
  • 30% HBr/HOAc 5 mL
  • Step 2 3-(4-ethylpyridin-3-yl)-5-fluorobenzoic acid
  • MeOH MeOH
  • H2O 6 mL
  • LiOH.H2O 65 mg, 1.54 mmol
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate
  • TBTU 216 mg, 0.67 mmol
  • DIPEA 174 mg, 1.35 mmol
  • Step 4 (R)-2-amino-3-(3-(4-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate 190 mg, 0.34 mmol
  • 30% HBr/ HOAc 5 mL
  • Step 2 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoate (300 mg, 1.15 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (73 mg, 1.73 mmol), the mixture was stirred at 25°C for 1 hour.
  • the precipitated solids was filtered to give 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoic acid (270 mg).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylpyrimidin-4-yl)-5- fluorobenzamido)propanoate
  • TBTU 528 mg, 1.64 mmol
  • DIPEA 425 mg, 3.29 mmol
  • Step 2 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid To a solution of methyl 3-(2-ethylpyridin-3-yl)-5-fluorobenzoate (200 mg, 0.77 mmol) in a mixture of H2O (2.5 mL) and MeOH (2.5 mL) was added LiOH.H2O (97 mg, 2.31 mmol). The mixture was stirred at 25°C for 3 hours.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate
  • 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid 280 mg, crude
  • (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (262 mg, 0.72 mmol, HCl)
  • HATU 410 mg, 1.08 mmol
  • DIPEA 279 mg, 2.16 mmol
  • Step 4 (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate 100 mg, 0.18 mmol
  • 30% HBr/HOAc 3 mL
  • Step 2 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid To a solution of methyl 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoate (290 mg, 1.11 mmol) in the mixture of MeOH (6 mL) and H 2 O (6 mL) was added LiOH.H 2 O (94 mg, 2.23 mmol). The mixture was stirred at 25°C for 1 hour. The mixture was concentrated.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate
  • 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid 150 mg, 0.61 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (267 mg, 0.73 mmol, HCl salt)
  • TBTU (293 mg, 0.91 mmol
  • DIPEA 236 mg, 1.83 mmol
  • Step 4 (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate 240 mg, 0.43 mmol
  • 30% HBr/HOAc 5 mL
  • Step 2 3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzoic acid
  • MeOH MeOH
  • THF THF
  • H2O 5 mL
  • LiOH.H2O 54 mg, 1.29 mmol
  • Step 4 (R)-2-amino-3-(3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-chloro-6-methylpyridin- 2-yl)-5-fluorobenzamido)propanoate 120 mg, 0.21 mmol
  • 30% HBr/HOAc 3 HBr/HOAc
  • Step 4 3-(2-ethylcyclohexyl)-5-fluorobenzoic acid
  • a solution of methyl 3-(2-ethylcyclohexyl)-5-fluorobenzoate (860 mg, 3.25 mmol) in a mixture of MeOH (10 mL) and H 2 O (5 mL) was added LiOH.H 2 O (410 mg, 9.76 mmol).
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was concentrated.
  • the residue was diluted with water (30 mL) and acidified with aq.
  • Step 5 (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate
  • TBTU 1-(2-ethylcyclohexyl)-5-fluorobenzoic acid
  • DIPEA 1,3-bis(trimethyl)-2-((benzyloxy)carbonyl)amino)propanoate
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL ⁇ 3). The combined organic layers were washed with brine (30 mL ⁇ 2), dried over Na2SO4, filtered and concentrated.
  • Step 2 ethyl 3-fluoro-5-(1-hydroxycyclobutyl)benzoate To a solution of ethyl 3-fluoro-5-iodobenzoate (1 g, 3.40 mmol) in THF (20 mL) was added i-PrMgCl-LiCl (1.3 M in THF, 3.66 mL, 1.4 eq) dropwise at -70 °C. The mixture was stirred at -70 °C for 30 min then cyclobutanone (238 mg, 3.40 mmol) was added. The mixture was warmed to 20 °C for 1 hour.
  • i-PrMgCl-LiCl 1.3 M in THF, 3.66 mL, 1.4 eq
  • Step 3 ethyl 3-fluoro-5-(1-methoxycyclobutyl)benzoate To a solution of ethyl 3-fluoro-5-(1-hydroxycyclobutyl)benzoate (300 mg, 1.26 mmol) in DMF (5 mL) was added NaH (76 mg, 1.89 mmol, 60% in mineral oil) at 0 °C and the mixture was stirred at 0 °C for 30 min. Then MeI (357 mg, 2.52 mmol) was added and the mixture was stirred at 60 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 4 3-fluoro-5-(1-methoxycyclobutyl)benzoic acid
  • ethyl 3-fluoro-5-(1-methoxycyclobutyl)benzoate 250 mg, crude
  • MeOH MeOH
  • H2O 5 mL
  • LiOH.H2O 125mg, 2.97 mmol
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was concentrated to remove MeOH.
  • the residue was diluted with water (10 mL) and acidified with 2N HCl to adjust pH to 4 and extracted with ethyl acetate (15 mLx3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoate
  • TBTU 430 mg, 1.34 mmol
  • DIPEA 346 mg, 2.68 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (325 mg, 0.89 mmol, HCl salt).
  • Step 6 (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclobutyl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoate 120 mg, 0.22 mmol
  • Pd/C 50 mg, 10% Pd, 50% water
  • Step 2 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoic acid To a solution of methyl 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoate (180 mg, 0.69 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (58 mg, 1.38 mmol), the mixture was stirred at 25°C for 1 hour. The mixture was concentrated.
  • Step 4 (R)-2-amino-3-(3-(4-ethylpyrimidin-5-yl)-5-fluorobenzamido)propanoic acid
  • HOAc 3 mL
  • Pd/C 10% Pd, 50% water, 20 mg
  • Step 2 methyl 3-fluoro-5-(1-hydroxycyclopentyl)benzoate
  • i-PrMgCl-LiCl 1.3 M in THF, 1.51 mL
  • cyclopentanone 150 mg, 1.79 mmol
  • THF 3 mL
  • Step 3 methyl 3-fluoro-5-(1-methoxycyclopentyl)benzoate
  • DMF dimethyl methoxycyclopentyl
  • NaH 53 mg, 1.32 mmol, 60% in mineral oil
  • MeI 250 mg, 1.76 mmol
  • the mixture was quenched with water (15 mL) and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 4 3-fluoro-5-(1-methoxycyclopentyl)benzoic acid
  • a solution of methyl 3-fluoro-5-(1-methoxycyclopentyl)benzoate (100 mg, 0.40 mmol) in a mixture of MeOH (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (50 mg, 1.19 mmol).
  • the mixture was stirred at 50 °C for 3 hours.
  • the mixture was concentrated to remove MeOH.
  • the residue was dissolved in water (8 mL) and washed with ethyl acetate (8 mL ⁇ 2). The organic layer was discarded.
  • aqueous layer was acidified with aqueous 2N HCl solution to adjust pH to 4 and extracted wth ethyl acetate (15 mL ⁇ 3).
  • the combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(1-methoxycyclopentyl)benzoic acid (60 mg, crude).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoate
  • TBTU 121 mg, 0.38 mmol
  • DIPEA 98 mg, 0.76 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (92 mg, 0.25 mmol, HCl salt).
  • the mixture was stirred at 25 °C for 2.5 hours.
  • Step 6 (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclopentyl)benzamido)propanoic acid and (R)-2-amino-3-(3-cyclopentyl-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoat 70 mg, 0.13 mmol
  • Pd/C 40 mg, 10% Pd, 50% water
  • Step 3 methyl 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoate 4-bromo-5-ethylisothiazole (160 mg, 0.40 mmol), (4-fluoro-3- (methoxycarbonyl)phenyl)boronic acid (159 mg, 0.8 mmol), Pd(dppf)Cl2.CH2Cl2 (66 mg, 0.08 mmol) and Na2CO3 (128 mg, 1.20 mmol) were taken up into a microwave tube in a mixture of dioxane (5 mL) and H2O (0.5 mL). The sealed tube was heated at 120 °C for 40 min under microwave.
  • Step 4 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoic acid
  • methyl 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoate 100 mg, 0.28 mmol
  • MeOH MeOH
  • H2O 2 mL
  • LiOH.H2O 35 mg, 0.83 mmol
  • the mixture was stirred at 35 °C for 1.5 hour.
  • the mixture was concentrated.
  • the residue was dissolved in water (10 mL) and acidified with 2N HCl solution to adjust pH to 4 and extracted with ethyl acetate (20 mL ⁇ 4).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-(5-ethylisothiazol-4-yl)-2- fluorobenzamido)propanoate
  • TBTU 125 mg, 0.39 mmol
  • DIPEA 101 mg, 0.78 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (95 mg, 0.26 mmol, HCl salt).
  • Step 6 (R)-2-amino-3-(5-(5-ethylisothiazol-4-yl)-2-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-(5-ethylisothiazol-4-yl)-2- fluorobenzamido)propanoate (50 mg, 0.089 mmol) in 30% HBr/HOAc (4 mL) was stirred at 50 °C for 3 hours. The mixture was concentrated.
  • Step 2 ethyl 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoate
  • DMF dimethyl methoxyethyl
  • NaH sodium sulfate
  • iodomethane 236 mg, 1.67 mmol
  • Step 3 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoic acid
  • MeOH MeOH
  • H2O 5 mL
  • LiOH.H2O 50 mg, 1.18 mmol
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(3-methoxyoxetan-3- yl)benzamido)propanoate
  • TBTU 2,3-fluoro-5-(3-methoxyoxetan-3-yl)benzoic acid
  • DIPEA DIPEA
  • Step 5 (R)-2-amino-3-(3-fluoro-5-(3-methoxyoxetan-3-yl)benzamido)propanoic acid
  • HOAc 4 mL
  • Pd/C 10% Pd, 50% water, 30 mg
  • Step 2 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
  • methyl 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate 500 mg, 1.91 mmol
  • H 2 O 3 mL
  • MeOH 3 mL
  • LiOH.H 2 O 240 mg, 5.72 mmol
  • Step 3 (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-3-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
  • 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid 500 mg, 1.76 mmol, HCl salt, crude
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (641 mg, 1.76 mmol, HCl salt) in DMF (10 mL) was added TBTU (677 mg, 2.11 mmol) and DIPEA (1.13 g, 8.78 mmol).
  • Step 2 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-5-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
  • 2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid 150 mg, 526.85 ⁇ mol, HCl salt, crude
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (198 mg, 543.83 ⁇ mol, HCl salt)
  • TBTU 291 mg, 906.34 ⁇ mol
  • DIPEA 234 mg, 1.81 mmol
  • Step 2 4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
  • Step 4 (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate 350 mg, 0.73 mmol
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate 350 mg, 0.73 mmol
  • the mixture was concentrated.
  • the residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-4- fluorobenzamido)propanoic acid (110 mg) as HCl salt.
  • Step 2 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H- pyrazole
  • NBS 3.55 g, 19.97 mmol
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • Step 4 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid To a solution of methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate (240 mg, 0.98 mmol) in a mixture of THF (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (82 mg, 1.96 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate
  • 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid (220 mg, 0.96 mmol)
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (383 mg, 1.05 mmol, HCl salt)
  • TBTU 460 mg, 1.43 mmol
  • DIPEA 370 mg, 2.87 mmol
  • Step 6 (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H- pyrazol-5-yl)benzamido)propanoate (380 mg, 0.70 mmol) in the solution of 30 % HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated.
  • Step 2 3-ethyl-5-fluoro-4-methylbenzoic acid To a solution of methyl 3-ethyl-5-fluoro-4-methylbenzoate (220 mg, 1.12 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (141 mg, 3.36 mmol) and the mixture was stirred at 20°C for 16 hours, then the reaction mixture was stirred at 35°C for another 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate
  • TBTU 264 mg, 0.82 mmol
  • DIPEA 213 mg, 1.65 mmol
  • Step 4 (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate 230 mg, 0.47 mmol
  • 30% HBr in AcOH 10 mL
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate
  • TBTU (344 mg, 1.07 mmol)
  • DIPEA 277 mg, 2.14 mmol
  • Step 4 (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate 120 mg, 0.25 mmol
  • HBr/HOAc 4 mL, 30%
  • the mixture was concentrated.
  • the residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(4-ethyl-3- fluorobenzamido)propanoic acid (50 mg) as HCl salt.
  • Step 2 methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate To a solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (180 mg, 0.73 mmol) in THF (10 mL) was added NCS (97 mg, 0.73 mmol), the mixture was stirred at 70 °C for 1 hour. The mixture was concentrated.
  • Step 3 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate 240 mg, 0.85 mmol
  • MeOH MeOH
  • H2O 5 mL
  • LiOH.H2O 53 mg, 1.27 mmol
  • Step 5 (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate 150 mg, 0.26 mmol
  • HBr/HOAc 4 mL, 30%
  • the mixture was concentrated.
  • the residue was added HOAc (3 mL), then added MTBE (5 mL).
  • Step 2 (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate and (R)-ethyl 3-fluoro-5-(1- hydroxyethyl)benzoate
  • ethyl 3-fluoro-5-iodobenzoate 5.2 g, 17.68 mmol
  • i-PrMgCl-LiCl 1.3 M in THF, 19.04 mL
  • the reaction mixture was quenched with saturated solution NH 4 Cl (20 mL) and concentrated.
  • the residue was diluted with water (50 mL) and extracted with ethyl acetate (50 mL ⁇ 3). The combined organic layers were dried over Na2SO4, filtered and concentrated.
  • the residue was further separated by SFC (Instrument: Thar200; Column: DAICEL CHIRALPAK AD 250 ⁇ 50 mm I.D.
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
  • (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid 200 mg, 0.942 mmol
  • DMF 10 mL
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
  • (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid 200 mg, 0.942 mmol
  • DMF 10 mL
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol).
  • the sealed tube was heated at 120 °C for 60 min under microwave.
  • the mixture was diluted with water (10 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.
  • the residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate (160 mg, crude).
  • Step 2 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid
  • methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate 160 mg, crude
  • MeOH MeOH
  • H2O 2 mL
  • LiOH.H2O 37 mg, 0.88 mmol
  • the mixture was stirred at 15 °C for 15 hours.
  • the mixture was concentrated.
  • the residue was dissolved in water (15 mL) and extracted with ethyl acetate (20 mL ⁇ 2). The organic layer was discarded.
  • Step 4 (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate 40 mg, 0.07 mmol
  • 30% HBr in AcOH (4 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated.
  • Step 2 methyl 3-fluoro-5-(5-methylisoxazol-4-yl)benzoate 4-bromo-5-methylisoxazole (1.0 g, 6.17 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.22 g, 6.17 mmol), Pd(t-Bu 3 P) 2 (316 mg, 0.62 mmol) and KF (897 mg, 15.43 mmol) were taken up into a microwave tube in a mixture of dioxane (12 mL) and H 2 O (1.2 mL) under N 2 atmosphere. The sealed tube was heated at 100 °C for 60 min under microwave.
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-methylisoxazol-4- yl)benzamido)propanoate
  • TBTU 218 mg, 0.68 mmol
  • DIPEA 146 mg, 1.13 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (165 mg, 0.45 mmol, HCl salt).
  • Step 5 (R)-2-amino-3-(3-fluoro-5-(5-methylisoxazol-4-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-methylisoxazol- 4-yl)benzamido)propanoate 160 mg, 0.30 mmol
  • 30% HBr in AcOH 5 mL
  • Step 2 methyl 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoate
  • 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid 70 mg, crude
  • (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 131 mg, 358.6 ⁇ mol, HCl salt
  • TBTU 144 mg, 448.3 ⁇ mol
  • DIPEA 116 mg, 896.6 ⁇ mol
  • NBS 512 mg, 2.88 mmol, 1.2 eq
  • the mixture was stirred at 15 °C for 16 hours.
  • the mixture was then stirred at 50 °C for 20 hours.
  • additional NBS 250 mg was added and the mixture was stirred at 50°C for 4 hours.
  • the mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 2), dried over Na2SO4, filtered and concentrated below 30 °C.
  • Step 2 methyl 3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzoate 4-bromo-5-(tert-butyl)isoxazole (400 mg, 1.96 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (388 mg, 1.96 mmol), Pd(t-Bu3P)2 (100 mg, 0.20 mmol) and KF (285 mg, 4.90 mmol) were taken up into a microwave tube in a mixture of dioxane (6 mL) and H2O (0.6 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 min under microwave.
  • Step 5 (R)-2-amino-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-(tert-butyl)isoxazol-4- yl)-5-fluorobenzamido)propanoate 180 mg, 0.31 mmol
  • 30% HBr in AcOH 5 mL
  • the mixture was concentrated.
  • the residue was added water (4 mL) and washed with MTBE (4 mL ⁇ 2).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate
  • 3-(5-ethylisoxazol-4-yl)benzoic acid (135 mg, 621.5 ⁇ mol)
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (227 mg, 621.5 ⁇ mol, HCl salt) was added TBTU (299 mg, 932.2 ⁇ mol) and DIPEA (24.0 mg, 1.86 mmol).
  • Step 4 (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate 130 mg, 246.4 ⁇ mol
  • 30% HBr in AcOH 5 mL
  • the reaction mixture was concentrated, the residue was dissolved in water (5 mL), washed with MTBE(5 mL).
  • Step 2 Methyl 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate 5-bromo-1-ethyl-1H-1,2,3-triazole (480 mg, 2.73 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (648 mg, 3.27 mmol), Pd(dppf)Cl2.CH2Cl2 (223 mg, 0.27 mmol) and K2CO3 (1.13 g, 8.18 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL). The sealed tube was heated at 100 °C for 60 min under microwave.
  • Step 3 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid
  • MeOH MeOH
  • LiOH.H2O LiOH.H2O
  • H2O H2O
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was concentrated.
  • the residue was added water (5 mL) and acidified with 2N HCl to adjust pH to 6 ⁇ 7 and extracted with ethyl acetate (20 mL ⁇ 3).
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate
  • TBTU 246 mg, 0.76 mmol
  • DIPEA 198 mg, 1.53 mmol
  • R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (186 mg, 0.51 mmol, HCl salt).
  • Step 5 (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5- yl)-5-fluorobenzamido)propanoate 70 mg, 0.13 mmol
  • 30% HBr in AcOH 5 mL
  • the mixture was concentrated.
  • the residue was added water (5 mL) and washed with MTBE (5 mL ⁇ 2).
  • Step 6 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropylisoxazol-4- yl)benzamido)propanoate
  • 3-fluoro-5-(5-isopropylisoxazol-4-yl)benzoic acid 270 mg, 1.08 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (435 mg, 1.19 mmol, HCl salt)
  • TBTU 522 mg, 1.62 mmol
  • DIPEA 420 mg, 3.25 mmol
  • Step 7 (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid
  • (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid (320 mg, 0.57 mmol) in 30 % HBr in AcOH (10 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was washed with a mixture of MeCN (1 mL) and MTBE (5 mL).
  • Step 3 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate (80 mg, 0.32 mmol) in a mixture of MeOH (4 mL) and H 2 O (1 mL) was added LiOH.H 2 O (41 mg, 0.97 mmol).
  • the mixture was stirred at 20 °C for 14 hours.
  • the mixture was concentrated.
  • the residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoate
  • TBTU 99 mg, 0.31 mmol
  • DIPEA 80 mg, 0.62 mmol
  • R R-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (125 mg, 0.34 mmol, HCl salt).
  • Step 3 cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3- yl)-5-fluorobenzamido)propanoate
  • TBTU 303 mg, 0.94 mmol
  • DIPEA 244 mg, 1.89 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (230 mg, 0.63 mmol, HCl salt).
  • Step 2 methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate A mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 3- bromo-4-iodoisothiazole (1.46 g, 5.05 mmol), Pd(dppf)Cl 2 (370 mg, 0.51 mmol) and K 2 CO 3 (2.09 g, 15.15 mmol) in a mixture of dioxane (20 mL) and H 2 O (2 mL) was degassed and purged with N 2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere.
  • Step 3 methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate
  • Step 4 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate (110 mg, 0.41 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (52 mg, 1.24 mmol) and the mixture was stirred at 10°C for 16 hours.
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
  • TBTU 86 mg, 0.27 mmol
  • DIPEA 69 mg, 0.54 mmol
  • Step 2 methyl 3-(4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoate
  • a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 53 mg, 0.27 mmol
  • 4,5- dibromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole 95 mg, 0.27 mmol
  • Pd(PPh 3 ) 4 31 mg, 0.03 mmol
  • Na 2 CO 3 85 mg, 0.80 mmol
  • the sealed tube was heated at 110°C for 30 minutes under microwave.
  • the mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL ⁇ 3).
  • the combined organic layers were washed with brine (20 mL ⁇ 3) and dried over Na 2 SO 4 , filtered and concentrated.
  • the residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoate (100 mg).
  • Step 3 methyl 3-fluoro-5-(1-((2-(trimethylsilyl)ethoxy)methyl)-4-vinyl-1H-imidazol-5- yl)benzoate
  • the reaction mixture was diluted with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • the combined organic layers were washed with brine (10 mL ⁇ 3), dried over Na2SO4 and concentrated to give 3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5-fluorobenzoic acid (24 mg).
  • Step 6 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate
  • Step 2 methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate
  • 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate 550 mg, 1.97 mmol
  • triethylborane 1 M in THF, 5.9 mL
  • Pd(dppf)Cl 2 144 mg, 196.64 ⁇ mol
  • CsOAc (1.13 g, 5.90 mmol) in THF (7 mL) was degassed and purged with N23 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was concentrated.
  • Step 3 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid
  • methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate 430 mg, 1.57 mmol
  • MeOH 3 mL
  • LiOH.H2O 198 mg, 4.72 mmol
  • the mixture was stirred at 10 °C for 16 hours.
  • the mixture was concentrated to remove MeOH.
  • Step 5 (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin- 3-yl)-5-fluorobenzamido)propanoate 140 mg, 245.78 ⁇ mol
  • 30% HBr in AcOH (3 mL) was stirred at 50 °C for 16 hours.
  • the mixture was concentrated.
  • the residue was dissolved in MeCN (3 mL) and MTBE (10 mL), and the solid was collected and lyophilizated.
  • Step 2 methyl 3-fluoro-5-(4-(trifluoromethyl)-2-vinylpyridin-3-yl)benzoate
  • a mixture of methyl 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (230 mg, 621.4 ⁇ mol, HCl salt), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (191 mg, 1.24 mmol), Pd(dppf)Cl 2 (45 mg, 62.1 umol), K 2 CO 3 (344 mg, 2.49 mmol)
  • Step 2 4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole
  • a mixture of 4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (500 mg, 1.80 mmol), triethylborane (5.40 mL, 1 M in THF), Pd(dppf)Cl 2 (132 mg, 0.18 mmol) and CsOAc (1.04 g, 5.40 mmol) in THF (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere.
  • Step 5 methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5- fluorobenzoate
  • Step 6 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)- 5-fluorobenzoate (280 mg, 0.74 mmol) in a mixture of THF (3 mL) and H 2 O (1 mL) was added LiOH.H2O (78 mg, 1.85 mmol) and the mixture was stirred at 20°C for 18 hours.
  • Step 7 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
  • a solution of a mixture of 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol- 5-yl)-5-fluorobenzoic acid 260 mg, 0.71 mmol
  • (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (286 mg, 0.78 mmol, HCl salt) in DMF (8 mL) was added TBTU (344 mg, 1.07 mmol) and DIPEA (277 mg, 2.14 mmol).
  • the reaction mixture was stirred at 20°C for 1 hour.
  • the mixture was added water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3).
  • the combined organic layers were washed with saturated brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • Step 8 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (420 mg, 0.62 mmol) in TFA (15 mL) was stirred at 20°C for 16 hours.

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Abstract

The present invention is directed to novel modulators of the NMDA receptor. Separate aspects of the inventions are directed to pharmaceutical compositions comprising said compounds and uses of the compounds to treat neurological disorders or neuropsychiatric disorders such as depression.

Description

NOVEL MODULATORS OF THE NMDA RECEPTOR
FIELD OF THE INVENTION
The present invention is directed to compounds which are modulators of the NMDA receptor, pharmaceutical compositions comprising the compounds, and their use in treatment of neurological disorders or neuropsychiatric disorders such as depression, in particular major depressive disorder (MDD) and treatment-resistant depression (TRD).
BACKGROUND OF THE INVENTION
The World Health Organization estimates 350 million people will be affected with Major Depressive Disorder (MDD) and has projected that depression will constitute the largest health burden on society worldwide by 2030. A rough working estimate of prevalence is that depression affects l/5th of the population at some point, affecting women in a higher proportion than men (5-9% and 2-3% incidence respectively in the US, representing an overall incidence of 6.6%). The North-American Center for Disease Control has reported that from 2005-2008, 8.9% of the US population was prescribed an antidepressant during any given month, antidepressants being also prescribed for anxiety, pain, and other non-mood disorders [Murray et al., Global Burden of Disease Study. Lancet. May 17, 1997; 349(9063): 1436-1442],
Antidepressants are marketed and thus known to the skilled person. Some examples of different types of antidepressant are selective serotonin reuptake inhibitors (SSRIs), Serotonin-norepinephrine reuptake inhibitors (SNRIs), Monoamine oxidase inhibitors (MAOIs), and Tricyclic antidepressants. Typical limitation of known antidepressants are delayed onset of efficacy and low remission rates after multiple courses of pharmacotherapy, and for some antidepressants severe side-effects [Jick et al., Antidepressants and the risk of suicidal behaviors. Jama. Jul. 21 2004; 292(3 ) :338-343] . In recent years, modulators of the N-Methyl-D-Aspartate (NMDA) receptors have received more attraction in treatment of MDD, in particular for treatment resistant depression (TRD). Especially, ketamine, a pore-blocking antagonist of the NMDA receptor, is used for treating MDD due to its antidepressant effect and fast onset. However, MDD treatment with ketamine has the drawbacks of sedation, psychotomimetic side effects and requirement of intravenous or intranasal administration.
NMDA receptors are tetrameric ligand-gated ion channels which are also involved in essential physiological processes such as synaptic plasticity and development. NMDA receptors are heterotetramers comprising two GluNl subunits and two GluN2/GluN3 subunits. This means that they assemble as either diheteromeric or triheteromeric receptors. The majority of native NMDA receptors consist of two GluNl subunits and two GluN2 subunits. Activation of the NMDA receptors requires simultaneous binding at two different binding sites. Glutamate, the major excitatory neurotransmitter in the central nervous system, binds to the GluN2 subunits and glycine binds to the GluNl and GluN3 subunits.
Another known modulator of the NMDA receptor is D-cycloserine, which is a partial glycine site agonist. D-cycloserine has been intensively studied due to its neuroactive properties and potential utility in treatment of depression and depression disorders such as MDD [Heresco-Levy, U., Javitt, D.C., Gelfin, Y., Gorelik, E., Bar, M., Blanaru, M., Kremer, I., 2006. Controlled trial of d-cycloserine adjuvant therapy for treatmentresistant major depressive disorder. J. Affect. Disord. 93, 239-243] and PTSD [Olden, M., Wyka, K., Cukor, J., Peskin, M., Altemus, M., Lee, F.S., Finkelstein-Fox, L., Rabinowitz, T., Difede, J., 2017. Pilot study of a telehealth-delivered medication augmented exposure therapy protocol for PTSD. J. Nerv. Ment. Dis. 205, 154-160], However, treatment of D- cycloserine suffers from frequent complaints of psychopathological stimulation such as anxiety, euphoria, agitation, feeling stimulated, dizziness/drowsiness, fatigue, headache, and gastrointestinal disturbance [Schade, S., Paulus, W., 2016. D-Cycloserine in neuropsychiatric diseases: a systematic review. Int. J. Neuropsychopharmacol]. Urwyler et al., J. Med. Chem. 2009, 52, 5093–5107 discloses 3-acylamino-2- amonipropionic acid derivatives with affinity for the glycine site of the NMDA receptor. Maolanon et. al., ACS Chem. Neurosci. 2017, 8, 1681-1687 discloses Subtype-Specific Agonists for NMDA Receptor Glycine Binding Sites. Despite the longstanding interest in the field, there is evidently still an unmet need as regards developing efficient, well tolerated and active drugs for the treatment of depression and depression disorders such as MDD. A compound which is a modulator of the NMDA receptor with affinity for the glycine site may fulfil such unmet needs. SUMMARY OF THE INVENTION With this background, it is an object of the invention to provide compounds which are modulators of the NMDA receptor with affinity to the glycine site. In a first aspect of the invention is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof wherein
Figure imgf000005_0001
R1 is selected from the group consisting of hydrogen and halogen; R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1- C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and - C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1- C4)alkoxy; or R1 and R2, or R2 and R3, together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaromatic ring is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl, and wherein said 5- or 6-membered heterocyclic ring is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl and oxo; with the proviso that at least one of R1, R2, R3 and R4 does not represent hydrogen; with the proviso that when R2 represents halogen or methyl, at least one of R1, R3 and R4 does not represent hydrogen; with the proviso that when R3 represents halogen or methyl, at least one of R1, R2 and R4 does not represent hydrogen; with the proviso that when R4 represents halogen or methyl, at least one of R1, R2 and R3 does not represent hydrogen; and with the proviso that when R3 represents methyl, R2 or R4 does not represent 3- trifluoromethylphenyl. In a further aspect is provided a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or diluents. In a further aspect is provided a method for the treatment of depression comprising the administration of a therapeutically effective amount of a compound of formula I, or acceptable salt thereof, or a pharmaceutical composition to a patient in need thereof.
In a further aspect is provided a compound of formula I, or a pharmaceutically acceptable salt thereof for use as a medicament comprising a compound of formula I, or a pharmaceutically acceptable salt thereof.
In a further aspect is provided a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of depression.
In a further aspect is provided a use of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression.
These and other aspects of the invention will become apparent upon reference to the following detailed description. It should be understood that the various aspects, embodiments, implementations and features of the invention mentioned herein may be claimed separately, or in any combination.
All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety.
Headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Effects of compound 3f in Resting state Electroencephalography (rsEEG) obtained in the Nucleus accumbens according to Example 3.
Y-axis: Baseline-normalized power (dB); X-axis: bars from left to right: 10% HPbetaCD (vehicle), 3 mg/kg compound 3f, 10 mg/kg compound 3f, and 30 mg/kg compound 3f.
Significance level for post-hoc comparison (relative to the vehicle group) is indicated: ***<0.001.
Figure 2: Effects of compound 3f in rat maximal electroshock threshold model (MEST) according to Example 4.
Y-axis: Estimated seizure threshold (CC50) current (mA); X-axis: bar to the left: 10% HPbetaCD (vehicle); bar to the right: 10 mg/kg compound 3f.
Significance level for post-hoc comparison (relative to the vehicle group) is indicated: ***<0.05.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have identified new compounds that has affinity for the glycine site of the NMDA receptor as seen in Table 2.
Further, the inventors observed that the administration of compound 3f dosed at 10 and 30 mg/kg showed significant effects as compared to vehicle in rsEEG as shown in Figure 1.
In addition, the inventors observed that administration of compound 3f dosed at 10 mg/kg showed significant effects as compared to vehicle in MEST as shown in Figure 2.
Definitions
As used herein, the term "(Cx-Cy) alkyl" refers to a hydrocarbyl radical obtained when one hydrogen atom is removed from a linear (i.e. unbranched) or branched saturated hydrocarbon. The number of carbon atoms in the hydrocarbyl substituent (i.e. alkyl) is indicated by the prefix “(Cx-Cy)”, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent. Said ”(Cx-Cy) alkyl” comprises 1-6, preferably 1-4, more preferably 1-3, such as 1-2 or such as 2-3 carbon atoms. Thus, for example, “(C1-C6) alkyl” refers to an alkyl substituent containing from 1 to 6 carbon atoms. Examples of alkyl groups include the subclasses normal alkyl (n-alkyl), secondary and tertiary alkyl. Thus “(C1-C6) alkyl” includes groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl, tert. -butyl, n-pentyl, isopentyl, neopentyl, n- hexyl and isohexyl. The term “alkylene,” as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms, for example, of 1 to 4 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2-, - CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, and - CH2CH2CH2CH2CH2-. As used herein, the term “(Cx-Cy) alkoxy” refers to a moiety of the formula –OR’, wherein R’ indicates (Cx-Cy)alkyl as defined above. Thus, for example “(C1-C4)alkoxy” refers to such moiety wherein the alkyl contains from one to four carbon atoms. Examples of “(C1- C4)alkoxy” include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy and tert- butoxy. As used herein, the term “phenoxy” refers refers to a moiety of the formula –O-phenyl, i.e. to a phenyl which is appended to the parent moiety via an oxygen linker. As used in the context of the present invention, the term “halogen” indicates a substituent selected from the 7th main group of the periodic table, such as fluoro, chloro, bromo and iodo (F, Cl, Br and I). As used herein, the term “cyano”, refers to a CN group appended to the parent molecule through the carbon atom of the CN group. The term "halo(Cx-Cy)alkyl" as used herein, means a (Cx-Cy)a I ky I group as defined herein, which is substituted with one or more halogen as defined herein, e.g. fluoro, chloro or bromo. Examples include, but are not limited to, trifluormethyl and difluoromethyl.
The term "hydroxyl" or "hydroxy," as used herein, means an -OH group.
The term "(Cz-Cw)alkoxy(Cx-Cy)alkyl", as used herein refers to an (Cz-Cw)alkoxy group, as defined herein, appended to the parent molecular moiety through an (Cx-Cy)a I ky I group, as defined herein. Examples include, but are not limited to, methoxymethyl and ethoxymethyl.
The term "phenyl" is intended to mean a benzene radical, with one H removed at the attachment point.
The term "hydroxy(Cx-Cy)alkyl" or "hydroxyl(Cx-Cy)alkyl" as used herein, means a (Cx- Cy)alkyl group as defined herein, which is substituted with one or more -OH group(s). Examples include, but are not limited to hydroxymethyl, hydroxyethyl, hydroxypropyl
The term "oxo" as used herein means an oxygen atom which is connected to the parent moiety via a double bond (=0).
The group C(O) is intended to represent a carbonyl group (C=O).
The term "heteroaryl," as used herein, refers to a radical of a heteroaromatic ring. As used herein the term "5- or 6-membered heteroaryl" means a radical of a five or six membered heteroaromatic monocyclic ring containing at least one heteroatom independently selected from the group consisting of N, O and S, such as containing 1 to 4 heteroatoms independently selected from O, S, and N, such as 1 to 3 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O and N. The five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. The heteroaryl radical may be connected to the parent moiety through a carbon atom or a nitrogen atom contained anywhere within the heteroaryl group. Representative examples of 5- or 6-membered heteroaryl include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl.
As used herein, the term '5- or 6-membered heteroaromatic rings comprising 1 or 2 nitrogen' comprises, but is not limited to, pyrrol, pyrazol, imidazole, pyridine, pyrimidin, pyridazine and pyrazine.
The term "heterocyclyl" as used herein, means a radical of an aliphatic heterocycle. The term '4 - 6 membered heterocyclyl' as used herein, means a radical of a four-, five- or six-membered monocyclic ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3- dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetra hydropyranyl.
As used herein, the term '5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen' comprises, but is not limited to, pyrrolidine, pyrazoline, piperidine, tetrahydropyrimidine dihydropyridine, imidazoline, imidazolidine.
The term "(Cx-Cy)cycloalkyl," as used herein, refers to a saturated cycloalkane hydrocarbon radical comprising from x to y carbon atoms, such as from 3-6 carbon atoms, such as 3-5 or such as 3-4 carbon atoms. The cycloalkyl may be monocyclic or bicyclic, wherein the two rings are bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
If substituents are described as being independently selected from a group, each substituent is selected independent of the other. Each substituent may therefore be identical or different from the other substituent(s).
The term "optionally substituted" means "unsubstituted or substituted", and therefore the general formulas described herein encompasses compounds containing the specified optional substituent(s) as well as compounds that do not contain the optional substituent(s).
For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the phrase "the compound" is to be understood as referring to various "compounds" of the invention or particular described aspect, unless otherwise indicated.
The description herein of any aspect or aspect of the invention using terms such as "comprising", "having," "including," or "containing" with reference to an element or elements is intended to provide support for a similar aspect or aspect of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
In the present context, the term "a modulator of the NMDA receptor with affinity to the glycine site" is a compound which modulates through binding the orthosteric glycine binding site on the NMDA receptor so as to increase or decrease the flux of Ca + + through the ligand-gated channel.
In the present context, the term "therapeutically effective amount" of a compound is intended to indicate an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease (e.g. depression) and its complications in a therapeutic intervention comprising the administration of said compound. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, e.g. by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician.
In the present context, the term "treatment" and "treating" means the management and care of a patient for the purpose of combating a disease. The term is intended to include the full spectrum of treatments for a given disease (e.g. depression) from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the depression, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the depression. The patient to be treated is preferably a mammal, in particular a human being. In the present context, "disease" can be used synonymous with disorder, condition, malfunction, dysfunction and the like.
Embodiments of the invention
In the following, embodiments of the invention are disclosed. In an embodiments, the invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof
Figure imgf000014_0001
wherein R1 is selected from the group consisting of hydrogen and halogen; R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1- C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and - C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1- C4)alkoxy; with the proviso that at least one of R1, R2, R3 and R4 does not represent hydrogen; with the proviso that when R2 represents halogen or methyl, at least one of R1, R3 and R4 does not represent hydrogen; with the proviso that when R3 represents halogen or methyl, at least one of R1, R2 and R4 does not represent hydrogen; with the proviso that when R4 represents halogen or methyl, at least one of R1, R2 and R3 does not represent hydrogen; and with the proviso that when R3 represents methyl, R2 or R4 does not represent 3- trifluoromethylphenyl. In a further aspect the invention provides a compound according to the above embodiment, or a pharmaceutically acceptable salt thereof, wherein R2 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; and wherein R4 is selected from the group consisting of hydrogen, halogen and (C1- C6)alkyl. In a further aspect the invention provides a compound according to the above embodiment, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; and wherein R2 is selected from the group consisting of hydrogen, halogen and (C1- C6)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R1 is selected from the group consisting of hydrogen and fluoro. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro and (C1-C3)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 is selected from the group consisting of hydrogen, halogen and (C1-C6)alkyl and wherein R4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6- membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3- C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 and R4 are not hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen; R2 is selected from the group consisting of halogen and (C1-C6)alkyl; R4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of 5- or 6-membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1- C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is pyrazolyl, wherein said pyrazolyl is optionally substituted with one or more substituents independently selected from the group consisting of fluor, chloro, (C1- C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, and halo(C1-C2)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 is fluoro. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 and R4 each independently are selected from the group consisting of halogen, -NRaRb, 5- or 6- membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3- C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1- C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; and R2 is selected from the group consisting of hydrogen, halogen and (C1-C6)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1- C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; and R2 is selected from the group consisting of halogen and (C1-C6)alkyl; and R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of 5- or 6-membered heteroaryl, phenyl, 4 - 6 membered heterocyclyl and (C3-C6)cycloalkyl, wherein said 5- or 6-membered heteroaryl, phenyl, 4-6 membered heterocyclyl and (C3-C6)cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1- C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2; and R2 is selected from the group consisting of halogen and (C1-C6)alkyl; and R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of, fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C2)alkyl, (C1-C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, - NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1- C2)alkyl, (C1-C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C1-C4)alkoxy, halo(C1-C2)alkyl, (C1-C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2; and wherein wherein R2 is selected from the group consisting of halogen and (C1- C6)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 5- or 6-membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy halo(C1- C2)alkyl, (C1-C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 5- or 6-membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C2)alkoxy(C1-C2)alkyl, halo(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 5- membered heteroaryl is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy, (C1- C2)alkoxy(C1-C2)alkyl, and halo(C1-C2)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C2)alkoxy(C1-C2)alkyl and halo(C1-C2)alkyl; and wherein R2 is selected from the group consisting of halogen and (C1-C6)alkyl; and wherein R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C1-C4)alkoxy, (C1-C2)alkoxy(C1-C2)alkyl and halo(C1-C2)alkyl; and wherein R2 is selected from the group consisting of halogen and (C1-C6)alkyl; and wherein R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 4 - 6 membered heterocyclyl is selected from the group consisting of morpholinyl, piperidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, wherein said 4 - 6 membered heterocyclyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy halo(C1- C2)alkyl, (C1-C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R2 is selected from the group consisting of morpholinyl, piperidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, wherein said 4 - 6 membered heterocyclyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy halo(C1-C2)alkyl, (C1- C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2; and wherein wherein R2 is selected from the group consisting of halogen and (C1- C6)alkyl; and wherein R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of (C3-C6)cycloalkyl, wherein said (C3-C6)cycloalkyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkyl, hydroxy(C1- C4)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein at least one of R2 or R4 does not represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 and R4 do not represent hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R1 represents hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, halogen and (C1-C6)alkyl. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro, chloro and (C1-C3)alkyl. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro, methyl and ethyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R3 is hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R1 is selected from the group consisting of hydrogen and fluoro. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R1 is selected from the group consisting of hydrogen and fluoro; R2 is selected from the group consisiting of hydrogen, fluoro, chloro and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, fluoro, (C1-C2)alkyl and (C1- C2)alkoxy; R4 is selected from the group consisiting of hydrogen, fluoro, -NRaRb, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl, thienyl, phenyl, (C1-C4)alkyl, morpholinyl, piperidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, (C3-C6)cycloalkyl and (C1- C2)alkoxy, wherein said pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl, thienyl, phenyl, (C1-C4)alkyl, morpholinyl, piperidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, (C3- C6)cycloalkyl and (C1-C2)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C1-C4)alkoxy halo(C1-C2)alkyl, (C1-C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C2)alkyl. In a further aspect the invention provides a compound according to formula I of the embodiment above, or a pharmaceutically acceptable salt thereof, wherein R1 and R2 together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaryl is optionally substituted with one or more substituents independently selected from (C1- C4)alkyl, and wherein said 5- or 6-membered heterocycle is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl and oxo; and wherein R3 and R4 each independently are selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1-C4)alkoxy; or R2 and R3, together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaryl is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl, and wherein said 5- or 6-membered heterocycle is optionally substituted with one or more substituents independently selected from (C1- C4)alkyl and oxo; and wherein R1 is selected from the group consisting of hydrogen and halogen and R4 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1-C4)alkoxy. In a further aspect the invention provides a compound according to the embodiment above, or a pharmaceutically acceptable salt thereof, wherein R1 and R2, or R2 and R3, together with the carbon atoms to which they are attached form a ring selected from the group consisting of pyridine, imidazole, dihydropyridine and tetrahydropyridine, wherein said pyridine and imidazole are optionally substituted with one or more substituents independently selected from (C1-C4)alkyl, and wherein said dihydropyridine and tetrahydropyridine are optionally substituted with one or more substituents independently selected from (C1-C4)alkyl and oxo. In a further aspect the invention provides a compound according to any one of the embodiments above, selected from the list consisting of (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid (R)-2-amino-3-(3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethylisoxazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid, (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(2-methoxypropan-2-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5- yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-methylcyclopropyl)benzamido)propanoic acid, (R)-2-amino-3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethylcyclopropyl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1,2-dimethylcyclopropyl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(diethylamino)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethylpyridin-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethylmorpholino)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(2-ethylpiperidin-1-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid, (R)-2-amino-3-(2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid, (R)-2-amino-3-(3-(4-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethylpyrimidin-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid, (-)(R)-2-amino-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoic acid, (+)(R)-2-amino-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclobutyl)benzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethylpyrimidin-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclopentyl)benzamido)propanoic acid, (R)-2-amino-3-(5-(5-ethylisothiazol-4-yl)-2-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(3-methoxyoxetan-3-yl)benzamido)propanoic acid, (R)-2-amino-3-(2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid, (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid, (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(5-methylisoxazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid, (-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid, (+)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(2-ethylthiophen-3-yl)-5-fluorobenzamido)propanoic acid (R)-2-amino-3-(3-(5-ethylthiazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4- yl)benzamido)propanoic acid, (R)-2-amino-3-(5-fluoro-2'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3- yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid, (R)-2-amino-3-(3-butylbenzamido)propanoic acid, (R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(4-methylquinoline-6-carboxamido)propanoic acid, (R)-2-amino-3-(1H-benzo[d]imidazole-5-carboxamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-methylpyrrolidin-2-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(4,4-difluoropyrrolidin-2-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(1-oxo-1,2-dihydroisoquinoline-7-carboxamido)propanoic acid, (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzamido)propanoic, (R)-2-amino-3-(3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(5-isopropylisothiazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-methylbenzamido)propanoic acid, (R)-2-amino-3-(3-ethylbenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-methoxybenzamido)propanoic acid, (R)-2-amino-3-(5-fluoro-2'-(methoxymethyl)-[1,1'-biphenyl]-3-carboxamido)propanoic acid, (R)-2-amino-3-(2'-carbamoyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(1,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(6-fluoro-1,2,3,4-tetrahydroquinoline-8-carboxamido)propanoic acid, (R)-2-amino-3-(5-fluoro-2'-methoxy-[1,1'-biphenyl]-3-carboxamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-isobutoxybenzamido)propanoic acid, (R)-2-amino-3-(5,5'-difluoro-2'-methoxy-[1,1'-biphenyl]-3-carboxamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-morpholinobenzamido)propanoic, (R)-2-amino-3-(3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(1-phenoxyethyl)benzamido)propanoic acid and (R)-2-amino-3-(3-fluoro-5-(methoxy(phenyl)methyl)benzamido)propanoic acid, or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, selected from the list consisting of (R)-2-amino-3-((3-(4-chloro-2-ethyl-pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 3f below
Figure imgf000034_0001
(R)-2-amino-3-((3-(2-ethylphenyl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 4l below
Figure imgf000034_0002
(R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid with the formula 3b below
Figure imgf000034_0003
or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, selected from the list consisting of (R)-2-amino-3-((3-(4-chloro-2-ethyl-pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 3f below
Figure imgf000035_0001
(R)-2-amino-3-((3-(2-ethylphenyl)-5-fluoro-benzoyl)amino)propanoic acid with the formula 4l below
Figure imgf000035_0002
or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above as a salt of hydrochloric acid. In a further aspect the invention provides a compound according to any one of the embodiments above as a salt of hydrobromic acid. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid, or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid hydrochloride. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid hydrobromide. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(4-chloro-2-ethyl- pyrazol-3-yl)-5-fluoro-benzoyl)amino)propanoic acid. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid, or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid hydrochloride. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid hydrobromide. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-2-amino-3-((3-(2-ethylphenyl)-5- fluoro-benzoyl)amino)propanoic acid. Reference to compounds encompassed by the invention includes the free substance (zwitter ion) of compounds of the invention, pharmaceutically acceptable salts of compounds of the invention, such as acid addition salts or base addition salts, and polymorphic and amorphic forms of compounds of the invention and of pharmaceutically acceptable salts thereof. Furthermore, the compounds of the invention and pharmaceutically acceptable salts thereof may potentially exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. Both solvated and unsolvated forms are encompassed by the present invention. Compound names can be assigned by using the Struct=Name naming algorithm as part of CHEMDRAW®. It should be understood that the compounds of the invention may possess tautomeric forms, stereoisomers, geometric isomers, and that these also constitute embodiments of the invention. Racemic forms may be resolved into the optical antipodes by known methods, for example, by separation of diastereomeric salts thereof with an optically active acid and liberating the optically active amine compound by treatment with a base. Separation of such diastereomeric salts can be achieved, e.g. by fractional crystallization. The optically active acids suitable for this purpose may include, but are not limited to, d- or l- tartaric, mandelic or camphorsulfonic acids. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix. The compounds of the present invention may also be resolved by the formation and chromatographic separation of diastereomeric derivatives from chiral derivatizing reagents, such as, chiral alkylating or acylating reagents, followed by cleavage of the chiral auxiliary. Any of the above methods may be applied either to resolve the optical antipodes of the compounds of the invention per se or to resolve the optical antipodes of synthetic intermediates, which can then be converted by methods described herein into the optically resolved final products which are the compounds of the invention. Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S. Wilen in Enantiomers, Racemates, and Resolutions, John Wiley and Sons, New York, 1981. Optically active compounds can also be prepared from optically active starting materials. Included in this invention are also isotopically labelled compounds, which are similar to those claimed in formula I, wherein one or more atoms are represented by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., 2H, 3H, 11C, 13C, 15N, 18F and the like). Particular mention is made of 2H substituted compounds i.e. compounds wherein one or more H atoms are represented by deuterium. In one embodiment of the invention one or more of the hydrogen atoms of the compound of formula I are represented by deuterium. It is recognized that elements are present in natural isotopic abundances in most synthetic compounds and result in inherent incorporation of deuterium. However, the natural isotopic abundance of hydrogen isotopes such as deuterium is immaterial (about 0.015%) relative to the degree of stable isotopic substitution of compounds indicated herein. Thus, as used herein, designation of an atom as deuterium at a position indicates that the abundance of deuterium is significantly greater than the natural abundance of deuterium. Any atom not designated as a particular isotope is intended to represent any stable isotope of that atom, as will be apparent to the ordinarily skilled artisan. Pharmaceutically acceptable salts The compounds of this invention are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof. When a compound of formula I contains a free base, such salts may be prepared in a conventional manner by treating a solution or suspension of a free base of formula I with a molar equivalent of a pharmaceutically acceptable acid. Representative examples of suitable organic and inorganic acids are described below. Pharmaceutically acceptable salts in the present context is intended to indicate non- toxic, i.e. physiologically acceptable salts. The term “pharmaceutically acceptable salts” include salts formed with inorganic and/or organic acids on the nitrogen atoms in the parent molecule. Said acids may be selected from for example hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fumaric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid, saccharin, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid. In an embodiment of the invention, the pharmaceutically acceptable salt is a hydrogen chloride salt. In an embodiment of the invention, the pharmaceutically acceptable salt is a hydrogen bromide salt. The term pharmaceutically acceptable salts also include salts formed with inorganic and/or organic bases on the acidic groups of compounds of formula I. Said bases may be selected from for example alkali metal bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, alkaline earth bases, such as calcium hydroxide and magnesium hydroxide, and organic bases, such as trimethylamine and trimethylamine. Additional examples of useful acids and bases to form pharmaceutically acceptable salts can be found e.g. in Stahl and Wermuth (Eds) “Handbook of Pharmaceutical salts. Properties, selection, and use”, Wiley-VCH, 2008. Conditions for treatment The invention encompasses compounds of the invention for use as a medicament, as well as for use in treatment of all diseases and disorders listed below. As described above compounds of the present invention may be useful in the treatment of depression and depressive disorders. Hence in one embodiment, a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of depression. The diagnosis of depression usually follows a clinical evaluation by a psychiatrist or other mental health professionals. The two most recognized sets of diagnostic criteria for major depressive disorder and other depressive, or mood disorders, are outlined in the DSM, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV) published by the American psychiatric association and the ICD (ICD-10: International Statistical Classification of Diseases and Related Health Problems – 10th Revision, published periodically by the World Health Organization) or any other psychiatric classification system. Signs and symptoms of depression are for example depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach. Thus in an embodiment of the invention, compounds of the invention are for use to prevent, alter, reduce or alleviate one or more signs or symptoms of depression selected from the group consisting of depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach. The skilled person is familiar with various test for measuring the improvement of depressive symptoms. Examples of tests for measuring the improvements are, but not limited to, the Hamilton Rating Scale for Depression (HAM-D) or Montgomery–Åsberg Depression Rating Scale (MADRS) scale. In an embodiment the depression is major depressive disorder. In a further embodiment the depression is treatment-resistant depression. In a further embodiment the depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. Clinically used NMDA antagonist such as ketamine and dextromethorphan are generally effective in patients with neurophatic pain [Hy et al.,Expert Rev Clin Pharmacol. 2011 May 1; 4(3): 379–388]. Hence, in an embodiment of the invention, compound of formula I or a pharmaceutically acceptable salt thereof is used in the treatment of pain. In a further embodiment the pain is neuropathic pain. Preclinical animal models have demonstrated pro-cognitive and antidepressant-like effects with the use of NDMA glycine site modulators [Peyrovian et al., Progress in Neuropsychopharmacology & Biological Psychiatry. 92 (2019) 387-404]. Hence, in an embodiment of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus. In a further embodiment a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of suicidal ideation. In an embodiment of the invention, compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of a neurological disorder or neuropsychiatric disorder. In an embodiment the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof to a patient (e.g. a human patient) in need thereof. In an embodiment the invention provides a method for the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof. In an embodiment the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof. In an embodiment the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. In an embodiment the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus. In an embodiment the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. In an embodiment the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus. Combination treatment In an embodiment of the invention, the compounds of formula I are for use as stand- alone treatment as the sole active compound. In another embodiment of the invention, the compounds of formula I may be used in combination with other agents useful in the treatment of disorders such as depression. The terms “combined use”, “in combination with” and “a combination of” and the like as used herein in the context of the method of the invention comprising the combined administration of therapeutically effective amounts of a compound of formula I, and another compound, which compound is useful in the treatment a neurodegenerative disease or disorder, is intended to mean the administration of a compound of formula I simultaneously or sequentially, in any order, together with said other compound. The two compounds may be administered simultaneously or sequentially with a time gap between the administrations of the two compounds. The two compounds may be administered either as part of the same pharmaceutical formulation or composition, or in separate pharmaceutical formulations or compositions. The two compounds may be administered on the same day or on different days. They may be administered by the same route, such for example by oral administration, by depot, by intramuscular injection or intravenous injection; or by different routes wherein one compound is for example administered orally or placed by depot and the other compound is for example injected. The two compounds may be administered by the same dosage regime or interval, such as once or twice daily, weekly, or monthly; or by different dosage regimes for example wherein one is administered once daily and the other is administered twice daily or weekly or monthly. In some instances, the patient to be treated may already be in treatment with one or more other compounds useful in the treatment of depression when treatment with a compound of the invention initiated. In other instances, the patient may already be in treatment with a compound of the invention when treatment with one or more other compounds useful in the treatment of a depression or psychosis is initiated. In other instances, the treatment with a compound of the invention and treatment with one or more other compounds useful in the treatment of psychosis initiated at the same time. Compounds for combination treatment Examples of therapeutically active compounds which may advantageously be combined with compounds of the invention include sedatives or hypnotics, such as benzodiazepines; anticonvulsants, such as lamotrigine, valproic acid, topiramate, gabapentin, carbamazepine; mood stabilizers such as lithium; dopaminergic drugs, such as dopamine agonists and L-Dopa; drugs to treat ADHD, such as atomoxetine; psychostimulants, such as modafinil, ketamine, methylphenidate and amphetamine; other antidepressants, such as mirtazapine, mianserin, vortioxetine, cipralex, and buproprion; hormones, such as T3, estrogen, DHEA and testosterone; atypical antipsychotics, such as olanzapine, brexpiprazole and aripiprazole; typical antipsychotics, such as haloperidol; drugs to treat Alzheimer's diseases, such as cholinesterase inhibitors and memantine, folate; S-Adenosyl-Methionine; immunmodulators, such as interferons; opiates, such as buprenorphins; angiotensin II receptor 1 antagonists (AT1 antagonists); ACE inhibitors; statins; and alpha1 adrenergic antagonist , such as prazosin. Administration routes The pharmaceutical compositions comprising a compound of formula I, either as the sole active compound or in combination with another active compound, may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, buccal, sublingual, pulmonal, transdermal and parenteral (e.g. subcutaneous, intramuscular, and intravenous) route. It will be appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient. Doses In one embodiment, the compound of the present invention is administered in an amount from about 0.5 mg/kg body weight to about 50 mg/kg body weight per day. In particular, daily dosages may be in the range of 1 mg/kg body weight to about 30 mg/kg body weight per day. The exact dosages will depend upon the frequency and mode of administration, the sex, the age, the weight, and the general condition of the subject to be treated, the nature and the severity of the condition to be treated, any concomitant diseases to be treated, the desired effect of the treatment and other factors known to those skilled in the art. In an embodiment the frequency of administration is 1, 2, 3, 4, or 5 times per day In an embodiment the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly. A typical oral dosage for adults will be in the range of 1-3000 mg/day of a compound of the present invention, such as 700-2800 mg/day, such as 1000-2000 mg/day or 1200- 1700mg/day. Conveniently, the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 100 to 1000 mg, such as 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 750 mg or up to 1000 mg of a compound of the present invention. In an embodiment the frequency of administration is 1, 2, 3, 4, or 5 times per day In an embodiment the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly. A typical IV dosage for adults will be in the range of 20-300 mg/day of a compound of the present invention, such as 50-200 mg/day, such as 70-150 mg/day or 75-125 mg/day. Conveniently, the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 10 to 300 mg, such as 10 mg, 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg of a compound of the present invention. In one embodiment the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly. Pharmaceutical formulations and excipients In the following, the term, “excipient” or “pharmaceutically acceptable excipient” refers to pharmaceutical excipients including, but not limited to, fillers, antiadherents, binders, coatings, colours, disintegrants, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, solvents, vehicles and adjuvants. The present invention also provides a pharmaceutical composition comprising a compound of formula I, such as one of the compounds disclosed in the Experimental Section herein, and one or more pharmaceutically acceptable carriers or diluents. The present invention also provides a process for making a pharmaceutical composition comprising a compound of formula I. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable excipients in accordance with conventional techniques such as those disclosed in Remington, “The Science and Practice of Pharmacy”, 22th edition (2013), Edited by Allen, Loyd V., Jr. Pharmaceutical compositions for oral administration include solid oral dosage forms such as tablets, capsules, powders and granules; and liquid oral dosage forms such as solutions, emulsions, suspensions and syrups as well as powders and granules to be dissolved or suspended in an appropriate liquid. Solid oral dosage forms may be presented as discrete units (e.g. tablets or hard or soft capsules), each containing a predetermined amount of the active ingredient, and preferably one or more suitable excipients. Where appropriate, the solid dosage forms may be prepared with coatings such as enteric coatings or they may be formulated so as to provide modified release of the active ingredient such as delayed or extended release according to methods well known in the art. Where appropriate, the solid dosage form may be a dosage form disintegrating in the saliva, such as for example an oral-dispersible tablet. Examples of excipients suitable for solid oral formulation include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, povidone, croscarmellose sodium, sucrose, cyclodextrin, talcum, gelatin, pectin, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Similarly, the solid formulation may include excipients for delayed or extended release formulations known in the art, such as glyceryl monostearate or hypromellose. If solid material is used for oral administration, the formulation may for example be prepared by mixing the active ingredient with solid excipients and subsequently compressing the mixture in a conventional tableting machine; or the formulation may for example be placed in a hard capsule e.g. in powder, pellet or mini tablet form. The amount of solid excipient will vary widely but will typically range from about 25 mg to about 1 g per dosage unit. Liquid oral dosage forms may be presented as for example elixirs, syrups, oral drops or a liquid filled capsule. Liquid oral dosage forms may also be presented as powders for a solution or suspension in an aqueous or non-aqueous liquid. Examples of excipients suitable for liquid oral formulation include, but are not limited to, ethanol, propylene glycol, glycerol, polyethylenglycols, poloxamers, sorbitol, poly-sorbate, mono and di- glycerides, cyclodextrins, coconut oil, palm oil, and water. Liquid oral dosage forms may for example be prepared by dissolving or suspending the active ingredient in an aqueous or non-aqueous liquid, or by incorporating the active ingredient into an oil-in-water or water-in-oil liquid emulsion. Further excipients may be used in solid and liquid oral formulations, such as colourings, flavourings and preservatives etc. Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous solutions, dispersions, suspensions or emulsions for injection or infusion, concentrates for injection or infusion as well as sterile powders to be reconstituted in sterile solutions or dispersions for injection or infusion prior to use. Examples of excipients suitable for parenteral formulation include, but are not limited to water, coconut oil, palm oil and solutions of cyclodextrins. Aqueous formulations should be suitably buffered if necessary and rendered isotonic with sufficient saline or glucose. Other types of pharmaceutical compositions include suppositories, inhalants, creams, gels, dermal patches, implants and formulations for buccal or sublingual administration. It is requisite that the excipients used for any pharmaceutical formulation comply with the intended route of administration and are compatible with the active ingredients. Compounds of the invention Table 1: Exemplified compounds of the invention
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
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Figure imgf000054_0001
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Figure imgf000056_0001
Figure imgf000057_0001
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Figure imgf000067_0001
(R)-2-amino-3-(3-fluoro-5- Compound 5r (methoxy(phenyl)methyl)benza mido)propanoic acid EXPERIMENTAL SECTION General Schemes for preparing compounds of the invention The compounds of the present invention of the general formula I, wherein R1, R2, R3 and R4 are as defined above can be prepared by the methods outlined in the following reaction Schemes 1-15 and in the examples. In the described methods, it is possible to make use of variants or modifications, which are themselves known to chemists skilled in the art or could be apparent to the person of ordinary skill in this art. Furthermore, other methods for preparing compounds of the invention will be readily apparent to the person skilled in the art in light of the following reaction schemes and examples. The schemes may involve the use of selective protecting groups during the synthesis of the compounds of the invention. One skilled in the art would be able to select the appropriate protecting group for a particular reaction. It may be necessary to incorporate protection and de-protection strategies for substituents such as amino, amido, carboxylic acid and hydroxyl groups in the synthetic methods described below to synthesize the compounds of Formula I. Methods for protection and de-protection of such groups are well known in the art, and may be found in T. Green, et al., Protective Groups in Organic Synthesis, 1991, 2nd Edition, John Wiley & Sons, New York. The schemes in this section are representative of methods useful in synthesizing the compounds of the present invention. They are not intended to constrain the scope of the invention in any way. Scheme 1
Figure imgf000069_0001
Compounds of general formula I (Scheme 1) may be prepared from compounds with general formula IV by standard de-protection procedures. As an example, compounds of general formula I (Scheme 1) may be prepared from compounds with general formula IV where Pg1 is Cbz (carboxybenzyl) and Pg2 is benzyl using conditions such as HBr in acetic acid. Compounds with general formula IV may be prepared by reacting protected amines of general formula III with carboxylic acids (or salt thereof) of general formula II by standard peptide coupling such as using O-(Benzotriazol-1-yl)-N,N,Nʹ,Nʹ- tetramethyluronium tetrafluoroborate in the presence of a base such as N,N- diisopropylethylamine in a solvent such as N,N-dimethylformamide.
Figure imgf000069_0002
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 can be hetaryl, aryl or alkenyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VI can be obtained from compounds of general formula V, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ- Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1ʹ-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as potassium triphosphate. Likewise, similar reaction conditions may be applied for introducing R4 substituents.
Figure imgf000070_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 3) where R2 is hetaryl or heterocycle as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained from compounds of general formula V, under reaction conditions such as in the presence of a catalyst such as copper (II) acetate and a base such as pyridine. Likewise, similar reaction conditions may be applied for introducing R4 substituents.
Figure imgf000070_0002
Benzoic acids of general formula II (or salt thereof), (Scheme 4) where R2 is NRaRb as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VI can be obtained by reacting compounds of general formula VIII together with amines of general formula RaRbNH. Likewise, similar reaction conditions may be applied for introducing R4 substituents.
Figure imgf000071_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 5) where R2 can be alkyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VI can be obtained by reacting compounds of general formula IX together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2-dicyclohexylphosphino-2ʹ,6ʹ-bis(N,N-dimethylamino)biphenyl (Cphos). Likewise, similar reaction conditions may be applied for introducing R4 substituents.
Figure imgf000072_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 6) where R3 can be alkyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VI can be obtained by reacting compounds of general formula X together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2-dicyclohexylphosphino-2ʹ,6ʹ-bis(N,N-dimethylamino)biphenyl (Cphos). Likewise, similar reaction conditions may be applied for introducing R1 substituents.
Figure imgf000072_0002
Benzoic acids of general formula II (or salt thereof), (Scheme 7) where R2 can be alkyloxy or cycloalkyoxy as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VI can be obtained by reacting compounds of general formula XI together with aldehydes or ketones, under reaction conditions such as in the presence of a base such as isopropylmagnesium chloride complex with lithium chloride. Likewise, similar reaction conditions may be applied for introducing R4 substituents.
Figure imgf000073_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 8) where R2 can be hetaryl, aryl or alkenyly as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VI by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VI can be obtained from compounds of general formula XII, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1ʹ-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as cesium carbonate. General Methods LC-MS methods Analytical LC-MS data were obtained using one of the methods identified below. Method A (550): A Waters Acquity UPLC-MS was used. Column: Acquity UPLC BEH C181.7µm; 2.1x50mm; Column temperature: 60 °C; Solvent system: A = water/trifluoroacetic acid (99.965:0.035) and B = acetonitrile /water/trifluoroacetic acid (94.965:5:0.035); Method: Linear gradient elution with A:B = 90:10 to 0:100 in 1.0 minutes and with a flow rate of 1.2 mL/min. Method B (551): A Waters Acquity UPLC-MS was used. Column: Acquity UPLC BEH C18 1.7µm; 2.1x50mm; Column temperature: 60 °C; Solvent system: A = water/trifluoroacetic acid (99.5:0.5) and B = acetonitrile /water/trifluoroacetic acid (94.965:5:0.035); Method: Linear gradient elution with A:B = 98:2 to 0:100 in 1.15 minutes and with a flow rate of 1.2 mL/min. Method C (CD05): An Agilent 1200 LCMS system with ELS detector was used. Column: Waters XBridge Sheild RP18 5 µm; 2.1x50mm; Column temperature: 40 °C; Solvent system: A = water/ammonia (99.95:0.05) and B = acetonitrile; Method: Linear gradient elution with A:B = 95:5 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method D (AB01): An Agilent 1200 LCMS system with ELS detector was used. Column: Agilent TC-C18 5 µm; 2.1x50mm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.9:0.1) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 99:1 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method E (AB10): An Agilent 1200 LCMS system with ELS detector was used. Column: Agilent TC-C18 5 µm; 2.1x50mm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.9:0.1) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 90:10 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method F (AB00): An Agilent 1200 LCMS system with ELS detector was used. Column: Phenomenex Luna-C18, 50×2mm, 5μm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.9:0.1) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 99:1 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Preparative HPLC Preparative-HPLC (Method AA): Instrument: Gilson GX-281 Liquid Handler, SHIMADZU LC-8A LCMS2010; Column: YMC-Actus Triart C18150*305μm; Mobile Phase A: water (0.05% HCl v/v); Mobile phase B: MeCN; Gradient: B from 5% to 35% in 10 minutes then hold at 100% for 3 minutes; FlowRate(ml/min): 25; Column temperature: 35°C and Wavelength: 220nm 254nm. Preparative HPLC (Method BB): Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Xtimate C18150*25mm*5μm; Mobile Phase A: water (0.05% ammonia hydroxide v/v); Mobile phase B: MeCN; Gradient: from 42% to 72% in 10 minutes then hold at 100% for 2.5 minutes; Flow Rate (ml/min): 25; Column temperature: 25°C and Wavelength: 220nm 254nm Preparative HPLC (Method CC): Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Agela Durashell C18150 mm × 25mm × 5µm; Mobile Phase A: water (0.225%TFA, v/v); Mobile phase B: MeCN; Gradient: B from 32% to 62% in 10 minutes, hold 100% B for 2 minutes; Flow Rate (ml/min): 25; Column temperature: 40°C and Wavelength: 220nm 254nm Preparative HPLC (Method DD): Instrument: Gilson GX-281, Gilson 322 Pump, Gilson 156 UV Detector; Column: Waters Xbridge 150*25*5μm; Mobile Phase A: water (10mM NH4HCO3); Mobile phase B: MeCN; Gradient: B from 40% to 66% in 8.4 minutes then hold at 100% for 2 minutes; FlowRate(ml/min): 25; Column temperature: 30°C and Wavelength: 220nm 254nm. NMR 1H NMR spectra were recorded at 300, 400, 500 or 600 MHz on Bruker Avance instruments. TMS was used as internal reference standard. Chemical shift values are expressed in ppm. The following abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, q = quartet, qui = quintet, h = heptet, dd = double doublet, dt = double triplet, dq = double quartet, tt = triplet of triplets, m = multiplet, br s = broad singlet and br = broad signal. Abbreviations are in accordance with to the ACS Style Guide: "The ACS Style guide – A manual for authors and editors" Janet S. Dodd, Ed.1997, ISBN: 0841234620 List of chemical abbreviations AcOH: Acetic Acid AcOK: Potassium acetate CuI: Copper(I) iodide Cu(OAc)2: Copper(II) acetate Cs2CO3: Cesium carbonate Cphos: 2-Dicyclohexylphosphino-2',6'-bis(N,N-dimethylamino)biphenyl CsOAc: Cesium acetate CPhos-Pd-G3: [(2-Dicyclohexylphosphino-2ʹ,6ʹ-bis(N,N-dimethylamino) -1,1ʹ-biphenyl)- 2-(2ʹ-amino-1,1ʹ-biphenyl)] palladium(II) methanesulfonate DCM: Dichloromethane DEA: Diethylamine DIBAL-H: diisobutylaluminum hydride DIPEA: Diisopropylethylamine DMA: N,N-dimethylacetamide DME: Dimethoxyethane DMF: N,N-dimethylformamide ee: Enantiomeric excess EDC: N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride EtOAc: Ethyl acetate EtOH: Ethanol HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate HBr/HOAc: HBr in acetic acid HOAc: Acetic acid HOBt: 1-Hydroxybenzotriazole HPLC: High performance liquid chromatography IPA: Isopropanol iPrOAc: Isopropyl acetate i-PrMgCl-LiCl: Isopropylmagnesium chloride-lithium chloride complex iPrOH: Isopropanol IR[DF(CF3)PPY]2(DTBPY))PF6: [4,4ʹ-Bis(1,1-dimethylethyl)-2,2ʹ-bipyridine-N1,N1ʹ]bis[3,5- difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-N]phenyl-C]Iridium(III) hexafluorophosphate MeCN: Acetonitrile MeI: Iodomethane MeOH: Methanol Me-THF: 2-Methyltetrahydrofuran MTBE: 2-Methoxy-2-methylpropane n-BuLi: n-Butyllithium NBS: N-Bromosuccinimide NCS: N-Chlorosuccinimide NaHMDS: Sodium bis(trimethylsilyl)amide NiCl2.glyme: Nickel(II) chloride ethylene glycol dimethyl ether complex NIS: N-Iodosuccinimide P2S5: Phosphorus(V) sulfide Pd/C: Palladium on carbon PEMB: 5-Ethyl-2-methylpyridine borane Pd(dtbpf)Cl2: [1,1ʹ-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) Pd(dppf)Cl2.CH2Cl2 : (1,1-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex) Pd(dppf)Cl2: [1,1ʹ-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(PPh3)4: Tetrakis(triphenylphosphine)palladium Pd(PPh3)2Cl2: dichloropalladium;triphenylphosphanium Pd(OAc)2: Palladium(II) acetate Pd(t-Bu3P)2: bis(tri-t-butylphosphine)palladium Py: SEM-Cl: 2-(Trimethylsilyl)ethoxymethyl chloride STAB: Sodium triacetoxyborohydride TBME: Tert-butyl methyl ether TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TEA: Triethylamine Tf2O: Trifluoromethanesulfonic anhydride TFA: Trifluoroacetic acid THF: Tetrahydrofuran TMSCHN2: Trimethylsilyldiazomethane TMSCl: Chlorotrimethylsilane T3P: Propylphosphonic anhydride Me-THF: 2-Methyltetrahydrofuran TsOH: p-Toluenesulfonic acid w/w: Weight per weight ZnEt2: Diethylzinc Preparation of the intermediates Intermediate 1 (R)-3-(Benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropan-1-aminium chloride
Figure imgf000079_0001
To phenylmethanol (56.0 mL) was added thionyl chloride (2.1 g, 17.6 mmol) dropwise at 28°C. After completion of the addition, (R)-3-amino-2- (((benzyloxy)carbonyl)amino)propanoic acid (3.8 g, 15.9 mmol) was added in several portions and the reaction was stirred for 24 hours at 28°C. The excess benzyl alcohol was removed at 80°C/0.02 bar and the residue was stirred in cyclohexane (35 mL) for 16 hours. Filtration and trituration of the filter cake with MTBE (50 mL) afforded the title compound (3.0 g). LCMS (MH+): (m/z) = 329.2, tR (min, Method A)= 0.52. Preparation of exemplified compounds of the invention Compound 1a (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethyl-1-methyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000080_0001
Step 1: methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000080_0002
A mixture of 5-bromo-4-ethyl-1-methyl-1H-pyrazole (200 mg, 1.06 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (251 mg, 1.27 mmol), Pd(dtbpf)Cl2 (69 mg, 0.11 mmol), K3PO4 (674 mg, 3.17 mmol) in the mixture of dioxane (3 mL) and H2O (0.5 mL) was degassed and purged with N2 (3 times), and then the mixture was stirred at 110 °C for 16 hours under N2 atmosphere. The mixture was concentrated, then added water (5 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzoate (230 mg). 1H NMR (400MHz, CDCl3) δ 7.82-7.79 (m, 2H), 7.44 (s, 1H), 7.24-7.21 (m, 1H), 3.96 (s, 3H), 3.77 (s, 3H), 2.41 (q, J = 7.6 Hz, 2H), 1.13 (t, J = 7.6 Hz, 3H). Step 2: 3-(4-Ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000081_0001
To a solution of methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (230 mg, 0.88 mmol) in the mixture of MeOH (2 mL), THF (2 mL) and H2O (2 mL) was added LiOH.H2O (110 mg, 2.63 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated to remove MeOH and THF, then added HCl (2 M) to adjust pH=3~4, and then extracted with ethyl acetate (10 mL × 3). The combined organic layer was washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (200 mg). 1H NMR (400MHz, CDCl3) δ 8.52 (br, 1H), 7.92-7.85 (m, 2H), 7.53 (s, 1H), 7.30-7.25 (m, 1H), 3.84 (s, 3H), 2.43 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). Step 3: (R)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate
Figure imgf000081_0002
To a solution of 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (200 mg, 0.81 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (353 mg, 0.97 mmol, HCl salt) in DMF (5 mL) was added TBTU (388 mg, 1.21 mmol) and DIPEA (312 mg, 2.42 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was added water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) twice to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (270 mg). 1H NMR (400MHz, CDCl3) δ 7.49-7.42 (m, 3H), 7.38-7.28 (m, 10H), 7.17-7.12 (m, 1H), 7.11-7.06 (m, 1H), 5.99-5.97 (m, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.63-4.56 (m, 1H), 3.99- 3.78 (m, 2H), 3.73 (s, 3H), 2.40 (q, J = 7.6 Hz, 2H), 1.12 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-Amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000082_0002
Figure imgf000082_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (270 mg, 0.48 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethyl-1-methyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.23-9.19 (m, 1H), 8.65 (br, 3H), 7.86-7.83 (m, 1H), 7.80 (s, 1H), 7.54-7.50 (m, 1H), 7.46 (s, 1H), 4.17-4.07 (m, 1H), 3.84-3.78 (m, 2H), 3.73 (s, 3H), 2.36 (q, J = 7.6 Hz, 2H), 1.05 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 335.0, tR (min, Method c) = 0.99. [α]20 ,D = 14 (c = 1 mg/mL,CH3OH). Compound 1b (R)-2-Amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-Amino-3-(3-(4-ethyl-1-methyl- 1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000083_0001
Step 1: methyl 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate
Figure imgf000083_0002
A mixture of 3-bromo-4-ethyl-1-methyl-1H-pyrazole (200 mg, 1.06 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (251 mg, 1.27 mmol), Pd(dtbpf)Cl2 (69 mg, 0.11 mmol), K3PO4 (674 mg, 3.17 mmol) in the mixture of dioxane (4 mL) and H2O (0.5 mL) was degassed and purged with N2 (3 times), and then the mixture was stirred at 110 °C for 16 hours under N2 atmosphere. The mixture was concentrated, then added water (5 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give methyl 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5- fluorobenzoate (130 mg) 1H NMR (400MHz, CDCl3) δ 8.20-8.10 (m, 1H), 7.69-7.63 (m, 1H), 7.62-7.57 (m, 1H), 7.25 (s, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 2.68 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). Step 2: 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid
Figure imgf000084_0001
To a solution of methyl 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate (130 mg, 0.5 mmol) in the mixture of MeOH (2 mL), THF (2 mL) and H2O (2 mL) was added LiOH.H2O (62 mg, 1.49 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated to remove MeOH and THF. Then HCl (2 M) was added to adjust to pH=3~4, followed by extraction with ethyl acetate (10 mL × 3). The combined organic layer was washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid (100 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3- yl)-5-fluorobenzamido)propanoate
Figure imgf000084_0002
To a solution of 3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid (100 mg, 402 µmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (159 mg, 0.44 mmol, HCl salt) in DMF (3 mL) was added TBTU (194 mg, 604 µmol) and DIPEA (156 mg, 1.21 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was added water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (4-ethyl-1-methyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoate (150 mg). 1H NMR (400MHz, CDCl3) δ 7.78 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.38-7.23 (m, 12H), 6.76 (br, 1H), 5.98 (d, J = 6.4 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.64-4.55 (m, 1H), 3.94-3.81 (m, 5H), 2.67 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-3-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000085_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H- pyrazol-3-yl)-5-fluorobenzamido)propanoate (150 mg, 0.27 mmol) in 30% HBr in AcOH (4 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethyl-1-methyl- 1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.10-9.05 (m, 1H), 8.62 (br, 3H), 8.00-7.98 (m, 1H), 7.74- 7.69 (m, 1H), 7.63 (s, 1H), 7.56-7.51 (m, 1H), 4.16-4.07 (m, 1H), 3.85 (s, 3H), 3.82-3.77 (m, 2H), 2.63 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 335.0, tR (min, Method c) = 1.054 [α]20 ,D = 2 (c = 5 mg/mL,CH3OH). Compound 1c (R)-2-amino-3-(3-(3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethylpyridin-2- yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000086_0001
Step 1: methyl 3-(3-ethylpyridin-2-yl)-5-fluorobenzoate
Figure imgf000086_0002
A mixture of 2-chloro-3-ethylpyridine (300 mg, 2.12 mmol), (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (503 mg, 2.54 mmol), Pd(dtpbf) Cl2(138 mg, 0.21 mmol), K3PO4 (1.35 g, 6.36 mmol) in a mixture of dioxane (3 mL) and H2O (0.5 mL) was degassed and purged with N2 (3 times), and then the mixture was stirred at 110 °C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated to give methyl 3-(3-ethylpyridin- 2-yl)-5-fluorobenzoate (420 mg). 1H NMR (400 MHz, CDCl3) δ 8.55-8.50 (m, 1H), 8.00-7.95 (m, 1H), 7.78-7.73 (m, 1H), 7.70-7.64 (m, 1H), 7.44-7.39 (m, 1H), 7.30-7.26 (m, 1H), 3.92 (s, 3H), 2.66 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). Step 2: 3-(3-ethylpyridin-2-yl)-5-fluorobenzoic acid
Figure imgf000087_0001
To a solution of methyl 3-(3-ethylpyridin-2-yl)-5-fluorobenzoate (400 mg, 1.54 mmol) in THF (5 mL) and H2O (2.5 mL) was added LiOH.H2O (78 mg, 1.85 mmol) and the mixture was stirred at 20°C for 1.5 hours. 5% HCl is used to adjust the pH to 3. The reaction mixture is concentrated. The residue was diluted with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-ethylpyridin-2-yl)-5-fluorobenzoic acid (320 mg). 1H NMR (400 MHz, CD3OD) δ 8.76-8.75 (m, 1H), 8.74-8.73 (m, 1H), 8.11-8.06 (m, 2H), 8.02-7.98 (m, 1H), 7.77-7.70 (m, 1H), 2.77 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-2-yl)-5- fluorobenzamido)propanoate
Figure imgf000087_0002
To a solution of 3-(3-ethylpyridin-2-yl)-5-fluorobenzoic acid (320 mg, 1.30 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (524 mg, 1.44 mmol, HCl salt) in DMF (10 mL) was added TBTU (628 mg, 1.96 mmol) and DIPEA (506 mg, 3.91 mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 50%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoate (590 mg). LC-MS (MH+): m/z = 556.1, tR (min, Method Method c) = 0.879 Step 4: (R)-2-amino-3-(3-(3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000088_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-2-yl)-5- fluorobenzamido)propanoate (590 mg, 1.06 mmol) in the solution of 30 % HBr in AcOH (10 mL) was stirred at 50°C for 3 hours. The reaction mixture was concentrated. The residue was washed with AcOH/MTBE=1/2 (30 mL). The resulting mixture was filtered, the filtration cake was washed with MTBE (2 mL) and collected to give (R)-2-amino-3-(3- (3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoic acid (350 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 9.00-8.95 (m, 1H), 8.74-8.73 (m, 1H), 8.43-8.27 (m, 4H), 7.96-7.87 (m, 2H), 7.87-7.80 (m, 1H), 7.76-7.73 (m, 1H), 4.19-4.09 (m, 1H), 3.85-3.77 (m, 1H), 3.76-3.67 (m, 1H), 2.70 (q, J = 7.6 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 332.0, tR (min, Method c) = 1.034 [α]20 ,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 1d and Compound 1e (R)-2-amino-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid and (R)- 2-amino-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of 1d: (R)-2-amino-3-(3-(5-ethyl-1H- pyrazol-1-yl)-5-fluorobenzamido)propanoic acid and 1e: (R)-2-amino-3-(3-(3-ethyl-1H- pyrazol-1-yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000089_0001
Step 1: Methyl 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoate and methyl 3-(3-ethyl-1H- pyrazol-1-yl)-5-fluorobenzoate
Figure imgf000089_0002
To a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.54 g, 7.8 mmol), 3- ethyl-1H-pyrazole (0.25 g, 2.6 mmol), Cu(OAc)2 (945 mg, 5.2 mmol) and Py (1.03 g, 13 mmol) in 1,2-dichloroethane (25 mL) was added 4A MS (0.2 g) under O2 (15 psi). The mixture was stirred at 85°C for 16 hours. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoate (70 mg) and methyl 3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoate (380 mg). methyl 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoate: 1H NMR (400MHz, CDCl3) δ 7.95- 7.91 (m, 1H), 7.76-7.71 (m, 1H), 7.61 (m, 1H), 7.45-7.42 (m, 1H), 6.28-6.22 (m, 1H), 3.94 (s, 3H), 2.74 (q, J = 7.6 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H). methyl 3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoate: 1H NMR (400MHz, CDCl3) δ 8.04 (s, 1H), 7.88 (d, J = 2.4 Hz, 1H), 7.72-7.68 (m, 1H), 7.58-7.54 (m, 1H), 6.31 (d, J = 2.4 Hz, 1H), 3.95 (s, 3H), 2.74 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). Step 2: 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid
Figure imgf000090_0001
A mixture of methyl 3-(5-ethylpyrazol-1-yl)-5-fluoro-benzoate (77 mg, 0.31 mmol) and LiOH.H2O (26 mg, 0.62 mmol) in a mixture of MeOH (5 mL) and H2O (5 mL) was stirred at 25°C for 15 hours. The mixture was extracted with MTBE (20 mL × 2). The aqueous phase was adjusted to pH=3~4 with 2M HCl. The mixture was concentrated to give 3-(5- ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid (72 mg). LC-MS (MH+): m/z = 233.1, tR (min, Method c) = 1.171 Step 2: 3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid
Figure imgf000091_0001
A mixture of methyl 3-(3-ethylpyrazol-1-yl)-5-fluoro-benzoate (450 mg, 1.81 mmol) and LiOH.H2O (152 mg, 3.63 mmol) in a mixture of MeOH (10 mL) and H2O (10 mL) was stirred at 25°C for 15 hours. The mixture was extracted with MTBE (20 mL × 2). The aqueous phase was adjusted to pH=3~4 with 2M HCl. The mixture was concentrated to give 3-(3- ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid (0.42 g). LC-MS (MH+): m/z = 233.1, tR (min, Method c) = 1.058 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate
Figure imgf000091_0002
A mixture of (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (112 mg, 0.31 mmol, HCl salt), 3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid (72 mg, 0.31 mmol), TBTU (148 mg, 0.46 mmol) and DIPEA (199 mg, 1.54 mmol) in DMF (10 mL) was stirred at 25°C for 16 hours. The mixture was poured into H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate (0.1 g, 59.7% yield). 1H NMR (400MHz, CDCl3) δ 7.60 (m, 1H), 7.58 (br, 1H), 7.40-7.26 (m, 12H), 7.00 (br, 1H), 6.27-6.23 (m, 1H), 5.95 (m, 1H), 5.19 (s, 2H), 5.09 (s, 2H), 4.62-4.52 (m, 1H), 3.95-3.85 (m, 1H), 3.84-3.76 (m, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate
Figure imgf000092_0001
A mixture of (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (654 mg, 1.79 mmol, HCl salt), 3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzoic acid (0.42 g, 1.79 mmol), TBTU (864 mg, 2.69 mmol) and DIPEA (1.16 g, 8.97 mmol) in DMF (20 mL) was stirred at 25°C for 16 hours. The mixture was poured into H2O (100 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate (0.5 g). 1H NMR (400MHz, CDCl3) δ 7.86 (d, J = 2.4 Hz, 1H), 7.76 (s, 1H), 7.60 (d, J = 9.6 Hz, 1H), 7.39 - 7.27 (m, 10H), 7.20 (d, J = 9.6 Hz, 1H), 6.94 (br s, 1H), 6.31 (d, J = 2.4 Hz, 1H), 5.98 (m, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.65-4.57 (m, 1H), 3.96 - 3.87 (m, 1H), 3.86-3.78 (m, 1H), 2.74 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000092_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoate (0.1 g, 0.18 mmol) and HBr in AcOH (2 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was diluted with AcOH (10 mL). The part of solvent was removed. MTBE (20 mL) was added to therein and stirred for 2 hours. The precipitate was filtered under N2. The filter cake was lyophilized to give (R)-2-amino-3-(3-(5-ethyl-1H-pyrazol-1-yl)-5- fluorobenzamido)propanoic acid (45 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.95-8.91 (m, 1H), 8.30 (br s, 3H), 7.87-7.81 (m, 1H), 7.75- 7.69 (m, 1H), 7.67-7.61 (m, 2H), 6.36-6.32 (m, 1H), 4.15-4.05 (m, 1H), 3.81-3.73 (m, 1H), 3.72-3.63 (m, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 321.0, tR (min, Method c) = 1.989 [α]20 ,D = 1.43 (c = 0.7 mg/mL, CH3OH). Step 4: (R)-2-amino-3-(3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000093_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1H-pyrazol-1-yl)- 5-fluorobenzamido)propanoate (150 mg, 0.28 mmol) and HBr in AcOH (3 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was diluted with H2O (20 mL). The precipitate was filtered and dried to give (R)-2-amino-3- (3-(3-ethyl-1H-pyrazol-1-yl)-5-fluorobenzamido)propanoic acid (60 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.06-8.95 (m, 1H), 8.46 (d, J = 2.4 Hz, 1H), 8.16-8.10 (m, 1H), 7.83-7.78 (m, 1H), 7.54-7.49 (m, 1H), 6.41 (d, J = 2.4 Hz, 1H), 3.80-3.71 (m, 1H), 3.56- 3.45 (m, 2H), 2.63 (q, J=7.6 Hz, 2H), 1.20 (t, J=7.6 Hz, 3H). COOH and NH3 is not seen. LC-MS (MH+): m/z = 321.0, tR (min, Method c) = 0.89 [α]20,D = 20.0 (c = 1.0 mg/mL, CH3OH). Compound 1f (R)-2-amino-3-(3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethyl-1H- imidazol-1-yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000094_0001
Step 1: 3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid and 3-(4-ethyl-1H-imidazol-1- yl)-5-fluorobenzoic acid
Figure imgf000094_0002
To a soluion of 5-ethyl-1H-imidazole (500 mg, 5.20 mmol) and methyl 3-bromo-5- fluorobenzoate (1.21 g, 5.20 mmol) in DMF (10 mL) were added Cs2CO3 (6.78 g, 20.81 mmol), CuI (99 mg, 0.52 mmol), L-Proline (120 mg, 1.04 mmol) and the mixture was stirred at 130°C for 16 hours under N2. The reaction mixture was quenched with water (30 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (2M), extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The sample was purified by preparation HPLC (Method CC). The residue was further purified by SFC separation (Instrument: Berger, MULTIGR AM-II; Column: Chiralpak AD 250 × 30 mm I.D.5 μm; Mobile phase: supercritical CO2/IPA (0.1% NH3`H2O, v%) = 65/35; Flow Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give 3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid (70 mg) and 3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid (700 mg). 3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid: 1H NMR (400 MHz, CD3OD) δ 8.41 (br s, 1H), 7.95-7.895 (m, 2H), 7.62-7.52 (m, 1H), 7.18 (br s, 1H), 2.61 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). 3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid : 1H NMR (400 MHz, CD3OD) δ 8.34 (s, 1H), 7.99 (s, 1H), 7.73-7.68 (m, 1H), 7.67-7.62 (m, 1H), 7.46 (s, 1H), 2.65 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). Step 2: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoate
Figure imgf000095_0001
To a solution of 3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid (70 mg, 0.30 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (120 mg, 0.33 mmol, HCl salt) in DMF (5 mL) was added TBTU (144 mg, 0.45 mmol) and DIPEA (116 mg, 0.90 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (3 × 20 mL), dried over Na2SO4 and concentrated. The residue was purified by preparation HPLC (Method BB) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoate (40 mg). LC-MS (MH+): m/z = 545.1, tR (min, Method c_1.5min) = 0.832 Step 3: (R)-2-amino-3-(3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000096_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1H-imidazol-1- yl)-5-fluorobenzamido)propanoate (40 mg, 0.73 mmol) in the solution of 30 % HBr in AcOH (3 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated and submitted to lyophilization. The residue was washed with AcOH/MTBE=1/2 (9 mL) and filtered, the filtration cake was washed with MTBE (2 mL) and collected to give (R)- 2-amino-3-(3-(5-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid (13 mg, 35.6% yield) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 9.16 (br s, 1H), 9.00 (t, J = 5.6Hz, 1H), 8.43 (br s, 3H), 7.98-7.87 (m, 3H), 7.58 (s, 1H), 4.12-4.08 (m, 1H), 3.83-3.74 (m, 1H), 3.71-3.63 (m, 1H), 2.50 (q, J = 7.6 Hz, 2H), 1.10 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 321.0, tR (min, Method D) = 1.044 [α]20,D = -4.0 (c = 1.0 mg/mL, CH3OH). Compound 1g (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethyl-1H- imidazol-1-yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000097_0001
Step 1: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoate
Figure imgf000097_0002
To a solution of 3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzoic acid (700 mg, 2.99 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.20 g, 3.29 mmol, HCl salt) in DMF (10 mL) was added TBTU (1.44 g, 4.48 mmol) and DIPEA (1.16 g, 8.97 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (30 mL), and extracted with ethyl acetate (40 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 100%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoate (470 mg). LC-MS (MH+): m/z = 545.1, tR (min, Method c) = 0.824 Step 2: (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000098_0001
A solution of (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-1-yl)-5- fluorobenzamido)propanoic acid (470 mg, 0.86 mmol) in the solution of 30 % HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The sample was purified by preparative HPLC (Method AA) twice to give (R)-2-amino-3- (3-(4-ethyl-1H-imidazol-1-yl)-5-fluorobenzamido)propanoic acid (200 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 9.42-9.38 (m, 1H), 8.64 (br s, 3H), 8.36 (s, 1H), 8.20 (s, 1H), 8.05-7.95 (m, 1H), 7.90-7.80 (m, 1H), 4.17-4.13 (m, 1H), 3.87-3.77 (m, 2H), 2.88 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 321.0 tR (min, Method c) = 2.221 [α]20 ,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 1h (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-1H- imidazol-2-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000098_0002
Step 1: 2-bromo-1-ethyl-1H-imidazole
Figure imgf000099_0001
To a solution of 2-bromo-1H-imidazole (2 g, 13.6 mmol) in DMF (40 mL) was added K2CO3 (3.76 g, 27.2 mmol) and iodoethane (2.76 g, 17.7 mmol). The mixture was stirred at 50°C for 16 hours. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (40 mL × 2). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, eluent of 0~20% ethyl acetate/Petroleum ether) to give 2-bromo-1-ethyl-1H- imidazole (1.8 g). 1H NMR (400MHz, DMSO-d6) δ 7.37 (d, J = 1.6 Hz, 1H), 6.92 (d, J = 1.6 Hz, 1H), 3.94 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoate
Figure imgf000099_0002
A mixture of 2-bromo-1-ethyl-1H-imidazole (300 mg, 1.71 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (509 mg, 2.57 mmol), Pd(dppf)Cl2 (251 mg, 0.34 mmol), K3PO4 (728 mg, 3.43 mmol) in 1,4-dioxane (10 mL) and H2O (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 110°C for 16 hours under N2 atmosphere. The reaction mixture was concentrated. The residue was purified by column chromatography (SiO2, eluent of 0~40% ethyl acetate/Petroleum ether) to give methyl 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoate (270 mg). 1H NMR (400MHz, CD3OD) δ 8.04 (t, J = 1.2 Hz, 1H), 7.85-7.80 (m, 1H), 7.64-7.59 (m, 1H), 7.33 (d, J = 1.2 Hz, 1H), 7.10 (d, J = 1.2 Hz, 1H), 4.15 (q, J = 7.6 Hz, 2H), 3.95 (s, 3H), 1.40 (t, J = 7.6 Hz, 3H). Step 3: 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoic acid
Figure imgf000100_0001
To a solution of methyl 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoate (270 mg, 1.09 mmol) in a mixture of MeOH (8 mL) and H2O (4 mL) was added LiOH.H2O (137 mg, 3.26 mmol). The mixture was stirred at 20°C for 16 hours. To the reaction mixture was added 6M HCl to pH=2~3, and then concentrated to give 3-(1-ethyl-1H-imidazol-2-yl)-5- fluorobenzoic acid (600 mg, crude). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-2-yl)-5- fluorobenzamido)propanoate
Figure imgf000100_0002
To a solution of 3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzoic acid (600 mg, crude) in DMF (8 mL) was added (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (841 mg, 2.56 mmol, HCl salt), HATU (974 mg, 2.56 mmol) and DIPEA (331 mg, 2.56 mmol). The mixture was stirred at 20°C for 16 hours. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 2). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, eluent of 0~80% ethyl acetate/Petroleum ether) to give a yellow oil. The yellow oil was diluted with ethyl acetate (50 mL) and washed with brine (25 mL × 2). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was further purified by Preparative HPLC (Method BB) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-2-yl)-5- fluorobenzamido)propanoate (190 mg). 1H NMR (400MHz, CDCl3) δ 7.71 (br s, 1H), 7.48-7.28 (m, 13H), 7.15 (d, J=1.2 Hz, 1H), 7.06 (d, J=1.2 Hz, 1H), 6.06 (br s, 1H), 5.20 (s, 2H), 5.11 (s, 2H), 4.61-4.59 (m, 1H), 4.08 (q, J=7.2 Hz, 2H), 3.92-3.82 (m, 2H), 1.43 (t, J=7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000101_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-2-yl)- 5-fluorobenzamido)propanoate (190 mg, 0.35 mmol), 30% HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by Preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(1-ethyl-1H- imidazol-2-yl)-5-fluorobenzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400MHz, CD3OD) δ 8.11 (m, 1H), 8.03-8.00 (m, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.81-7.77 (m, 1H), 7.75 (d, J = 2.0 Hz, 1H), 4.35-4.29 (m, 3H), 4.08-4.02 (m, 1H), 3.92-3.87 (m, 1H), 1.50 (t, J = 7.2 Hz, 3H). NH and COOH were not observed. LC-MS (MH+): m/z = 321.0 tR (min, Method F) = 2.013 [α]20,D = +8.0 (c = 1.0 mg/mL, CH3OH). Compound 1i (R)-2-amino-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethyl-2- methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000102_0001
Step 1: methyl 3-(but-1-yn-1-yl)-5-fluorobenzoate
Figure imgf000102_0002
To a solution of methyl 3-bromo-5-fluorobenzoate (2.0 g, 8.58 mmol) in DMF (30 mL) was bubbled but-1-yne (929 mg, 17.16 mmol) (15 psi) at 0 °C. Then Pd(PPh3)2Cl2 (301 mg, 0.43 mmol), CuI (490 mg, 2.57 mmol) and TEA (2.17 g, 21.46 mmol) was added. The mixture was stirred at 80 °C for 16 hours in a sealed tube. The mixture was diluted with water (40 mL) and extracted with ethyl acetate (40 mL × 3). The combined organic layer was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-(but- 1-yn-1-yl)-5-fluorobenzoate (1.7 g). 1H NMR (400 MHz, CDCl3) δ 7.87-7.84 (m, 1H), 7.64-7.59 (m, 1H), 7.28-7.24 (m, 1H), 3.92 (s, 3H), 2.43 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). Step 2: methyl 3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzoate
Figure imgf000103_0001
To a solution of methyl 3-(but-1-yn-1-yl)-5-fluorobenzoate (400 mg, 1.94 mmol) in MeCN (10 mL) was added iodosylbenzene (768 mg, 3.49 mmol) and 1,1,1- trifluoro-N-(trifluoromethylsulfonyl)methanesulfonamide (818 mg, 2.91 mmol) at 0 °C. The mixture was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(5-ethyl-2-methyloxazol-4-yl)-5- fluorobenzoate (70 mg). 1H NMR (400 MHz, CDCl3) δ 8.07-8.05 (m, 1H), 7.67-7.61 (m, 1H), 7.58-7.53 (m, 1H), 3.95 (s, 3H), 2.90 (q, J = 7.6 Hz, 2H), 2.49 (s, 3H), 1.33 (t, J = 7.6 Hz, 3H). Step 3: 3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000103_0002
To a solution of methyl 3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzoate (100 mg, 0.38 mmol) in MeOH (5 mL) was added LiOH.H2O (48 mg, 1.14 mmol) in H2O (1 mL). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (10 mL) and acidified with aqueous 2N HCl to adjust pH to 4 and extracted with ethyl acetate (10 mL × 3). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-(5-ethyl-2- methyloxazol-4-yl)-5-fluorobenzoic acid (90 mg). 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 2.93 (q, J = 7.6 Hz, 2H), 2.66 (s, 3H), 1.36 (t, J = 7.6 Hz, 3H). Step 4. (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000104_0001
To a solution of 3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzoic acid (90 mg, 0.36 mmol) in DMF (5 mL) was added TBTU (174 mg, 0.54 mmol), DIPEA (140 mg, 1.08 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (132 mg, 0.36 mmol, HCl salt). The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5- fluorobenzamido)propanoate (150 mg). 1H NMR (400 MHz, CDCl3) δ 7.76-7.73 (m, 1H), 7.48-7.43 (m, 1H), 7.39-7.27 (m, 11H), 6.82 (br s, 1H), 5.96 (br s, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.65-4.56 (m, 1H), 3.96-3.81 (m, 2H), 2.89 (q, J = 7.6 Hz, 2H), 2.47 (s, 3H), 1.32 (t, J = 7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000105_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-2-methyloxazol-4- yl)-5-fluorobenzamido)propanoate (140 mg, 0.25 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (6 mL) and washed with MTBE (6 mL × 2). The organic layer was lyophilized to give (R)-2-amino-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid (70 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.90-8.86 (m, 1H), 8.32 (br, 3H), 7.95-7.93 (m, 1H), 7.63- 7.57 (m, 1H), 7.57-7.52 (m, 1H), 4.15-4.08 (m, 1H), 3.81-3.79 (m, 2H), 2.92 (q, J = 7.6 Hz, 2H), 2.43 (s, 3H), 1.23 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 336.0 tR (min, Method c) = 1.094 [α]20,D = 2.5, (c = 3.7 mg/mL, MeOH). Compound 1j (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisoxazol-4- yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000106_0001
Step 1: methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate
Figure imgf000106_0002
To a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (877 mg, 4.43 mmol), 4-bromo-5-ethyl-isoxazole (0.6 g, 3.41 mmol) and Na2CO3 (723 mg, 6.82 mmol) in a mixture of H2O (4 mL) and dioxane (16 mL) was added Pd(dppf)Cl2.CH2Cl2 (557 mg, 0.68 mmol) under N2. The mixture was purged with N2, sealed and heated at 120°C for 40 minutes in the microwave. The mixture was concentrated. The residue was diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 5%) to give methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.36 (s, 1H), 7.85-7.81 (m, 1H), 7.71-7.63 (m, 1H), 7.28-7.20 (m, 1H), 3.94 (s, 3H), 2.95 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 2: 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000107_0001
A mixture of methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate (0.125 g, 0.5 mmol) and LiOH.H2O (42 mg, 1.0 mmol) in a mixture of THF (2 mL) and H2O (2 mL) was stirred at 25°C for 0.5 hour. The mixture was diluted with H2O (20 mL). The aqueous phase was adjusted to pH=3~4 with sat. aq. KHSO4 at 0°C and extracted with ethyl acetate (10 mL × 2). The organic phase was dried and concentrated to give 3-(5-ethylisoxazol-4-yl)-5- fluorobenzoic acid (0.11 g, crude). LC-MS (MH+): m/z = 236.0 tR (min, Method c) = 0.685 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000107_0002
A mixture of 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid (0.11 g, crude), (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (171 mg, 0.47 mmol, HCl salt), DIPEA (302 mg, 2.34 mmol) and TBTU (225 mg, 0.7 mmol) in DMF (5 mL) was stirred at 25°C for 16 hours. The mixture was poured into H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (0.12 g). 1H NMR (400MHz, CDCl3) δ 8.35 (s, 1H), 7.53 (s, 1H), 7.42-7.27 (m, 11H), 7.23-7.15 (m, 1H), 7.05 (br s, 1H), 5.97 (br s, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.67-4.52 (m, 1H), 3.99-3.87 (m, 1H), 3.86-3.76 (m, 1H), 2.95 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000108_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (60 mg, 0.11 mmol) and HBr in AcOH (2 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by Preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.14 (br s, 1H), 8.98 (s, 1H), 8.52 (br s, 3H), 7.91 (s, 1H), 7.71-7.64 (m, 1H), 7.60-7.54 (m, 1H), 4.13-4.06 (m, 1H), 3.84-3.72 (m, 2H), 3.02 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 322.1 tR (min, Method c) = 0.995 [α]20,D = 6.0 (c = 0.5 mg/mL, CH3OH). Compound 1k (R)-2-amino-3-(3-(4-ethylisoxazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(4-ethylisoxazol-5- yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000109_0001
To a solution of 3-bromo-5-fluorobenzoic acid (5.0 g, 22.8 mmol) in THF (100 mL) was added n-BuLi (2.5 M, 19.2 mL) at -78°C under N2. The mixture was stirred at -78°C for 0.5 hour. N-methoxy-N-methylbutyramide (2.99 g, 22.8 mmol, 2.2 mL) in THF (10 mL) was added to the reaction mixture at -78°C, and the mixture was stirred at -78°C for 5 hours. The mixture was quenched with NH4Cl (20 mL) and the aqueous phase was separated and adjusted to pH=3~4 with aq. KHSO4. The mixture was extracted with MTBE (20 mL × 2). The organic phase was dried over Na2SO4 and concentrated to give 3- butyryl-5-fluorobenzoic acid (4.5 g, crude). Step 2: methyl 3-butyryl-5-fluorobenzoate
Figure imgf000110_0001
To a mixture of 3-butanoyl-5-fluoro-benzoic acid (4.5 g, crude) in MeOH (50 mL) was added SOCl2 (5.09 g, 42.8 mmol, 3.1 mL) at 0°C. The mixture was stirred at 70°C for 16 hours. The mixture was concentrated and the residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-butyryl-5-fluorobenzoate (1.0 g). 1H NMR (400MHz, CDCl3) δ 8.37 (m, 1H), 7.93-7.87 (m, 1H), 7.85-7.81 (m, 1H), 3.96 (s, 3H), 2.97 (t, J = 7.2 Hz, 2H), 1.82-1.73 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). Step 3: (E)-methyl 3-(2-((dimethylamino)methylene)butanoyl)-5-fluorobenzoate
Figure imgf000110_0002
A mixture of methyl 3-butyryl-5-fluorobenzoate (0.5 g, 2.23 mmol) and DMF-DMA (17.9 g, 150 mmol) were stirred at 110°C for 16 hours. The mixture was concentrated to give (E)-methyl 3-(2-((dimethylamino)methylene)butanoyl)-5-fluorobenzoate (0.6 g, crude). LC-MS (MH+): m/z = 279.9 tR (min, Method c) = 0.735 Step 4: methyl 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoate
Figure imgf000110_0003
A mixture of (E)-methyl 3-(2-((dimethylamino)methylene)butanoyl)-5-fluorobenzoate (0.6 g, crude) and NH2OH.HCl (0.35 g, 5.04 mmol) in EtOH (10 mL) was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%). The crude product was purified by Preparative-HPLC (HCl) to give methyl 3-(4-ethylisoxazol-5-yl)- 5-fluorobenzoate (0.1 g, crude). 1H NMR (400MHz, CDCl3) δ 8.25 (s, 1H), 8.18 (m, 1H), 7.83-7.75 (m, 1H), 7.66-7.59 (m, 1H), 3.97 (s, 3H), 2.73 (q, J = 7.6 Hz, 2H), 1.32 (t, J = 7.6 Hz, 3H). Step 5: 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoic acid
Figure imgf000111_0001
A mixture of methyl 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoate (50 mg, crude) and LiOH.H2O (17 mg, 0.4 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was stirred at 25°C for 0.5 hour. The mixture was adjusted to pH=3~4 with aq. KHSO4. The mixture was concentrated to give 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoic acid (47 mg, crude). LC-MS (MH+): m/z = 235.9 tR (min, Method c) = 0.695 Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylisoxazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000111_0002
A mixture of 3-(4-ethylisoxazol-5-yl)-5-fluorobenzoic acid (47 mg, crude), (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (73 mg, 0.2 mmol, HCl salt), TBTU (128 mg, 0.4 mmol) and DIPEA (129 mg, 1.0 mmol) in DMF (5 mL) was stirred at 25°C for 2 hours. The mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (10 mL × 2). The organic phase was washed with brine (10 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-ethylisoxazol-5-yl)-5- fluorobenzamido)propanoate (50 mg). 1H NMR (400MHz, CDCl3) δ 8.25 (s, 1H), 7.83 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.38-7.28 (m, 10H), 7.04 (br s, 1H), 6.00-5.95 (m, 1H), 5.21 (s, 2H), 5.11 (s, 2H), 4.66-4.57 (m, 1H), 3.98-3.89 (m, 1H), 3.87-3.79 (m, 1H), 2.71 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). Step 7: (R)-2-amino-3-(3-(4-ethylisoxazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000112_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylisoxazol-5-yl)-5- fluorobenzamido)propanoate (50 mg, 0.09 mmol) and HBr in AcOH (2 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by Preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(4-ethylisoxazol-5-yl)-5- fluorobenzamido)propanoic acid (18 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.20-9.14 (m, 1H), 8.68 (s, 1H), 8.49 (br s, 2H), 8.04 (m, 1H), 7.91-7.83 (m, 1H), 7.74-7.66 (m, 1H), 4.12-4.04 (m, 1H), 3.86-3.70 (m, 2H), 2.68 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 322.0 tR (min, Method c) = 1.307 [α]20,D = 7.5 (c = 0.4 mg/mL, CH3OH). Compound 1l (R)-2-amino-3-(3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-fluoro-5-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid is shown below
Figure imgf000113_0001
Step 1: methyl 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate
Figure imgf000113_0002
To a solution of 4-bromo-1,3,5-trimethyl-1H-pyrazole (500 mg, 2.64 mmol) and (3- fluoro-5-(methoxycarbonyl)phenyl)boronic acid (524 mg, 2.64 mmol) in dioxane (30 mL) was added Pd(dtbpf)Cl2 (173 mg, 0.26 mmol) and K3PO4 (1.68 g, 7.93 mmol) under N2. The mixture was stirred at 100°C for 16 hours. The reaction was concentrated. The residue was purified by Combi Flash (silica gel, Petroleum ether: ethyl acetate, ethyl acetate from 0% to 40%) to give methyl 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzoate (130 mg). 1H NMR (400MHz, CDCl3) δ 7.71 (m, 1H), 7.65-7.59 (m, 1H), 7.16-7.09 (m, 1H), 3.93 (s, 3H), 3.78 (s, 3H), 2.25 (s, 3H), 2.24 (s, 3H). Step 2: 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
Figure imgf000114_0001
To a solution of methyl 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (130 mg, 0.50 mmol) in a mixture of MeOH (10 mL) and H2O (2 mL) was added LiOH.H2O (62 mg, 1.49 mmol) under N2. The mixture was stirred at 20°C for 14 hours. The reaction was added 2N aq. HCl to pH=3~4, and extracted with ethyl acetate (30 mL × 3). The organic layers were concentrated to give 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (100 mg). 1H NMR (400MHz, CDCl3) δ 7.78 (m, 1H), 7.71-7.66 (m, 1H), 7.20-7.15 (m, 1H), 3.81 (s, 3H), 2.27 (s, 6H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
Figure imgf000114_0002
To a solution of 3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (100 mg, 0.40 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (176 mg, 0.48 mmol, HCl salt) in DMF (10 mL) was added T3P (0.4 mL, 50% in DMF) and DIPEA (0.2 mL,1.15 mmol) under N2. The mixture was stirred at 20°C for 16 hours. The reaction was added water (50 mL) and extracted with ethyl acetate (30 ml × 3). The organic layers were concentrated. The residue was purified by Combi Flash (silica gel, Petroleum ether: ethyl acetate, ethyl acetate from 0% to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoate (180 mg). LC-MS (MH+): m/z = 559.2 tR (min, Method c) = 0.865 Step 4: (R)-2-amino-3-(3-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid
Figure imgf000115_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1,3,5-trimethyl- 1H-pyrazol-4-yl)benzamido)propanoate (180 mg, 0.32 mmol) in 30% HBr in AcOH (10 mL) was stirred at 50°C for 14 hours. The reaction was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-fluoro-5-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid (85 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.06-9.01 (m, 1H), 8.57 (br s, 3H), 7.70-7.61 (m, 2H), 7.33- 7.23 (m, 1H), 4.13-4.06 (m, 1H), 3.80-3.70 (m, 2H), 3.72 (s, 3H), 2.23 (s, 3H), 2.15 (s, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method c) = 0.848 [α]20 ,D = 5.0 (c = 1.0 mg/mL, CH3OH). Compound 1m (R)-2-amino-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(4-ethyl-2- methyloxazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000116_0001
Step 1: methyl 3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzoate
Figure imgf000116_0002
To a solution of 5-bromo-4-ethyl-2-methyloxazole (120 mg, 0.63 mmol) and (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (125 mg, 0.63 mmol) in a mixture of dioxane (5 mL) and H2O (0.5 mL) was added Pd(dppf)Cl2.CH2Cl2 (52 mg, 0.063 mmol) and Na2CO3 (201 mg, 1.90 mmol) under N2 atmosphere. The mixture was stirred at 120 °C for 40 min under microwave. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzoate (60 mg).1H NMR (400 MHz, CDCl3) δ 8.05-8.02 (m, 1H), 7.66-7.62 (m, 1H), 7.47-7.42 (m, 1H), 3.97 (s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 2.52 (s, 3H), 1.33 (t, J = 7.6 Hz, 3H). Step 2: 3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzoic acid
Figure imgf000117_0001
To a solution of methyl 3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzoate (60 mg, 0.23 mmol) in MeOH (5 mL) was added LiOH.H2O (29 mg, 0.68 mmol) in H2O (2 mL). The mixture was stirred at 25 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (10 mL) and washed with ethyl acetate (15 mL). The organic layer was discarded. The aqueous layer was aicidied with aqueous 2N HCl solution to adjust pH to 4 and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give 3-(4-ethyl-2-methyloxazol-5-yl)-5- fluorobenzoic acid (35 mg).1H NMR (400 MHz, DMSO-d6) δ 13.74-13.01 (m, 1H), 7.94- 7.91 (m, 1H), 7.66-7.51 (m, 2H), 2.71 (q, J = 7.6 Hz, 2H), 2.46 (s, 3H), 1.21 (t, J = 7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000117_0002
To a solution of 3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzoic acid (35 mg, 0.14 mmol) in DMF (4 mL) was added TBTU (68 mg, 0.21 mmol), DIPEA (54 mg, 0.42 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (51 mg, 0.14 mmol, HCl salt). The mixture was stirred at 25 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-2-methyloxazol-5- yl)-5-fluorobenzamido)propanoate (60 mg). 1H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.39-7.31 (m, 12H), 6.85 (br s, 1H), 5.95-5.93 (m, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 4.65-4.56 (m, 1H), 3.99-3.90 (m, 1H), 3.85 - 3.80 (m, 1H), 2.77 (q, J = 7.6 Hz, 2H), 2.51 (s, 3H), 1.31 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000118_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-2-methyloxazol-5- yl)-5-fluorobenzamido)propanoate (60 mg, 0.11 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was purified by Preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethyl-2-methyloxazol-5-yl)- 5-fluorobenzamido)propanoic acid (17 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.15-9.05 (m, 1H), 8.55 (br s, 3H), 7.89 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 4.18-4.10 (m, 1H), 3.76-3.75 (m, 2H), 2.72 (q, J = 7.6 Hz, 2H), 2.46 (s, 3H), 1.21 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 336.0 tR (min, Method D) = 1.535 [α]20,D = 2.5, (c = 1.6 mg/mL, MeOH). Compound 1n (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(5-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000119_0001
Step 1: 4-Bromo-5-ethyl-1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H- pyrazole
Figure imgf000119_0002
To a solution of 4-bromo-5-ethyl-1H-pyrazole (300 mg, 1.71 mmol) in DMF (5 mL) were added NaH (82 mg, 2.06 mmol, 60% in mineral oil) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. CH3I (268 mg, 1.89 mmol) was added to the reaction mixture and stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give a mixture of 4-bromo-5-ethyl-1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H-pyrazole (300 mg, crude). Step 2: Methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate and methyl 3-(3- ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate
Figure imgf000119_0003
To a solution of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (300 mg, 1.52 mmol) in dioxane (10 mL) were added a mixture of 4-bromo-5-ethyl-1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H-pyrazole (300 mg, crude). The reaction mixture was stirred at 100°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give the mixture of two isomers (200 mg), which were seperated by SFC separation to give methyl 3-(3-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro- benzoate (70 mg), and methyl 3-(5-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate (90 mg). Methyl 3-(3-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate: 1H NMR (400 MHz, CDCl3) δ 7.84 (t, J=1.2 Hz, 1H), 7.61 - 7.56 (m, 1H), 7.47 (s, 1H), 7.27 - 7.24 (m, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 2.80 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H) Methyl 3-(5-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate: 1H NMR (400 MHz, CDCl3) δ 7.84 (t, J=1.2 Hz, 1H), 7.62-7.59 (m, 1H), 7.58 (s, 1H), 7.26 - 7.22 (m, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 2.80 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). Step 3: 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid
Figure imgf000120_0001
To a solution of methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate (90 mg, 0.34 mmol) in a mixture of MeOH (3 mL) and H2O (2 mL) were added LiOH.H2O (43 mg, 1.03 mmol). The reaction mixture was stirred at 25°C for 1 hour. The MeOH was removed. The residue was added 1M HCl to adjust pH =3. The reaction mixture was extracted with ethyl acetate (10 mL × 3), and the combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(5-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzoic acid (70 mg). LC-MS (MH+): m/z = 249.1 tR (min, Method c) = 0.706 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4- yl)-5-fluorobenzamido)propanoate
Figure imgf000121_0001
To a solution of 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid (70 mg, 0.28 mmol) in DMF (5 mL) were added (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (123 mg, 0.34 mmol, HCl salt), TBTU (136 mg, 0.44 mmol) and DIPEA (109 mg, 0.85 mmol). The reaction mixture was stirred at 20°C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 60% to 70%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzamido)propanoate (100 mg). LC-MS (MH+): m/z = 559.3 tR (min, Method c) = 0.882 Step 5: (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000121_0002
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1-methyl-1H- pyrazol-4-yl)-5-fluorobenzamido)propanoate (100 mg, 0.18 mmol) in 30% HBr in AcOH (5 mL) were stirred at 50°C for 16 hours. The solvent was removed. The residue was purified by preparative HPLC (HCl condition) to give (R)-2-amino-3-(3-(5-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid (65 mg) as 2HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.04 (br s, 1H), 8.51 (br, 2H), 7.77 (s, 1H), 7.70 (s, 1H), 7.60 - 7.54 (m, 1H), 7.39 - 7.33 (m, 1H), 4.11 - 4.04 (m, 1H), 3.81 (s, 3H), 3.79 - 3.69 (m, 2H), 2.81 (q, J = 7.6 Hz, 2H), 1.15 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 335.0 tR (min, CD05) = 0.967 [α]20,D = +4.0 (c = 1.0 mg/mL, CH3OH). Compound 1o (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(1,4-diethyl-1H- pyrazol-3-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000122_0001
Step 1: 3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid
Figure imgf000122_0002
To a solution of methyl 3-(1,4-diethylpyrazol-3-yl)-5-fluoro-benzoate (as prepared in compound 1u, 50 mg, 0.18 mmol) in a mixture of MeOH (3 mL) and H2O (3 mL) were added LiOH (13 mg, 0.54 mmol). The reaction mixture was stirred at 20°C for 2 hours. The MeOH was removed. The residue was added 1M HCl to adjust pH =3. The reaction mixture was extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid (40 mg). LC-MS (MH+): m/z = 263.1 tR (min, Method c) = 0.755 Step 2: 3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoyl chloride
Figure imgf000123_0001
To a solution of 3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoic acid (40 mg, 0.15 mmol) in DCM (1 mL) were added oxalyl dichloride (0.5 mL). The reaction mixture was stirred at 20°C for 2 hours. The solvent was removed to give 3-(1,4-diethyl-1H-pyrazol-3-yl)-5- fluorobenzoyl chloride (40 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,4-diethyl-1H-pyrazol-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000123_0002
To a solution of 3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoyl chloride (40 mg, 0.14 mmol) in DCM (3 mL) were added TEA (43 mg, 0.43 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (57 mg, 0.16 mmol, HCl salt). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoate (50 mg). LC-MS (MH+): m/z = 573.3 tR (min, Method c) = 0.904 Step 4: (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000124_0001
A solution of benzyl (2R)-2-(benzyloxycarbonylamino)-3-[[3-(1,4-diethylpyrazol-3-yl)-5- fluoro-benzoyl]amino]propanoate (50 mg, 0.08 mmol) in 30% HBr in AcOH (3 mL) was stirred at 50°C for 16 hours. The solvent was removed. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-3-yl)-5- fluorobenzamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ9.01 (m, 1H), 8.54 (br s, 3H), 7.98 (s, 1H), 7.72-7.68 (m, 2H), 7.57-7.52 (m, 1H), 4.19-4.07 (m, 3H), 3.80-3.78 (m, 2H), 2.64 (q, J=7.6 Hz, 2H), 1.40 (t, J=7.2 Hz, 3H), 1.17 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 349.0 tR (min, Method c) = 1.238 [α]20 ,D = -2.0 (c = 1.0 mg/mL, CH3OH). Compound 1p (R)-2-amino-3-(3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(5-ethyl-3- methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000125_0001
Step 1: 3-ethyl-5-methylisoxazole and 5-ethyl-3-methylisoxazole
Figure imgf000125_0002
To a solution of NH2OH.HCl (2.5 g, 36.0 mmol) in a mixture of MeOH (10 mL) and H2O (20 mL) were added Na2CO3 (1.91 g, 17.99 mmol). When bubbles were absent, hexane- 2,4-dione (4.93 g, 43.2 mmol, 1.2 eq) was added dropwise. The mixture was heated at 100°C for 16 hours. The reaction mixture was extracted with DCM (40 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated to give a mixture of 3-ethyl-5-methylisoxazole and 5-ethyl-3- methylisoxazole (4.0 g, crude). Step 2: 4-bromo-3-ethyl-5-methylisoxazole and 4-bromo-5-ethyl-3-methylisoxazole
Figure imgf000126_0001
A mixture of 3-ethyl-5-methylisoxazole and 5-ethyl-3-methylisoxazole (4.0 g, crude) dissolved in DMF (20 mL) was added NBS (7.05 g, 39.59 mmol) and the reaction mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give a mixture of 4-bromo-3-ethyl-5-methylisoxazole and 4-bromo-5- ethyl-3-methylisoxazole (2.5 g, crude). LC-MS (MH+): m/z = 192.0 tR (min, Method c) = 0.763 Step 3: methyl 3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzoate and methyl 3-(5- ethyl-3-methylisoxazol-4-yl)-5-fluorobenzoate
Figure imgf000126_0002
To a solution of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.04 g, 5.26 mmol) in dioxane (5 mL) and H2O (1 mL) were added a mixture of 4-bromo-3-ethyl-5- methylisoxazole and 4-bromo-5-ethyl-3-methylisoxazole (1.0 g, 5.26 mmol), Pd(dppf)Cl2 (385 mg, 0.53 mmol) and Na2CO3 (1.67 g, 15.79 mmol). The reaction mixture was stirred at 120°C for 40 minutes under microwave. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 10%) to give methyl 3-(3-ethyl-5-methylisoxazol-4-yl)-5- fluorobenzoate and methyl 3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzoate (1.1 g). LC-MS (MH+): m/z = 264.2 tR (min, Method c) = 1.137 Step 4: 3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzoic acid and 3-(5-ethyl-3- methylisoxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000127_0001
To a solution of a mixture of methyl 3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzoate and methyl 3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzoate (1.1 g, 4.18 mmol) in a mixture of THF (10 mL) and H2O (4 mL) was added LiOH.H2O (351 mg, 8.36 mmol) and the mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzoic acid and 3-(5-ethyl-3- methylisoxazol-4-yl)-5-fluorobenzoic acid (1.0 g). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-3-methylisoxazol-4-yl)- 5-fluorobenzamido)propanoate and (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-((3-(3- ethyl-5-methylisoxazol-4-yl)-5-fluorobenzyl)amino)propanoate
Figure imgf000128_0001
To a solution of a mixture of 3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzoic acid and 3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzoic acid (1 g, 4.01 mmol) and (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.61 g, 4.41 mmol, HCl salt) in DMF (10 mL) was added TBTU (1.93 g, 6.02 mmol) and DIPEA (1.56 g, 12.04 mmol). The reaction mixture was stirred at 20°C for 15 hours. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) twice and further purified by SFC separation (Instrument: Berger, MULTIGR SFC-10; Column: DAICEL CHIRALCEL OJ 250 × 50 mm I.D.10 μm; Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, v%) = 75/25; Flow Rate: 200 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-3- methylisoxazol-4-yl)-5-fluorobenzamido)propanoate (700 mg) and (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-((3-(3-ethyl-5-methylisoxazol-4-yl)-5- fluorobenzyl)amino)propanoate (280 mg). (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-3-methylisoxazol-4-yl)-5- fluorobenzamido)propanoate: 1H NMR (400 MHz, CDCl3) δ 7.42-7.40 (m, 1H), 7.39-7.28 (m, 11H), 7.11-7.04 (m, 2H), 5.98 (br s, 1H), 5.22 (s, 2H), 5.10 (s, 2H), 4.64-4.54 (m, 1H), 4.00-3.91 (m, 1H), 3.85-3.75 (m, 1H), 2.75 (q, J = 7.6 Hz, 2H), 2.26 (s, 3H), 1.27 (t, J = 7.6 Hz, 3H). (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-((3-(3-ethyl-5-methylisoxazol-4-yl)-5- fluorobenzyl)amino)propanoate: 1H NMR (400 MHz, CDCl3) δ 7.45-7.28 (m, 12H), 7.15- 7.05 (m, 2H), 5.99 (br s, 1H), 5.21(s, 2H), 5.10 (s, 2H), 4.65-4.55 (m, 1H), 4.02-3.90 (m, 1H), 3.85-3.78 (m, 1H), 2.66 (q, J = 7.6 Hz, 2H), 2.38 (s, 3H), 1.18 (t, J = 7.6 Hz, 3H). Step: 6 (R)-2-amino-3-(3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000129_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-3-methylisoxazol- 4-yl)-5-fluorobenzamido)propanoate (200 mg, 0.36 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 2 hours. The reaction mixture was concentrated. The sample was purified by Prep-HPLC (Method CC). The sample was further purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(5-ethyl-3-methylisoxazol-4-yl)-5- fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.11-9.05 (m, 1H), 8.52 (br s, 3H), 7.78-7.70 (m, 2H), 7.52-7.46 (m, 1H), 4.17-4.07 (m, 1H), 3.85-3.72 (m, 2H), 2.80 (q, J = 7.6 Hz, 2H), 2.24 (s, 3H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 336.0 tR (min, Method c) = 1.168 [α]20,D = +5.0 (c = 2.0 mg/mL, CH3OH). Compound 1q (R)-2-amino-3-(3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid Preparation of (R)-2-amino-3-(3-(3-ethyl-5-methylisoxazol-4-yl)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000130_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-((3-(3-ethyl-5-methylisoxazol- 4-yl)-5-fluorobenzyl)amino)propanoate (280 mg, 0.50 mmol) in the solution of 30% HBr in AcOH (5 mL) was heated at 50°C for 2 hours. The reaction mixture was concentrated. The sample was purified by Prep-HPLC (Method CC), and further purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethyl-5-methylisoxazol-4-yl)-5- fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.06-8.95 (m, 1H), 8.45 (br s, 3H), 7.78-7.69 (m, 2H), 7.53-7.47 (m, 1H), 4.18-4.07 (m, 1H), 3.84-3.70 (m, 2H), 2.69 (q, J = 7.6 Hz, 2H), 2.41 (s, 3H), 1.07 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 336.0 tR (min, Method c) = 1.133 [α]20,D = +4.0 (c = 2.0 mg/mL, CH3OH). Compound 1r (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-4-methyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000131_0001
Step 1: 1-ethyl-4-methyl-1H-pyrazole
Figure imgf000131_0002
To a solution of 4-methyl-1H-pyrazole (2.0 g, 24.4 mmol) in DMF (10 mL) was added NaH (1.27 g, 31.7 mmol, 60% in mineral oil) at 0°C and stirred at 0°C for 0.5 hour. Then iodoethane (4.56 g, 29.2 mmol) was added at 0°C and stirred at 30°C for 3 hours. The reaction mixture was quenched with water (20 mL), and then extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 20% to 30%) to give 1-ethyl-4-methyl-1H-pyrazole (1.3 g). 1H NMR (400 MHz, CDCl3) δ 7.29 (s, 1H), 7.16 (s, 1H), 4.10 (q, J = 7.2 Hz, 2H), 2.07 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H). Step 2: 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H- pyrazole
Figure imgf000132_0001
To a solution of 1-ethyl-4-methyl-1H-pyrazole (650 mg, 5.90 mmol) in CHCl3 (10 mL) was added NBS (1.16 g, 6.49 mmol) and the reaction mixture was stirred at 30°C for 3 hours. The reaction mixture was quenched with water (10 mL), and then extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 10%) to give a mixture of 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1- ethyl-4-methyl-1H-pyrazole (260 mg, crude). LC-MS (MH+): m/z = 189.0 tR (min, Method c) = 0.734 Step 3: methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate and methyl 3-(1- ethyl-4-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate
Figure imgf000132_0002
To a solution of (3-fluoro-5-methoxycarbonyl-phenyl)boronic acid (272 mg, 1.38 mmol) in dioxane (5 mL) were added a mixture of 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H-pyrazole (260 mg, crude), Pd(dtbpf)Cl2 (90 mg, 0.14 mmol) and K3PO4 (876 mg, 4.13 mmol). The reaction was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 15%). The sample was further purified by Preparative HPLC (Method AA) to give methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (100 mg) and methyl 3-(1-ethyl-4-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate (10 mg). methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate: 1H NMR (400 MHz, CDCl3) δ 7.81-7.74 (m, 2H), 7.41 (s, 1H), 7.24-7.18 (m, 1H), 4.04 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 1.99 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H). methyl 3-(1-ethyl-4-methyl-1H-pyrazol-3-yl)-5-fluorobenzoate: LC-MS (MH+): m/z = 263.1 tR (min, Method c) = 0.851 Step 4: 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000133_0001
A mixture of 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (100 mg, 0.38 mmol) and LiOH.H2O (32 mg, 0.76 mmol) in a mixture of MeOH (4 mL) and H2O (2 mL) was stirred at 30°C for 1 hour. The reaction mixture was quenched with water (5 mL), and extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), and then extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (90 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate
Figure imgf000133_0002
To a solution of 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (90 mg, 0.36 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (145 mg, 0.40 mmol, HCl salt) in DMF (5 mL) was added TBTU (175 mg, 0.54 mmol) and DIPEA (141 mg, 1.09 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 50%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (140 mg). LC-MS (MH+): m/z = 559.3 tR (min, Method c) = 0.895 Step 6: (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000134_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (140 mg, 0.25 mmol) in 30 % HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The sample was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(1-ethyl- 4-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.18-9.11 (m, 1H), 8.58 (br s, 3H), 7.81 (d, J = 9.6 Hz, 1H), 7.74 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.39 (s, 1H), 4.13-4.07 (m, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.84-3.72 (m, 2H), 1.93 (s, 3H), 1.19 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method D) = 1.523 [α]20,D = -4.0 (c = 1.0 mg/mL, CH3OH). Compound 1s (R)-2-amino-3-(3-(3-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000135_0001
Step 1: 3-(3-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid
Figure imgf000135_0002
To a solution of methyl 3-(3-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate (70 mg, 0.27 mmol) in a mixture of MeOH (3 mL) and H2O (3 mL) were added LiOH.H2O (33.60 mg, 0.8 mmol). The reaction mixture was stirred at 20°C for 1hour. The MeOH was removed. To the residue was added 1M HCl to adjust pH =3, and the aqueous mixture was extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzoic acid (60 mg). LC-MS (MH+): m/z = 249.1 tR (min, Method c) = 0.706 Step 2: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1-methyl-1H-pyrazol-4- yl)-5-fluorobenzamido)propanoate
Figure imgf000136_0001
To a solution of 3-(3-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid (60 mg, 0.24 mmol) in DMF (5 mL) was added (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (106 mg, 0.29 mmol, HCl salt), TBTU (116 mg, 0.36mol) and DIPEA (94 mg, 0.73 mmol). The reaction mixture was stirred at 25°C for 3 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1-methyl- 1H-pyrazol-4-yl)-5-fluorobenzamido)propanoate (90 mg). LC-MS (MH+): m/z = 559.3 tR (min, Method c) = 0.881 Step 3: (R)-2-amino-3-(3-(3-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000136_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethyl-1-methyl-1H- pyrazol-4-yl)-5-fluorobenzamido)propanoate (90 mg, 0.16 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The solvent was removed. The residue was purified by Preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethyl-1-methyl-1H- pyrazol-4-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ9.07 (br s, 1H), 8.59 (br s, 3H), 8.05 (s, 1H), 7.82 (s, 1H), 7.59 (d, J = 10 Hz, 1H), 7.40 (d, J = 10.0 Hz, 1H), 4.17-4.08 (m, 1H), 3.82-3.78 (m, 5H), 2.75 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method D) = 1.487 [α]20 ,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 1t (R)-2-amino-3-(2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(2'-chloro-6'-ethyl-5- fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid is shown below
Figure imgf000137_0001
Step 1: Preparation of methyl 2'-bromo-6'-chloro-5-fluoro-[1,1'-biphenyl]-3-carboxylate
Figure imgf000137_0002
A mixture of 1-bromo-3-chloro-2-iodobenzene (500 mg, 1.58 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (312 mg, 1.58 mmol), Pd(PPh3)2Cl2 (111 mg, 0.16 mmol) and K2CO3 (653 mg, 4.73 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 15%) to give methyl 2'-bromo-6'-chloro-5-fluoro-[1,1'-biphenyl]-3- carboxylate (260 mg). 1H NMR (400 MHz, CDCl3) δ 7.82-7.76 (m, 1H), 7.74 (s, J = 1.2 Hz, 1H), 7.60 (dd, J = 8.0 Hz, 0.8 Hz, 1H), 7.45 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 7.24-7.14 (m, 2H), 3.94 (s, 3H). Step 2: methyl 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate
Figure imgf000138_0001
A mixture of methyl 2'-bromo-6'-chloro-5-fluoro-[1,1'-biphenyl]-3-carboxylate (240 mg, 0.70 mmol), triethylborane (1 M in hexane, 0.84 mL), Pd(dppf)Cl2 (51 mg, 0.07 mmol) and K2CO3 (290 mg, 2.10 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) twice to give methyl 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3- carboxylate (100 mg). 1H NMR (400 MHz, CDCl3) δ 7.78-7.73 (m, 1H), 7.70 (t, J = 1.6 Hz, 1H), 7.31 (dd, J = 7.2 Hz, 1.6 Hz, 1H), 7.30-7.25 (m, 1H), 7.21 (dd, J = 7.2 Hz, 1.6 Hz, 1H), 7.16-7.12 (m, 1H), 3.93 (s, 3H), 2.37 (q, J = 7.6 Hz, 2H), 1.04 (t, J = 7.6 Hz, 3H). Step 3: 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid
Figure imgf000139_0001
To a solution of methyl 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate (100 mg, 0.34 mmol) in a mixture of MeOH (4 mL) and H2O (2 mL) was added LiOH.H2O (29 mg, 0.68 mmol) and the mixture was stirred at 30 °C for 1 hour. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (90 mg). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-6'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000139_0002
To a solution of 2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (90 mg, 0.32 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (117 mg, 0.32 mmol, as HCl salt) in DMF (5 mL) was added TBTU (156 mg, 0.48 mmol) and DIPEA (125 mg, 0.97 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (110 mg). LC-MS (MH+): m/z = 589.2 tR (min, Method c) = 1.003 Step 5: (R)-2-amino-3-(2'-chloro-6'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoic acid
Figure imgf000140_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-6'-ethyl-5-fluoro- [1,1'-biphenyl]-3-ylcarboxamido)propanoate (110 mg, 0.19 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(2'-chloro- 6'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.00-8.92 (m, 1H), 8.35 (br s, 2H), 7.80-7.74 (m, 1H), 7.56 (s, 1H), 7.44-7.30 (m, 4H), 4.05-3.98 (m, 1H), 3.82-3.63 (m, 2H), 2.33 (q, J = 7.6 Hz, 2H), 0.97 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 365.0 tR (min, Method D) = 2.090 [α]20 ,D = +8.0 (c = 1.0 mg/mL, CH3OH). Compound 1u (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1,4-diethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000141_0001
Step 1: 1,4-diethyl-1H-pyrazole
Figure imgf000141_0002
To a solution of 4-ethyl-1H-pyrazole (1.0 g, 10.4 mmol) in DMF (10 mL) were added NaH (624 mg, 15.6 mmol, 60% in mineral oil) at 0°C. The reaction mixture was stirred at 0°C for 30 min. CH3CH2I (1.95 g, 12.5 mmol) was added to the reaction mixture and stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 10%) to give 1,4-diethyl-1H-pyrazole (1.2 g). 1H NMR (400 MHz, CDCl3) δ 7.32 (s, 1H), 7.17 (s, 1H), 4.12 (q, J=7.2 Hz, 2H), 2.48 (q, J=7.6 Hz, 2H), 1.46 (t, J=7.2 Hz, 3H), 1.19 (t, J=7.6 Hz, 3H). Step 2: 5-bromo-1,4-diethyl-1H-pyrazole and 3-bromo-1,4-diethyl-1H-pyrazole
Figure imgf000142_0001
To a solution of 1,4-diethylpyrazole (500 mg, 4.03 mmol) in CHCl3 (15 mL) were added NBS (860 mg, 4.83 mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give a mixture of 5-bromo-1,4-diethyl-1H-pyrazole and 3-bromo-1,4-diethyl-1H-pyrazole (600 mg,crude). LC-MS (MH+): m/z = 205.0 tR (min, Method c) = 0.796 Step 3: methyl 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoate and methyl 3-(1,4- diethyl-1H-pyrazol-3-yl)-5-fluorobenzoate
Figure imgf000142_0002
To a solution of 5-bromo-1,4-diethyl-1H-pyrazole and 3-bromo-1,4-diethyl-1H-pyrazole (500 mg, crude) in a mixture of dioxane (10 mL) and H2O (0.5 mL) were added (3-fluoro- 5-(methoxycarbonyl)phenyl)boronic acid (536 mg, 2.71 mmol), Pd(dtbpf)Cl2 (160 mg, 0.25 mmol) and K3PO4 (1.57 g, 7.39 mmol). The reaction mixture was stirred at 100°C for 16 hours under N2. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 10%). The crude product was further purified by Preparation HPLC (Method AA) to give methyl 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (300 mg) and methyl 3- (1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoate (50 mg). methyl 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoate: 1H NMR (400 MHz, CDCl3) δ7.84 - 7.80 (m, 2H), 7.51 (s, 1H), 7.22 (br s, 1H), 4.09 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 2.38 (q, J = 7.6 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H), 1.12 (t, J = 7.6 Hz, 3H). methyl 3-(1,4-diethyl-1H-pyrazol-3-yl)-5-fluorobenzoate: 1H NMR (400 MHz, CDCl3) δ8.17 - 8.13 (m, 1H), 7.69-7.58 (m, 2H), 7.28 (s, 1H), 4.19 (q, J = 7.2 Hz, 2H), 3.94 (s, 3H), 2.69 (q, J = 7.6 Hz, 2H), 1.53 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.6 Hz, 3H). Step 4: 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000143_0001
To a solution of methyl 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (150 mg, 0.54 mmol) in a mixture of MeOH (3 mL) and H2O (3 mL) were added LiOH.H2O (39 mg, 0.93 mmol). The reaction mixture was stirred at 20°C for 2 hours. The MeOH was removed. To the residue was added 1M HCl to adjust to pH =3 and the reaction mixture was extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1,4-diethyl-1H-pyrazol- 5-yl)-5-fluorobenzoic acid (120 mg) Step 5: 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoyl chloride
Figure imgf000144_0001
To a solution of 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (100 mg, 0.38 mmol) in DCM (3 mL) was added oxalyl dichloride (1 mL). The reaction mixture was stirred at 20°C for 1.5 hours. The solvent was removed to give 3-(1,4-diethyl-1H-pyrazol- 5-yl)-5-fluorobenzoyl chloride (107 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,4-diethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000144_0002
To a solution of 3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzoyl chloride (100 mg, 0.36 mmol) in DCM (3 mL) were added TEA (108 mg, 1.07 mmol), (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (143 mg, 0.39 mmol, HCl salt). The reaction mixture was stirred at 20°C for 16 hours. The solvent was removed. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (120 mg). LC-MS (MH+): m/z = 573.3 tR (min, Method c) = 0.886 Step 7: (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000145_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,4-diethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (100 mg, 0.17 mmol) in 30% HBr in AcOH (3 mL) was stirred at 50°C for 16 hours. The solvent was removed. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(1,4-diethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.12-9.04 (m, 1H), 8.52 (br s, 3H), 7.85-7.77 (m, 1H), 7.71 (s, 1H), 7.50 - 7.45 (m, 1H), 7.43 (s, 1H), 4.14-4.06 (m, 1H), 4.00-3.90 (m, 4H), 2.31 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 349.0 tR (min, Method D) = 1.633 [α]20 ,D = -10.0 (c = 1.0 mg/mL, CH3OH). Compound 1v (R)-2-amino-3-(3-fluoro-5-(2-methoxypropan-2-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(2- methoxypropan-2-yl)benzamido)propanoic acid is shown below
Figure imgf000146_0001
Step 1: 3-fluoro-5-(2-hydroxypropan-2-yl)benzoic acid
Figure imgf000146_0002
To a solution of 3-bromo-5-fluorobenzoic acid (2.0 g, 9.13 mmol) in THF (40 mL) was added n-BuLi (2.5 M in hexane, 8.04 mL) at -78 °C and the mixture was stirred at - 78 °C for 30 min. Then acetone (1.06 g, 18.26 mmol) was added and the mixture was allowed to warm to 25 °C for 1 hour. The reaction was quenched with saturated NH4Cl solution (20 mL) and 2N HCl (8 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give 3-fluoro-5-(2-hydroxypropan-2-yl)benzoic acid (300 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.21 (br s, 1H), 7.90 (t, J = 1.2 Hz, 1H), 7.54 - 7.46 (m, 2H), 5.29 (br s, 1H), 1.43 (s, 6H). Step 2: methyl 3-fluoro-5-(2-hydroxypropan-2-yl)benzoate
Figure imgf000147_0001
To a solution of 3-fluoro-5-(2-hydroxypropan-2-yl)benzoic acid (300 mg, 1.11 mmol) in a mixture of DCM (10 mL) and MeOH (2 mL) was added TMSCHN2 (2 M in hexane, 1.67 mL). The mixture was stirred at 25 °C for 1 hour. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-fluoro-5-(2-hydroxypropan-2-yl)benzoate (250 mg). 1H NMR (400 MHz, CDCl3) δ 7.93 (t, J = 1.2 Hz, 1H), 7.62 - 7.56 (m, 1H), 7.49 - 7.42 (m, 1H), 3.94 (s, 3H), 1.84 (s, 1H), 1.60 (s, 6H). Step 3: methyl 3-fluoro-5-(2-methoxypropan-2-yl)benzoate
Figure imgf000147_0002
To a solution of methyl 3-fluoro-5-(2-hydroxypropan-2-yl)benzoate (150 mg, 0.71 mmol) in DMF (5 mL) was added NaH (42 mg, 1.06 mmol, 60% in mineral oil) at 0 °C and the mixture was stirred at 0 °C for 30 min. Then MeI (201 mg, 1.41 mmol) was added and the mixture was stirred at 60 °C for 16 hours. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (15 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 15%) to give methyl 3-fluoro-5-(2-methoxypropan-2-yl)benzoate (90 mg). 1H NMR (400 MHz, CDCl3) δ 7.86 (t, J = 1.6 Hz, 1H), 7.64 - 7.59 (m, 1H), 7.38 - 7.33 (m, 1H), 3.94 (s, 3H), 3.10 (s, 3H), 1.54 (s, 6H). Step 4: 3-fluoro-5-(2-methoxypropan-2-yl)benzoic acid
Figure imgf000148_0001
To a solution of methyl 3-fluoro-5-(2-methoxypropan-2-yl)benzoate (90 mg, 0.40 mmol) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (51 mg, 1.19 mmol). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated. The residue was added water (8 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(2-methoxypropan-2-yl)benzoic acid (80 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.29 (br s, 1H), 7.80 (t, J = 1.6 Hz, 1H), 7.57 - 7.52 (m, 1H), 7.48 - 7.42 (m, 1H), 3.02 (s, 3H), 1.46 (s, 6H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(2-methoxypropan-2- yl)benzamido)propanoate
Figure imgf000148_0002
To a solution of 3-fluoro-5-(2-methoxypropan-2-yl)benzoic acid (80 mg, 0.38 mmol) in DMF (5 mL) was added TBTU (182 mg, 0.57 mmol), DIPEA (146 mg, 1.13 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (138 mg, 0.38 mmol, HCl salt). The mixture was stirred at 25 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(2-methoxypropan-2- yl)benzamido)propanoate (140 mg) 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.41 - 7.27 (m, 11H), 7.24 (br d, J = 9.2 Hz, 1H), 6.80 (br s, 1H), 5.94 (br d, J = 6.8 Hz, 1H), 5.23 (s, 2H), 5.13 (s, 2H), 4.64-4.57 (m, 1H), 3.98 - 3.88 (m, 1H), 3.86 - 3.75 (m, 1H), 3.10 (s, 3H), 1.53 (s, 6H). Step 6: (R)-2-amino-3-(3-fluoro-5-(2-methoxypropan-2-yl)benzamido)propanoic acid
Figure imgf000149_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(2- methoxypropan-2-yl)benzamido)propanoate (140 mg, 0.27 mmol) in AcOH (10 mL) was added Pd/C (50 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture was filtered and the filtrate was concentrated. The residue was added water (30 mL) and lyophilized to give (R)-2-amino-3-(3-fluoro-5-(2- methoxypropan-2-yl)benzamido)propanoic acid (55 mg). 1H NMR (400 MHz, DMSO-d6) δ 9.02-8.91 (m, 1H), 7.75-7.71 (m, 1H), 7.58-7.53 (m, 1H), 7.36-7.31 (m, 1H), 3.75-3.67 (m, 1H), 3.52-3.50 (m, 2H), 3.00 (s, 3H), 1.46 (s, 6H). LC-MS (MH+): m/z = 299.0 tR (min, Method D) = 1.471 [α]20 ,D = 27.7, (c = 1.3 mg/mL, MeOH). Compound 1w (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(4- (methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzamido)propanoic acid is shown below
Figure imgf000150_0001
Step 1: (3-bromo-1-methyl-1H-pyrazol-4-yl)methanol
Figure imgf000150_0002
To a solution of ethyl 3-bromo-1-methyl-1H-pyrazole-4-carboxylate (360 mg, 1.54 mmol) in THF (4 mL) was added DIBAL-H (1 M in toluene, 4.63 mL) at -70 °C. The mixture was warmed to 25 °C for 16 hours. The reaction was quenched with saturated aqueous sodium potassium tartrate (10 mL) and stirred for 1 hour and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give (3-bromo-1-methyl-1H-pyrazol-4- yl)methanol (290 mg). 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 1H), 4.52 (s, 2H), 3.87 (s, 3H), 1.79 (br s, 1H). Step 2: 3-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole
Figure imgf000151_0001
To a solution of (3-bromo-1-methyl-1H-pyrazol-4-yl)methanol (290 mg, 1.52 mmol) in THF (8 mL) was added NaH (121 mg, 3.04 mmol, 60% in mineral oil ) at 0 °C and stirred at 0 °C for 30 min. Then MeI (431 mg, 3.04 mmol) was added and the mixture was stirred at 25 °C for 3 hours. The reaction was quenched with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give 3-bromo-4-(methoxymethyl)-1- methyl-1H-pyrazole (220 mg). 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 1H), 4.28 (s, 2H), 3.86 (s, 3H), 3.36 (s, 3H). Step 3: methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoate
Figure imgf000151_0002
To a solution of 3-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole (140 mg, 0.68 mmol) and (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (135 mg, 0.68 mmol) in a mixture of dioxane (5 mL) and H2O (0.5 mL) was added Pd(dppf)Cl2 (50 mg, 0.068 mmol) and K2CO3 (283 mg, 2.05 mmol) under N2 atmosphere. The mixture was stirred at 90 °C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoate (135 mg). 1H NMR (400 MHz, CDCl3) δ 8.26 (t, J = 1.6 Hz, 1H), 7.76 - 7.71 (m, 1H), 7.71 - 7.66 (m, 1H), 7.46 (s, 1H), 4.40 (s, 2H), 3.94 (s, 6H), 3.46 (s, 3H). Step 4: 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoic acid
Figure imgf000152_0001
To a solution of methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzoate (130 mg, 0.47 mmol) in a mixture of MeOH (5 mL) and H2O (1 mL) was added LiOH.H2O (59 mg, 1.40 mmol). The mixture was stirred at 25 °C for 16 hours. The mixture was concentrated. The residue was added water (8 mL) and adjusted pH to 5 with 2N HCl and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4 filtered and concentrated to give 3- fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoic acid (110 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.21 (t, J = 1.6 Hz, 1H), 7.85 (s, 1H), 7.75 - 7.70 (m, 1H), 7.63 - 7.57 (m, 1H), 4.35 (s, 2H), 3.89 (s, 3H), 3.30 (s, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-(methoxymethyl)-1- methyl-1H-pyrazol-3-yl)benzamido)propanoate
Figure imgf000152_0002
To a solution of 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzoic acid (110 mg, 0.42 mmol) in DMF (5 mL) was added TBTU (201 mg, 0.62 mmol), DIPEA (161 mg, 1.25 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (152 mg, 0.42 mmol, HCl salt). The mixture was stirred at 25 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 50%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol- 3-yl)benzamido)propanoate (160 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (br t, J = 6.0 Hz, 1H), 8.04 (s, 1H), 7.88 - 7.80 (m, 2H), 7.67 - 7.61 (m, 1H), 7.56 - 7.50 (m, 1H), 7.36 - 7.23 (m, 10H), 5.15 - 5.00 (m, 4H), 4.44 - 4.37 (m, 1H), 4.35 (s, 2H), 3.88 (s, 3H), 3.74 - 3.60 (m, 2H), 3.28 (s, 3H). Step 6: (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzamido)propanoic acid
Figure imgf000153_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4- (methoxymethyl)-1-methyl-1H-pyrazol-3-yl)benzamido)propanoate (80 mg, 0.14 mmol) in AcOH (10 mL) was added Pd/C (40 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 1 hour. The reaction mixture was filtered and the filtrate was concentrated. The residue was added water (30 mL) and lyophilized to give (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-3- yl)benzamido)propanoic acid (40 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.84 (br s, 1H), 8.06 (s, 1H), 7.83 (s, 1H), 7.65-7.59 (m, 2H), 4.36 (s, 2H), 3.88 (s, 3H), 3.72-3.62 (m, 1H), 3.48-3.47 (m, 2H), 3.29 (s, 3H). LC-MS (MH+): m/z = 351.0 tR (min, Method D) = 1.427 [α]20 ,D = 16.9, (c = 1.3 mg/mL, MeOH). Compound 1x (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(4- (methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid is shown below
Figure imgf000154_0001
Step 1: ethyl 5-bromo-1-methyl-1H-pyrazole-4-carboxylate and ethyl 3-bromo-1- methyl-1H-pyrazole-4-carboxylate
Figure imgf000154_0002
To a solution of ethyl 5-bromo-1H-pyrazole-4-carboxylate (1.0 g, 4.57 mmol) in DMF (10 mL) was added NaH (274 mg, 6.85 mmol, 60% NaH in mineral oil) at 0°C. The mixture was stirred under nitrogen atmosphere at 0°C for 0.5 hour, then the reaction mixture was added iodomethane (778 mg, 5.48 mmol) at 0°C, and the mixture was stirred at 30°C for 1 hour. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 20%) to give ethyl 5-bromo-1-methyl-1H-pyrazole-4-carboxylate (400 mg) and ethyl 3-bromo-1-methyl-1H-pyrazole-4-carboxylate (500 mg). Step 2: (5-bromo-1-methyl-1H-pyrazol-4-yl)methanol
Figure imgf000155_0001
To a solution of ethyl 5-bromo-1-methyl-1H-pyrazole-4-carboxylate (400 mg, 1.72 mmol) in THF (10 mL) was added DIBAL-H (1 M, 5.15 mL) (1 M solution in toluene) at - 70 °C. The mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with saturated aqueous sodium potassium tartrate (10 mL), extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated to give (5-bromo-1-methyl-1H-pyrazol-4-yl)methanol (300 mg). 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 4.51 (d, J = 5.2 Hz, 2H), 3.88 (s, 3H), 1.80 - 1.74 (m, 1H). Step 3: 5-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole
Figure imgf000156_0001
To a solution of (5-bromo-1-methyl-1H-pyrazol-4-yl)methanol (300 mg, 1.57 mmol) in THF (4 mL) was added NaH (126 mg, 3.14 mmol, 60% NaH in mineral oil) at 0°C. The mixture was stirred under nitrogen atmosphere at 0°C for 0.5 hours, then the reaction mixture was added MeI (446 mg, 3.14 mmol) at 0°C, and the mixture was stirred at 30°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated to give 5-bromo-4-(methoxymethyl)-1-methyl-1H- pyrazole (320 mg). 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 1H), 4.09 (s, 2H), 3.68 (s, 3H), 3.14 (s, 3H). Step 4: methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzoate
Figure imgf000156_0002
A mixture of 5-bromo-4-(methoxymethyl)-1-methyl-1H-pyrazole (320 mg, 1.56 mmol), (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (309 mg, 1.56 mmol), K2CO3 (647 mg, 4.68 mmol) and Pd(dppf)Cl2 (114 mg, 0.16 mmol) in a mixture of dioxane (5 mL) and H2O (0.5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 90°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give methyl 3-fluoro-5-(4-(methoxymethyl)-1- methyl-1H-pyrazol-5-yl)benzoate (240 mg). 1H NMR (400 MHz, CDCl3) δ 7.89 (t, J = 1.2 Hz, 1H), 7.82-7.77 (m, 1H), 7.58 (s, 1H), 7.40- 7.34 (m, 1H), 4.18 (s, 2H), 3.95 (s, 3H), 3.83 (s, 3H), 3.32 (s, 3H). Step 5: 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzoic acid
Figure imgf000157_0001
To a solution of methyl 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5- yl)benzoate (240 mg, 0.86 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (72 mg, 1.72 mmol) and the mixture was stirred at 30°C for 15 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5-(4-(methoxymethyl)-1-methyl- 1H-pyrazol-5-yl)benzoic acid (220 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-(methoxymethyl)-1- methyl-1H-pyrazol-5-yl)benzamido)propanoate
Figure imgf000157_0002
To a solution of 3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzoic acid (100 mg, 0.38 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (138 mg, 0.38 mmol, HCl salt) in DMF (4 mL) was added TBTU (182 mg, 0.57 mmol) and DIPEA (147 mg, 1.14 mmol). The reaction mixture was stirred at 30°C for 2 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 60% to 70%) thrice to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol- 5-yl)benzamido)propanoate (70 mg). LC-MS (MH+): m/z = 543.2 tR (min, Method c) = 0.829 Step 7: (R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5- yl)benzamido)propanoic acid
Figure imgf000158_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4- (methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzamido)propanoate (50 mg, 0.09 mmol) and Pd/C (20 mg, 10% Pd, 50% water) in AcOH (3 mL) was degassed and purged with H2 for 3 times, and then the mixture was stirred at 25°C for 1 hour under H2 atmosphere (15psi). The reaction mixture was filtered and concentrated to give (R)-2-amino-3-(3- fluoro-5-(4-(methoxymethyl)-1-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid (28 mg). 1H NMR (400 MHz, DMSO-d6) δ 9.02 (br s, 1H), 7.82-7.75 (m, 2H), 7.58-7.52 (m, 2H), 4.16 (s, 2H), 3.79 (s, 3H), 3.74-3.68 (m, 1H), 3.50-3.45 (m, 2H), 3.17 (s, 3H). LC-MS (MH+): m/z = 351.0 tR (min, Method E) = 0.982 [α]20 ,D = +24.0 (c = 1.0 mg/mL, CH3OH). Compound 1y (R)-2-amino-3-(3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3,5- dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000159_0001
Step 1: methyl 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoate
Figure imgf000159_0002
To a mixture of 4-bromo-3,5-dimethylisoxazole (800 mg, 4.55 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (900 mg, 4.55 mmol) and K3PO4 (2.89 g, 13.64 mmol) in the mixture of dioxane (30 mL) and H2O (3 mL) was added Pd(dtbpf)Cl2 (296 mg, 0.45 mmol), the mixture was stirred at 100°C for 16 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 10%) to give methyl 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoate (640 mg). 1H NMR (400MHz, CDCl3) δ 7.75-7.71 (m, 2H), 7.19-7.15 (m, 1H), 3.96 (s, 3H), 2.44 (s, 3H), 2.30 (s, 3H). Step 2: methyl 3-(2-amino-4-oxopent-2-en-3-yl)-5-fluorobenzoate
Figure imgf000160_0001
To a solution of methyl 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoate (540 mg, 2.17 mmol) in MeOH (10 mL) was added Raney-Ni (250 mg) under N2 atmosphere. The suspension was degassed and purged with H23 times. The mixture was stirred under H2 (1 atm) at 25°C for 6 hours. The mixture was filtered and concentrated to give methyl 3- (2-amino-4-oxopent-2-en-3-yl)-5-fluorobenzoate (530 mg). 1H NMR (400MHz, CDCl3) δ 10.61 (s, 1H), 7.69-7.64 (m, 2H), 7.14-7.09 (m, 1H), 5.19 (s, 1H), 3.94 (s, 3H), 1.85 (s, 3H), 1.72 (s, 3H). Step 3: methyl 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoate
Figure imgf000160_0002
To a solution of methyl 3-(2-amino-4-oxopent-2-en-3-yl)-5-fluorobenzoate (530 mg, 2.11 mmol) in toluene (5 mL) was added P2S5 (1.41 g, 6.33 mmol), chloranil (519 mg, 2.11 mmol,) under N2 atmosphere. The mixture was stirred at 110°C for 30 minutes under N2 atmosphere. The mixture was filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 10%) and further purified by preparative-TLC (Petroleum ether: ethyl acetate=5:1) to give methyl 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoate (150 mg). 1H NMR (400MHz, CDCl3) δ 7.78-7.74 (m, 1H), 7.71 (s, 1H), 7.17-7.13 (m, 1H), 3.95 (s, 3H), 2.40 (s, 3H), 2.34 (s, 3H). Step 4: 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoic acid
Figure imgf000161_0001
To a solution of methyl 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoate (150 mg, 0.57 mmol) in the mixture of THF (8 mL) and H2O (4 mL) was added LiOH.H2O (36 mg, 0.85 mmol), the mixture was stirred at 25°C for 1 hour. The mixture was concentrated, then added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and precipitate solids, filtered to give 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoic acid (120 mg). LC-MS (MH+): m/z = 252.1 tR (min, Method c) = 0.633 Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000161_0002
To a mixture of 3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzoic acid (120 mg, 0.47 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (209 mg, 0.57 mmol, HCl salt) in DMF (8 mL) was added TBTU (230 mg, 0.72 mmol) and DIPEA (185 mg, 1.43 mmol), the mixture was stirred at 25°C for 15 hours. The mixture was added water (100 mL) and extracted with ethyl acetate (60 mL × 3), the combined organic layers were washed with brine (100 mL), dried over Na2SO4 and concentrated. The mixture was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (3,5-dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoate (180 mg). 1H NMR (400MHz, CDCl3) δ 7.43-7.30 (m, 12H), 7.10-7.06 (m, 1H), 6.94 (br, 1H), 5.94 (br d, J=6.8 Hz, 1H), 5.22 (s, 2H), 5.10 (s, 2H), 4.63-4.56 (m, 1H), 4.00-3.92 (m, 1H), 3.86-3.75 (m, 1H), 2.39 (s, 3H), 2.34 (s, 3H). Step 6: (R)-2-amino-3-(3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000162_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethylisothiazol-4- yl)-5-fluorobenzamido)propanoate (170 mg, 0.30 mmol) in 30% HBr/HOAc (6 mL) was stirred at 50°C for 15 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(3,5-dimethylisothiazol-4-yl)-5- fluorobenzamido)propanoic acid (106 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.92-8.87 (m, 1H), 8.27 (br s, 2H), 7.71 (d, J = 9.2 Hz, 1H), 7.66 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 3.99-3.89 (m, 1H), 3.81-3.74 (m, 1H), 3.72-3.63 (m, 1H), 2.41 (s, 3H), 2.29 (s, 3H). LC-MS (MH+): m/z = 338.0 tR (min, Method c) = 1.078 [α]20,D = +10 (c = 1.0 mg/mL, CH3OH). Compound 1z (R)-2-amino-3-(3-fluoro-5-(1-methylcyclopropyl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(1- methylcyclopropyl)benzamido)propanoic acid is shown below
Figure imgf000162_0002
Step 1: methyl 3-fluoro-5-(prop-1-en-2-yl)benzoate
Figure imgf000163_0001
To a solution of methyl 3-bromo-5-fluorobenzoate (1.5 g, 6.44 mmol) in a mixture of dioxane (30 mL) and H2O (6 mL) was added 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)- 1,3,2-dioxaborolane (1.62 g, 9.66 mmol), K2CO3 (2.67 g, 19.31 mmol) and Pd(dppf)Cl2 (706 mg, 0.966 mmol). The mixture was stirred at 90°C for 16 hours under N2. The reaction mixture was concentrated. The residue was diluted with ethyl acetate (80 mL) and washed with brine (30 mL × 2), dried over MgSO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether/ethyl acetate=100/0 to 90:10) to give methyl 3-fluoro-5-(prop-1-en-2-yl)benzoate (800 mg). 1H NMR (400MHz, CDCl3) δ 7.96-7.93 (m, 1H), 7.64-7.60 (m, 1H), 7.37-7.31 (m, 1H), 5.47 (s, 1H), 5.20 (s, 1H), 3.94 (s, 3H), 2.17 (s, 3H). Step 2: methyl 3-fluoro-5-(1-methylcyclopropyl)benzoate
Figure imgf000163_0002
To a solution of ZnEt2 (1 M in Hexane, 15.45 mL) in DCM (20 mL) was added TFA (0.2 mL) in DCM (5 mL). The mixture was stirred at 0°C for 20 minutes. Then diiodomethane (4.14 g, 15.45 mmol) in DCM (5 mL) was added. The mixture was stirred at 0°C for another 20 minutes. Then a solution of methyl 3-fluoro-5-isopropenyl-benzoate (600 mg, 3.09 mmol) in DCM (5 mL) was added at 0°C. After addition, the mixture was stirred at 20°C for 16 hours. The reaction mixture was filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether/ethyl acetate=100/0 to 80:20) to give methyl 3-fluoro-5-(1-methylcyclopropyl)benzoate (550 mg). 1H NMR (400MHz, CDCl3) δ 7.71-7.70 (m, 1H), 7.53-7.49 (m, 1H), 7.16-7.12 (m, 1H), 3.92 (s, 3H), 1.43 (s, 3H), 0.93-0.90 (m, 2H), 0.83-0.80 (m, 2H). Step 3: 3-fluoro-5-(1-methylcyclopropyl)benzoic acid
Figure imgf000164_0001
To a solution of methyl 3-fluoro-5-(1-methylcyclopropyl)benzoate (550 mg, 2.64 mmol) in a mixture of THF (15 mL) and H2O (10 mL) was added LiOH.H2O (554 mg, 13.21 mmol). The mixture was stirred at 20°C for 16 hour. The reaction mixture was concentrated. The residue was added 2M HCl to pH=2~3. The mixture was extracted with ethyl acetate (20 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(1- methylcyclopropyl)benzoic acid (500 mg). 1H NMR (400MHz, CDCl3) δ 7.78 (s, 1H), 7.60-7.57 (m, 1H), 7.21-7.18 (m, 1H), 1.45 (s, 3H), 0.95-0.91 (m, 2H), 0.86-0.82 (m, 2H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methylcyclopropyl)benzamido)propanoate
Figure imgf000164_0002
To a solution of 3-fluoro-5-(1-methylcyclopropyl)benzoic acid (150 mg, 0.772 mmol) in DMF (20 mL) was added (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (338 mg, 0.927 mmol, HCl salt), TBTU (372 mg, 1.16 mmol) and DIPEA (299 mg, 2.32 mmol). The mixture was stirred at 20°C for 3 hours. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (30 mL × 2). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether/ethyl acetate=100/0 to 70:30) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methylcyclopropyl)benzamido)propanoate (300 mg). LC-MS (MH+): m/z = 505.2 tR (min, Method c_1.5min) = 0.834 Step 5: (R)-2-amino-3-(3-fluoro-5-(1-methylcyclopropyl)benzamido)propanoic acid
Figure imgf000165_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methylcyclopropyl)benzamido)propanoate (300 mg, 0.59 mmol) in AcOH (15 mL) was added Pd/C (60 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 2 hours. The reaction mixture was filtered through celite and concentrated. The residue was purified by Preparative HPLC (Method AA) to give (R)-2-amino-3-(3-fluoro- 5-(1-methylcyclopropyl)benzamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.05-8.95 (m, 1H), 8.52 (br s, 3H), 7.58-7.57 (m, 1H), 7.49- 7.46 (m, 1H), 7.24-7.20 (m, 1H), 4.11-4.06 (m, 1H), 3.78-3.35 (m, 2H), 1.41 (s, 3H), 0.97- 0.93 (m, 2H), 0.84-0.80 (m, 2H). LC-MS (MH+): m/z = 281.0 tR (min, Method c) = 1.238 [α]20,D = +2.7 (c = 3.0 mg/mL, CH3OH). Compound 2a (R)-2-amino-3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3,5-dimethyl-1H- pyrazol-4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000166_0001
Step 1: methyl 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoate
Figure imgf000166_0002
A mixture of 4-bromo-3,5-dimethyl-1H-pyrazole (300 mg, 1.71 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (339 mg, 1.71 mmol), Pd(dtbpf)Cl2 (112 mg, 0.17 mmol) and K3PO4 (1.09 g, 5.14 mmol) in a mixture of dioxane (5 mL) and H2O (0.5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 5 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 50%) to give methyl 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoate (330 mg). 1H NMR (400 MHz, CD3Cl) δ 7.76 (s, 1H), 7.66-7.61 (m, 1H), 7.20-7.15 (m, 1H), 3.94 (s, 3H), 2.33 (s, 6H). Step 2: 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid
Figure imgf000167_0001
To a solution of methyl 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoate (330 mg, 1.33 mmol) in a mixture of THF (5 mL) and H2O (2.5 mL) was added LiOH.H2O (112 mg, 2.66 mmol) and the mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid (300 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)- 5-fluorobenzamido)propanoate
Figure imgf000167_0002
To a solution of 3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid (300 mg, 1.28 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (514 mg, 1.41 mmol, HCl salt) in DMF (10 mL) was added TBTU (617 mg, 1.92 mmol) and DIPEA (497 mg, 3.84 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 50% to 60%) twice to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate (400 mg). LC-MS (MH+): m/z = 545.2 tR (min, Method c) = 0.789 Step 4: (R)-2-amino-3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000168_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-pyrazol- 4-yl)-5-fluorobenzamido)propanoate (400 mg, 0.74 mmol) in 30% HBr in AcOH (10 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(3,5-dimethyl- 1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid (160 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.07-9.00 (m, 1H), 8.55 (br s, 3H), 7.68 (s, 1H), 7.60 (d, J=9.2 Hz, 1H), 7.31 (d, J=9.2 Hz, 1H), 4.13-4.13 (m, 1H), 3.79-3.73 (m, 2H), 2.23 (s, 6H). LC-MS (MH+): m/z = 321.0 tR (min,Method c) = 2.310 [α]20 ,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 2b (R)-2-amino-3-(3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3,5-dimethyl-4H- 1,2,4-triazol-4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000169_0001
Step 1: 3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzoic acid
Figure imgf000169_0002
To a solution of 2,5-dimethyl-1,3,4-oxadiazole (300 mg, 3.06 mmol) in xylene (5 mL) was added 3-amino-5-fluorobenzoic acid (474 mg, 3.06 mmol), 4-methylbenzenesulfonic acid (132 mg, 0.76 mmol) and the mixture was stirred at 140°C for 16 hours under N2. The reaction mixture was concentrated and the residue was washed thoroughly twice with 4 ml of a 1:1 (v/v) ethyl acetate : ethanol mixture. The remaining suspension was filtered and concentrated to give 3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzoic acid (280 mg). 1H NMR (400 MHz, CD3Cl) δ 7.90-7.80 (m, 3H), 2.19 (s, 6H). Step 2: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-4H-1,2,4-triazol-4- yl)-5-fluorobenzamido)propanoate To a solution of 3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5-fluorobenzoic acid (100 mg, 0.43 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (171 mg, 0.47 mmol, HCl salt) in DMF (5 mL) was added TBTU (205 mg, 0.64 mmol) and DIPEA (165 mg, 1.28 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 20%) and further purified by preparation HPLC (Method BB) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5- fluorobenzamido)propanoate (100 mg). LC-MS (MH+): m/z = 546.2 tR (min,Method c) = 0.822 Step 3: (R)-2-amino-3-(3-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000170_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-4H-1,2,4- triazol-4-yl)-5-fluorobenzamido)propanoate (100 mg, 0.18 mmol) in 30% HBr in AcOH (5 mL) was heated at 50°C for 3 hours. The reaction mixture was concentrated. The residue was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(3,5-dimethyl- 4H-1,2,4-triazol-4-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.44-9.37 (m, 1H), 8.63 (br s, 3H), 8.14-8.07 (m, 2H), 7.88-7.84 (m, 1H), 4.17-4.07 (m, 1H), 3.84-3.75 (m, 2H), 2.40 (s, 6H). LC-MS (MH+): m/z = 322.0 tR (min, Method F) = 2.097 [α]20 ,D = +8.0 (c = 1.0 mg/mL, CH3OH). Compound 2c (R)-2-amino-3-(3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3,5-dimethyl-1H- 1,2,4-triazol-1-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000171_0001
Step 1: methyl 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoate
Figure imgf000171_0002
To a solution of 3,5-dimethyl-4H-1,2,4-triazole (300 mg, 3.09 mmol) in DCM (30 mL) was added (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.22 g, 6.18 mmol), Cu(OAc)2.H2O (740 mg, 3.71 mmol), 4A MS (240 mg), TEA (625 mg, 6.18 mmol) under N2. The suspension was degassed under vacuum and purged with O2 several times. The mixture was stirred under O2 (15psi) at 30°C for 16 hours. The reaction mixture was filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 50%) to give methyl 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoate (90 mg). 1H NMR (400 MHz, CD3Cl) δ 7.93 (s, 1H), 7.81-7.74 (m, 1H), 7.47-7.41 (m, 1H), 3.96 (s, 3H), 2.55 (s, 3H) , 2.41 (s, 3H). Step 2: 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoic acid
Figure imgf000172_0001
To a solution of methyl 3-(3,5-dimethyl-1,2,4-triazol-1-yl)-5-fluoro-benzoate (90 mg, 0.36 mmol) in a mixture of THF (2 mL) and H2O (1 mL) was added LiOH.H2O (30 mg, 0.72 mmol) and the mixture was stirred at 30 °C for 1 hour. The reaction mixture was diluted with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoic acid (80 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-1,2,4-triazol- 1-yl)-5-fluorobenzamido)propanoate
Figure imgf000172_0002
To a solution of 3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzoic acid (80 mg, 0.34 mmol and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (136 mg, 0.37 mmol, HCl salt) in DMF (3 mL) was added TBTU (164 mg, 0.51 mmol) and DIPEA (132 mg, 1.02 mmol). The reaction mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 70% to 80%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5- fluorobenzamido)propanoate (130 mg). LC-MS (MH+): m/z = 546.3 tR (min, Method c) = 0.790 Step 4: (R)-2-amino-3-(3-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000173_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethyl-1H-1,2,4- triazol-1-yl)-5-fluorobenzamido)propanoate (130 mg, 0.24 mmol) in 30% HBr in AcOH (5 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(3,5- dimethyl-1H-1,2,4-triazol-1-yl)-5-fluorobenzamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.22-9.16 (m, 1H), 8.55 (br s, 3H), 7.97 (s, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.75 (d, J = 9.2 Hz, 1H), 4.18-4.08 (m, 1H), 3.85-3.75 (m, 2H), 2.53 (s, 3H), 2.31 (s, 3H). LC-MS (MH+): m/z = 322.0 tR (min, Method D) = 1.149 [α]20 ,D = +4.0 (c = 2.0 mg/mL, CH3OH). Compound 2d (R)-2-amino-3-(3-(1-ethylcyclopropyl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1- ethylcyclopropyl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000174_0001
Step 1: methyl 3-(but-1-en-2-yl)-5-fluorobenzoate and (E)-methyl 3-(but-2-en-2-yl)-5- fluorobenzoate
Figure imgf000174_0002
To a solution of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol) in a mixture of dioxane (40 mL) and H2O (8 mL) was added 2-bromobut-1-ene (1.02 g, 7.58 mmol), K2CO3 (2.09 g, 15.15 mmol) and Pd(dppf)Cl2 (554 mg, 0.76 mmol). The mixture was stirred at 90°C for 15 hours under N2. The reaction mixture was filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether) to give a mixture of compounds methyl 3-(but-1-en-2-yl)-5-fluorobenzoate and (E)-methyl 3-(but-2-en-2-yl)-5-fluorobenzoate (670 mg, crude). LC-MS (MH+): m/z = 209.1 tR (min, Method c_1.5min) = 0.796 Step 2: methyl 3-(1-ethylcyclopropyl)-5-fluorobenzoate and methyl 3-(1,2- dimethylcyclopropyl)-5-fluorobenzoate
Figure imgf000175_0001
To a solution of ZnEt2 (1 M in toluene, 8.64 mL) in DCM (20 mL) was added TFA (0.1 mL). The mixture was stirred at 0°C for 30 minutes. Then diiodomethane (2.31 g, 8.64 mmol) was added. The mixture was stirred at 0°C for 30 minutes. A mixture of compounds methyl 3-(but-1-en-2-yl)-5-fluorobenzoate and (E)-methyl 3-(but-2-en-2- yl)-5-fluorobenzoate (600 mg, crude) in DCM (8 mL) was added. The mixture was stirred at 20°C for 16 hours. The reaction mixture was filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether). The residue was further purified by Preparative HPLC (Method BB) to give a mixture of compounds methyl 3-(1-ethylcyclopropyl)-5-fluorobenzoate and methyl 3-(1,2- dimethylcyclopropyl)-5-fluorobenzoate ( 180 mg, crude). LC-MS (MH+): m/z = 223.1 tR (min, Method c) = 0.824 Step 3: 3-(1-ethylcyclopropyl)-5-fluorobenzoic acid and 3-(1,2-dimethylcyclopropyl)-5- fluorobenzoic acid
Figure imgf000175_0002
To a solution of methyl 3-(1-ethylcyclopropyl)-5-fluorobenzoate and methyl 3-(1,2- dimethylcyclopropyl)-5-fluorobenzoate (180 mg, crude) in a mixture of THF (5 mL) and H2O (5 mL) was added LiOH.H2O (170 mg, 4.05 mmol). The mixture was stirred at 20°C for 14 hours. The reaction mixture was concentrated. The residue was diluted with water (20 mL) and added 2M HCl to pH=2~3. The mixture was extracted with ethyl acetate (30 mL × 2). The combined organic layers were dried over MgSO4, filtered and concentrated to give a mixture of 3-(1-ethylcyclopropyl)-5-fluorobenzoic acid and 3- (1,2-dimethylcyclopropyl)-5-fluorobenzoic acid ( 160 mg, crude). LC-MS (MH+): m/z = 209.1 tR (min, Method c) = 0.706 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethylcyclopropyl)-5- fluorobenzamido)propanoate and (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (1,2-dimethylcyclopropyl)-5-fluorobenzamido)propanoate
Figure imgf000176_0001
To a solution of 3-(1-ethylcyclopropyl)-5-fluorobenzoic acid and 3-(1,2- dimethylcyclopropyl)-5-fluorobenzoic acid (150 mg, crude) in DMF (20 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (394 mg, 1.08 mmol, HCl salt), TBTU (347 mg, 1.08 mmol) and DIPEA (279 mg, 2.16 mmol). The mixture was stirred at 20°C for 14 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (50 mL × 2), dried over MgSO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether/ethyl acetate=100/0 to 70:30). The residue was further separated by SFC (condition: Instrument: Berger, MULTIGR AM-II; Column: Chiralpak AD 250 × 30 mm I.D. 5 μm; Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O) = 60/40; Flow Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethylcyclopropyl)-5- fluorobenzamido)propanoate (150 mg) and (2R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(1,2-dimethylcyclopropyl)-5- fluorobenzamido)propanoate (60 mg). (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethylcyclopropyl)-5- fluorobenzamido)propanoate: 1H NMR (400MHz, CDCl3) δ 7.45 (s, 1H), 7.35-7.32 (m, 10H), 7.17-7.11 (m, 2H), 6.81 (br, 1H), 6.01 (br d, J=6.4 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.61-4.59 (m, 1H), 3.93-3.79 (m, 2H), 1.62 (q, J=7.2 Hz, 2H), 0.84 (t, J=7.2 Hz, 3H), 0.80- 0.78 (m, 2H), 0.74-0.70 (m, 2H). (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,2-dimethylcyclopropyl)-5- fluorobenzamido)propanoate: 1H NMR (400MHz, CDCl3) δ 7.37-7.32 (m, 11H), 7.11-7.02 (m, 2H), 6.77 (br, 1H), 6.00 (br d, J=6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.61-4.58 (m, 1H), 3.93-3.78 (m, 2H), 1.38 (s, 3H), 1.22 (d, J=6.0 Hz, 3H), 1.12-1.05 (m, 2H), 0.45-0.41 (m, 1H). Step 5: (R)-2-amino-3-(3-(1-ethylcyclopropyl)-5-fluorobenzamido)propanoic acid
Figure imgf000177_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethylcyclopropyl)-5- fluorobenzamido)propanoate (140 mg, 0.27 mmol) in AcOH (10 mL) was added Pd/C (40 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 1 hour. Then Pd/C (40 mg, 10% Pd, 50% water) was added. The mixture was stirred at 20°C for another 1 hour. The reaction mixture was filtered through celite and concentrated. The residue was purified by Preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(1- ethylcyclopropyl)-5-fluorobenzamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.03-8.97 (m, 1H), 8.52 (br s, 3H), 7.65-7.63 (m, 1H), 7.54- 7.50 (m, 1H), 7.28-7.24 (m, 1H), 4.10-4.07 (m, 1H), 3.79-3.70 (m, 2H), 1.64 (q, J = 7.2 Hz, 2H), 0.85-0.82 (m, 2H), 0.79 (t, J = 7.2 Hz, 3H), 0.75-0.71 (m, 2H). LC-MS (MH+): m/z = 295.0 tR (min, Method D) = 1.813 [α]20,D = +4.0 (c = 1.0 mg/mL, CH3OH). Compound 2e (R)-2-amino-3-(3-(1,2-dimethylcyclopropyl)-5-fluorobenzamido)propanoic acid Preparation of (R)-2-amino-3-(3-(1,2-dimethylcyclopropyl)-5- fluorobenzamido)propanoic acid
Figure imgf000178_0001
To a solution of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1,2- dimethylcyclopropyl)-5-fluorobenzamido)propanoate (60 mg, 0.12 mmol) in AcOH (15 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 1 hour. Then Pd/C (20 mg, 10% Pd, 50% water) was added. The mixture was stirred at 20°C for another 1 hour. The reaction mixture was filtered through celite and concentrated. The residue was purified by Preparative HPLC (Method AA) to give (2R)-2-amino-3-(3-(1,2-dimethylcyclopropyl)-5- fluorobenzamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.92-8.85 (m, 1H), 7.53-7.52 (m, 1H), 7.45-7.41 (m, 1H), 7.20-7.16 (m, 1H), 3.88-3.85 (m, 1H), 3.78-3.74 (m, 2H), 1.37 (s, 3H), 1.19 (m, 3H), 1.09- 1.01 (m, 2H), 0.49-0.46 (m, 1H). LC-MS (MH+): m/z = 295.0 tR (min, Method D) = 1.854 [α]20 ,D = +8.0 (c = 1.0 mg/mL, CH3OH). Compound 2f (R)-2-amino-3-(3-(diethylamino)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(diethylamino)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000179_0001
Step 1: ethyl 3-(diethylamino)-5-fluorobenzoate
Figure imgf000179_0002
A solution of ethyl 3,5-difluorobenzoate (2 g, 10.74 mmol) and diethylamine (943 mg, 12.89 mmol) in DMSO (10 mL) was stirred at 100°C for 64 hours under N2 atmosphere. The reaction mixture was added water (200 mL) and extracted with ethyl acetate (100 mL × 3). The organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 0%) and further purified by preparative-TLC (ethyl acetate: Petroleum ether = 0:1) to give ethyl 3-(diethylamino)-5- fluorobenzoate (160 mg). 1H NMR (400MHz, CDCl3) δ 7.13 (s, 1H), 6.97-6.93 (m, 1H), 6.51-6.47 (m, 1H), 4.36 (q, J=6.8 Hz, 2H), 3.37 (q, J=7.2 Hz, 4H), 1.39 (t, J=7.2 Hz, 3H), 1.18 (t, J=7.2 Hz, 6H). Step 2: 3-(diethylamino)-5-fluorobenzoic acid
Figure imgf000180_0001
To a solution of ethyl 3-(diethylamino)-5-fluorobenzoate (130 mg, 0.54 mmol) in the mixture of MeOH (10 mL), THF (8 mL) and H2O (4 mL) was added LiOH.H2O (34 mg, 0.81mmol), the mixture was stirred at 30°C for 23 hours. The reaction mixture was concentrated, then added water (50 mL) and dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3). The organic layers were dried over Na2SO4, filtered and concentrated to give 3-(diethylamino)-5-fluorobenzoic acid (110 mg). 1H NMR (400MHz, CDCl3) δ 7.18 (s, 1H), 7.04-7.00 (m, 1H), 6.57-6.53 (m, 1H), 3.39 (q, J = 7.2 Hz, 4H), 1.19 (t, J = 7.2 Hz, 6H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(diethylamino)-5- fluorobenzamido)propanoate
Figure imgf000180_0002
To a mixture of 3-(diethylamino)-5-fluorobenzoic acid (130 mg, 0.62 mmol) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (269 mg, 0.74 mmol) in DMF (8 mL) was added T3P (587 mg, 0.92 mmol, 50% in DMF) and DIPEA (239 mg, 1.85 mmol), the mixture was stirred at 30°C for 16 hours. The mixture was added water (200 mL) and extracted with ethyl acetate (80 mL × 3), the combined organic layers were washed with brine (100 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(diethylamino)-5-fluorobenzamido)propanoate (170 mg). 1H NMR (400MHz, CDCl3) δ 7.36-7.30 (m, 10H), 6.85 (t, J=1.6 Hz, 1H), 6.62 (br, 1H), 6.46- 6.41 (m, 2H), 6.00 (d, J=6.4 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.61-4.56 (m, 1H), 3.93- 3.75 (m, 2H), 3.35(q, J=7.2 Hz, 4H), 1.17 (t, J=7.2 Hz, 6H). Step 4: (R)-2-amino-3-(3-(diethylamino)-5-fluorobenzamido)propanoic acid
Figure imgf000181_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(diethylamino)-5- fluorobenzamido)propanoate (170 mg, 0.33 mmol) in HBr/ HOAc (5 mL, 30% purity), the mixture was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was added MTBE (20 mL) and stirred for 5 hours and filtered to give a yellow solid. The solid was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3- (diethylamino)-5-fluorobenzamido)propanoic acid (29 mg) as HCl salt. 1H NMR (400MHz, D2O) δ 7.87 (s, 1H), 7.85-7.82 (m, 1H), 7.73-7.70 (m, 1H), 4.45-4.39 (m, 1H), 4.15-4.00 (m, 2H), 3.77 (q, J=7.2 Hz, 4H), 1.19 (t, J=7.2 Hz, 6H). LC-MS (MH+): m/z = 298.0 tR (min, Method D) = 1.271 [α]20,D = -2.0 (c = 1.0 mg/mL, CH3OH). Compound 2g (R)-2-amino-3-(3-(3-ethylpyridin-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethylpyridin-4- yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000182_0001
Step 1: methyl 3-(3-ethylpyridin-4-yl)-5-fluorobenzoate
Figure imgf000182_0002
A mixture of 4-bromo-3-ethylpyridine (200 mg, 1.07 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (213 mg, 1.07 mmol), Pd(dppf)Cl2 (79 mg, 0.11 mmol) and K2CO3 (446 mg, 3.22 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 5 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give methyl 3-(3-ethylpyridin-4-yl)-5-fluorobenzoate (210 mg). 1H NMR (400MHz, CDCl3) δ 8.56 (s, 1H), 8.48 (d, J = 5.2 Hz, 1H), 7.80 – 7.74 (m, 2H), 7.24 – 7.19 (m, 1H), 7.09 (d, J = 4.8 Hz, 1H),3.94 (s, 3H), 2.61 (q, J = 7.6 Hz, 2H), 1.09 (t, J = 7.6 Hz, 3H). Step 2: 3-(3-ethylpyridin-4-yl)-5-fluorobenzoic acid
Figure imgf000183_0001
To a solution of methyl 3-(3-ethylpyridin-4-yl)-5-fluorobenzoate (210 mg, 0.81 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (68 mg, 1.62 mmol) and the mixture was stirred at 25°C for 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-ethylpyridin-4-yl)-5-fluorobenzoic acid (108 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000183_0002
To a solution of 3-(3-ethylpyridin-4-yl)-5-fluorobenzoic acid (108 mg, 0.44 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (177 mg, 0.48 mmol, HCl salt) in DMF (5 mL) was added TBTU (212 mg, 0.66 mmol) and DIPEA (171 mg, 1.32 mmol). The reaction mixture was stirred at 20°C for 16 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 60% to 70%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-4-yl)-5-fluorobenzamido)propanoate (210 mg). LC-MS (MH+): m/z = 556.3 tR (min, Method c) = 0.767 Step 4: (R)-2-amino-3-(3-(3-ethylpyridin-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000184_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyridin-4-yl)-5- fluorobenzamido)propanoate (210 mg, 0.38 mmol) in the solution of 30% HBr/HOAc (5 mL) was heated at 50°C for 1 hour. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethylpyridin-4-yl)- 5-fluorobenzamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.23-9.18 (m, 1H), 8.90 (s, 1H), 8.83-8.80 (m, 1H), 8.54 (br s, 3H), 7.90 (d, J = 9.2 Hz, 1H), 7.88-7.81 (m, 2H), 7.64 (d, J = 9.2 Hz, 1H), 4.17-4.08 (m, 1H), 3.85-3.72 (m, 2H), 2.75 (q, J = 7.6 Hz, 2H), 1.07 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 332.0 tR (min, Method c) = 1.199 [α]20,D = +4.0 (c = 2 mg/mL,CH3OH). Compound 2h (R)-2-amino-3-(3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-chloro-5- methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000185_0001
Step 1: methyl 3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzoate
Figure imgf000185_0002
A mixture of 2,3-dichloro-5-methylpyridine (200 mg, 1.23 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (244 mg, 1.23 mmol), Pd(dppf)Cl2 (90 mg, 0.12 mmol) and K2CO3 (512 mg, 3.70 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 5% to 10%) to give methyl 3-(3-chloropyridin-2-yl)-5-fluorobenzoate (250 mg). 1H NMR (400MHz, CDCl3) δ 8.44 (d, J = 1.2 Hz, 1H), 8.24 (d, J = 1.2 Hz, 1H), 7.81 – 7.76 (m, 1H), 7.69 – 7.64 (m, 2H), 3.95 (s, 3H), 2.41 (s, 3H). Step 2: 3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzoic acid
Figure imgf000186_0001
To a solution of methyl 3-(3-chloropyridin-2-yl)-5-fluorobenzoate (250 mg, 0.89 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (75 mg, 1.79 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzoic acid (230 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-chloro-5-methylpyridin-2-yl)- 5-fluorobenzamido)propanoate
Figure imgf000186_0002
To a solution of 3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzoic acid (100 mg, 0.38 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (151 mg, 0.41 mmol, HCl salt) in DMF (5 mL) was added TBTU (181 mg, 0.56 mmol) and DIPEA (146 mg, 1.13 mmol). The reaction mixture was stirred at 20°C for 16 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 30% to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(3-chloro-5-methylpyridin-2-yl)-5- fluorobenzamido)propanoate (130 mg). LC-MS (MH+): m/z = 576.3 tR (min, Method c) = 0.917 Step 4: (R)-2-amino-3-(3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000187_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-chloro-5-methylpyridin- 2-yl)-5-fluorobenzamido)propanoate (130 mg, 0.23 mmol) in the solution of 30% HBr/HOAc (5 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(3- chloro-5-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid (85 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.06-9.00 (m, 1H), 8.50-8.40 (m, 4H), 8.00 (s, 1H), 7.96- 7.93 (m, 1H), 7.85-7.80 (m, 1H), 7.69-7.64 (m, 1H), 4.12-4.05 (m, 1H), 3.82-3.70 (m, 2H), 2.35 (s, 3H). LC-MS (MH+): m/z = 351.9 tR (min, Method c) = 1.269 [α]20,D = +2.0 (c = 2 mg/mL,CH3OH). Compound 2i (R)-2-amino-3-(3-(3-ethylmorpholino)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3- ethylmorpholino)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000188_0001
Step 1: methyl 3-(3-ethylmorpholino)-5-fluorobenzoate
Figure imgf000188_0002
A mixture of methyl 3-bromo-5-fluorobenzoate (200 mg, 0.86 mmol), 3- ethylmorpholine (143 mg, 0.94 mmol, HCl salt) ,CPhos-Pd-G3 (69 mg, 0.08 mmol) and Cs2CO3 (839 mg, 2.57 mmol) in dioxane (4 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 90°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(3- ethylmorpholino)-5-fluorobenzoate (200 mg). 1H NMR (400MHz, CDCl3) δ 7.29 (s, 1H), 7.14 - 7.09 (m, 1H), 6.69 - 6.64 (m, 1H), 4.03 - 3.94 (m, 2H), 3.91 (s, 3H), 3.75 - 3.70 (m, 1H), 3.53 - 3.47 (m, 1H), 3.25 - 3.20 (m, 2H), 2.48 (d, J = 3.2 Hz, 1H), 1.93 - 1.80 (m, 1H), 1.51 - 1.40 (m, 1H), 0.92 (t, J = 7.2 Hz, 3H). Step 2: 3-(3-ethylmorpholino)-5-fluorobenzoic acid
Figure imgf000189_0001
To a solution of methyl 3-(3-ethylmorpholino)-5-fluorobenzoate (200 mg, 0.75 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (63 mg, 1.50 mmol) and the mixture was stirred at 15°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-ethylmorpholino)-5-fluorobenzoic acid (150 mg). Step 3: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylmorpholino)-5- fluorobenzamido)propanoate
Figure imgf000189_0002
To a solution of a mixture of 3-(3-ethylmorpholino)-5-fluorobenzoic acid (150 mg, 0.59 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (238 mg, 0.65 mmol, HCl salt) in DMF (5 mL) was added TBTU (285 mg, 0.89 mmol) and DIPEA (230 mg, 1.78 mmol). The reaction mixture was stirred at 10°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (2R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(3-ethylmorpholino)-5- fluorobenzamido)propanoate (250 mg). 1H NMR (400MHz, CDCl3) δ 7.36-7.30 (m, 10H), 7.03 (s, 1H), 6.69 - 6.56 (m, 3H), 5.96 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 4.63 - 4.55 (m, 1H), 4.01 - 3.86 (m, 3H), 3.84 - 3.60 (m, 3H), 3.50 (d, J = 10.0 Hz, 1H), 3.25 - 3.14 (m, 2H), 1.92 - 1.79 (m, 1H), 1.49 - 1.38 (m, 1H), 0.91 (t, J = 7.6 Hz, 3H). Step 4: (2R)-2-amino-3-(3-(3-ethylmorpholino)-5-fluorobenzamido)propanoic acid
Figure imgf000190_0001
A solution of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylmorpholino)-5- fluorobenzamido)propanoate (250 mg, 0.44 mmol) in the solution of 30% HBr/HOAc (10 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (2R)-2-amino-3-(3-(3- ethylmorpholino)-5-fluorobenzamido)propanoic acid (100 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.98-8.90 (m, 1H), 8.56 (br s, 3H), 7.24 (s, 1H), 7.04-6.98 (m, 1H), 6.90-6.83 (m, 1H), 4.12-4.06 (m, 1H), 3.94-3.84 (m, 2H), 3.79-3.69 (m, 3H), 3.60- 3.47 (m, 2H), 3.52-3.33 (m, 1H), 3.10-3.01 (m, 1H), 1.79-1.65 (m, 1H), 1.39-1.27 (m, 1H), 0.85 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 340.0 tR (min, Method c) = 1.004 [α]20 ,D = +2.0 (c = 2 mg/mL,CH3OH). Compound 2j (R)-2-amino-3-(3-(2-ethylpiperidin-1-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethylpiperidin- 1-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000191_0001
Step 1: methyl 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoate
Figure imgf000191_0002
To a mixture of methyl 3-bromo-5-fluorobenzoate (200 mg, 0.86 mmol) and 2- ethylpiperidine (117 mg, 1.03 mmol) in dioxane (4 mL) was added Cs2CO3 (839 mg, 2.57 mmol) and CPhos-Pd-G3 (69 mg, 0.086 mmol) in glovebox, the mixture was stirred at 90°C for 16 hours in glovebox. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 5%) to give methyl 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoate (190 mg, crude). 1H NMR (400MHz, CDCl3) δ 7.32 (s, 1H), 7.03 (d, J=8.8 Hz, 1H), 6.69 (d, J=12.4 Hz, 1H), 3.90 (s, 3H), 3.80-3.76 (m, 1H), 3.48-3.43 (m, 1H), 3.05 - 2.94 (m, 1H), 1.79 - 1.50 (m, 8H), 0.87 (t, J=7.6 Hz, 3H). Step 2: 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoic acid
Figure imgf000192_0001
To a solution of methyl 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoate (190 mg, crude) in the mixture of MeOH (3 mL), H2O (3 mL) and THF (3 mL) was added LiOH.H2O (60 mg, 1.43 mmol), the mixture was stirred at 15°C for 14 hours. The mixture was concentrated. The residue was added water (10 mL), then drop-wise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (15 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoic acid (170 mg, crude). LC-MS (MH+): m/z = 252.1 tR (min, Method c) = 0.706 Step 3: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpiperidin-1-yl)-5- fluorobenzamido)propanoate
Figure imgf000192_0002
To a mixture of 3-(2-ethylpiperidin-1-yl)-5-fluorobenzoic acid (170 mg, crude) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (296 mg, 0.81 mmol, HCl salt) in DMF (5 mL) was added TBTU (326 mg, 1.01 mmol) and DIPEA (262 mg, 2.03 mmol), the mixture was stirred at 15°C for 15 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) and further purified by preparative-HPLC (Method AA) to give (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpiperidin-1-yl)-5- fluorobenzamido)propanoate (220 mg). Step 4: (2R)-2-amino-3-(3-(2-ethylpiperidin-1-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000193_0001
A solution of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpiperidin-1-yl)-5- fluorobenzamido)propanoate (100 mg, 0.18 mmol) in 30% HBr/HOAc (3 mL) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (2R)-2-amino-3-(3-(2-ethylpiperidin-1-yl)-5- fluorobenzamido)propanoic acid (10 mg) HCl salt. 1H NMR (400MHz, D2O) δ 7.88 (s, 1H), 7.78 (d, J=8.4 Hz, 1H), 7.73 (d, J=8.4 Hz, 1H), 4.32- 4.27 (m, 1H), 4.12-4.05 (m, 1H), 4.00-3.93 (m, 1H), 3.83-3.65 (m, 3H), 2.37-2.30 (m, 1H), 2.12-1.98 (m, 3H), 1.84-1.71 (m, 2H), 1.56-1.39 (m, 2H), 0.86 (t, J=7.2 Hz, 3H). LC-MS (MH+): m/z = 338.0 tR (min, Method c) = 1.441 [α]20 ,D = +2.0 (c = 2.0 mg/mL, CH3OH). Compound 2k (R)-2-amino-3-(2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(2'-chloro-5-fluoro- 5'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid is shown below
Figure imgf000194_0001
Step 1: methyl 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-carboxylate
Figure imgf000194_0002
A mixture of 2-bromo-1-chloro-4-methylbenzene (300 mg, 1.46 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (289 mg, 1.46 mmol), Pd(dppf)Cl2 (107 mg, 0.15 mmol) and K2CO3 (605 mg, 4.38 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3- carboxylate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.91 (d, J = 1.2 Hz, 1H), 7.77 - 7.71 (m, 1H), 7.41 - 7.35 (m, 2H), 7.17 - 7.11 (m, 2H), 3.95 (s, 3H), 2.38 (s, 3H). Step 2: 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-carboxylic acid
Figure imgf000195_0001
To a solution of methyl 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-carboxylate (350 mg, 1.26 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (105 mg, 2.51 mmol) and the mixture was stirred at 25°C for 1 hour. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-carboxylic acid (310 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-5'-methyl-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000195_0002
To a solution of 2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-carboxylic acid (100 mg, 0.38 mmol and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (152 mg, 0.42 mmol, HCl salt) in DMF (5 mL) was added TBTU (182 mg, 0.57 mmol) and DIPEA (146 mg, 1.13 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (200 mg). LC-MS (MH+): m/z = 575.2 tR (min, Method c) = 0.979 Step 4: (R)-2-amino-3-(2'-chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoic acid
Figure imgf000196_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-5'-methyl- [1,1'-biphenyl]-3-ylcarboxamido)propanoate (200 mg, 0.35 mmol) in the solution of 30% HBr/HOAc (5 mL) was heated at 50°C for 16 hours. The mixture was concentrate. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(2'- chloro-5-fluoro-5'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.06-9.00 (m, 1H), 8.50 (br s, 3H), 7.81-7.74 (m, 2H), 7.54- 7.45 (m, 2H), 7.34-7.25 (m, 2H), 4.15-4.06 (m, 1H), 3.85-3.71 (m, 2H), 2.35 (s, 3H). LC-MS (MH+): m/z = 350.9 tR (min, Method D) = 2.036 [α]20,D = -4.0 (c = 2 mg/mL,CH3OH). Compound 2l (R)-2-amino-3-(2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(2'-chloro-5-fluoro- 4'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid is shown below
Figure imgf000197_0001
A mixture of 1-bromo-2-chloro-4-methylbenzene (200 mg, 0.97 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (193 mg, 0.97 mmol), Pd(dppf)Cl2 (71 mg, 0.10 mmol) and K2CO3 (404 mg, 2.92 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3- carboxylate (200 mg). 1H NMR (400MHz, CDCl3) δ 7.91 (d, J = 1.2 Hz, 1H), 7.75 - 7.70 (m, 1H), 7.40 - 7.35 (m, 1H), 7.32 (s, 1H), 7.25 - 7.21 (m, 1H), 7.17 - 7.13 (m, 1H), 3.95 (s, 3H), 2.40 (s, 3H). Step 2: 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-carboxylic acid
Figure imgf000198_0001
To a solution of methyl 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-carboxylate (200 mg, 0.72 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (60 mg, 1.44 mmol) and the mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-carboxylic acid (180 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-4'-methyl-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000198_0002
To a solution of a mixture of 2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-carboxylic acid (180 mg, 0.68 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (273 mg, 0.75 mmol, HCl salt) in DMF (5 mL) was added TBTU (328 mg, 1.02 mmol) and DIPEA (264 mg, 2.04 mmol). The reaction mixture was stirred at 20°C for 16 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'- chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (250 mg). 1H NMR (400MHz, CDCl3) δ 7.51 (s, 1H), 7.42 - 7.25 (m, 13H), 7.22 - 7.11 (m, 2H), 6.83 (br s, 1H), 6.00 (br d, J = 6.8 Hz, 1H), 5.27 - 5.05 (m, 4H), 4.63 - 4.55 (m, 1H), 3.95 - 3.76 (m, 2H), 2.39 (s, 3H). Step 4: (R)-2-amino-3-(2'-chloro-5-fluoro-4'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoic acid
Figure imgf000199_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-chloro-5-fluoro-4'-methyl- [1,1'-biphenyl]-3-ylcarboxamido)propanoate (200 mg, 0.35 mmol) in the solution of 30% HBr/HOAc (5 mL) was heated at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(2'-chloro- 5-fluoro-4'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid (120 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.05-9.00 (m, 1H), 7.77-7.67 (m, 2H), 7.52-7.42 (m, 2H), 7.39-7.34 (m, 1H), 7.30-7.25 (m, 1H), 3.82-3.71 (m, 2H), 3.66-3.57 (m, 1H), 2.37 (s, 3H). LC-MS (MH+): m/z = 350.9 tR (min, Method D) = 2.034 [α]20,D = +7.0 (c = 2 mg/mL,CH3OH). Compound 2m The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethylpyridin-3- yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000200_0001
To a mixture of 3-bromo-4-ethylpyridine (300 mg, 1.61 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (319 mg, 1.61 mmol) in the mixture of dioxane (10 mL) and H2O (1 mL) was added K2CO3 (669 mg, 4.84 mmol) and Pd(dppf)Cl2 (118 mg, 0.16 mmol), the mixture was stirred at 90°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give methyl 3-(4-ethylpyridin-3-yl)-5-fluorobenzoate (380 mg). 1H NMR (400MHz, CDCl3) δ 8.54 (d, J=5.6 Hz, 1H), 8.40 (s, 1H), 7.81 (t, J=0.6 Hz, 1H), 7.79 - 7.74 (m, 1H), 7.27 - 7.21 (m, 2H), 3.95 (s, 3H), 2.60 (q, J=7.6 Hz, 2H), 1.14 (t, J=7.6 Hz, 3H). Step 2: 3-(4-ethylpyridin-3-yl)-5-fluorobenzoic acid
Figure imgf000201_0001
To a solution of methyl 3-(4-ethylpyridin-3-yl)-5-fluorobenzoate (200 mg, 0.77 mmol) in the mixture of MeOH (6 mL) and H2O (6 mL) was added LiOH.H2O (65 mg, 1.54 mmol), the mixture was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (20 mL × 4), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(4-ethylpyridin-3-yl)-5-fluorobenzoic acid (110 mg). 1H NMR (400MHz, DMSO-d6) δ 8.57 (d, J=5.2 Hz, 1H), 8.45 (s, 1H), 7.76 - 7.72 (m, 2H), 7.63 - 7.58 (m, 1H), 7.49 (d, J=4.8 Hz, 1H), 2.60 (q, J=7.6 Hz, 2H), 1.08 (t, J=7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000201_0002
To a mixture of 3-(4-ethylpyridin-3-yl)-5-fluorobenzoic acid (110 mg, 0.45 mmol) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (196 mg, 0.54 mmol, HCl salt) in DMF (5 mL) was added TBTU (216 mg, 0.67 mmol) and DIPEA (174 mg, 1.35 mmol), the mixture was stirred at 25°C for 14 hours. The mixture was added water (60 mL) and extracted with ethyl acetate (30 mL × 3), the organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyridin- 3-yl)-5-fluorobenzamido)propanoate (190 mg). 1H NMR (400MHz, CDCl3) δ 8.54 (d, J=5.2 Hz, 1H), 8.38 (s, 1H), 7.45 - 7.40 (m, 2H), 7.37 - 7.29 (m, 10H), 7.26 (s, 1H), 7.18 - 7.14 (m, 1H), 6.94 (br s, 1H), 5.96 (br d, J=6.4 Hz, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.63 - 4.55 (m, 1H), 3.99 - 3.78 (m, 2H), 2.59 (q, J=7.6 Hz, 2H), 1.14 (t, J=7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(4-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000202_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate (190 mg, 0.34 mmol) in 30% HBr/ HOAc (5 mL) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethylpyridin-3-yl)-5- fluorobenzamido)propanoic acid (75 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.34-9.27 (m, 1H), 8.86 (d, J=6.0 Hz, 1H), 8.81 (s, 1H), 8.69 (br s, 3H), 8.03 (d, J=6.0 Hz, 1H), 7.95-7.89 (m, 2H), 7.68-7.62 (m, 1H), 4.18-4.08 (m, 1H), 3.86-3.77 (m, 2H), 2.81 (q, J = 7.6 Hz, 2H), 1.12 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 332.0 tR (min, Method D) = 1.138 [α]20,D = +1.67 (c = 4.8 mg/mL, CH3OH). Compound 2n (R)-2-amino-3-(3-(5-ethylpyrimidin-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylpyrimidin- 4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000203_0001
To a mixture of 4-chloro-5-ethylpyrimidine (200 mg, 1.4 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (278 mg, 1.4 mmol) in the mixture of dioxane (10 mL) and H2O (1 mL) was added K2CO3 (582 mg, 4.21 mmol) and Pd(dppf)Cl2 (103 mg, 0.14 mmol). The mixture was stirred at 90°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give methyl 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoate (300 mg). 1H NMR (400MHz, CDCl3) δ 9.14 (s, 1H), 8.73 (s, 1H), 8.10-8.00 (m, 1H), 7.88 - 7.81 (m, 1H), 7.53 - 7.48 (m, 1H), 3.95 (s, 3H), 2.76 (q, J=7.6 Hz, 2H), 1.22 (t, J=7.6 Hz, 3H). Step 2: 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoic acid
Figure imgf000203_0002
To a solution of methyl 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoate (300 mg, 1.15 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (73 mg, 1.73 mmol), the mixture was stirred at 25°C for 1 hour. The mixture was concentrated and added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4. The precipitated solids was filtered to give 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoic acid (270 mg). 1H NMR (400MHz, DMSO-d6) δ 9.12 (s, 1H), 8.86 (s, 1H), 7.98-7.95 (m, 1H), 7.83 - 7.79 (m, 1H), 7.78 - 7.74 (m, 1H), 2.72 (q, J=7.6 Hz, 2H), 1.12 (t, J=7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylpyrimidin-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000204_0001
To a mixture of 3-(5-ethylpyrimidin-4-yl)-5-fluorobenzoic acid (270 mg, 1.1 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (480 mg, 1.32 mmol, HCl salt) in DMF (8 mL) was added TBTU (528 mg, 1.64 mmol) and DIPEA (425 mg, 3.29 mmol). The mixture was stirred at 25°C for 14 hours. The mixture was added water (80 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5- ethylpyrimidin-4-yl)-5-fluorobenzamido)propanoate (380 mg). LC-MS (MH+): m/z = 557.1 tR (min, Method c) = 0.918. Step 4: (R)-2-amino-3-(3-(5-ethylpyrimidin-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000205_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylpyrimidin-4-yl)-5- fluorobenzamido)propanoate (160 mg, 0.29 mmol) in 30% HBr/HOAc (3 mL) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(5-ethylpyrimidin-4-yl)-5- fluorobenzamido)propanoic acid (65 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.24-9.16 (m, 1H), 9.15 (s, 1H), 8.88 (s, 1H), 8.65 (br s, 3H), 7.97-7.92 (m, 2H), 7.68-7.63 (m, 1H), 4.15-4.08 (m, 1H), 3.83-3.78 (m, 2H), 2.73 (q, J = 7.6 Hz, 2H), 1.09 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 333.0 tR (min, Method D) = 1.400. [α]20,D = +3.2 (c = 5.0 mg/mL, CH3OH). Compound 2o (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethylpyridin-3- yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000205_0002
Figure imgf000206_0001
To a solution of 3-bromo-2-ethyl-pyridine (200 mg, 1.07 mmol) and (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (213 mg, 1.07 mmol) in a mixture of dioxane (5 mL) and H2O (0.5 mL) was added Pd(dppf)Cl2 (79 mg, 0.11 mmol) and K2CO3 (446 mg, 3.22 mmol). The mixture was heated to 90°C and stirred for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layer was dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20%) to give methyl 3-(2-ethylpyridin-3-yl)-5-fluorobenzoate (0.2 g). 1H NMR (400MHz, CDCl3) δ 8.61 (dd, J=1.2, 4.8 Hz, 1H), 7.81 (dd, J=1.2, 1.2 Hz, 1H), 7.78 - 7.73 (m, 1H), 7.50 (dd, J=1.6, 7.6 Hz, 1H), 7.26 - 7.18 (m, 2H), 3.95 (s, 3H), 2.76 (q, J=7.6 Hz, 2H), 1.21 (t, J=7.6 Hz, 3H). Step 2: 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid
Figure imgf000206_0002
To a solution of methyl 3-(2-ethylpyridin-3-yl)-5-fluorobenzoate (200 mg, 0.77 mmol) in a mixture of H2O (2.5 mL) and MeOH (2.5 mL) was added LiOH.H2O (97 mg, 2.31 mmol). The mixture was stirred at 25°C for 3 hours. The mixture was concentrated, diluted with water (5 mL) then acidified with aqueous HCl (2M) to pH=4-5, the mixture was concentrated, the residue was washed with ethyl acetate/MeOH(10 mL, 5:1), filtered, the filtrate was concentrated to give 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid (300 mg, crude). LC-MS (MH+): m/z = 246.1 tR (min, 5.95Method c) = 0.505. Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000207_0001
To a solution of 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid (280 mg, crude) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (262 mg, 0.72 mmol, HCl) in DMF (5 mL) was added HATU (410 mg, 1.08 mmol) and DIPEA (279 mg, 2.16 mmol). The mixture was stirred at 25°C for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3), the organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (Petroleum ether /ethyl acetate with ethyl acetate from 10 to 50%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.62 (dd, J=1.6, 4.8 Hz, 1H), 7.50-7.30 (m, 13H), 7.28-7.18 (m, 2H), 6.83 (br, 1H), 5.92 (br d, J=5.6 Hz, 1H), 5.22 (s, 2H), 5.10 (s, 2H), 4.64 - 4.56 (m, 1H), 3.98 - 3.92 (m, 1H), 3.88 - 3.79 (m, 1H), 2.76 (q, J=7.2 Hz, 2H), 1.22 (t, J=7.2 Hz, 3H). Step 4: (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000207_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate (100 mg, 0.18 mmol) in 30% HBr/HOAc (3 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by Preparative HPLC (HCl condiiton) to give (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.32-9.25 (m, 1H), 8.85-8.80 (m, 1H), 8.66 (br s, 3H), 8.43- 8.37 (m, 1H), 7.98-7.88 (m, 3H), 7.68-7.61 (m, 1H), 4.15-4.13 (m, 1H), 3.84-3.79 (m, 2H), 3.00 (q, J=7.6 Hz, 2H), 1.16 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 332.0 tR (min, Method D) = 1.076. [α]20 ,D = +3.3 (c = 1.8 mg/mL, CH3OH). Compound 2p (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethylpyrazin-2- yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000208_0001
Figure imgf000209_0001
To a mixture of 2-chloro-3-ethylpyrazine (200 mg, 1.40 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (278 mg, 1.40 mmol) in the mixture of dioxane (5 mL) and H2O (0.5 mL) was added K2CO3 (582 mg, 4.21 mmol) and Pd(dppf)Cl2 (103 mg, 0.14 mmol), the mixture was stirred at 90°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 10%) to give methyl 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoate (290 mg). 1H NMR (400MHz, CDCl3) δ 8.56 (d, J=2.0 Hz, 1H), 8.50 (d, J=2.0 Hz, 1H), 8.04 (d, J=1.6 Hz, 1H), 7.82 (dd, J=1.2 Hz, 8.4 Hz,1H), 7.49 (dd, J=2.4 Hz, 7.2 Hz,1H), 3.96 (s, 3H), 2.91 (q, J=7.6 Hz, 2H), 1.30 (t, J=7.6 Hz, 2H). Step 2: 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid
Figure imgf000209_0002
To a solution of methyl 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoate (290 mg, 1.11 mmol) in the mixture of MeOH (6 mL) and H2O (6 mL) was added LiOH.H2O (94 mg, 2.23 mmol). The mixture was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid (270 mg). LC-MS (MH+): m/z = 247.1 tR (min, 5.95Method c) = 0.595. Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate
Figure imgf000210_0001
Figure imgf000210_0002
To a mixture of 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid (150 mg, 0.61 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (267 mg, 0.73 mmol, HCl salt) in DMF (6 mL) was added TBTU (293 mg, 0.91 mmol) and DIPEA (236 mg, 1.83 mmol). The mixture was stirred at 20°C for 14 hours. The mixture was added water (60 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3- ethylpyrazin-2-yl)-5-fluorobenzamido)propanoate (240 mg). 1H NMR (400MHz, CDCl3) δ 8.57 (d, J=2.0 Hz, 1H), 8.49 (d, J=2.0 Hz, 1H), 7.69 (s, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.37 - 7.28 (m, 10H), 6.85 (br s, 1H), 5.92 (br d, J=6.4 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.64 - 4.56 (m, 1H), 3.98 - 3.79 (m, 2H), 2.91 (q, J=7.2 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H). Step 4: (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000210_0003
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate (240 mg, 0.43 mmol) in 30% HBr/HOAc (5 mL) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoic acid (116 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.18-9.11 (m, 1H), 8.65 (s, 1H), 8.64 - 8.57 (m, 4H), 7.95 (s, 1H), 7.89 (d, J=8.8 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H), 4.15-4.08 (m, 1H), 3.84-3.76 (m, 2H), 2.86 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 333.0 tR (min, Method D) = 1.446 [α]20 ,D = +3.5 (c = 4.0 mg/mL, CH3OH). Compound 2q (R)-2-amino-3-(3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-chloro-6- methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000211_0001
To a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (244 mg, 1.23 mmol) and 2,3-dichloro-6-methylpyridine (200 mg, 1.23 mmol) in the mixture of dioxane (5 mL) and H2O (0.5 mL) was added K2CO3 (512 mg, 3.7 mmol) and Pd(dppf)Cl2 (90 mg, 0.12 mmol), the mixture was stirred at 90°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 5%) to give methyl 3-(3-chloro- 6-methylpyridin-2-yl)-5-fluorobenzoate (180 mg). 1H NMR (400MHz, CDCl3) δ 8.22 (t, J=1.2 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.68 - 7.63 (m, 1H), 7.14 (d, J=8.0 Hz, 1H), 3.95 (s, 3H), 2.60 (s, 3H). Step 2: 3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzoic acid
Figure imgf000212_0001
To a solution of methyl 3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzoate (180 mg, 0.64 mmol) in the mixture of MeOH (5 mL), THF (5 mL) and H2O (5 mL) was added LiOH.H2O (54 mg, 1.29 mmol), the mixture was stirred at 20°C for 2 hours. The mixture was concentrated. The residue was added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(3-chloro-6-methylpyridin- 2-yl)-5-fluorobenzoic acid (170 mg). LC-MS (MH+): m/z = 266.0 tR (min, 5.95Method c) = 0.660 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-chloro-6-methylpyridin-2-yl)- 5-fluorobenzamido)propanoate
Figure imgf000213_0001
To a mixture of 3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzoic acid (170 mg, 0.64 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (280mg, 0.77 mmol, HCl salt) in DMF (5 mL) was added TBTU (308 mg, 0.96 mmol) and DIPEA (248 mg, 1.92 mmol), the mixture was stirred at 20°C for 15 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (3-chloro-6-methylpyridin-2-yl)-5-fluorobenzamido)propanoate (240 mg). 1H NMR (400MHz, CDCl3) δ 7.82 (s, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.37 - 7.28 (m, 10H), 7.15 (d, J=8.0 Hz, 1H), 6.77 (br s, 1H), 5.96 (br d, J=6.4 Hz, 1H), 5.21 (s, 2H), 5.11 (s, 2H), 4.63 - 4.54 (m, 1H), 3.96 - 3.80 (m, 2H), 2.60 (s, 3H). Step 4: (R)-2-amino-3-(3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000213_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-chloro-6-methylpyridin- 2-yl)-5-fluorobenzamido)propanoate (120 mg, 0.21 mmol) in 30% HBr/HOAc (3 mL) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(3-chloro-6-methylpyridin-2- yl)-5-fluorobenzamido)propanoic acid (48 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.18-9.10 (m, 1H), 8.65 (br s, 3H), 8.03-7.99 (m, 2H), 7.94- 7.89 (m, 1H), 7.69-7.65 (m, 1H), 7.45-7.38 (m, 1H), 4.15-4.07 (m, 1H), 3.85-3.78 (m, 2H), 2.54 (s, 3H). LC-MS (MH+): m/z = 351.9 tR (min, Method D) = 1.664 [α]20 ,D = +2.0 (c = 2.0 mg/mL, CH3OH). Compound 2r (-) (2R)-2-amino-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (-)(2R)-2-amino-3-(3-(2- ethylcyclohexyl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000215_0001
To a solution of 2-ethylcyclohexanone (4 g, 31.70 mmol) and Na2CO3 (6.72 g, 63.39 mmol) in DCM (40 mL) was added dropwise Tf2O (17.89 g, 63.39 mmol) in DCM (20 mL) at 10 °C. The mixture was stirred at 20 °C for 16 hours. The mixture was filtered and the filtration cake was washed with DCM (30 mL), the combined organic layers were washed with saturated NaHCO3 (3 × 40 mL) and brine (2 × 30 mL), dried with Na2SO4 and concentrated to give 2-ethylcyclohex-1-en-1-yl trifluoromethanesulfonate (4.5 g, crude). Step 2: methyl 6'-ethyl-5-fluoro-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-3-carboxylate
Figure imgf000216_0001
To a solution of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.2 g, 6.06 mmol) and 2-ethylcyclohex-1-en-1-yl trifluoromethanesulfonate (4.50 g, crude) in a mixture of DME (20 mL) and H2O (2 mL) was added Pd(PPh3)4 (701 mg, 0.61 mmol) and Na2CO3 (1.93 g, 18.18 mmol) under N2 atmosphere. The mixture was stirred at 90 °C for 14 hours. The mixture was diluted with water (30 mL) and ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 5%) to give methyl 6'-ethyl-5-fluoro-2',3',4',5'-tetrahydro-[1,1'- biphenyl]-3-carboxylate (960 mg). LC-MS (MH+): m/z = 263.1 tR (min, Method c) = 1.023 Step 3: methyl 3-(2-ethylcyclohexyl)-5-fluorobenzoate
Figure imgf000216_0002
To a solution of methyl 6'-ethyl-5-fluoro-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-3- carboxylate (960 mg, 3.66 mmol) in MeOH (15 mL) was added Pd/C (200 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 3 hours. The mixture was filtered and the filtrate was concentrated to give methyl 3-(2-ethylcyclohexyl)-5- fluorobenzoate (860 mg). Step 4: 3-(2-ethylcyclohexyl)-5-fluorobenzoic acid
Figure imgf000217_0001
To a solution of methyl 3-(2-ethylcyclohexyl)-5-fluorobenzoate (860 mg, 3.25 mmol) in a mixture of MeOH (10 mL) and H2O (5 mL) was added LiOH.H2O (410 mg, 9.76 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was diluted with water (30 mL) and acidified with aq. 2N HCl to adjust pH to 4~5 and extracted with ethyl acetate (20 mL × 3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-(2-ethylcyclohexyl)-5- fluorobenzoic acid (700 mg, crude). Step 5: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate
Figure imgf000217_0002
To a solution of 3-(2-ethylcyclohexyl)-5-fluorobenzoic acid (700 mg, crude) in DMF (15 mL) was added TBTU (1.35 g, 4.20 mmol), DIPEA (1.09 g, 8.40 mmol) and (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.02 g, 2.80 mmol, HCl salt). The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) followed by SFC (Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALPAK IC(250mm*30mm,5 μm); Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, v%) = 55/45; Flow Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give 350 mg of the mixture product.350 mg of the product was separated by SFC: (Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALPAK AD-H(250mm*30mm,5 μm); Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, v%) = 60/40; Flow Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (2R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoate (140 mg), and (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate (140 mg). (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate: 1H NMR (400 MHz, CD3OD) δ 7.44 (s, 1H), 7.39 - 7.19 (m, 11H), 7.14 - 7.08 (m, 1H), 5.21 - 5.11 (m, 2H), 5.10 - 5.03 (m, 2H), 4.58 - 4.51 (m, 1H), 3.85 - 3.71 (m, 2H), 2.97 - 2.89 (m, 1H), 1.95 - 1.77 (m, 4H), 1.70 - 1.55 (m, 2H), 1.53 - 1.42 (m, 3H), 1.39 - 1.29 (m, 1H), 0.94 - 0.83 (m, 1H), 0.69 (t, J = 7.2 Hz, 3H) (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate: 1H NMR (400 MHz, CD3OD) δ 7.44 (s, 1H), 7.37 - 7.19 (m, 11H), 7.14 - 7.08 (m, 1H), 5.21 - 5.10 (m, 2H), 5.10 - 5.03 (m, 2H), 4.58 - 4.51 (m, 1H), 3.84 - 3.70 (m, 2H), 2.97 - 2.89 (m, 1H), 1.95 - 1.75 (m, 4H), 1.70 - 1.55 (m, 2H), 1.53 - 1.40 (m, 3H), 1.38 - 1.28 (m, 1H), 0.93 - 0.82 (m, 1H), 0.68 (t, J = 7.2 Hz, 3H) Step 6: (-) (R)-2-amino-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoic acid
Figure imgf000218_0001
A mixture of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate (60 mg, 0.11 mmol) in 30% HBr/HOAc (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (HCl) to give (-)(2R)-2-amino-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoic acid (18 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.99-8.90 (m, 1H), 8.52 (br s, 3H), 7.59 (s, 1H), 7.53 (d, J = 9.2 Hz, 1H), 7.21 (d, J = 9.2 Hz, 1H), 4.14-4.04 (m, 1H), 3.79-3.69 (m, 2H), 2.96-2.87 (m, 1H), 1.89-1.74 (m, 4H), 1.64-1.49 (m, 2H), 1.46-1.22 (m, 4H), 0.83-0.71 (m, 1H), 0.68-0.61 (m, 3H). LC-MS (MH+): m/z = 337.1 tR (min, Method D) = 2.008 [α]20,D = -44.6 (c = 1.3 mg/mL, CH3OH). Compound 2s The scheme for the preparation of (+) (R)-2-amino-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000219_0001
A mixture of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoate (70 mg, 0.12 mmol) in 30% HBr/HOAc (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (HCl) twice to give (+)(2R)-2-amino-3-(3-(2-ethylcyclohexyl)-5- fluorobenzamido)propanoic acid (16 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.92-8.84 (m, 1H), 8.41 (br s, 3H), 7.57 (s, 1H), 7.50 (d, J = 9.6 Hz, 1H), 7.22 (d, J = 9.6 Hz, 1H), 4.11-4.04 (m, 1H), 3.77-3.71 (m, 2H), 2.96-2.87 (m, 1H), 1.88-1.74 (m, 4H), 1.65-1.49 (m, 2H), 1.47-1.25 (m, 4H), 0.84-0.71 (m, 1H), 0.68-0.59 (m, 3H). LC-MS (MH+): m/z = 337.0 tR (min, Method D) = 2.220 [α]20,D = 22.2 (c = 1.8 mg/mL, CH3OH). Compound 2t (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclobutyl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoic acid is shown below
Figure imgf000220_0001
To a solution of 3-fluoro-5-iodobenzoic acid (5.0 g, 18.80 mmol) in EtOH (50 mL) was added SOCl2 (4.47 g, 37.59 mmol) at 0 °C. The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 5%) to give ethyl 3- fluoro-5-iodobenzoate (4.75 g). 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 1.2 Hz, 1H), 7.72 - 7.66 (m, 1H), 7.64 - 7.59 (m, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). Step 2: ethyl 3-fluoro-5-(1-hydroxycyclobutyl)benzoate
Figure imgf000221_0001
To a solution of ethyl 3-fluoro-5-iodobenzoate (1 g, 3.40 mmol) in THF (20 mL) was added i-PrMgCl-LiCl (1.3 M in THF, 3.66 mL, 1.4 eq) dropwise at -70 °C. The mixture was stirred at -70 °C for 30 min then cyclobutanone (238 mg, 3.40 mmol) was added. The mixture was warmed to 20 °C for 1 hour. The reaction was quenched with saturated solution NH4Cl (10 mL) and diluted with water (20 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. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 15%) to give ethyl 3-fluoro-5-(1-hydroxycyclobutyl)benzoate (670 mg). 1H NMR (400 MHz, CDCl3) δ 7.97 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 7.64 - 7.58 (m, 1H), 7.45 - 7.38 (m, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.60 - 2.51 (m, 2H), 2.45 - 2.36 (m, 2H), 2.35 (s, 1H), 2.13 - 2.05 (m, 1H), 1.83 - 1.73 (m, 1H), 1.40 (t, J = 7.2 Hz, 3H). Step 3: ethyl 3-fluoro-5-(1-methoxycyclobutyl)benzoate
Figure imgf000221_0002
To a solution of ethyl 3-fluoro-5-(1-hydroxycyclobutyl)benzoate (300 mg, 1.26 mmol) in DMF (5 mL) was added NaH (76 mg, 1.89 mmol, 60% in mineral oil) at 0 °C and the mixture was stirred at 0 °C for 30 min. Then MeI (357 mg, 2.52 mmol) was added and the mixture was stirred at 60 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give ethyl 3-fluoro-5-(1-methoxycyclobutyl)benzoate (250 mg, crude). Step 4: 3-fluoro-5-(1-methoxycyclobutyl)benzoic acid
Figure imgf000222_0001
To a solution of ethyl 3-fluoro-5-(1-methoxycyclobutyl)benzoate (250 mg, crude) in a mixture of MeOH (10 mL) and H2O (5 mL) was added LiOH.H2O (125mg, 2.97 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated to remove MeOH. The residue was diluted with water (10 mL) and acidified with 2N HCl to adjust pH to 4 and extracted with ethyl acetate (15 mLx3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3- fluoro-5-(1-methoxycyclobutyl)benzoic acid (200 mg). 1H NMR (400 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.62 - 7.56 (m, 1H), 7.52 - 7.45 (m, 1H), 2.86 (s, 3H), 2.32 (t, J =8.0 Hz, 4H), 1.93 - 1.83 (m, 1H), 1.68 - 1.56 (m, 1H), COOH not observed. Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoate
Figure imgf000222_0002
To a solution of 3-fluoro-5-(1-methoxycyclobutyl)benzoic acid (200 mg, 0.89 mmol) in DMF (8 mL) was added TBTU (430 mg, 1.34 mmol), DIPEA (346 mg, 2.68 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (325 mg, 0.89 mmol, HCl salt). The mixture was stirred at 20 °C for 3 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoate (350 mg). 1H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H), 7.40 - 7.28 (m, 12H), 6.93 - 6.82 (m, 1H), 5.96 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 4.65 - 4.56 (m, 1H), 3.99 - 3.89 (m, 1H), 3.86 - 3.78 (m, 1H), 2.95 (s, 3H), 2.42 - 2.35 (m, 4H), 2.02 - 1.93 (m, 1H), 1.76 - 1.68 (m, 1H). Step 6: (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclobutyl)benzamido)propanoic acid
Figure imgf000223_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoate (120 mg, 0.22 mmol) in AcOH (5 mL) was added Pd/C (50 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture was filtered and the filtrate was concentrated. To the residue was added water (30 mL) and lyophilized to give (R)-2-amino-3-(3-fluoro-5-(1- methoxycyclobutyl)benzamido)propanoic acid (60 mg). 1H NMR (400 MHz, DMSO-d6) δ 9.06 (br s, 1H), 7.77 (s, 1H), 7.61 (d, J = 9.6 Hz, 1H), 7.37 (d, J = 9.6 Hz, 1H), 3.79-3.69 (m, 1H), 3.55-3.45 (m, 2H), 2.85 (s, 3H), 2.40-2.27 (m, 4H), 1.91-1.82 (m, 1H), 1.70-1.57 (m, 1H). LC-MS (MH+): m/z = 311.0 tR (min, Method D) = 1.586 [α]20 ,D = 19.0 (c = 2.0 mg/mL, CH3OH). Compound 2u (R)-2-amino-3-(3-(4-ethylpyrimidin-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethylpyrimidin- 5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000224_0001
To a mixture of 5-bromo-4-ethylpyrimidine (300 mg, 1.60 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (349 mg, 1.76 mmol) in the mixture of dioxane (10 mL) and H2O (1 mL) was added K2CO3 (665 mg, 4.81 mmol) and Pd(dppf)Cl2 (117 mg, 0.16 mmol). The mixture was stirred at 90°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give methyl 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoate (380 mg). 1H NMR (400MHz, CDCl3) δ 9.18 (s, 1H), 8.53 (s, 1H), 7.84 - 7.80 (m, 2H), 7.27 - 7.23 (m, 1H), 3.97 (s, 3H), 2.78 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). Step 2: 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoic acid
Figure imgf000225_0001
To a solution of methyl 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoate (180 mg, 0.69 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (58 mg, 1.38 mmol), the mixture was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (20 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoic acid (160 mg, 94.0% yield). LC-MS (MH+): m/z = 247.1 tR (min, Method c) = 0.571 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyrimidin-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000225_0002
To a mixture of 3-(4-ethylpyrimidin-5-yl)-5-fluorobenzoic acid (160 mg, 0.65 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (284 mg, 0.78 mmol, HCl salt) in DMF (6 mL) was added TBTU (313 mg, 0.97 mmol) and DIPEA (252 mg, 1.95 mmol), the mixture was stirred at 25°C for 14 hours. The mixture was added water (60 mL) and extracted with ethyl acetate(40 mL × 3), the organic layers were washed with brine (40 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- ethylpyrimidin-5-yl)-5-fluorobenzamido)propanoate (250 mg). 1H NMR (400MHz, CDCl3) δ 9.18 (s, 1H), 8.50 (s, 1H), 7.48 - 7.41 (m, 2H), 7.38 - 7.30 (m, 10H), 7.19 - 7.15 (m, 1H), 6.97 (br s, 1H), 5.92 (br d, J=6.0 Hz, 1H), 5.22 (s, 2H), 5.15-5.05 (m, 2H), 4.63 - 4.55 (m, 1H), 3.99 - 3.77 (m, 2H), 2.76 (q, J=7.6 Hz, 2H), 1.27 - 1.22 (t, J=7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(4-ethylpyrimidin-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000226_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethylpyrimidin-5-yl)- 5-fluorobenzamido)propanoate (80 mg, 0.14 mmol) in HOAc (3 mL) was added Pd/C (10% Pd, 50% water, 20 mg) under N2 atmosphere. The suspension was degassed and purged with H23 times. The mixture was stirred under H2 (15 Psi) at 20 °C for 1 hour. The mixture was filtered and concentrated. To the residue was added HOAc (2 mL) and MTBE (8 mL), filtered and concentrated. The residue was purified by preparative-HPLC (Method DD) to give (R)-2-amino-3-(3-(4-ethylpyrimidin-5-yl)-5- fluorobenzamido)propanoic acid (10 mg). 1H NMR (400MHz, DMSO-d6) δ 9.15 (s, 1H), 9.00-8.93 (m, 1H), 8.63 (s, 1H), 7.77-7.73 (m, 2H), 7.63-7.54 (m, 1H), 3.76-3.68 (m, 1H), 3.54-3.42 (m, 2H), 2.75 (q, J=7.6 Hz, 2H), 1.15 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 333.0 tR (min, Method D) = 1.359 [α]20,D = +21.4 (c = 1.4 mg/mL, CH3OH). Compound 2v (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclopentyl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoic acid is shown below
Figure imgf000227_0001
To a solution of 3-fluoro-5-iodobenzoic acid (2 g, 7.52 mmol) in a mixture of DCM (30 mL) and MeOH (6 mL) was added TMSCHN2 (2 M in hexane, 5.64 mL). The mixture was stirred at 20 °C for 1 hour. Then additional TMSCHN2 (3 mL, 2 M in hexane) was added and the mixture was stirred at 20 °C for 1 hour. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-fluoro-5-iodobenzoate (2.05 g). 1H NMR (400 MHz, CDCl3) δ 8.17 (dd, J = 1.2 Hz, J = 1.2 Hz, 1H), 7.72 - 7.66 (m, 1H), 7.65 - 7.59 (m, 1H), 3.93 (s, 3H). Step 2: methyl 3-fluoro-5-(1-hydroxycyclopentyl)benzoate
Figure imgf000227_0002
To a solution of methyl 3-fluoro-5-iodobenzoate (500 mg, 1.79 mmol) in THF (17 mL) was added i-PrMgCl-LiCl (1.3 M in THF, 1.51 mL) dropwise at -70 °C and stirred at - 70 °C for 30 min. Then cyclopentanone (150 mg, 1.79 mmol) dissolved in THF (3 mL) was added dropwise and the mixture was warmed to 20 °C for 1 hour. The residue was quenched with saturated NH4Cl solution (10 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. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3- fluoro-5-(1-hydroxycyclopentyl)benzoate (210 mg). 1H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 1.6 Hz, J = 1.6 Hz, 1H), 7.63 - 7.55 (m, 1H), 7.50 - 7.42 (m, 1H), 3.94 (s, 3H), 2.04 - 1.94 (m, 6H), 1.93 - 1.85 (m, 2H), 1.65 (s, 1H). Step 3: methyl 3-fluoro-5-(1-methoxycyclopentyl)benzoate
Figure imgf000228_0001
To a solution of methyl 3-fluoro-5-(1-hydroxycyclopentyl)benzoate (210 mg, 0.88 mmol) in DMF (5 mL) was added NaH (53 mg, 1.32 mmol, 60% in mineral oil) at 0 °C and the mixture was stirred at 0 °C for 30 min. Then MeI (250 mg, 1.76 mmol) was added and the mixture was stirred at 60 °C for 16 hours. The mixture was quenched with water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-fluoro-5-(1-methoxycyclopentyl)benzoate (100 mg) 1H NMR (400 MHz, CDCl3) δ 7.86 (dd, J = 1.2 Hz, J = 1.2 Hz, 1H), 7.66 - 7.59 (m, 1H), 7.38 - 7.34 (m, 1H), 3.94 (s, 3H), 2.99 (s, 3H), 2.21 - 2.12 (m, 2H), 1.92 - 1.76 (m, 6H). Step 4: 3-fluoro-5-(1-methoxycyclopentyl)benzoic acid
Figure imgf000229_0001
To a solution of methyl 3-fluoro-5-(1-methoxycyclopentyl)benzoate (100 mg, 0.40 mmol) in a mixture of MeOH (4 mL) and H2O (2 mL) was added LiOH.H2O (50 mg, 1.19 mmol). The mixture was stirred at 50 °C for 3 hours. The mixture was concentrated to remove MeOH. The residue was dissolved in water (8 mL) and washed with ethyl acetate (8 mL × 2). The organic layer was discarded. The aqueous layer was acidified with aqueous 2N HCl solution to adjust pH to 4 and extracted wth ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(1-methoxycyclopentyl)benzoic acid (60 mg, crude). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoate
Figure imgf000229_0002
To a solution of 3-fluoro-5-(1-methoxycyclopentyl)benzoic acid (60 mg, crude) in DMF (5 mL) was added TBTU (121 mg, 0.38 mmol), DIPEA (98 mg, 0.76 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (92 mg, 0.25 mmol, HCl salt). The mixture was stirred at 25 °C for 2.5 hours. The mixture was diluted with water (10 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. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoate (70 mg). 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.38 - 7.29 (m, 11H), 7.26 - 7.21 (m, 1H), 6.81 (br s, 1H), 5.95 (br d, J = 6.8 Hz, 1H), 5.22 (s, 2H), 5.13 (s, 2H), 4.65 - 4.57 (m, 1H), 3.99 - 3.89 (m, 1H), 3.86 - 3.77 (m, 1H), 2.98 (s, 3H), 2.20 - 2.11 (m, 2H), 1.90 - 1.75 (m, 6H). Step 6: (R)-2-amino-3-(3-fluoro-5-(1-methoxycyclopentyl)benzamido)propanoic acid and (R)-2-amino-3-(3-cyclopentyl-5-fluorobenzamido)propanoic acid
Figure imgf000230_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoat (70 mg, 0.13 mmol) in AcOH (5 mL) was added Pd/C (40 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 15 °C for 1 hour. The mixture was filtered and the filtrate was concentrated. The residue was purified by preparative HPLC (neutral) to give (R)-2-amino-3-(3-fluoro-5-(1- methoxycyclopentyl)benzamido)propanoic acid (12 mg). 1H NMR (400 MHz, DMSO-d6, WX-4OG_22_001) δ 8.96 (br s, 1H), 7.72 (s, 1H), 7.60-7.54 (m, 1H), 7.38-7.32 (m, 1H), 3.79-3.64 (m, 1H), 3.54-3.50 (m, 2H), 2.88 (s, 3H), 2.15-2.06 (m, 2H), 1.85-1.69 (m, 6H). LC-MS (MH+): m/z = 325.0 tR (min, Method D) = 1.707 [α]20 ,D = 17.5 (c = 0.8 mg/mL, CH3OH). Compound 2w (R)-2-amino-3-(5-(5-ethylisothiazol-4-yl)-2-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(5-(5-ethylisothiazol- 4-yl)-2-fluorobenzamido)propanoic acid is shown below
Figure imgf000231_0001
To a solution of 5-bromoisothiazole (500 mg, 3.05 mmol) and triethylborane (1 M in hexane, 9.15 mL) in THF (15 mL) was added Pd(dppf)Cl2 (223 mg, 0.30 mmol) and caesium acetate (1.76 g, 9.15 mmol) under N2 atmosphere. The mixture was stirred at 65°C for 16 hours. The mixture was diluted with water (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 below 30 °C under reduced pressure. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give 5-ethylisothiazole (300 mg). 1H NMR (400MHz, CDCl3) δ 8.36 (s, 1H), 7.00 (s, 1H), 2.98 (q, J = 7.6 Hz, 2H), 1.37 (t, J = 7.6 Hz, 3H).
Figure imgf000231_0002
To a solution of 5-ethylisothiazole (200 mg, 1.77 mmol) in AcOH (8 mL) was added AcOK (277 mg, 2.83 mmol) and Br2 (847 mg, 5.30 mmol). The mixture was stirred at 100 °C for 3 hours. The mixture was diluted with water (15 mL), added saturated Na2SO3 solution (5 mL) and adjusted to pH to 7 with 2N NaOH solution. The aqueous solution was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give 4-bromo-5-ethylisothiazole (170 mg). Step 3: methyl 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoate
Figure imgf000232_0001
4-bromo-5-ethylisothiazole (160 mg, 0.40 mmol), (4-fluoro-3- (methoxycarbonyl)phenyl)boronic acid (159 mg, 0.8 mmol), Pd(dppf)Cl2.CH2Cl2 (66 mg, 0.08 mmol) and Na2CO3 (128 mg, 1.20 mmol) were taken up into a microwave tube in a mixture of dioxane (5 mL) and H2O (0.5 mL). The sealed tube was heated at 120 °C for 40 min under microwave. The mixture was diluted with water (15 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. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.42 (s, 1H), 7.98 - 7.93 (m, 1H), 7.56 - 7.50 (m, 1H), 7.25 - 7.21 (m, 1H), 3.97 (s, 3H), 2.98 (q, J = 7.6 Hz, 2H), 1.36 (t, J = 7.6 Hz, 3H). Step 4: 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoic acid
Figure imgf000232_0002
To a solution of methyl 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoate (100 mg, 0.28 mmol) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (35 mg, 0.83 mmol). The mixture was stirred at 35 °C for 1.5 hour. The mixture was concentrated. The residue was dissolved in water (10 mL) and acidified with 2N HCl solution to adjust pH to 4 and extracted with ethyl acetate (20 mL × 4). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 5-(5- ethylisothiazol-4-yl)-2-fluorobenzoic acid (95 mg). 1H NMR (400MHz, DMSO-d6) δ 13.44 (br s, 1H), 8.63 (s, 1H), 7.92 - 7.87 (m, 1H), 7.78 - 7.74 (m, 1H), 7.45 - 7.40 (m, 1H), 2.99 (q, J = 7.6 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-(5-ethylisothiazol-4-yl)-2- fluorobenzamido)propanoate
Figure imgf000233_0001
To a solution of 5-(5-ethylisothiazol-4-yl)-2-fluorobenzoic acid (90 mg, 0.26 mmol) in DMF (5 mL) was added TBTU (125 mg, 0.39 mmol), DIPEA (101 mg, 0.78 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (95 mg, 0.26 mmol, HCl salt). The mixture was stirred at 15 °C for 15 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate(20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(5-(5-ethylisothiazol-4-yl)-2- fluorobenzamido)propanoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.41 (s, 1H), 8.08 - 8.03 (m, 1H), 7.51 - 7.47 (m, 1H), 7.37 - 7.31 (m, 10H), 7.23 - 7.16 (m, 1H), 7.13 - 7.03 (m, 1H), 5.92 (br d, J = 6.8 Hz, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 4.67 - 4.59 (m, 1H), 3.98 - 3.87 (m, 2H), 2.97 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 6: (R)-2-amino-3-(5-(5-ethylisothiazol-4-yl)-2-fluorobenzamido)propanoic acid
Figure imgf000234_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-(5-ethylisothiazol-4-yl)-2- fluorobenzamido)propanoate (50 mg, 0.089 mmol) in 30% HBr/HOAc (4 mL) was stirred at 50 °C for 3 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(5-(5-ethylisothiazol-4-yl)-2- fluorobenzamido)propanoic acid (19 mg). 1H NMR (400MHz, DMSO-d6) δ 8.68 (br s, 1H), 8.62 (s, 1H), 8.39 (br s, 3H), 7.80-7.55 (m, 1H), 7.71-7.65 (m, 1H), 7.47-7.40 (m, 1H), 4.15-4.05 (m, 1H), 3.83-3.69 (m, 2H), 3.01 (q, J = 7.6 Hz, 2H), 1.27 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 338.0 tR (min, Method D) = 1.440 [α]20,D = 8.2 (c = 1.7 mg/mL, CH3OH). Compound 2x (R)-2-amino-3-(3-fluoro-5-(3-methoxyoxetan-3-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(3- methoxyoxetan-3-yl)benzamido)propanoic acid is shown below
Figure imgf000234_0002
Figure imgf000235_0001
To a solution of ethyl 3-fluoro-5-iodobenzoate (1 g, 3.4 mmol) in THF (20 mL) was added i-PrMgCl-LiCl (1.3 M in THF, 3.66 mL) at -70 °C. The mixture was stirred at -70 °C for 30 min then oxetan-3-one (245 mg, 3.4 mmol) was added. The mixture was warmed to 20 °C for 1 hour. The reaction mixture was quenched with saturated solution NH4Cl (10 mL) and diluted with water (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. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give ethyl 3-fluoro-5- (3-hydroxyoxetan-3-yl)benzoate (500 mg). 1H NMR (400MHz, CDCl3) δ 80.15-8.10 (m, 1H), 7.71 - 7.65 (m, 1H), 7.59 - 7.54 (m, 1H), 4.94 (d, J=7.2 Hz, 2H), 4.88 (d, J=7.2 Hz, 2H), 4.40 (q, J=7.2 Hz, 2H), 3.44 ( s, 1H), 1.41 (t, J=7.2 Hz, 3H). Step 2: ethyl 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoate
Figure imgf000235_0002
To a solution of ethyl 3-fluoro-5-(3-hydroxyoxetan-3-yl)benzoate (200 mg, 0.83 mmol) in DMF (5 mL) was added NaH (49.95 mg, 1.25 mmol, 60% in mineral oil) at 0 °C and the mixture was stirred at 0 °C for 30 min. Then iodomethane (236 mg, 1.67 mmol) was added and the mixture was stirred at 60 °C for 16 hours. The mixture was quenched with water (20 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (15 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 10%) to give ethyl 3-fluoro-5-(3-methoxyoxetan- 3-yl)benzoate (150 mg, crude). Step 3: 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoic acid
Figure imgf000236_0001
To a solution of ethyl 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoate (150 mg, crude) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (50 mg, 1.18 mmol), the mixture was stirred at 20°C for 2 hours. The mixture was concentrated. The residue was added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 4), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoic acid (130 mg). 1H NMR (400MHz, CDCl3) δ 8.10-8.03 (m, 1H), 7.81 - 7.76 (m, 1H), 7.50 - 7.44 (m, 1H), 5.00 (d, J=7.2 Hz, 2H), 4.81 (d, J=7.2 Hz, 2H), 3.21 (s, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(3-methoxyoxetan-3- yl)benzamido)propanoate
Figure imgf000236_0002
To a mixture of 3-fluoro-5-(3-methoxyoxetan-3-yl)benzoic acid (130 mg, 0.57 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (251.60 mg, 0.69 mmol, HCl salt) in DMF (5 mL) was added TBTU (277 mg, 0.86 umol) and DIPEA (223 mg, 1.72 mmol), the mixture was stirred at 20°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro- 5-(3-methoxyoxetan-3-yl)benzamido)propanoate (240 mg). 1H NMR (400MHz, CDCl3) δ 7.69 (s, 1H), 7.40 - 7.31 (m, 12H), 6.97 (br s, 1H), 5.95 (br d, J=6.4 Hz, 1H), 5.23 (s, 2H), 5.13 (s, 2H), 4.94 (d, J=6.8 Hz, 2H), 4.75 ( t, J=7.6 Hz, 2H), 4.66- 4.59 (m, 1H), 3.98 - 3.77 (m, 2H), 3.17 (s, 3H). Step 5: (R)-2-amino-3-(3-fluoro-5-(3-methoxyoxetan-3-yl)benzamido)propanoic acid
Figure imgf000237_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(3- methoxyoxetan-3-yl)benzamido)propanoate (120 mg, 0.22 mmol) in HOAc (4 mL) was added Pd/C (10% Pd, 50% water, 30 mg) under N2 atmosphere. The suspension was degassed and purged with H23 times. The mixture was stirred under H2 (15 Psi ) at 20 °C for 1 hour. The mixture was filtered and concentrated. The residue was added water (10 mL) and freeze-drying twice to give (R)-2-amino-3-(3-fluoro-5-(3-methoxyoxetan-3- yl)benzamido)propanoic acid (15 mg). 1H NMR (400MHz, DMSO-d6) δ 8.98-8.91 (m, 1H), 7.79 (s, 1H), 7.65 (d, J=8.8 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 4.81-4.75 (m, 4H), 3.76-3.67 (m, 1H), 3.55-3.44 (m, 2H), 3.05 (s, 3H). LC-MS (MH+): m/z = 313.0 tR (min, Method F) = 2.59 [α]20 ,D = +27.0 (c = 2.0 mg/mL, CH3OH). Compound 2y (R)-2-amino-3-(2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(2-fluoro-3-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid is shown below
Figure imgf000238_0001
A mixture of (2-fluoro-3-(methoxycarbonyl)phenyl)boronic acid (500 mg, 2.53 mmol), 4- bromo-1,3,5-trimethyl-1H-pyrazole (478 mg, 2.53 mmol), Pd(t-Bu3P)2 (129 mg, 252.58 µmol), KF (440 mg, 7.58 mmol) in the mixture of dioxane (6 mL) and H2O (2 mL) was degassed and purged with N23 times, and then the mixture was stirred at 100 °C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) to give methyl 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (500 mg). 1H NMR (400MHz, CDCl3) δ 7.91 - 7.82 (m, 1H), 7.40 - 7.32 (m, 1H), 7.25 - 7.20 (m, 1H), 3.93 (s, 3H), 3.78 (s, 3H), 2.19 - 2.14 (m, 6H). Step 2: 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
Figure imgf000239_0001
To a solution of methyl 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (500 mg, 1.91 mmol) in the mixture of THF (3 mL), H2O (3 mL) and MeOH (3 mL) was added LiOH.H2O (240 mg, 5.72 mmol). The mixture was stirred at 20 °C for 2 hours. The mixture was concentrate to remove MeOH and THF, Then HCl (2 M) was added to adjust pH=3~4 and concentrated to give 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (1.5 g, crude) as HCl salt. Step 3: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-3-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
Figure imgf000239_0002
To a solution of 2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (500 mg, 1.76 mmol, HCl salt, crude)and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (641 mg, 1.76 mmol, HCl salt) in DMF (10 mL) was added TBTU (677 mg, 2.11 mmol) and DIPEA (1.13 g, 8.78 mmol). The mixture was stirred at 20 °C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 80%) to give (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-3-(1,3,5-trimethyl- 1H-pyrazol-4-yl)benzamido)propanoate (90 mg). LC-MS (MH+): m/z = 559.1 tR (min, Method c) = 0.890 Step 4: (2R)-2-amino-3-(2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid
Figure imgf000240_0001
A solution of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-3-(1,3,5-trimethyl- 1H-pyrazol-4-yl)benzamido)propanoate (90 mg, 161.1 µmol) in 30% HBr/HOAc (3 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. Then AcOH (2 mL) was added and the reaction mixture was stirred at 20°C for 30 minutes. The solid was collected and dried to give (2R)-2-amino-3-(2-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid (60 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.54 (br s, 1H), 8.34 (br s, 3H), 7.69-7.61 (m, 1H), 7.47- 7.39 (m, 1H), 7.37-7.30 (m, 1H), 4.12-4.10 (m, 1H), 3.82-3.66 (m, 5H), 2.14 (s, 3H), 2.06 (s, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method c) = 0.923 [α]20,D = -3 (c = 2 mg/mL, CH3OH). Compound 2z (R)-2-amino-3-(2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(2-fluoro-5-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid is shown below
Figure imgf000240_0002
Step 1: 2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
Figure imgf000241_0001
To a solution of methyl 2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (200 mg, 762.55 µmol) in the mixture of MeOH (2 mL) , THF (2 mL) and H2O (2 mL) was added LiOH.H2O (96 mg, 2.29 mmol). The mixture was stirred at 20 °C for 2 hours. The mixture was concentrate to remove MeOH and THF, Then HCl (2 M) was added to adjust pH=3~4 and concentrated to give 2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (300 mg, crude) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 7.65 (dd, J=2.4, 7.2 Hz, 1H), 7.54 - 7.47 (m, 1H), 7.35 (dd, J=8.4, 10.8 Hz, 1H), 3.74 (s, 3H), 2.20 (s, 3H), 2.13 (s, 3H). Step 2: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-5-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
Figure imgf000241_0002
To a solution of 2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (150 mg, 526.85 µmol, HCl salt, crude) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (198 mg, 543.83 µmol, HCl salt) in DMF (5 mL) was added TBTU (291 mg, 906.34 µmol) and DIPEA (234 mg, 1.81 mmol). The mixture was stirred at 20 °C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) twice to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2- fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoate (100 mg). LC-MS (MH+): m/z = 559.1 tR (min, Method c) = 0.767 Step 3: (R)-2-amino-3-(2-fluoro-5-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid
Figure imgf000242_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2-fluoro-5-(1,3,5-trimethyl- 1H-pyrazol-4-yl)benzamido)propanoate (100 mg, 179.0 µmol) in 30% HBr/HOAc (3 mL) was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(2-fluoro-5-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.73-8.56 (m, 4H), 7.60-7.54 (m, 1H), 7.48-7.42 (m, 1H), 7.40-7.33 (m, 1H), 4.12-4.03 (m, 1H), 3.84-3.72 (m, 5H), 2.25 (s, 3H), 2.18 (s, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method D) = 1.252 [α]20,D = 4 (c = 2 mg/mL, CH3OH). Compound 3a (R)-2-amino-3-(4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(4-fluoro-3-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid is shown below
Figure imgf000243_0001
A mixture of (2-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (500 mg, 2.53 mmol), 4- bromo-1,3,5-trimethyl-1H-pyrazole (478 mg, 2.53 mmol), Pd(t-Bu3P)2 (129 mg, 252.58 µmol), KF (440 mg, 7.58 mmol) in the mixture of dioxane (6 mL) and H2O (2 mL) was degassed and purged with N23 times, and then the mixture was stirred at 100 °C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give 4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (380 mg). 1H NMR (400MHz, CDCl3) δ 8.02 - 7.96 (m, 1H), 7.92 (dd, J=2.4, 7.2 Hz, 1H), 7.18 (dd, J=9.2, 8.8 Hz, 1H), 3.91 (s, 3H), 3.79 (s, 3H), 2.17 (s, 6H). Step 2: 4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
Figure imgf000244_0001
To a solution of methyl 4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (380 mg, 1.45 mmol) in the mixture of MeOH (3 mL), THF (3 mL) and H2O (3 mL) was added LiOH.H2O (182 mg, 4.35 mmol,). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrate to remove MeOH and THF, Then HCl(2 M) was added to adjust pH=5~6 and extracted with ethyl acetate (10 mL × 3). The combined organic washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated to give 4- fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (340 mg). Step 3: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-fluoro-3-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
Figure imgf000244_0002
To a solution of 4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (140 mg, 563.94 µmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (226 mg, 620.34 µmol, HCl salt) in DMF (5 mL) was added TBTU (217 mg, 676.73 µmol) and DIPEA (219 mg, 1.69 mmol). The mixture was stirred at 20 °C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) to give (2R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoate (220 mg). LC-MS (MH+): m/z = 559.3 tR (min, Method c) = 0.835 Step 4: (2R)-2-amino-3-(4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid
Figure imgf000245_0001
A solution of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-fluoro-3-(1,3,5-trimethyl- 1H-pyrazol-4-yl)benzamido)propanoate (220 mg, 393.8 µmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. Then AcOH:MTBE=1:2(15 mL) was added and stirred at 20°C for 30 minutes, and the reaction mixture was collected and lyophilized. The residue was purified by preparative HPLC (Method AA) to give (2R)-2-amino-3-(4-fluoro-3-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid (100 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.03-8.92 (m, 1H), 8.58 (br s, 3H), 8.00-7.93 (m, 1H), 7.90- 7.83 (m, 1H), 7.47-7.33 (m, 1H), 4.12-4.10 (m, 1H), 3.80-3.74 (m, 5H), 2.15 (s, 3H), 2.07 (s, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method D) = 1.356 [α]20,D = 0.57 (c = 3.5 mg/mL, CH3OH). Compound 3b (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-ethyl-4- fluorobenzamido)propanoic acid is shown below
Figure imgf000246_0001
A mixture of methyl 3-bromo-4-fluorobenzoate (1 g, 4.29 mmol), triethylborane (12.9 mL, 1M in hexane), Pd(dppf)Cl2 (314 mg, 0.43 mmol) and CsOAc (2.47 g, 12.87 mmol) in THF (10 mL) was degassed and purged with N23 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-ethyl-4-fluorobenzoate (510 mg). 1H NMR (400MHz, CDCl3) δ 7.95 - 7.85 (m, 2H), 7.08 - 7.02 (m, 1H), 3.91 (s, 3H), 2.70 (q, J = 7.6 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H). Step 2: 3-ethyl-4-fluorobenzoic acid
Figure imgf000246_0002
To a solution of methyl 3-ethyl-4-fluorobenzoate (200 mg, 1.10 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (138 mg, 3.29 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-ethyl- 4-fluorobenzoic acid (160 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate
Figure imgf000247_0001
To a solution of a mixture of 3-ethyl-4-fluorobenzoic acid (160 mg, 0.95 mmol) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (382 mg, 1.05 mmol, HCl salt) in DMF (5 mL) was added TBTU (458 mg, 1.43 mmol) and DIPEA (369 mg, 2.85 mmol). The reaction mixture was stirred at 20 °C for 16 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- ethyl-4-fluorobenzamido)propanoate (400 mg). 1H NMR (400MHz, DMSO-d6) δ 7.61 (d, J = 6.0 Hz, 1H), 7.44 - 7.29 (m, 11H), 7.03 - 6.96 (m, 1H), 6.65 (br s, 1H), 5.99 (br d, J = 6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.63 - 4.56 (m, 1H), 3.94 - 3.88 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid
Figure imgf000248_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate (350 mg, 0.73 mmol) in the solution of 30% HBr in AcOH (10 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-4- fluorobenzamido)propanoic acid (110 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.84-8.74 (m, 1H), 7.89-7.80 (m, 1H), 7.79-7.74 (m, 1H), 7.28-7.20 (m, 1H), 3.81-3.70 (m, 2H), 3.65-3.57 (m, 1H), 2.65 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 255.0 tR (min, Method c) = 0.945 [α]20 ,D = +6.0 (c = 2 mg/mL,CH3OH). Compound 3c (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-4-methyl- 1H-pyrazol-5-yl)benzamido)propanoic acid is shown below
Figure imgf000249_0001
To a solution of 4-methyl-1H-pyrazole (3.0 g, 36.5 mmol) in DMF (10 mL) was added NaH (1.90 g, 47.50 mmol, 60% in mineral oil) at 0°C and stirred at 0°C for 0.5 hour. Then iodoethane (6.84 g, 43.85 mmol) was added at 0°C and stirred at 20°C for 3 hours. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 10%) to give 1-ethyl- 4-methyl-1H-pyrazole (4.0 g). 1H NMR (400 MHz, CD3Cl) δ 7.29 (s, 1H), 7.16 (s, 1H), 4.10 (q, J = 7.6 Hz, 2H), 2.07 (s, 3H), 1.45 (t, J = 7.6 Hz, 3H). Step 2: 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H- pyrazole
Figure imgf000250_0001
To a solution of 1-ethyl-4-methyl-1H-pyrazole (2.0 g, 18.16 mmol) in CHCl3 (10 mL) was added NBS (3.55 g, 19.97 mmol) and the reaction mixture was stirred at 30°C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give a mixture of 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H-pyrazole (1.9 g). LC-MS (MH+): m/z = 191.0 tR (min, Method c) = 0.718 Step 3: methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate
Figure imgf000250_0002
To a solution of (3-(methoxycarbonyl)phenyl)boronic acid (952 mg, 5.29 mmol) in DME (10 mL) were added a mixture of 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo- 1-ethyl-4-methyl-1H-pyrazole (1.0 g, 5.29 mmol), Pd(PPh3)4 (611 mg, 0.53 mmol) and Na2CO3 (1.68 g, 15.87 mmol). The reaction was degassed and purged with N23 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 15%) to give methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate (240 mg). 1H NMR (400 MHz, CD3Cl) δ 8.10 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.60 - 7.54 (m, 1H), 7.53 - 7.48 (m, 1H), 7.42 (s, 1H), 4.04 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 1.99 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). Step 4: 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid
Figure imgf000251_0001
To a solution of methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate (240 mg, 0.98 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (82 mg, 1.96 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid (220 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate
Figure imgf000251_0002
To a solution of 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid (220 mg, 0.96 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (383 mg, 1.05 mmol, HCl salt) in DMF (5 mL) was added TBTU (460 mg, 1.43 mmol) and DIPEA (370 mg, 2.87 mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 45%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate (380 mg). 1H NMR (400 MHz, CDCl3) δ 7.69-7.65 (m, 2H), 7.53 - 7.48 (m, 1H), 7.45 - 7.40 (m, 2H), 7.38 - 7.28 (m, 10H), 6.88 (br, 1H), 5.99 (br, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.63 - 4.56 (m, 1H), 4.01 (q, J = 7.2 Hz, 2H), 3.98 - 3.78 (m, 2H), 1.98 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H). Step 6: (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid
Figure imgf000252_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H- pyrazol-5-yl)benzamido)propanoate (380 mg, 0.70 mmol) in the solution of 30 % HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The sample was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(1- ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid (140 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.94-8.84 (m, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.86 (s, 1H), 7.64 (dd, J = 7.6 Hz, 7.6 Hz, 1H ), 7.55 (d, J = 7.6 Hz, 1H), 7.38 (s, 1H), 4.02-3.96 (m, 3H), 3.81-3.66 (m, 2H), 1.94 (s, 3H), 1.20 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 317.0 tR (min, Method c) = 1.027 [α]20 ,D = +9.0 (c = 2.0 mg/mL, CH3OH). Compound 3d (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoic acid is shown below
Figure imgf000253_0001
A mixture of methyl 3-bromo-5-fluoro-4-methylbenzoate (300 mg, 1.21 mmol), triethylborane (1 M in hexane, 3.64 mL), Pd(dppf)Cl2 (89 mg, 0.12 mmol) and CsOAc (699 mg, 3.64 mmol) in THF (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-ethyl-5- fluoro-4-methylbenzoate (220 mg). 1H NMR (400MHz, CDCl3) δ 7.65 (s, 1H), 7.51 (d, J = 10.0 Hz, 1H), 3.91 (s, 3H), 2.68 (q, J = 7.6 Hz, 2H), 2.26 (s, 3H), 1.23 (t, J = 7.6 Hz, 3H). Step 2: 3-ethyl-5-fluoro-4-methylbenzoic acid
Figure imgf000253_0002
To a solution of methyl 3-ethyl-5-fluoro-4-methylbenzoate (220 mg, 1.12 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (141 mg, 3.36 mmol) and the mixture was stirred at 20°C for 16 hours, then the reaction mixture was stirred at 35°C for another 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-ethyl-5- fluoro-4-methylbenzoic acid (200 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate
Figure imgf000254_0001
To a solution of a mixture of 3-ethyl-5-fluoro-4-methylbenzoic acid (100 mg, 0.55 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (220 mg, 0.60 mmol, HCl salt) in DMF (5 mL) was added TBTU (264 mg, 0.82 mmol) and DIPEA (213 mg, 1.65 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoate (230 mg). 1H NMR (400MHz, CDCl3) δ 7.40 - 7.30 (m, 11H), 7.14 (d, J = 9.6 Hz, 1H), 6.64 (br, 1H), 5.99 (br d, J = 7.2 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.65 - 4.55 (m, 1H), 3.95 - 3.65 (m, 2H), 2.65 (q, J = 7.6 Hz, 2H), 2.25 (s, 3H), 1.21 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid
Figure imgf000255_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate (230 mg, 0.47 mmol) in the solution of 30% HBr in AcOH (10 mL) was heated at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-5-fluoro- 4-methylbenzamido)propanoic acid (80 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.82 (m, 1H), 8.46 (br s, 2H), 7.59 (s, 1H), 7.55-7.49 (m, 1H), 4.04-4.01 (m, 1H), 3.81-3.70 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 2.21 (s, 3H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 269.0 tR (min, Method c) = 1.096 [α]20 ,D = +6.0 (c = 2 mg/mL,CH3OH). Compound 3e (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(4-ethyl-3- fluorobenzamido)propanoic acid is shown below
Figure imgf000255_0002
Figure imgf000256_0001
To a mixture of methyl 4-bromo-3-fluorobenzoate (500 mg, 2.15 mmol) and triethylborane (6.44 mL, 1M in hexane) in THF (10 mL) was added caesium acetate (1.24 g, 6.44 mmol) and Pd(dppf)Cl2 (157 mg, 0.21 mmol), the mixture was stirred at 65 °C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 5%) to give methyl 4-ethyl-3-fluorobenzoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.75 (dd, J = 1.6, 8.0 Hz, 1H), 7.66 (dd, J = 1.6, 11.2 Hz, 1H), 7.29 - 7.24 (m, 1H), 3.91 (s, 3H), 2.72 (q, J=7.6 Hz, 2H), 1.25 (t, J=7.6 Hz, 3H). Step 2: 4-ethyl-3-fluorobenzoic acid
Figure imgf000256_0002
To a solution of methyl 4-ethyl-3-fluorobenzoate (350 mg, 1.92 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (161 mg, 3.84 mmol), the mixture was stirred at 20°C for 3 hours. The mixture was concentrated. The residue was added water (30 mL), then drop-wise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 4-ethyl-3-fluorobenzoic acid (300 mg). 1H NMR (400MHz, CDCl3) δ 7.84 (dd, J = 1.6, 8.0 Hz, 1H), 7.66 (dd, J = 1.6, 10.4 Hz, 1H), 7.32 (dd, J = 7.2, 8.0 Hz, 1H), 2.76 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate
Figure imgf000257_0001
To a mixture of 4-ethyl-3-fluorobenzoic acid (120 mg, 0.71 mmol) and (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (312 mg, 0.86 mmol, HCl salt) in DMF (5 mL) was added TBTU (344 mg, 1.07 mmol) and DIPEA (277 mg, 2.14 mmol), the mixture was stirred at 20°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate(40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate (120 mg). 1H NMR (400MHz, CDCl3) δ 7.39 - 7.31 (m, 12H), 7.22 (t, J = 7.6 Hz, 1H), 6.65 (br s, 1H), 5.97 (br d, J = 6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.64 - 4.54 (m, 1H), 3.95 - 3.77 (m, 2H), 2.71 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid
Figure imgf000257_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate (120 mg, 0.25 mmol) in HBr/HOAc (4 mL, 30%) was stirred at 50 °C for 14 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(4-ethyl-3- fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.98-8.90 (m, 1H), 8.49 (br s, 2H), 7.72-7.66 (m, 2H), 7.46- 7.37 (m, 1H), 4.04-3.99 (m, 1H), 3.82-3.67 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 255.0 tR (min, Method D) = 1.531 [α]20 ,D = +6.0 (c = 2.0 mg/mL, CH3OH). Compound 3f (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000258_0001
To a mixture of 5-bromo-1-ethyl-1H-pyrazole (200 mg, 1.14 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (271 mg, 1.37 mmol) in the mixture of dioxane (5 mL) and H2O (0.5 mL) was added K3PO4 (728 mg, 3.43 mmol) and Pd(dtbpf)Cl2 (74 mg, 0.11 mmol). The mixture was stirred at 100 °C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (230 mg). 1H NMR (400MHz, CDCl3) δ 7.89 (d, J = 1.6 Hz, 1H), 7.80 - 7.75 (m, 1H), 7.57 (d, J = 1.6 Hz, 1H), 7.33 - 7.28 (m, 1H), 6.34 (d, J = 2.0 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000259_0001
To a solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (180 mg, 0.73 mmol) in THF (10 mL) was added NCS (97 mg, 0.73 mmol), the mixture was stirred at 70 °C for 1 hour. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 10%) to give methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (190 mg). 1H NMR (400MHz, CDCl3) δ 7.87 (s, 1H), 7.86 - 7.82 (m, 1H), 7.56 (s, 1H), 7.36 - 7.32 (m, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.38 (t, J = 7.2 Hz, 3H). Step 3: 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000259_0002
To a solution of methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (240 mg, 0.85 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (53 mg, 1.27 mmol), the mixture was stirred at 20°C for 1 hour. The mixture was concentrated. The residue was added water (30 mL), then drop-wise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (210 mg). LC-MS (MH+): m/z = 269.0 tR (min, Method c) = 0.659 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate
Figure imgf000260_0001
To a mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (210 mg, 0.78 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (342 mg, 0.94 mmol, HCl salt) in DMF (6 mL) was added TBTU (376 mg, 1.17 mmol) and DIPEA (303 mg, 2.34 mmol), the mixture was stirred at 20°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.56 (s, 1H), 7.52 - 7.44 (m, 2H), 7.37 - 7.25 (m, 11H), 6.92 (br s, 1H), 5.91 (br d, J = 6.4 Hz, 1H), 5.22 (d, J = 3.6 Hz, 2H), 5.11 (s, 2H), 4.65 - 4.55 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 4.03 - 3.75 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000261_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (150 mg, 0.26 mmol) in HBr/HOAc (4 mL, 30%) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was added HOAc (3 mL), then added MTBE (5 mL). The formed solid was collected by filtration and added water (10 mL) for freeze-drying. The obtained solid was further purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (28 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.96 (br t, J = 5.6 Hz, 1H), 8.34 (br d, J = 3.6 Hz, 3H), 7.84 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 4.17 - 4.03 (m, 3H), 3.84 - 3.66 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H) LC-MS (MH+): m/z = 355.0 tR (min, Method D) = 1.674 [α]20,D = +3.0 (c = 2.0 mg/mL, CH3OH). Compound 3g (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (+) (2R)-2-amino-3-(3-(1- ethoxyethyl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000262_0001
To a solution of 3-fluoro-5-iodobenzoic acid (5 g, 18.80 mmol) in EtOH (40 mL) was added sulfurous dichloride (6.71 g, 56.39 mmol). The mixture was stirred at 50°C for 14 hours. The reaction mixture was concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:ethyl acetate=100:0 to 98:2) to give ethyl 3- fluoro-5-iodobenzoate (5.2 g). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.72-7.69 (m, 1H), 7.64-7.61 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). Step 2: (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate and (R)-ethyl 3-fluoro-5-(1- hydroxyethyl)benzoate
Figure imgf000263_0001
To a solution of ethyl 3-fluoro-5-iodobenzoate (5.2 g, 17.68 mmol) in THF (60 mL) was added i-PrMgCl-LiCl (1.3 M in THF, 19.04 mL) at -70°C. The mixture was stirred at -70°C for 30 minutes. Then acetaldehyde (935 mg, 21.22 mmol) was added. The mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with saturated solution NH4Cl (20 mL) and concentrated. The residue was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether : ethyl acetate=100:0 to 90:10). The residue was further separated by SFC (Instrument: Thar200; Column: DAICEL CHIRALPAK AD 250 × 50 mm I.D. 5 μm; Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, 15%) = 85/15; Flow Rate: 180 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.7 g) and (R)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.6 g). (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate: 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.63-7.59 (m, 1H), 7.34-7.31 (m, 1H), 4.97 (q, J = 6.4 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.04 (s, 1H), 1.52 (d, J = 6.4 Hz, 3H), 1.40 (t, J = 7.2 Hz, 3H). (R)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate: 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.63-7.59 (m, 1H), 7.34-7.31 (m, 1H), 4.97 (q, J = 6.4 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.05 (d, J = 4.0 Hz, 1H), 1.52 (d, J = 6.4 Hz, 3H), 1.40 (t, J = 7.2 Hz, 3H). Note: The (S) and (R) configuration was assigned arbitrarily after SFC separation. Step 3: (S)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate
Figure imgf000264_0001
To a solution of (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.7 g, 8.01 mmol) in DMF (30 mL) was added NaH (481 mg, 12.02 mmol, 60% in mineral oil). The mixture was stirred at 0°C for 30 minutes. Then iodoethane (3.75 g, 24.03 mmol) was added. The mixture was stirred at 60°C for 15 hours. The reaction mixture was quenched by addition H2O (10 mL), and then concentrated under reduced pressure to give a residue. The residue was diluted with ethyl acetate (60 mL) and 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 (SiO2, Petroleum ether : ethyl acetate=100:0 to 95:5) to give (S)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate (1.45 g). 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.63-7.59 (m, 1H), 7.29-7.25 (m, 1H), 4.46 (q, J = 6.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.43-3.33 (m, 2H), 1.45-1.39 (m, 6H), 1.21 (t, J = 7.2 Hz, 3H). Step 4: (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid
Figure imgf000264_0002
To a solution of (S)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate (600 mg, 2.50 mmol) in a mixture of THF (10 mL) and H2O (5 mL) was added LiOH.H2O (315 mg, 7.50 mmol). The mixture was stirred at 30°C for 16 hours. The reaction mixture was added 2 M HCl to pH=2~3 and concentrated. The residue was diluted with ethyl acetate (40 mL) and washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated to give (S)- 3-(1-ethoxyethyl)-5-fluorobenzoic acid (480 mg). 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.72-7.68 (m, 1H), 7.36-7.33 (m, 1H), 4.50 (q, J = 6.4 Hz, 1H), 3.44-3.37 (m, 2H), 1.46 (d, J = 6.4 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
Figure imgf000265_0001
To a solution of (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid (200 mg, 0.942 mmol) in DMF (10 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol). The mixture was stirred at 30°C for 16 hours. 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 × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether : ethyl acetate=100:0 to 60:40) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (400 mg). LC-MS (MH+): m/z = 523.2 tR (min, Method c_220&254) = 0.831 Step 6: (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid
Figure imgf000265_0002
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (200 mg, 0.383 mmol) in AcOH (8 mL) was added Pd/C (50 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 1 hour. The reaction mixture was filtered and concentrated. The residue was purified by preparation HPLC (Method AA) to give (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5- fluorobenzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.07-8.99 (m, 1H), 8.56 (br s, 3H), 7.73 (s, 1H), 7.66-7.63 (m, 1H), 7.35-7.31 (m, 1H), 4.51 (q, J = 6.4 Hz, 1H), 4.12-4.09 (m, 1H), 3.78-3.76 (m, 2H), 3.48-3.24 (m, 2H), 1.35 (d, J = 6.4 Hz, 3H), 1.10 (t, J = 6.8 Hz, 3H). LC-MS (MH+): m/z = 299.1 tR (min, Method D) = 1.534 [α]20,D = +56.0 (c = 1.0 mg/mL, CH3OH). Compound 3h (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (-) (2R)-2-amino-3-(3-(1- ethoxyethyl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000266_0001
Figure imgf000267_0001
To a solution of (R)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.6 g, 7.54 mmol) in DMF (30 mL) was added NaH (452 mg, 11.31 mmol, 60% in mineral oil). The mixture was stirred at 0°C for 30 minutes. Then iodoethane (3.53 g, 22.62 mmol) was added. The mixture was stirred at 60°C for 15 hours. The reaction mixture was quenched by addition H2O (10 mL), and then concentrated. The residue was diluted with ethyl acetate (60 mL) and washed with brine (30 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:ethyl acetate=100:0 to 95:5) to give (R)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate (1.3 g). 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.64-7.59 (m, 1H), 7.29-7.25 (m, 1H), 4.46 (q, J = 6.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.43-3.33 (m, 2H), 1.60-1.39 (m, 6H), 1.21 (t, J = 7.2 Hz, 3H). Step 2: (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid
Figure imgf000267_0002
Figure imgf000267_0003
To a solution of (R)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate (600 mg, 2.50 mmol) in a mixture of THF (10 mL) and H2O (5 mL) was added LiOH.H2O (315 mg, 7.50 mmol). The mixture was stirred at 30°C for 16 hours. The reaction mixture was added 2 M HCl to pH=2~3 and concentrated. The residue was diluted with ethyl acetate (40 mL) and washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated to give (R)- 3-(1-ethoxyethyl)-5-fluorobenzoic acid (480 mg). 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.72-7.68 (m, 1H), 7.36-7.33 (m, 1H), 4.50 (q, J = 6.4 Hz, 1H), 3.44-3.37 (m, 2H), 1.46 (d, J = 6.4 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
Figure imgf000268_0001
To a solution of (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid (200 mg, 0.942 mmol) in DMF (10 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol). The mixture was stirred at 30°C for 16 hours. 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 × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:ethyl acetate=100:0 to 60:40) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1- ethoxyethyl)-5-fluorobenzamido)propanoate (400 mg). LC-MS (MH+): m/z = 523.2 tR (min, Method c_220&254) = 0.831 Step 4: of (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid
Figure imgf000268_0002
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (200 mg, 0.383 mmol) in AcOH (8 mL) was added Pd/C (50 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 0.5 hour. The reaction mixture was filtered and concentrated. The residue was purified by preparation HPLC (Method AA) to give (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5- fluorobenzamido)propanoic acid (30 mg) as HCl 1H NMR (400MHz, DMSO-d6) δ 9.10-9.01 (m, 1H), 8.50 (br s, 3H), 7.73 (s, 1H), 7.66-7.63 (m, 1H), 7.35-7.30 (m, 1H), 4.51 (q, J = 6.4 Hz, 1H), 4.01-3.98 (m, 1H), 3.84-3.67 (m, 2H), 3.38-3.23 (m, 2H), 1.35 (d, J = 6.4 Hz, 3H), 1.10 (t, J = 6.8 Hz, 3H). LC-MS (MH+): m/z = 299.1 tR (min, Method D) = 1.524 [α]20,D = -34.0 (c = 1.0 mg/mL, CH3OH). Compound 3i (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisothiazol- 4-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000269_0001
4-bromo-5-ethylisothiazole (500 mg, 1.11 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (286 mg, 1.44 mmol), Pd(dppf)Cl2.CH2Cl2 (91 mg, 0.11 mmol) and Na2CO3 (353 mg, 3.33 mmol) were taken up into a microwave tube in a mixture of dioxane (6 mL) and H2O (0.7 mL). The sealed tube was heated at 120 °C for 60 min under microwave. The mixture was diluted with water (10 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. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate (160 mg, crude). Step 2: 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid
Figure imgf000270_0001
To a solution of methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate (160 mg, crude) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (37 mg, 0.88 mmol). The mixture was stirred at 15 °C for 15 hours. The mixture was concentrated. The residue was dissolved in water (15 mL) and extracted with ethyl acetate (20 mL × 2). The organic layer was discarded. The aqueous layer was acidified with 2N HCl to adjust pH to 4 and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-(5-ethylisothiazol- 4-yl)-5-fluorobenzoic acid (60 mg). LC-MS (MH+): m/z = 252.1 tR (min, Method D) = 0.797 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000271_0001
To a solution of 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid (60 mg, 0.12 mmol) in DMF (5 mL) was added TBTU (60 mg, 0.19 mmol), DIPEA (48 mg, 0.37 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (55 mg, 0.15 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3- (3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate (45 mg. 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.52 (s, 1H), 7.38-7.32 (m, 11H), 7.25-7.19 (m, 1H), 6.87 (br s, 1H), 5.91 (br d, J = 6.0 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.65-4.56 (m, 1H), 4.01-3.92 (m, 1H), 3.86-3.78 (m, 1H), 3.00 (q, J = 7.6 Hz, 2H), 1.36 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000271_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (40 mg, 0.07 mmol) in 30% HBr in AcOH (4 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (HCl) to give (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoic acid (18 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.16-9.10 (m, 1H), 8.71 (s, 1H), 8.54 (br s, 3H), 7.89 (s, 1H), 7.76-7.69 (m, 1H), 7.63-7.56 (m, 1H), 4.16-4.08 (m, 1H), 3.82-3.76 (m, 2H), 3.07 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 338.0 tR (min, Method D) = 1.543 [α]20 ,D = 1.0 (c = 2.5 mg/mL, CH3OH). Compound 3j (R)-2-amino-3-(3-fluoro-5-(5-methylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- methylisoxazol-4-yl)benzamido)propanoic acid is shown below
Figure imgf000272_0001
To a solution of 5-methylisoxazole (5.0 g, 60.2 mmol) in DMF (50 mL) was added NBS (12.9 g, 72.2 mmol). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (60 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (40 mL × 2), dried over Na2SO4, filtered and concentrated below 30 °C. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 3%) to give 4-bromo-5- methylisoxazole (5.8 g). 1H NMR (400MHz, CDCl3) δ 8.17 (s, 1H), 2.45 (s, 3H). Step 2: methyl 3-fluoro-5-(5-methylisoxazol-4-yl)benzoate
Figure imgf000273_0001
4-bromo-5-methylisoxazole (1.0 g, 6.17 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.22 g, 6.17 mmol), Pd(t-Bu3P)2 (316 mg, 0.62 mmol) and KF (897 mg, 15.43 mmol) were taken up into a microwave tube in a mixture of dioxane (12 mL) and H2O (1.2 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 min under microwave. The mixture was diluted with water (40 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. The residue was purified by Combi flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 15%) to give methyl 3-fluoro-5-(5-methylisoxazol-4-yl)benzoate (2.3 g). 1H NMR (400MHz, CDCl3) δ 8.40 (s, 1H), 7.88-7.84 (m, 1H), 7.73-7.65 (m, 1H), 7.31-7.27 (m, 1H), 3.96 (s, 3H), 2.62 (s, 3H). Step 3: 3-fluoro-5-(5-methylisoxazol-4-yl)benzoic acid
Figure imgf000273_0002
A mixture of methyl 3-fluoro-5-(5-methylisoxazol-4-yl)benzoate (450 mg, 1.91 mmol) in conc. HCl (15 mL) was stirred at 80 °C for 16 hours. To the mixture was added 2N NaOH (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(5-methylisoxazol-4-yl)benzoic acid (400 mg). 1H NMR (400MHz, DMSO-d6) δ 13.3 (br s, 1H), 9.01 (s, 1H), 7.90 (s, 1H), 7.74-7.67 (m, 1H), 7.66-7.59 (m, 1H), 2.62 (s, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-methylisoxazol-4- yl)benzamido)propanoate
Figure imgf000274_0001
To a solution of 3-fluoro-5-(5-methylisoxazol-4-yl)benzoic acid (100 mg, 0.45 mmol) in DMF (5 mL) was added TBTU (218 mg, 0.68 mmol), DIPEA (146 mg, 1.13 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (165 mg, 0.45 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 35%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-methylisoxazol-4- yl)benzamido)propanoate (160 mg). 1H NMR (400MHz, CDCl3) δ 8.38 (s, 1H), 7.54 (s, 1H), 7.39-7.29 (m, 11H), 7.24-7.18 (m, 1H), 6.99 (br s, 1H), 5.93 (br d, J = 6.0 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.66-4.56 (m, 1H), 4.02-3.91 (m, 1H), 3.85-3.73 (m, 1H), 2.60 (s, 3H). Step 5: (R)-2-amino-3-(3-fluoro-5-(5-methylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000274_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-methylisoxazol- 4-yl)benzamido)propanoate (160 mg, 0.30 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was washed with MeCN (3 mL × 2) and the solid was dried to give (R)-2-amino-3-(3-fluoro-5-(5- methylisoxazol-4-yl)benzamido)propanoic acid (90 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 14.0 (br s, 1H), 8.98 (s, 1H), 8.96-8.90 (m, 1H), 8.34 (br s, 3H), 7.86 (s, 1H), 7.68-7.60 (m, 2H), 4.18-4.08 (m, 1H), 3.85-3.66 (m, 2H), 2.64 (s, 3H). LC-MS (MH+): m/z = 308.1 tR (min, Method D) = 1.442 [α]20 ,D = 1.4 (c = 3.3 mg/mL, CH3OH). Compound 3k (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-1H- imidazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000275_0001
Figure imgf000276_0001
1-ethyl-1H-imidazole (5.0 g, 52.0 mmol) was dissolved in DCM (70 mL), and 1,3- dibromo-5,5-dimethylimidazolidine-2,4-dione (7.58 g, 26.5 mmol) dissolved in DCM (50 ml) was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 3 hours. The reaction mixture was poured into 100 mL of sat. Na2SO3, extracted with DCM (80 mL × 3), the organic layers were washed with brine (50 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 10%) to give 5-bromo-1-ethyl-1H-imidazole (1.55 g). 1H NMR (400MHz, CDCl3) δ 7.53 (s, 1H), 6.97 (s, 1H), 3.95 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoate
Figure imgf000276_0002
A mixture of 5-bromo-1-ethyl-1H-imidazole (800 mg, 4.57 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.09 g, 5.48 mmol), Pd(dppf)Cl2 (298 mg, 457.0 µmol), K3PO4 (2.91 g, 13.71 mmol) in the mixture of H2O (2 mL) and dioxane (10 mL) was degassed by purging with N2, and then the mixture was stirred at 100 °C for 14 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 5%). The obtained material was further purified by preparative HPLC (Method BB) to give methyl 3-(1- ethyl-1H-imidazol-5-yl)-5-fluorobenzoate (150 mg). 1H NMR (400MHz, CDCl3) δ 7.87 (s, 1H), 7.76-7.70 (m, 1H), 7.63 (s, 1H), 7.31-7.27 (m, 1H), 7.16 (s, 1H), 4.06 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H) Step 3: 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid
Figure imgf000277_0001
To a solution of methyl 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoate (150 mg, 604.2 µmol) in MeOH (2 mL) and H2O (2 mL) was added LiOH.H2O (76.0 mg, 1.81 mmol). The mixture was stirred at 5 °C for 16 hours. The mixture was concentrated to remove MeOH, then HCl (2 M) was added to adjust pH=5~6. Following concentration on the rotavapor afforded 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid (141 mg, crude). Step: 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000277_0002
To a solution of 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid (70 mg, crude) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (131 mg, 358.6 µmol, HCl salt) in DMF (3 mL) was added TBTU (144 mg, 448.3 µmol) and DIPEA (116 mg, 896.6 µmol). The mixture was stirred at 5 °C for 16 hours. To the reaction mixture was added water (5 mL), extracted with ethyl acetate (10 mL × 4), and the organic layers were washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 10%) and further purified by preparative HPLC to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)- 3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate (60 mg). LC-MS (MH+): m/z = 545.3 tR (min, Method c) = 0.801. Step 5: (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000278_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-5-yl)- 5-fluorobenzamido)propanoate (60.0 mg, 110.2 µmol) in 30% HBr in AcOH (2 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoic acid (15 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.33 (br s, 1H), 9.18 (br s, 1H), 8.64 (br s, 3H), 8.03 (s, 1H), 7.96-7.87 (m, 2H), 7.72 (d, J = 8.8 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 4.19-4.09 (m, 1H), 3.86- 3.76 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 321.0 tR (min, Method D) = 1.107. [α]20,D = 4 (c = 1 mg/mL, MeOH). Compound 3l (R)-2-amino-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-(tert- butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000279_0001
To a solution of 5-(tert-butyl)isoxazole (300 mg, 2.40 mmol) in DMF (5 mL) was added NBS (512 mg, 2.88 mmol, 1.2 eq). The mixture was stirred at 15 °C for 16 hours. The mixture was then stirred at 50 °C for 20 hours. Then additional NBS (250 mg) was added and the mixture was stirred at 50°C for 4 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated below 30 °C. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 5%) to give 4-bromo-5-(tert-butyl)isoxazole (400 mg). 1H NMR (400MHz, CDCl3) δ 8.13 (s, 1H), 1.46 (s, 9H). Step 2: methyl 3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzoate
Figure imgf000279_0002
4-bromo-5-(tert-butyl)isoxazole (400 mg, 1.96 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (388 mg, 1.96 mmol), Pd(t-Bu3P)2 (100 mg, 0.20 mmol) and KF (285 mg, 4.90 mmol) were taken up into a microwave tube in a mixture of dioxane (6 mL) and H2O (0.6 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 min under microwave. The mixture was diluted with water (15 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. The residue was purified by Combi flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 15%) to give methyl 3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzoate (400 mg). 1H NMR (400MHz, CDCl3) δ 8.08 (s, 1H), 7.80-7.71 (m, 2H), 7.24-7.18 (m, 1H), 3.95 (s, 3H), 1.31 (s, 9H). Step 3: 3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000280_0001
A mixture of methyl 3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzoate (400 mg, 1.44 mmol) in conc. HCl (15 mL) was stirred at 80 °C for 16 hours. The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-(5-(tert-butyl)isoxazol-4-yl)- 5-fluorobenzoic acid (240 mg). 1H NMR (400MHz, DMSO-d6) δ 8.55 (s, 1H), 7.73-7.67 (m, 2H), 7.60-7.54 (m, 1H), 1.25 (s, 9H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000281_0001
To a solution of 3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzoic acid (120 mg, 0.46 mmol) in DMF (5 mL) was added TBTU (220 mg, 0.68 mmol), DIPEA (177 mg, 1.37 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (166 mg, 0.46 mmol, HCl salt). The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5- fluorobenzamido)propanoate (180 mg). 1H NMR (400MHz, CDCl3) δ 8.05 (s, 1H), 7.45 (s, 1H), 7.39-7.31 (m, 11H), 7.17-7.11 (m, 1H), 6.91 (br s, 1H), 5.92 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.64-4.55 (m, 1H), 4.01-3.89 (m, 1H), 3.86-3.75 (m, 1H), 1.30 (s, 9H). Step 5: (R)-2-amino-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000281_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-(tert-butyl)isoxazol-4- yl)-5-fluorobenzamido)propanoate (180 mg, 0.31 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was added water (4 mL) and washed with MTBE (4 mL × 2). The aqueous layer was dried to give (R)- 2-amino-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid (100 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.86 (m, 1H), 8.52 (s, 1H), 8.30 (br s, 3H), 7.76-7.67 (m, 2H), 7.54-7.48 (m, 1H), 4.16-4.06 (m, 1H), 3.82-3.75 (m, 1H), 3.71-3.65 (m, 1H), 1.26 (s, 9H). LC-MS (MH+): m/z = 350.1 tR (min, Method D) = 1.842. [α]20 ,D = 1.5 (c = 6.8 mg/mL, CH3OH). Compound 3m (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoic acid
Figure imgf000282_0001
4-bromo-5-ethyl-isoxazole (500 mg, 2.84 mmol), (3-ethoxycarbonylphenyl)boronic acid (551 mg, 2.84 mmol), Pd(t-Bu3P)2 (145 mg, 284.1 μmol) and KF (413 mg, 7.10 mmol) were taken up into a microwave tube in a mixture of dioxane (6 mL) and H2O (0.6 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 min under microwave. The reaction mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether /ethyl acetate with ethyl acetate from 0% to 10%) to give ethyl 3-(5-ethylisoxazol-4-yl)benzoate (340 mg).1H NMR (400MHz, CDCl3) δ 8.39 (s, 1H), 8.06-8.00 (m, 2H), 7.56-7.50 (m, 2H), 4.42 (q, J = 7.2Hz, 2H), 2.97 (q, J = 7.6Hz, 2H), 1.42 (t, J = 7.2Hz, 3H), 1.37 (t, J = 7.6Hz, 3H). Step 2: 3-(5-ethylisoxazol-4-yl)benzoic acid
Figure imgf000283_0001
A solution of ethyl 3-(5-ethylisoxazol-4-yl)benzoate (430 mg, 1.75 mmol) in conc. HCl (10 mL) was stirred at 80 °C for 16 hours. The reaction mixture was extracted with ethyl acetate (20 mL × 3). The organic layer was concentrated to give 3-(5-ethylisoxazol-4- yl)benzoic acid (270 mg).1H NMR (400MHz, DMSO-d6) δ 13.0 (br, 1H), 8.90 (s, 1H), 7.98 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.56 (dd, J = 8.0 Hz, 7.6Hz, 1H), 2.94 (q, J = 7.6Hz, 2H), 1.23 (t, J = 7.6Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate
Figure imgf000283_0002
To a solution of 3-(5-ethylisoxazol-4-yl)benzoic acid (135 mg, 621.5 μmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (227 mg, 621.5 μmol, HCl salt) in DMF (5 mL) was added TBTU (299 mg, 932.2 μmol) and DIPEA (24.0 mg, 1.86 mmol). The mixture was stirred at 15°C for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3), the organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 38%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate (230 mg). 1H NMR (400MHz, CDCl3) δ 8.37 (s, 1H), 7.77 (s, 1H), 7.58-7.52 (m, 1H), 7.51-7.44 (m, 2H), 7.39-7.29 (m, 10H), 6.85 (br s, 1H), 5.95 (br d, J = 6.8 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.62 (br d, J = 4.0 Hz, 1H), 4.02-3.90 (m, 1H), 3.88-3.76 (m, 1H), 2.96 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000284_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate (130 mg, 246.4 μmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated, the residue was dissolved in water (5 mL), washed with MTBE(5 mL). The water phase was lyophizised to give (R)- 2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid (85 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.89 (s, 1H), 8.85-8.78 (m, 1H), 8.31 (br s, 3H), 7.96 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.63-7.56 (m, 1H), 4.20-4.10 (m, 1H), 3.83-3.66 (m, 2H), 3.00 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 304.1 tR (min, Method D) = 1.53 [α]20,D = +1.2 (c = 5 mg/mL,CH3OH). Compound 3n (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3- triazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000285_0001
To a solution of 1-ethyl-1H-1,2,3-triazole (300 mg, 3.09 mmol) in THF (5 mL) was added n-BuLi (2.5 M in hexane, 1.50 mL) at -40 °C and stirred for 1 hour. Then Br2 (592 mg, 3.71 mmol) was added and the mixture was stirred at 20 °C for 2 hours. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (15 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 5-bromo-1-ethyl-1H-1,2,3-triazole (530 mg). 1H NMR (400MHz, CDCl3) δ 7.66 (s, 1H), 4.43 (q, J = 7.2 Hz, 2H), 1.54 (t, J = 7.2 Hz, 3H). Step 2: Methyl 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate
Figure imgf000285_0002
5-bromo-1-ethyl-1H-1,2,3-triazole (480 mg, 2.73 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (648 mg, 3.27 mmol), Pd(dppf)Cl2.CH2Cl2 (223 mg, 0.27 mmol) and K2CO3 (1.13 g, 8.18 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL). The sealed tube was heated at 100 °C for 60 min under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-(1-ethyl-1H- 1,2,3-triazol-5-yl)-5-fluorobenzoate (540 mg). 1H NMR (400MHz, CDCl3) δ 7.91 - 7.87 (m, 1H), 7.87-7.81 (m, 1H), 7.76 (s, 1H), 7.35-7.29 (m, 1H), 4.44 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.51 (t, J = 7.2 Hz, 3H). Step 3: 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid
Figure imgf000286_0001
To a solution of methyl 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate (540 mg, 2.17 mmol) in MeOH (10 mL) was added LiOH.H2O (273 mg, 6.50 mmol) in H2O (2 mL). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was added water (5 mL) and acidified with 2N HCl to adjust pH to 6~7 and extracted with ethyl acetate (20 mL × 3). The combined organic layer was concentrated to give 3-(1- ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid (480 mg). 1H NMR (400MHz, DMSO-d6) δ 8.00 (s, 1H), 7.90-7.86 (m, 1H), 7.82-7.75 (m, 2H), 4.44 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000287_0001
To a solution of 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid (120 mg, 0.51 mmol) in DMF (5 mL) was added TBTU (246 mg, 0.76 mmol), DIPEA (198 mg, 1.53 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (186 mg, 0.51 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 50%) twice to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate (70 mg). 1H NMR (400MHz, CDCl3) δ 7.70 (s, 1H), 7.58-7.49 (m, 2H), 7.39-7.28 (m, 11H), 7.24-7.19 (m, 1H), 6.00 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.65-4.57 (m, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.02-3.92 (m, 1H), 3.88-3.77 (m, 1H), 1.47 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000287_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5- yl)-5-fluorobenzamido)propanoate (70 mg, 0.13 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 5 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with MTBE (5 mL × 2). The aqueous layer was lyophilized to give (R)- 2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.00-8.93 (m, 1H), 8.32 (br s, 3H), 7.98 (s, 1H), 7.85 (s, 1H), 7.82-7.77 (m, 1H), 7.76-7.70 (m, 1H), 4.47 (q, J = 7.2 Hz, 2H), 4.17-4.08 (m, 1H), 3.82- 3.75 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 322.1 tR (min, Method D) = 1.271 [α]20,D = 0.9 (c = 4.4 mg/mL, CH3OH). Compound 3o (R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000288_0001
A mixture of pentan-2-one (10 g, 116.10 mmol), DMF-DMA (16.6 g, 139.32 mmol) were stirred at 110°C for 16 hours. The reaction mixture was concentrated to give (E)-1- (dimethylamino)hex-1-en-3-one (2.5 g). 1H NMR (400 MHz, CD3Cl) δ 7.54 (d, J = 12.4 Hz, 1H), 5.02 (d, J = 12.4 Hz, 1H), 3.07 (s, 6H), 2.48-2.38 (m, 2H), 1.67-1.58 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H).
Figure imgf000289_0001
A mixture of (E)-1-(dimethylamino)hex-1-en-3-one (2.5 g, 17.7 mmol) and NH2OH.HCl (2.46 g, 35.4 mmol) in EtOH (20 mL) was stirred at 80°C for 16 hours. The mixture was diluted with H2O (20 mL) and extracted with pentane (30 mL × 3). The organic phase was distilled to give 5-propylisoxazole (1.6 g, crude). 1H NMR (400 MHz, CD3Cl) δ 8.14 (d, J = 1.2 Hz, 1H), 5.98 (d, J = 1.2 Hz, 1H), 2.78 (t, J = 7.6 Hz, 2H), 1.80-1.69 (m, 2H), 0.99 (t, J = 7.6 Hz, 3H).
Figure imgf000289_0002
To a solution of 5-propylisoxazole (1.6 g, crude) in DMF (15 mL) was added NBS (3.33 g, 18.7 mmol) and the reaction mixture was stirred at 15°C for 16 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give 4-bromo- 5-propylisoxazole (1.6 g). 1H NMR (400 MHz, CD3Cl) δ 8.17 (s, 1H), 2.78 (t, J = 7.2 Hz, 2H), 1.81-1.71 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H).
Figure imgf000290_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 4-bromo-5- propylisoxazole (960 mg, 5.05 mmol), KF (880 mg, 15.15 mmol) and Pd(t-Bu3P)2 (258 mg, 0.51 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 10%) to give methyl 3-fluoro-5-(5-propylisoxazol-4-yl)benzoate (180 mg). 1H NMR (400 MHz, CD3Cl) δ 8.38 (s, 1H), 7.86-7.84 (m, 1H), 7.72-7.68 (m, 1H), 7.27-7.24 (m, 1H), 3.97 (s, 3H), 2.92 (t, J = 7.6 Hz, 2H), 1.85-1.78 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H).
Figure imgf000290_0002
A mixture of methyl 3-fluoro-5-(5-propylisoxazol-4-yl)benzoate (310 mg, 1.18 mmol) in conc.HCl (10 mL) was stirred at 80°C for 14 hours. The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(5-propylisoxazol-4- yl)benzoic acid (180 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoate
Figure imgf000291_0001
To a solution of 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid (90 mg, 0.36 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (145 mg, 0.40 mmol, HCl salt) in DMF (5 mL) was added TBTU (174 mg, 0.54 mmol) and DIPEA (140 mg, 1.08 mmol). The reaction mixture was stirred at 10°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoate (90 mg). 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.54 (s, 1H), 7.38-7.29 (m, 11H), 7.22-7.18 (m, 1H), 6.95 (br, 1H), 5.91 (br d, J = 6.4 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.64-4.58 (m, 1H), 4.00-3.76 (m, 2H), 2.91 (t, J = 7.6 Hz, 2H), 1.84-1.77 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). Step 7: (R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000292_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol- 4-yl)benzamido)propanoat (90 mg, 0.16 mmol) in 30 % HBr in AcOH (5 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was washed with a mixture of MeCN (1 mL) and MTBE (5 mL). The organic layer was decanted, then the residue was submitted to lyophilization to give (R)-2-amino-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoic acid (45 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.93-8.87 (m, 1H), 8.25 (br s, 2H), 7.82 (s, 1H), 7.68-7.58 (m, 2H), 4.07-4.03 (m, 1H), 3.84-3.64 (m, 2H), 2.97 (t, J = 7.6 Hz, 2H), 1.76- 1.66 (m, 2H), 0.91 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 336.1 tR (min, Method D) = 1.757 [α]20 ,D = +4.0 (c = 2.0 mg/mL, CH3OH). Compound 3p (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- isopropylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000293_0001
A mixture of 3-methylbutan-2-one (10.0 g, 116.1 mmol), DMF-DMA (16.6 g, 139.3 mmol) were stirred at 110°C for 4 days. The reaction mixture was concentrated to give (E)-1- (dimethylamino)-4-methylpent-1-en-3-one (6.0 g). 1H NMR (400 MHz, CD3Cl) δ 7.54 (d, J = 12.4 Hz, 1H), 5.02 (d, J = 12.4 Hz, 1H), 3.08-2.80 (m, 6H), 2.60-2.50 (m, 1H), 1.08 (d, J = 6.8 Hz, 6H).
Figure imgf000293_0002
A mixture of (E)-1-(dimethylamino)-4-methylpent-1-en-3-one (6.0 g, 42.5 mmol) and NH2OH.HCl (5.91 g, 85.0 mmol) in EtOH (30 mL) was stirred at 80°C for 16 hours. The mixture was diluted with H2O (20 mL) and extracted with pentane (30 mL × 3). The organic phase was distilled to give 5-isopropylisoxazole (5.0 g, crude). 1H NMR (400 MHz, CD3Cl) δ 8.13 (d, J = 1.6 Hz, 1H), 5.95 (d, J = 0.8 Hz, 1H), 3.15-3.07 (m, 1H), 1.31 (d, J = 6.8 Hz, 6H).
Figure imgf000294_0001
To a solution of 5-isopropylisoxazole (5.0 g, crude) in DMF (20 mL) was added NBS (10.4 g, 58.5 mmol) and the reaction mixture was stirred at 15°C for 16 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give 4-bromo- 5-isopropylisoxazole (5.0 g). 1H NMR (400 MHz, CD3Cl) δ 8.15 (s, 1H), 3.30-3.22 (m, 1H), 1.34 (d, J = 7.2 Hz, 6H).
Figure imgf000294_0002
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 4-bromo-5- isopropylisoxazole (960 mg, 5.05 mmol), KF (881 mg, 15.15 mmol) and Pd(t-Bu3P)2 (258 mg, 0.51 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give methyl 3-fluoro-5-(5-isopropylisoxazol-4-yl)benzoate (580 mg). 1H NMR (400 MHz, CD3Cl) δ 8.29 (s, 1H), 7.84-7.61 (m, 2H), 7.26-7.15 (m, 1H), 3.92 (s, 3H), 3.39-3.25 (m, 1H), 1.34 (d, J = 6.8 Hz, 6H).
Figure imgf000295_0001
A mixture of methyl 3-fluoro-5-(5-isopropylisoxazol-4-yl)benzoate (580 mg, 2.20 mmol) in conc.HCl (15 mL) was stirred at 80°C for 16 hours. The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(5-isopropylisoxazol-4- yl)benzoic acid (540 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropylisoxazol-4- yl)benzamido)propanoate
Figure imgf000295_0002
To a solution of 3-fluoro-5-(5-isopropylisoxazol-4-yl)benzoic acid (270 mg, 1.08 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (435 mg, 1.19 mmol, HCl salt) in DMF (6 mL) was added TBTU (522 mg, 1.62 mmol) and DIPEA (420 mg, 3.25 mmol). The reaction mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropylisoxazol-4- yl)benzamido)propanoate (320 mg). 1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.53 (s, 1H), 7.38-7.28 (m, 11 H), 7.20-7.15 (m, 1H), 6.95 (br s, 1H), 5.91 (br d, J = 6.0 Hz, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 4.64-4.57 (m, 1H), 4.00-3.92 (m, 1H), 3.85-3.76 (m, 1H), 3.39-3.31 (m, 1H), 1.37 (d, J = 7.2 Hz, 6H). Step 7: (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000296_0001
A solution of (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid (320 mg, 0.57 mmol) in 30 % HBr in AcOH (10 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was washed with a mixture of MeCN (1 mL) and MTBE (5 mL). The organic layer was decanted, then the residue was submitted to lyophilization to give (R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4- yl)benzamido)propanoic acid (140 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.95-8.89 (m, 1H), 8.88 (s, 1H), 8.29 (br s, 3H), 7.78 (s, 1H), 7.68-7.64 (m, 1H), 7.59-7.55 (m, 1H), 4.11-4.07 (m, 1H), 3.84-3.65 (m, 2H), 3.50-3.42 (m, 1H), 1.30 (d, J = 6.8 Hz, 6H). LC-MS (MH+): m/z = 336.0 tR (min, Method D) = 1.597 [α]20 ,D = +2.0 (c = 2.0 mg/mL, CH3OH). Compound 3q (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethylfuran-3- yl)-5-fluorobenzamido)propanoic acid
Figure imgf000297_0001
To a solution of 2,3-dibromofuran (2.3 g, 10.2 mmol) in THF (50 mL) was added Pd(dppf)Cl2 (745 mg, 1.02 mmol), cesium acetate (4.89 g, 25.46 mmol) and triethylborane (1 M in THF, 12.2 mL). The mixture was stirred at 65 °C for 7 hours. The mixture was added water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were concentrated below 30 °C. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 1%) and concentrated below 30 °C to give 3-bromo-2-ethylfuran (1.5 g). 1H NMR (400MHz, CDCl3) δ 7.27 (d, J = 1.2 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 2.67 (q, J = 7.6 Hz, 2H), 1.24 - 1.20 (m, 3H). Step 2: Methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate
Figure imgf000298_0001
To a solution of 3-bromo-2-ethylfuran (1.50 g, 8.57 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.70 g, 8.57 mmol) in a mixture of DME (40 mL) and H2O (4 mL) was added Pd(PPh3)4 (990 mg, 0.86 mmol) and Na2CO3 (3.55 g, 25.7 mmol) under N2 atmosphere. The mixture was stirred at 85 °C for 16 hours. The mixture was diluted with water (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. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give 1.1 g of the crude product. The crude product was further purified by preparative-HPLC (HCl) to give methyl 3-(2- ethylfuran-3-yl)-5-fluorobenzoate (630 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.84 (m, 1H), 7.65-7.58 (m, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.28-7.24 (m, 1H), 6.52 (d, J = 1.6 Hz, 1H), 3.95 (s, 3H), 2.83 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). Step 3: 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid
Figure imgf000298_0002
To a solution of methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate (80 mg, 0.32 mmol) in a mixture of MeOH (4 mL) and H2O (1 mL) was added LiOH.H2O (41 mg, 0.97 mmol). The mixture was stirred at 20 °C for 14 hours. The mixture was concentrated. The residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid (68 mg). 1H NMR (400MHz, DMSO-d6) δ 13.39 (br s, 1H), 7.79 (s, 1H), 7.65 (d, J = 1.6 Hz, 1H), 7.59- 7.48 (m, 2H), 6.80 (d, J = 1.6 Hz, 1H), 2.82 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000299_0001
To a solution of 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid (80 mg, 0.21 mmol, ~60% purity) in DMF (5 mL) was added TBTU (99 mg, 0.31 mmol), DIPEA (80 mg, 0.62 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (125 mg, 0.34 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give 150 mg of the crude product. The crude product was further purified by preparative-HPLC (HCl) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoate (65 mg). 1H NMR (400MHz, CDCl3) δ 7.51 (s, 1H), 7.39-7.29 (m, 11H), 7.26-7.17 (m, 2H), 6.79 (br s, 1H), 6.51 (d, J = 1.6 Hz, 1H), 5.93 (br d, J = 6.8 Hz, 1H), 5.27-5.17 (m, 2H), 5.16-5.08 (m, 2H), 4.65-4.57 (m, 1H), 3.99-3.89 (m, 1H), 3.86-3.77 (m, 1H), 2.82 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000300_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoate (40 mg, 0.07 mmol) in ethyl acetate (2 mL) was added Pd/C (40 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by preparative-HPLC (HCl) to give (R)-2-amino- 3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid (15 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.95-8.93 (m, 1H), 8.34 (br s, 2H), 7.75 (s, 1H), 7.66 (d, J=2.0 Hz, 1H), 7.60-7.54 (m, 1H), 7.47-7.41 (m, 1H), 6.78 (d, J = 2.0 Hz, 1H), 4.10-4.04 (m, 1H), 3.83-3.75 (m, 1H), 3.74-3.68 (m, 1H), 2.83 (t, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 320.9 tR (min, Method D) = 1.758 [α]20,D = 3.1 (c = 1.3 mg/mL, CH3OH). Compound 3r (-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (-)cis-(2R)-2-amino-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000301_0001
To a solution of methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate (500 mg, 2.01 mmol) in AcOH (5 mL) was added Pd/C (200 mg, 10% Pd, <1% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 18 hours. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give cis- methyl 3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoate (400 mg). 1H NMR (400MHz, CDCl3) δ 7.65 (s, 1H), 7.60-7.55 (m, 1H), 7.14-7.08 (m, 1H), 4.23-4.15 (m, 1H), 3.93 (s, 3H), 3.90-3.79 (m, 2H), 3.42-3.35 (m, 1H), 2.53-2.42 (m, 1H), 2.14-2.05 (m, 1H), 1.31-1.21 (m, 1H), 1.15-1.05 (m, 1H), 0.84 (t, J = 7.6 Hz, 3H). Step 2: cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid
Figure imgf000302_0001
To a solution of cis-methyl 3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoate (350 mg, 1.39 mmol) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (175 mg, 4.16 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give cis-3-(2-ethyltetrahydrofuran- 3-yl)-5-fluorobenzoic acid (310 mg). 1H NMR (400MHz, CDCl3) δ 13.26 (br s, 1H), 7.60 (s, 1H), 7.52-7.47 (m, 1H), 7.30-7.25 (m, 1H), 4.10-4.02 (m, 1H), 3.78-3.70 (m, 2H), 3.53-3.44 (m, 1H), 2.45-2.34 (m, 1H), 2.05-1.95 (m, 1H), 1.08-0.90 (m, 2H), 0.79-0.71 (m, 3H). Step 3: cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3- yl)-5-fluorobenzamido)propanoate
Figure imgf000302_0002
To a solution of cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid (150 mg, 0.63 mmol) in DMF (5 mL) was added TBTU (303 mg, 0.94 mmol), DIPEA (244 mg, 1.89 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (230 mg, 0.63 mmol, HCl salt). The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give cis-(2R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (100 mg). LC-MS (MH+): m/z = 549.3 tR (min, Method c),) = 0.924 Step 4: cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3- yl)-5-fluorobenzamido)propanoate and cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)- 3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate
Figure imgf000303_0001
cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (100 mg, 0.18 mmol) was separated by SFC (Instrument: Waters; Column: DAICEL CHIRALCEL OJ-H(250mm*30mm,5μm); Mobile phase: supercritical CO2/EtOH (0.1% NH3`H2O, v%) = 80/20; Flow Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give cis-(2R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (30 mg, peak 1, tR = 2.700 min) and the other diastereomer of cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (32 mg, peak 2, tR = 2.870 min). 1H NMR (400MHz, CDCl3) δ 7.38-7.32 (m, 11H), 7.23-7.17 (m, 1H), 7.07-7.02 (m, 1H), 6.78 (br s, 1H), 5.93 (br s, 1H), 5.22 (s, 2H), 5.12 (s, 2H), 4.64-4.55 (m, 1H), 4.23-4.17 (m, 1H), 3.94-3.86 (m, 2H), 3.84-3.77 (m, 2H), 3.38-3.32 (m, 1H), 2.51-2.43 (m, 1H), 2.09 (s, 1H), 1.25-1.18 (m, 1H), 1.11-1.05 (m, 1H), 0.86-0.82 (m, 3H). Step 5: (-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000304_0001
To a solution of cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (30 mg, 0.05 mmol) in MeOH (5 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by prep-HPLC (HCl) to give (- )cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid (10 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.84 (m, 1H), 8.39 (br s, 3H), 7.59-7.50 (m, 2H), 7.24- 7.17 (m, 1H), 4.15-4.03 (m, 2H), 3.81-3.66 (m, 4H), 3.50-3.40 (m, 1H), 2.43-2.32 (m, 1H), 2.08-1.96 (m, 1H), 1.09-0.89 (m, 2H), 0.80-0.72 (m, 3H). LC-MS (MH+): m/z = 325.2 tR (min, Method D) = 1.580 [α]20,D = -40 (c = 1.5 mg/mL, CH3OH). Compound 3s (+)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid The synthesis scheme for the preparation of (+)cis-(2R)-2-amino-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000304_0002
To a solution of cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (32 mg, 0.03 mmol, 48.6% purity) in MeOH (5 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 0.5 hour. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by prep-HPLC (HCl) to give (+)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoic acid (7 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.84 (m, 1H), 8.39 (br s, 3H), 7.57-7.51 (m, 2H), 7.24- 7.18 (m, 1H), 4.12-4.03 (m, 2H), 3.82-3.68 (m, 4H), 3.50-3.40 (m, 1H), 2.44-2.39 (m, 1H), 2.07-1.96 (m, 1H), 1.09-0.90 (m, 2H), 0.81-0.71 (m, 3H). LC-MS (MH+): m/z = 325.0 tR (min, Method D) = 1.429 for (+)cis-(2R)-2-amino-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid [α]20,D = 34.5 (c = 1.1 mg/mL, CH3OH). Compound 3t (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethylisothiazol- 4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000305_0001
Figure imgf000306_0001
To a solution of 3-bromoisothiazole (1.0 g, 6.10 mmol) in H2SO4 (20 mL) was added I2 (1.08 g, 4.27 mmol). After 30 minutes, NaIO4 (652 mg, 3.05 mmol) was added in small portions to the reaction mixture and the mixture was stirred at 30°C for 16 hours. The reaction mixture was poured onto ice (~100 g), decolorized with Na2SO3 (20 mL), and extracted with CHCl3 (20 mL × 3). The combined organic extracts were washed with water (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give 3-bromo-4-iodoisothiazole (1.46 g). 1H NMR (400MHz, CDCl3) δ 8.63 (s, 1H). Step 2: methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate
Figure imgf000306_0002
A mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 3- bromo-4-iodoisothiazole (1.46 g, 5.05 mmol), Pd(dppf)Cl2 (370 mg, 0.51 mmol) and K2CO3 (2.09 g, 15.15 mmol) in a mixture of dioxane (20 mL) and H2O (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate (1.2 g). 1H NMR (400MHz, CDCl3) δ 8.63 (s, 1H), 7.99-7.97 (m, 1H), 7.81-7.77 (m, 1H), 7.50-7.45 (m, 1H), 3.97 (s, 3H). Step 3: methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate
Figure imgf000307_0001
A mixture of methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate (500 mg, 1.58 mmol), triethylborane (1 M in THF, 4.74 mL), Pd(dppf)Cl2 (116 mg, 0.16 mmol) and CsOAc (911 mg, 4.74 mmol) in THF (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(3- ethylisothiazol-4-yl)-5-fluorobenzoate (110 mg). 1H NMR (400MHz, CDCl3) δ 8.56 (s, 1H), 7.87-7.86 (m, 1H), 7.76-7.73 (m, 1H), 7.31-7.28 (m, 1H), 3.96 (s, 3H), 2.86 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H). Step 4: 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid
Figure imgf000307_0002
To a solution of methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate (110 mg, 0.41 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (52 mg, 1.24 mmol) and the mixture was stirred at 10°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid (90 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000308_0001
To a solution of a mixture of 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid (45 mg, 0.18 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (72 mg, 0.20 mmol, HCl salt) in DMF (3 mL) was added TBTU (86 mg, 0.27 mmol) and DIPEA (69 mg, 0.54 mmol). The reaction mixture was stirred at 10°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(7- (pentan-3-yl)benzo[d]thiazole-2-carboxamido)propanoate (45 mg). LC-MS (MH+): m/z = 562.3 tR (min, Method c) = 0.953 Step 6: (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000308_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(7-(pentan-3- yl)benzo[d]thiazole-2-carboxamido)propanoate (45 mg, 0.08 mmol) in 30% HBr in AcOH (3 mL) was heated at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethylisothiazol-4- yl)-5-fluorobenzamido)propanoic acid (10 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.13 (s, 1H), 9.10 (br, 1H), 8.52 (br s, 3H), 7.84 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 4.15-4.09 (m, 1H), 3.81-3.77 (m, 2H), 2.84 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 338.1 tR (min, Method c) = 1.105 [α]20,D = +1.0 (c = 2 mg/mL,CH3OH). Compound 3u (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethyl-1H- imidazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000309_0001
Figure imgf000310_0001
To a solution of 4,5-dibromo-1H-imidazole (100 mg, 0.44 mmol) in DMF (3 mL) was added NaH (19 mg, 0.49 mmol, 60% in mineral oil) at 0°C. The mixture was stirred at 0°C for 15 minutes and SEM-Cl (74 mg, 0.44 mmol) was added. Then the mixture was stirred at 20°C for 3 hours. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered and concentrated to give 4,5-dibromo-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazole (150 mg). 1H NMR (400MHz, CDCl3) δ 7.64 (s, 1H), 5.29 (s, 2H), 3.57-3.52 (m, 2H), 0.95-0.90 (m, 2H), 0.00 (s, 9H). Step 2: methyl 3-(4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoate
Figure imgf000310_0002
A mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (53 mg, 0.27 mmol), 4,5- dibromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole (95 mg, 0.27 mmol), Pd(PPh3)4 (31 mg, 0.03 mmol) and Na2CO3 (85 mg, 0.80 mmol) in a mixture of H2O (0.3 mL) and DME (3 mL) under N2 atmosphere. The sealed tube was heated at 110°C for 30 minutes under microwave. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.05 (s, 1H), 7.81-7.77 (m, 1H), 7.65 (s, 1H), 7.55-7.50 (m, 1H), 5.18 (s, 2H), 3.96 (s, 3H), 3.53 (t, J = 8.0 Hz, 2H), 0.94 (t, J = 8.0 Hz, 2H), 0.00 (s, 9H). Step 3: methyl 3-fluoro-5-(1-((2-(trimethylsilyl)ethoxy)methyl)-4-vinyl-1H-imidazol-5- yl)benzoate
Figure imgf000311_0001
A mixture of methyl 3-(4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)- 5-fluorobenzoate (100 mg, 20.23 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2- dioxaborolane (54 mg, 0.35 mmol), Pd(dppf)Cl2 (17 mg, 0.02 mmol) and K2CO3 (97 mg, 0.70 mmol) in a mixture of dioxane (3 mL) and H2O (0.3 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) to give methyl 3-fluoro-5-(1-((2-(trimethylsilyl)ethoxy)methyl)-4-vinyl-1H-imidazol-5- yl)benzoate (35 mg). LC-MS (MH+): m/z = 377.2 tR (min, Method c) = 0.888 Step 4: methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoate
Figure imgf000312_0001
A mixture of methyl 3-fluoro-5-(1-((2-(trimethylsilyl)ethoxy)methyl)-4-vinyl-1H- imidazol-5-yl)benzoate (35 mg, 0.09 mmol) and Pd/C (10 mg, 10% Pd, 50% water) in MeOH (3 mL) was degassed and purged with H2 for 3 times, and then the mixture was stirred at 20°C for 16 hours under H2 atmosphere (15 Psi). The reaction mixture was filtered and concentrated to give methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)- 1H-imidazol-5-yl)-5-fluorobenzoate (25 mg). Step 5: 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoic acid
Figure imgf000312_0002
To a solution of methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5- yl)-5-fluorobenzoate (25 mg, 0.07 mmol) in a mixture of THF (3 mL) and H2O (1 mL) was added LiOH.H2O (7 mg, 0.17 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was diluted with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5-fluorobenzoic acid (24 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000313_0001
To a solution of 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5- fluorobenzoic acid (24 mg, 0.07 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (26 mg, 0.07 mmol, HCl salt) in DMF (3 mL) was added TBTU (32 mg, 0.10 mmol) and DIPEA (26 mg, 0.20 mmol). The reaction mixture was stirred at 20°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 70% to 75%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate (40 mg). LC-MS (MH+): m/z = 675.4 tR (min, Method c_) = 0.898 Step 7: (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000313_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate (40 mg, 0.06 mmol) in the solution of 30% HBr in AcOH (3 mL) was heated at 50°C for 16 hours. Then to the mixture was added TFA (2 mL) and heated at 30°C for 1 hour and heated at 50°C for another hour. Then the mixture was heated at 80°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid (15 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 14.82 (br s, 1H), 9.24 (s, 1H), 9.20-9.14 (m, 1H), 8.54 (br s, 3H), 7.99 (s, 1H), 7.87 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H), 4.17-4.11 (m, 1H), 3.84-3.75 (m, 2H), 2.85 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 321.3 tR (min, Method c) = 0.878 [α]20,D = +8.0 (c = 1 mg/mL,CH3OH). Compound 3v (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethyl-4- methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000314_0001
Figure imgf000315_0001
A mixture of 3-bromo-2-chloro-4-(trifluoromethyl)pyridine (1.5 g, 7.27 mmol), (3-fluoro- 5-methoxycarbonylphenyl)boronic acid (1.44 g, 7.27 mmol), Pd(dppf)Cl2 (532 mg, 726.5 µmol), Na2CO3 (2.31 g, 21.80 mmol) in the mixture of H2O (2 mL) and Dioxane (18 mL) was degassed and purged with N23 times, and then the mixture was stirred at 80 °C for 3 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (1.6 g). 1H NMR (400MHz, CDCl3) δ 8.30 (d, J = 5.2 Hz, 1H), 7.84-7.77 (m, 1H), 7.73-7.69 (m, 1H), 7.22-7.13 (m, 2H), 3.95 (s, 3H), 2.13 (s, 3H). Step 2: methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate
Figure imgf000315_0002
A mixture of 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (550 mg, 1.97 mmol), triethylborane (1 M in THF, 5.9 mL), Pd(dppf)Cl2 (144 mg, 196.64 µmol), CsOAc (1.13 g, 5.90 mmol) in THF (7 mL) was degassed and purged with N23 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) to give methyl 3-(2-ethyl- 4-methylpyridin-3-yl)-5-fluorobenzoate (430 mg). 1H NMR (400MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1H), 7.79-7.73 (m, 1H), 7.67 (dd, J = 1.6, 1.6 Hz, 1H), 7.13-7.08 (m, 1H), 7.05 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 2.52 (q, J = 7.6 Hz, 2H), 2.02 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). Step 3: 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid
Figure imgf000316_0001
To a solution of methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate (430 mg, 1.57 mmol) in the mixture of H2O (3 mL) and MeOH (3 mL) was added LiOH.H2O (198 mg, 4.72 mmol). The mixture was stirred at 10 °C for 16 hours. The mixture was concentrated to remove MeOH. Then HCl (2 M) was added to adjust pH=5~6 and concentrated to give 3- (2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid (465 mg, crude) as HCl salt. LC-MS (MH+): m/z = 260.1 tR (min, Method c_) = 0.584 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000316_0002
To a solution of 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid (116 mg, HCl salt, crude) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (157 mg, 431.47 µmol, HCl salt) in DMF (3 mL) was added TBTU (189 mg, 588.4 µmol) and DIPEA (253 mg, 1.96 mmol). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was quenched with H2O (5 mL), extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) twice to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate (140 mg). 1H NMR (400MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1H), 7.49-7.28 (m, 12H), 7.09-7.01 (m, 2H), 6.93 (m, 1H), 5.95 (d, J = 6.0 Hz, 1H), 5.29-5.15 (m, 2H), 5.08 (s, 2H), 4.64-4.52 (m, 1H), 4.02-3.75 (m, 2H), 2.51 (q, J = 7.6 Hz, 2H), 2.02 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000317_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin- 3-yl)-5-fluorobenzamido)propanoate (140 mg, 245.78 µmol) in 30% HBr in AcOH (3 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was dissolved in MeCN (3 mL) and MTBE (10 mL), and the solid was collected and lyophilizated. The residue was dissolved in AcOH (3 mL) and MTBE (10 mL), and the solid was collected and lyophilizated to give (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)- 5-fluorobenzamido)propanoic acid (70 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99 (br s, 1H), 8.81 (br s, 1H), 8.37 (br s, 3H), 8.06-7.77 (m, 2H), 7.76-7.49 (m, 2H), 4.20-4.07 (m, 1H), 3.80-3.66 (m, 2H), 2.74-2.63 (m, 2H), 2.26 (s, 3H), 1.09 (m, 3H). LC-MS (MH+): m/z = 346.0 tR (min, Method D) = 0.983 [α]20,D = 1.5 (c = 4 mg/mL, MeOH). Compound 3w (R)-2-amino-3-(3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethyl-4- (trifluoromethyl)pyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000318_0001
A mixture of 3-bromo-2-chloro-4-(trifluoromethyl)pyridine (1.50 g, 5.76 mmol), methyl 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (1.61 g, 5.76 mmol), Pd(dppf)Cl2 (421.4 mg, 575.9 µmol), Na2CO3 (1.83 g, 17.3 mmol) in the mixture of H2O (2 mL) and dioxane (20 mL) was degassed and purged with N23 times, and then the mixture was stirred at 80 °C for 2 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) and further purified by preparative HPLC (Method AA) to give 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzoate (260 mg) as HCl salt. 1H NMR (400MHz, CDCl3) δ 8.66 (d, J = 5.2 Hz, 1H), 7.85 (dd, J = 1.2 Hz, 8.8 Hz, 1H), 7.75 (s, 1H), 7.65 (d, J = 5.2 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 3.95 (s, 3H) Step 2: methyl 3-fluoro-5-(4-(trifluoromethyl)-2-vinylpyridin-3-yl)benzoate
Figure imgf000319_0001
A mixture of methyl 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (230 mg, 621.4 µmol, HCl salt), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (191 mg, 1.24 mmol), Pd(dppf)Cl2 (45 mg, 62.1 umol), K2CO3 (344 mg, 2.49 mmol) in the mixture of H2O (0.5 mL) and dioxane (5 mL) was degassed and purged with N23 times, and then the mixture was stirred at 80 °C for 19 hours under N2 atmosphere. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 15%) to give methyl 3-fluoro-5-(4-(trifluoromethyl)-2- vinylpyridin-3-yl)benzoate (120 mg). 1H NMR (400MHz, CDCl3) δ 8.82 (d, J = 4.8 Hz, 1H), 7.87-7.80 (m, 1H), 7.73 (s, 1H), 7.55 (d, J = 4.8 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 6.53-6.44 (m, 1H), 6.39 - 6.29 (m, 1H), 5.44 (dd, J = 2.0 Hz, 10.4 Hz, 1H), 3.95 (s, 3H) Step 3: methyl 3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate
Figure imgf000320_0001
A mixture of methyl 3-fluoro-5-(4-(trifluoromethyl)-2-vinylpyridin-3-yl)benzoate (110 mg, 338.2 µmol), Pd/C (50 mg, 10% Pd, 50% water) in MeOH (5 mL) was degassed and purged with H23 times, and then the mixture was stirred at 15 °C for 16 hours under H2 (15 Psi) atmosphere. The reaction mixture was filtered through celite and the filtrate was concentrated to give methyl 3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzoate (110 mg). Step 4: 3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoic acid
Figure imgf000320_0002
To a solution of methyl 3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (110 mg, 336.1 µmol) in the mixture of H2O (3 mL) and MeOH (3 mL) was added LiOH.H2O (42 mg, 1.01 mmol). The mixture was stirred at 15 °C for 16 hours. The mixture was concentrated to remove MeOH, Then HCl (2 M) was added to adjust pH=5~6 and the solid was collected to give 3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzoic acid (100 mg). 1H NMR (400MHz, DMSO-d6) δ 13.53 (br s, 1H), 8.86 (d, J = 4.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 5.2 Hz, 1H), 7.65 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 2.49-2.44 (m, 2H), 1.08 (t, J = 7.6 Hz, 3H) Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4- (trifluoromethyl)pyridin-3-yl)-5-fluorobenzamido)propanoate
Figure imgf000321_0001
To a solution of 3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoic acid (50 mg, 159.6 µmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (64 mg, 175.6 µmol, HCl salt) in DMF (3 mL) was added TBTU (77 mg, 239.4 µmol) and DIPEA (62 mg, 478.9 µmol). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzamido)propanoate (80 mg). LC-MS (MH+): m/z = 624.3 tR (min, Method c) = 0.946 Step 6: (R)-2-amino-3-(3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000322_0001
A solution of (R)-2-amino-3-(3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzamido)propanoic (70 mg, 112.3 µmol) in 30% HBr in AcOH (3 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(2-ethyl-4- (trifluoromethyl)pyridin-3-yl)-5-fluorobenzamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.13-8.99 (m, 1H), 8.86 (d, J = 5.2 Hz, 1H), 8.51 (br s, 3H), 7.88 (d, J = 9.6 Hz, 1H), 7.73 (d, J = 5.2 Hz, 1H), 7.66 (s, 1H), 7.50 (d, J = 9.6 Hz, 1H), 4.16- 4.06 (m, 1H), 3.80-3.77 (m, 2H), 2.49-2.45 (m, 2H), 1.09 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 399.9 tR (min, Method D) = 1.625 [α]20,D = 2.67 (c = 3 mg/mL, MeOH). Compound 3x (R)-2-amino-3-(3-(4-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000323_0001
To a solution of 4-bromo-1H-pyrazole (2.00 g, 13.61 mmol) in DMF (20 mL) was added NaH (653 mg, 16.33 mmol, 60% in mineral oil) at 0°C. The mixture was stirred at 0°C for 15 minutes and SEM-Cl (2.27 g, 13.61 mmol) was added then the mixture was stirred at 20°C for 3 hours. The mixture was quenched with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated to give 4-bromo-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazole (3.7 g). 1H NMR (400MHz, CDCl3) δ 7.60 (s, 1H), 7.50 (s, 1H), 5.39 (s, 2H), 3.55 (t, J = 8.0 Hz, 2H), 0.89 (t, J = 8.0 Hz, 2H), 0.01 (s, 9H). Step 2: 4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole
Figure imgf000324_0001
A mixture of 4-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (500 mg, 1.80 mmol), triethylborane (5.40 mL, 1 M in THF), Pd(dppf)Cl2 (132 mg, 0.18 mmol) and CsOAc (1.04 g, 5.40 mmol) in THF (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give 4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazole (320 mg). 1H NMR (400MHz, CDCl3) δ 7.39 (s, 1H), 7.35 (s, 1H), 5.37 (s, 2H), 3.55 (t, J = 8.0 Hz, 2H), 2.50 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H), 0.98 (t, J = 8.0 Hz, 2H), 0.02 (s, 9H).
Figure imgf000324_0002
To a stirred solution of 4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (500 mg, 2.21 mmol) in THF (10 mL) was added n-BuLi (2.5 M in hexane, 1.06 mL) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (673 mg, 2.65 mmol) dissolved in 5 mL THF was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 20°C for 2 hours. The mixture was quenched with ice water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give 4-ethyl-5-iodo-1- ((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (400 mg). 1H NMR (400MHz, CDCl3) δ 7.46 (s, 1H), 5.49 (s, 2H), 3.57 (t, J = 8.4 Hz, 2H), 2.42 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H), 0.93 (t, J = 8.4 Hz, 2H), 0.02 (s, 9H). Step 5: methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5- fluorobenzoate
Figure imgf000325_0001
A mixture of ((3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (190 mg, 0.96 mmol), 4-ethyl-5-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (338 mg, 0.96 mmol), Pd(dppf)Cl2 (70 mg, 0.10 mmol) and K3PO4 (611 mg, 2.88 mmol) in a mixture of H2O (0.6 mL) and dioxane (6 mL) under N2 atmosphere in a sealed tube was heated at 85°C for 15 hours under N2. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 10%) to give methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5- fluorobenzoate (280 mg). 1H NMR (400MHz, CDCl3) δ 7.94 (t, J = 1.2 Hz, 1H), 7.81-7.76 (m, 1H), 7.50 (s, 1H), 7.46- 7.42 (m, 1H), 5.30 (s, 2H), 3.96 (s, 3H), 3.65 (t, J = 8.4 Hz, 2H), 2.47 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H), 0.93 (t, J = 8.4 Hz, 2H), 0.01 (s, 9H). Step 6: 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000326_0001
To a solution of methyl 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)- 5-fluorobenzoate (280 mg, 0.74 mmol) in a mixture of THF (3 mL) and H2O (1 mL) was added LiOH.H2O (78 mg, 1.85 mmol) and the mixture was stirred at 20°C for 18 hours. The reaction mixture was diluted with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid (260 mg). Step 7: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000326_0002
To a solution of a mixture of 3-(4-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol- 5-yl)-5-fluorobenzoic acid (260 mg, 0.71 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (286 mg, 0.78 mmol, HCl salt) in DMF (8 mL) was added TBTU (344 mg, 1.07 mmol) and DIPEA (277 mg, 2.14 mmol). The reaction mixture was stirred at 20°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (420 mg). 1H NMR (400MHz, CDCl3) δ 7.60 (s, 1H), 7.50 (s, 1H), 7.38-7.30 (m, 12H), 6.92 (br, 1H), 5.95 (br d, J = 6.4 Hz, 1H), 5.28 (s, 2H), 5.21 (s, 2H), 5.10 (s, 2H), 4.64-4.56 (m, 1H), 3.97- 3.81 (m, 2H), 3.67 (t, J = 8.4 Hz, 2H), 2.45 (q, J = 7.2 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H), 0.91 (t, J = 8.4 Hz, 2H), 0.02 (s, 9H). Step 8: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000327_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-((2- (trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (420 mg, 0.62 mmol) in TFA (15 mL) was stirred at 20°C for 16 hours. The mixture was concentrated to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (330 mg) as TFA salt. Step 9: (R)-2-amino-3-(3-(4-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000327_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoate (330 mg, 0.50 mmol, TFA salt) in the solution of 30% HBr in AcOH (15 mL) was heated at 50°C for 2 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.08-9.02 (m, 1H), 8.58 (br s, 3H), 8.01 (s, 1H), 7.71 (d, J = 9.6 Hz, 1H), 7.66 (s, 1H), 7.59 (d, J = 9.6 Hz, 1H), 4.15-4.08 (m, 1H), 3.83-3.76 (m, 2H), 2.64 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 321.2 tR (min, Method D) = 1.554 [α]20,D = +2.0 (c = 1 mg/mL,CH3OH). Compound 3y (R)-2-amino-3-(3-(2-ethylthiophen-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethylthiophen- 3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000328_0001
To a solution of 2,3-dibromothiophene (2.0 g, 8.27 mmol) in THF (50 mL) was added Pd(dppf)Cl2 (605 mg, 0.83 mmol), cesium acetate (3.97 g, 20.67 mmol) and triethylborane (1 M in THF, 9.92 mL). The mixture was stirred at 65 °C for 16 hours. The mixture was added water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were concentrated below 30 °C. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 1%) and concentrated below 30 °C to give 3-bromo-2-ethylthiophene (200 mg, crude). Step 2: 3-(2-ethylthiophen-3-yl)-5-fluorobenzoic acid
Figure imgf000329_0001
To a solution of 3-bromo-2-ethylthiophene (200 mg, crude) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (207 mg, 1.05 mmol) in a mixture of DME (5 mL) and H2O (0.5 mL) was added Pd(PPh3)4 (121 mg, 0.10 mmol) and K2CO3 (434 mg, 3.14 mmol) under N2 atmosphere. The mixture was stirred at 85 °C for 16 hours. The mixture was diluted with water (10 mL) and acidified with 2N HCl to adjust pH to 4. The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give 3- (2-ethylthiophen-3-yl)-5-fluorobenzoic acid (110 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.39 (br s, 1H), 7.76 (s, 1H), 7.66-7.59 (m, 1H), 7.56- 7.50 (m, 1H), 7.45 (d, J = 5.2 Hz, 1H), 7.16 (d, J = 5.2 Hz, 1H), 2.89 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylthiophen-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000329_0002
To a solution of 3-(2-ethylthiophen-3-yl)-5-fluorobenzoic acid (100 mg, 0.40 mmol) in DMF (5 mL) was added TBTU (192 mg, 0.60 mmol), DIPEA (155 mg, 1.20 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (146 mg, 0.4 mmol, HCl salt). The mixture was stirred at 25 °C for 3 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethylthiophen-3-yl)-5- fluorobenzamido)propanoate (160 mg). 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 1H), 7.38-7.29 (m, 11H), 7.23-7.17 (m, 2H), 7.01 (d, J = 4.8 Hz, 1H), 6.78 (br s, 1H), 5.94 (br d, J = 6.4 Hz, 1H), 5.26-5.16 (m, 2H), 5.11 (s, 2H), 4.64-4.55 (m, 1H), 3.99-3.88 (m, 1H), 3.86-3.78 (m, 1H), 2.90 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(2-ethylthiophen-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000330_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylthiophen-3-yl)-5- fluorobenzamido)propanoate (80 mg, 0.14 mmol) in 30% HBr in AcOH (4 mL) was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(2-ethylthiophen-3-yl)-5- fluorobenzamido)propanoic acid (15 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.99-8.92 (m, 1H), 8.32 (br s, 2H), 7.75 (s, 1H), 7.66-7.61 (m, 1H), 7.48-7.41 (m, 2H), 7.16 (d, J = 5.6 Hz, 1H), 4.02-3.97 (m, 1H), 3.82-3.77 (m, 1H), 3.73-3.65 (m, 1H), 2.90 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 337.0 tR (min, Method D) = 1.843 [α]20,D = 2.0 (c = 1.0 mg/mL, CH3OH). Compound 3z (R)-2-amino-3-(3-(5-ethylthiazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylthiazol-4- yl)-5-fluorobenzamido)propanoic acid
Figure imgf000331_0001
A mixture of 4-bromothiazole (800 mg, 4.88 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.16 g, 5.86 mmol), Pd(dppf)Cl2 (357 mg, 487.9 µmol) and K2CO3 (2.02 g, 14.64 mmol) in dioxane (10 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80°C for 2 hours under N2 atmosphere. H2O (10 ml) was added and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~20%) to give methyl 3-fluoro- 5-(thiazol-4-yl)benzoate (760 mg). 1H NMR (400 MHz, CDCl3) δ 8.90 (d, J = 2.0 Hz, 1H), 8.36 (t, J = 1.2 Hz, 1H), 7.94-7.87 (m, 1H), 7.72 - 7.66 (m, 2H), 3.96 (s, 3H). Step 2: methyl 3-(5-bromothiazol-4-yl)-5-fluorobenzoate
Figure imgf000332_0001
To a solution of methyl 3-fluoro-5-(thiazol-4-yl)benzoate (760 mg, 3.20 mmol) and AcONa (526 mg, 6.41 mmol) in AcOH (10 mL) was added Br2 (3.1 g, 19.4 mmol) at 0°C and the resulting mixture was stirred at 80°C for 4 hours. The reaction was cooled to room temperature (25°C) and poured into H2O (40 ml). The solid was filtered, washed with H2O (5 mL) and dried in vacuum to give methyl 3-(5-bromothiazol-4-yl)-5- fluorobenzoate (900 mg). 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.39 (s, 1H), 8.05-7.99 (m, 1H), 7.81-7.74 (m, 1H), 3.90 (s, 3H).
Figure imgf000332_0002
A mixture of methyl 3-(5-bromothiazol-4-yl)-5-fluorobenzoate (450 mg, 1.42 mmol), triethylborane (1 M in THF, 8.54 mL), CsOAc (820 mg, 4.27 mmol), Pd(dtbpf)Cl2 (93 mg, 142.67 µmol) in THF (50 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere. THF was removed under reduced pressure. H2O (20 ml) was added and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine (20 mL×2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~15%) to give 100 mg of desired product and 255 mg of the crude product which was purified further by preparative HPLC (Method CC) to give another 120 mg of the desired product.
Figure imgf000333_0001
To a solution of methyl 3-(5-ethylthiazol-4-yl)-5-fluorobenzoate (120 mg, 452.3 µmol) in MeOH (8 mL) was added a solution of LiOH.H2O (60 mg, 1.43 mmol) in H2O (2 mL). The mixture was stirred at 25°C for 3 hours. The solvent was removed under reduced pressure. The residue was suspended in water (10 mL), acidified with 2N aqueous HCl to pH=5 and extracted with ethyl acetate (10 mL × 4). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 3-(5- ethylthiazol-4-yl)-5-fluorobenzoic acid (100 mg). 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.07 (s, 1H), 7.75-7.64 (m, 2H), 3.03 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylthiazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000334_0001
A mixture of 3-(5-ethylthiazol-4-yl)-5-fluorobenzoic acid (30 mg, 119.4 µmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (52 mg, 143.3 µmol, HCl salt), TBTU (58 mg, 180.6 umol) and DIPEA (63 µL, 361.7 µmol) in DMF (1 mL) was stirred at 25 °C for 16 hours. H2O (2 ml) was added to quench the reaction and then extracted with ethyl acetate (5 mL×3). The combined organic layers were washed with brine (5 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by preparative HPLC (Method BB) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylthiazol-4- yl)-5-fluorobenzamido)propanoate (45 mg). 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.78 (s, 1H), 7.51-7.28 (m, 12H), 7.01 (br s, 1H), 6.06 (br d, J = 6.4 Hz, 1H), 5.20 (s, 2H), 5.10 (s, 2H), 4.70-7.79 (m, 1H), 3.97-3.83 (m, 2H), 3.02 (q, J = 7.6 Hz, 2H), 1.36 (t, J = 7.6 Hz, 3H).
Figure imgf000334_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylthiazol-4-yl)-5- fluorobenzamido)propanoate (45 mg, 80.1 µmol) in 33% HBr in AcOH (3 mL) was stirred at 50°C for 16 hours. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(5-ethylthiazol-4-yl)- 5-fluorobenzamido)propanoic acid (17 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.12-9.04 (m, 2H), 8.56 (br s, 3H), 8.02 (s, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 9.2 Hz, 1H), 4.15-4.06 (m, 1H), 3.85-3.68 (m, 2H), 3.03 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 338.1 tR (min, Method D) = 1.657 [α]20,D = 4.0 (c = 1 mg/mL, MeOH). Compound 4a (R)-2-amino-3-(3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- (hydroxymethyl)-1-methyl-1H-pyrazol-4-yl)benzamido)propanoic acid
Figure imgf000335_0001
A mixture of (4-bromo-1-methyl-1H-pyrazol-5-yl)methanol (200 mg, 1.05 mmol), (3- fluoro-5-(methoxycarbonyl)phenyl)boronic acid (228 mg, 1.15 mmol), Pd(dppf)Cl2 (77 mg, 104.7 µmol), K2CO3 (434 mg, 3.14 mmol) in the mixture of H2O (0.5 mL) and dioxane (4 mL) was degassed and purged with N23 times, and then the mixture was stirred at 80 °C for 3 hours under N2 atmosphere. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel ( ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) to give methyl 3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4-yl)benzoate (200 mg). LC-MS (MH+): m/z = 265.1 tR (min, Method c) = 0.695 Step 2: 3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4-yl)benzoic acid
Figure imgf000336_0001
To a solution of methyl 3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4- yl)benzoate (200 mg, 756.9 µmol) in the mixture of H2O (3 mL) and MeOH (3 mL) was added LiOH.H2O (95 mg, 2.27 mmol). The mixture was stirred at 15 °C for 16 hours. The mixture was concentrated to remove MeOH. Then HCl (2 M) was added to adjust pH=5~6 and the solid was collected to give 3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H- pyrazol-4-yl)benzoic acid (170 mg). 1H NMR (400MHz, DMSO-d6) δ 7.90 (s, 1H), 7.74 (s, 1H), 7.60 (d, J = 9.6 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 5.55 (br, 1H), 4.55 (s, 2H), 3.89 (s, 3H) Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-(hydroxymethyl)-1- methyl-1H-pyrazol-4-yl)benzamido)propanoate
Figure imgf000337_0001
To a solution of (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (273 mg, 747.3 µmol, HCl salt) and 3-fluoro-5-(5-(hydroxymethyl)-1- methyl-1H-pyrazol-4-yl)benzoic acid (170 mg, 679.4 µmol) in DMF (5 mL) was added TBTU (327 mg, 1.02 mmol) and DIPEA (263 mg, 2.04 mmol). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel ( ethyl acetate/Petroleum ether with ethyl acetate from 0% to 80%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5- (hydroxymethyl)-1-methyl-1H-pyrazol-4-yl)benzamido)propanoate (290 mg). 1H NMR (400MHz, CDCl3) δ 7.57-7.47 (m, 2H), 7.39-7.20 (m, 13H), 6.12 (d, J = 7.6 Hz, 1H), 5.27-4.97 (m, 4H), 4.73-4.54 (m, 3H), 3.95 (s, 3H), 3.91-3.76 (m, 2H), 3.71 (s, 1H) Step 4: (R)-2-amino-3-(3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4- yl)benzamido)propanoic acid
Figure imgf000337_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5- (hydroxymethyl)-1-methyl-1H-pyrazol-4-yl)benzamido)propanoate (150 mg, 267.6 µmol) and Pd/C (70 mg, 10% Pd, 50% water) in MeOH (5 mL) was degassed and purged with H2 3 times, and then the mixture was stirred at 25 °C for 1 hour under H2 atmosphere. The reaction mixture was filtered through celite and the filtrate was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2- amino-3-(3-fluoro-5-(5-(hydroxymethyl)-1-methyl-1H-pyrazol-4- yl)benzamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.04-8.98 (m, 1H), 8.49 (br s, 3H), 7.88 (s, 1H), 7.76 (s, 1H), 7.63-7.51 (m, 2H), 4.57 (s, 2H), 4.17-4.08 (m, 1H), 3.90 (s, 3H), 3.83-3.78 (m, 2H). LC-MS (MH+): m/z = 337.1 tR (min, Method c) = 2.216 [α]20,D = 2.4 (c = 2.5 mg/mL, MeOH). Compound 4b (R)-2-amino-3-(5-fluoro-2'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(5-fluoro-2'-methyl- [1,1'-biphenyl]-3-ylcarboxamido)propanoic acid
Figure imgf000338_0001
Step 1: methyl 5-fluoro-2'-methyl-[1,1'-biphenyl]-3-carboxylate
Figure imgf000338_0002
A mixture of methyl 3-bromo-5-fluorobenzoate (200 mg, 858.2 µmol), o-tolylboronic acid (128 mg, 944.1 µmol), Pd(dtbpf)Cl2 (56 mg, 85.82 µmol), K3PO4 (547 mg, 2.57 mmol) in the mixture of H2O (0.5 mL) and dioxane (4 mL) was degassed and purged with N23 times, and then the mixture was stirred at 100 °C for 2 hours under N2 atmosphere. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 10%) to give methyl 5-fluoro-2'- methyl-[1,1'-biphenyl]-3-carboxylate (180 mg). 1H NMR (400MHz, CDCl3) δ 7.82 (dd, J = 1.2, 1.2 Hz, 1H), 7.74-7.67 (m, 1H), 7.32-7.22 (m, 5H), 3.94 (s, 3H), 2.28 (s, 3H) Step 2: Preparation of 5-fluoro-2'-methyl-[1,1'-biphenyl]-3-carboxylic acid
Figure imgf000339_0001
To a solution of methyl 5-fluoro-2'-methyl-[1,1'-biphenyl]-3-carboxylate (180 mg, 736.9 µmol) in the mixture of H2O (2 mL) and MeOH (2 mL) was added LiOH.H2O (93 mg, 2.21 mmol). The mixture was stirred at 20 °C for 2 hours. The mixture was concentrated to remove MeOH, then added HCl (2 M) to adjust pH=4~5. The solid was collected and dried to give 5-fluoro-2'-methyl-[1,1'-biphenyl]-3-carboxylic acid (160 mg). 1H NMR (400MHz, DMSO-d6) δ 7.71-7.64 (m, 2H), 7.54-7.49 (m, 1H), 7.35-7.23 (m, 4H), 2.24 (s, 3H) Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-methyl-[1,1'-biphenyl]- 3-ylcarboxamido)propanoate
Figure imgf000340_0001
To a solution of 5-fluoro-2'-methyl-[1,1'-biphenyl]-3-carboxylic acid (160 mg, 694.9 µmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (304 mg, 833.9 µmol, HCl salt) in DMF (5 mL) was added TBTU (335 mg, 1.04 mmol) and DIPEA (269 mg, 2.08 mmol). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-methyl-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (330 mg). 1H NMR (400MHz, CDCl3) δ 7.44-7.28 (m, 15H), 7.21-7.15 (m, 2H), 6.80 (br, 1H), 5.97 (d, J = 6.8 Hz, 1H), 5.25-5.16 (m, 2H), 5.09 (s, 2H), 4.63-4.54 (m, 1H), 3.98-3.77 (m, 2H), 2.26 (s, 3H). Step 4: (R)-2-amino-3-(5-fluoro-2'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid
Figure imgf000340_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-methyl-[1,1'- biphenyl]-3-ylcarboxamido)propanoate (150 mg, 277.5 µmol) in 30% HBr in AcOH (3 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was dissolved in water (2 mL), extracted with ethyl acetate (5 mL × 3). The water layer was lyophilized. The residue was purified by preparative HPLC (Method AA) to give (R)-2- amino-3-(5-fluoro-2'-methyl-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.03-8.96 (m, 1H), 8.44 (br s, 3H), 7.73-7.67 (m, 2H), 7.44 (d, J = 9.2 Hz, 1H), 7.35-7.24 (m, 4H), 4.12-4.05 (m, 1H), 3.86-3.66 (m, 2H), 2.25 (s, 3H). LC-MS (MH+): m/z = 317.0 tR (min, Method c) = 1.145 [α]20,D = 3 (c = 2 mg/mL, MeOH). Compound 4c (R)-2-amino-3-(3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(4- methyl-2-(trifluoromethyl)pyridin-3-yl)benzamido)propanoic acid
Figure imgf000341_0001
Step 1: methyl 3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3-yl)benzoate
Figure imgf000341_0002
A mixture of 3-bromo-4-methyl-2-(trifluoromethyl)pyridine (200 mg, 0.83 mmol), (3- fluoro-5-(methoxycarbonyl)phenyl)boronic acid (165 mg, 0.83 mmol), Pd(dppf)Cl2 (61 mg, 0.08 mmol) and K2CO3 (346 mg, 2.50 mmol) in a mixture of dioxane (4 mL) and H2O (1 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine (20 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 15%) to give methyl 3-fluoro-5-(4-methyl-2- (trifluoromethyl)pyridin-3-yl)benzoate (150 mg). 1H NMR (400MHz, CDCl3) δ 8.61 (d, J = 4.8 Hz, 1H), 7.84-7.79 (m, 1H), 7.70 (s, 1H), 7.43 (d, J = 4.8 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H), 2.11 (s, 3H). Step 2: 3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3-yl)benzoic acid
Figure imgf000342_0001
To a solution of methyl 3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3-yl)benzoate (150 mg, 0.48 mmol) in a mixture of THF (6 mL) and H2O (3 mL) was added LiOH.H2O (40 mg, 0.96 mmol) and the mixture was stirred at 20°C for 15 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3-yl)benzoic acid (140 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-methyl-2- (trifluoromethyl)pyridin-3-yl)benzamido)propanoate
Figure imgf000343_0001
To a solution of a mixture of 3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3- yl)benzoic acid (140 mg, 0.47 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (188 mg, 0.51 mmol, HCl salt) in DMF (5 mL) was added TBTU (255 mg, 0.70 mmol) and DIPEA (181 mg, 1.40 mmol). The reaction mixture was stirred at 25°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 30% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-methyl-2- (trifluoromethyl)pyridin-3-yl)benzamido)propanoate (220 mg). 1H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 4.8 Hz, 1H), 7.49-7.29 (m, 13H), 7.06 (d, J = 8.0 Hz, 1H), 6.94-6.82 (m, 1H), 5.90 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.09 (s, 2H), 4.63-4.56 (m, 1H), 3.99- 3.74 (m, 2H), 2.10 (s, 3H). Step 4: (R)-2-amino-3-(3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3- yl)benzamido)propanoic acid
Figure imgf000343_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(4-methyl-2- (trifluoromethyl)pyridin-3-yl)benzamido)propanoate (220 mg, 0.36 mmol) in 30% HBr in AcOH (10 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with MTBE (5 mL × 2). The aqueous layer was lyophilized to give (R)-2-amino-3-(3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin- 3-yl)benzamido)propanoic acid (100 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.90 (br s, 1H), 8.69-8.64 (m, 1H), 8.34 (br s, 3H), 7.83- 7.71 (m, 2H), 7.60 (s, 1H), 7.48-7.44 (m, 1H), 4.15-4.05 (m, 1H), 3.85-3.60 (m, 2H), 2.08 (s, 3H). LC-MS (MH+): m/z = 386.0 tR (min, Method D) = 1.562 [α]20,D = -1.3 (c = 1.5 mg/mL,CH3OH). Compound 4d (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoic acid
Figure imgf000344_0001
Step 1: methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000344_0002
To a solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (600 mg, 2.29 mmol) in THF (20 mL) was added NIS (1.03 g, 4.58 mmol). The mixture was stirred at 70°C for 2 hours. The reaction mixture was concentrated. The residue was added water (15 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (10 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20 %) to give methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5- yl)benzoate (660 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.81 (m, 2H), 7.61 (s, 1H), 7.32-7.27 (m, 1H), 4.04 (t, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.81-1.71 (m, 2H), 0.80 (t, J = 7.6 Hz, 3H). Step 2: methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoate
Figure imgf000345_0001
To a mixture of methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate (590 mg, 1.52 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (1.46 g, 7.60 mmol) in DMF (15 mL) was added CuI (29 mg, 0.15 mmol). The mixture was stirred at 120°C for 15 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers were washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20%) twice to give methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzoate (280 mg). 1H NMR (400MHz,CDCl3) δ 7.88-7.79 (m, 3H), 7.29-7.26 (m, 1H), 3.96 (s, 3H), 3.95-3.90 (m, 2H), 1.84-1.75 (m, 2H), 0.82 (t, J = 7.6 Hz, 3H). Step 3: 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid
Figure imgf000346_0001
To a solution of methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzoate (280 mg, 0.85 mmol) in a mixture of MeOH (10 mL) and H2O (2 mL) was added LiOH.H2O (90 mg, 2.14 mmol). The mixture was stirred at 30°C for 1 hour. The reaction mixture was concentrated. The residue was added water (10 mL), adjusted pH to 5-6 with 1N HCl and extracted with ethyl acetate (15 mL × 3). The organic layers was dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid (255 mg). 1H NMR (400MHz, DMSO-d6) δ 7.99 (s, 1H), 7.80-7.71 (m, 2H), 7.38 (d, J = 8.8 Hz, 1H), 3.93 (t, J = 7.2 Hz, 2H), 1.72-1.63 (m, 2H), 0.70 (t, J = 7.2 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate
Figure imgf000346_0002
To a mixture of 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid (100 mg, 0.32 mmol), TBTU (152 mg, 0.47 mmol) and DIPEA (123 mg, 0.95 mmol) in DMF (15 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (138 mg, 0.38 mmol, HCl salt). The mixture was stirred at 35°C for 16 hours. The reaction mixture was added water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoate (110 mg). 1H NMR (400MHz, CDCl3) δ 7.81 (s, 1H), 7.52-7.47 (m, 2H), 7.37-7.31 (m, 10H), 7.25-7.20 (m, 1H), 6.99-6.69 (m, 1H), 5.90 (br d, J = 6.0 Hz, 1H), 5.28-5.17 (m, 2H), 5.11 (s, 2H), 4.60-4.55 (m, 1H), 4.01-3.91 (m, 3H), 3.85-3.74 (m, 1H), 1.85-1.75 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid
Figure imgf000347_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate (110 mg, 0.18 mmol) in 30% HBr in AcOH (6 mL) was stirred at 20°C for 16 hours. The reaction mixture was concentrated. The residue was added water and extracted with MTBE (10 ml × 3). The aqueous layer was lyophilized. The product was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid (14 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.10-9.04 (m, 1H), 8.44 (br s, 3H), 8.04 (s, 1H), 7.94 (d, J = 9.6 Hz, 1H), 7.79 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 4.17-4.08 (m, 1H), 3.95 (t, J = 7.6 Hz, 2H), 3.85-3.78 (m, 1H), 3.76-3.70 (m, 1H), 1.72-1.63 (m, 2H), 0.71 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 403.2 tR (min, Method D) = 1.891 [α]20,D = 11.3 (c = 1.0 mg/mL, CH3OH). Compound 4e (R)-2-amino-3-(3-butylbenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3- butylbenzamido)propanoic acid
Figure imgf000348_0001
Step 1: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-butylbenzamido) propanoate
Figure imgf000348_0002
To a mixture of 3-butylbenzoic acid (400 mg, 2.24 mmol), (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (817 mg, 2.24 mmol, HCl salt), DIPEA (868 mg, 6.72 mmol) and TBTU (1.08 g, 3.36 mmol) in DMF (5 mL) was stirred at 30°C for 16 hours. The mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mLx3). The combined organic phase was washed by brine (10 mLx3), dried over anhydrous Na2SO4, concentrated. The residue was purified by combiFlash (ethyl acetate: Petroleum ether = 0~50%, then hold, after that 50~0%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-butylbenzamido) propanoate (300 mg). 1H NMR (400MHz, DMSO-d6) δ 8.52-8.49 (m, 1H), 7.81-7.61 (d, J = 8.0 Hz, 1H), 7.63-7.55 (m, 2H), 7.37-7.18 (m, 12H), 5.12-4.95 (m, 4H), 4.40-4.33 (m, 1H), 3.68-3.56 (m, 2H), 2.59 (t, J=7.6 Hz, 2H), 1.49-1.59 (m, 2H), 1.24-1.33 (m, 2H), 0.87 (t, J=7.6 Hz, 3H). Step 2: (R)-2-amino-3-(3-butylbenzamido)propanoic acid
Figure imgf000349_0001
To a mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3- (3- butylbenzamido)propanoate (150 mg, 0.31 mmol) in HBr/AcOH (3 mL, 30%) was stirred at 50°C for 16 hours. The mixture was concentrated on vacuo. The residue was washed by the mixture solvent of MBTE (10 mL) and MeOH (1 mL), followed by filtration. The residue was purified by preparative HPLC (HCl) to afford (R)-2-amino-3-(3- butylbenzamido)propanoic acid (35 mg) as HCl salt. 1H NMR (400MHz, CD3OD) δ 7.70 (s, 1H), 7.68-7.64 (m, 1H), 7.42-7.34 (m, 2H), 4.21 (m, 1H), 3.96 (dd, J = 14.4, 3.6 Hz, 1H), 3.83 (dd, J = 14.4, 6.4 Hz, 1H), 2.67 (t, J = 3.6 Hz, 2H), 1.57-1.68 (m, 2H), 1.31-1.42 (m, 2H), 0.94 (t, J=7.2 Hz, 3H). LC-MS (MH+): m/z = 265.1 tR (min, WXL-Method D) = 2.009 [α]20,D = 2.5 (c = 0.8 mg/mL,CH3OH). Compound 4f (R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-ethyl-5- fluorobenzamido)propanoic acid
Figure imgf000349_0002
Step 1: methyl 3-fluoro-5-vinylbenzoate
Figure imgf000350_0001
To a solution of methyl 3-bromo-5-fluorobenzoate (400 mg, 1.72 mmol) and 4,4,5,5- tetramethyl-2-vinyl-1,3,2-dioxaborolane (318 mg, 2.06 mmol) in dioxane (10 mL) and H2O (1 mL) was added Pd(dppf)Cl2 (126 mg, 0.17 mmol) and K2CO3 (713 mg, 5.16 mmol) under N2 atmosphere. The mixture was stirred at 100°C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3), the combined organic layers were concentrated under reduced pressure. The residue was purified by Combi Flash (petroleum ether: ethyl acetate with ethyl acetate from 0 to 15%) to give methyl 3-fluoro-5-vinylbenzoate (150 mg). 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.64-7.56 (m, 1H), 7.33-7.27 (m, 1H), 6.71 (dd, J = 17.6, 11.2 Hz, 1H), 5.84 (d, J = 17.6 Hz, 1H), 5.39 (d, J = 11.2 Hz, 1H), 3.94 (s, 3H). Step 2: methyl 3-ethyl-5-fluorobenzoate
Figure imgf000350_0002
To a solution of methyl 3-fluoro-5-vinylbenzoate (140 mg, 0.78 mmol) in MeOH (8 mL) was added Pd/C (10% w.t, 50% water, 80 mg) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (balloon) at 20°C for 3 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure to give methyl 3-ethyl-5-fluorobenzoate (130 mg). The product was used next step directly. Step 3: 3-ethyl-5-fluorobenzoic acid
Figure imgf000351_0001
To a solution of methyl 3-ethyl-5-fluorobenzoate (130 mg, 0.71 mmol) in MeOH (10 mL) was added LiOH.H2O (90 mg, 2.14 mmol) in H2O (3 mL). The mixture was stirred at 15°C for 16 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in water (5 mL) and washed with ethyl acetate (6 mL), and the organic layer was discarded. The aqueous layers was acidified with 2N HCl to adjust pH to 2 and extracted with DCM (10 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 3-ethyl-5-fluoro-benzoic acid (100 mg). 1H NMR (400 MHz, CD3OD) δ 7.70 (s, 1H), 7.52-7.47 (m, 1H), 7.23-7.18 (m, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate
Figure imgf000351_0002
To a solution of 3-ethyl-5-fluoro-benzoic acid (100 mg, 0.59 mmol) in DMF (6 mL) was added TBTU (286 mg, 0.89 mmol), DIPEA (231 mg, 1.78 mmol) and (R)-benzyl 3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (217 mg, 0.59 mmol, HCl salt). The mixture was stirred at 20°C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL) and concentrated under reduced pressure. The residue was purified by Combi Flash (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino) -3-(3-ethyl-5- fluorobenzamido)propanoate (220 mg). 1H NMR (400 MHz, CDCl3) δ 7.38-7.30 (m, 11H), 7.15 (d, J = 9.2 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 6.63 (br s, 1H), 5.94 (d, J = 6.8 Hz, 1H), 5.22 (s, 2H), 5.13 (s, 2H), 4.63-4.57 (m, 1H), 3.96-3.78 (m, 2H), 2.68 (q, J=7.6 Hz, 2H), 1.25 (t, J=7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid
Figure imgf000352_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (100 mg, 0.21 mmol) in 33% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The mixture was concentrated under reduced pressure. The residue was purified by Preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-5- fluorobenzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.96-8.90 (m, 1H), 8.49 (br s, 3H), 7.63 (s, 1H), 7.51 (d, J = 9.6 Hz, 1H), 7.28 (d, J = 9.6 Hz, 1H), 4.12-4.06 (m, 1H), 3.90-3.64 (m, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.20 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 255.0 tR (min, WXL-Method D) = 1.641 [α]20,D = 6.0, (c = 1 mg/mL, MeOH). Compound 4g (R)-2-amino-3-(4-methylquinoline-6-carboxamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(4-methylquinoline-6- carboxamido)propanoic acid
Figure imgf000353_0001
To a solution of 4-methylquinoline-6-carboxylic acid (200 mg, 1.07 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (386 mg, 1.18 mmol, HCl salt) in DMF (10 mL) was added TBTU (515 mg, 1.61 mmol) and DIPEA (415 mg, 3.21 mmol) under N2. The mixture was stirred at 20°C for 16 hours. The reaction was added water (50 mL) and extracted with ethyl acetate (30 mL × 3). The organic layers were concentrated to give yellow oil. The oil was purified by Combi Flash (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 50%) to (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(4-methylquinoline-6-carboxamido)propanoate (400 mg). LC-MS (MH+): m/z = 498.4 tR (min, Method c) = 1.007 Step 2: (R)-2-amino-3-(4-methylquinoline-6-carboxamido)propanoic acid
Figure imgf000353_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-methylquinoline-6- carboxamido)propanoate (400 mg, 0.80 mmol) in 33% wt HBr in AcOH (10 mL) was stirred at 50°C for 15 hours. The reaction was concentrated to give a yellow solid. The solid was added AcOH (20 mL) and stirred at 20°C for 1 hour. Filtered and dried to give a white solid. The solid was purified by Preparative HPLC (Method AA) to give (R)-2- amino-3-(4-methylquinoline-6-carboxamido)propanoic acid (80 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.65-9.58 (m, 1H), 9.13 (d, J = 5.2 Hz, 1H), 9.00 (s, 1H), 8.70 (br s, 3H), 8.46 (d, J = 9.2 Hz, 1H), 8.37 (d, J=9.2 Hz, 1H), 7.89 (d, J=5.2 Hz, 1H), 4.20- 4.17 (m, 1H), 3.91-3.86 (m, 2H), 2.99 (s, 3H). LC-MS (MH+): m/z = 274.0 tR (min, WXL-Method D) = 1.040 [α]20,D = -11.89(c = 1.43 mg/mL,CH3OH). Compound 4h (R)-2-amino-3-(1H-benzo[d]imidazole-5-carboxamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(1H-benzo[d]imidazole- 5-carboxamido)propanoic acid
Figure imgf000354_0001
To a solution of (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (250 mg, 0.69 mmol, HCl salt) and 1H-benzo[d]imidazole-5-carboxylic acid (111 mg, 0.69 mmol) in DMF (10 mL) was added TBTU (330 mg, 1.03 mmol) and DIPEA (443 mg, 3.43 mmol). The mixture was stirred at 20-30°C for 2 hours. The residue was diluted with water (40 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic layers were washed with brine (15 mL), dried over MgSO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of 0~100% ethyl acetate/Petroleum ether) to give (R)-benzyl3- (1H-benzo[d]imidazole-5-carboxamido)-2-(((benzyloxy)carbonyl)amino)propanoate (120 mg). LC-MS (MH+): m/z = 473.2 tR (min, Method c) = 0.596 Step 2: (R)-2-amino-3-(1H-benzo[d]imidazole-5-carboxamido)propanoic acid
Figure imgf000355_0001
A solution of (R)-benzyl 3-(1H-benzo[d]imidazole-5-carboxamido)-2-(((benzyloxy) carbonyl)amino)propanoate (110 mg, 0.23 mmol) in HBr/AcOH (2 mL, 30%) was stirred at 50 °C for 16 hours . The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was washed with MeCN (10 mL × 2), filtered and concentrated under reduced pressure to give (R)-2-amino-3-(1H-benzo[d] imidazole-5- carboxamido)propanoic acid (45 mg) as HBr salt. 1HNMR (400MHz, DMSO-d6) δ 9.54 (s, 1H), 9.00-8.96 (m, 1H), 8.46-8.24 (m, 4H), 8.01 (dd, J= 8.4, 1.2 Hz, 1H), 7.89 (d, J=8.4 Hz, 1H), 4.15-4.10 (m, 1H), 3.82-3.67 (m, 2H). LC-MS (MH+): m/z = 249.0 tR (min, Method c) = 3.223 [α]20,D = -41(c = 2 mg/mL, CH3OH). Compound 4i (2R)-2-amino-3-(3-fluoro-5-(1-methylpyrrolidin-2-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (2R)-2-amino-3-(3-fluoro-5-(1- methylpyrrolidin-2-yl)benzamido)propanoic acid
Figure imgf000356_0001
Step 1: tert-butyl 2-(3-fluoro-5-(methoxycarbonyl)phenyl)pyrrolidine-1-carboxylate
Figure imgf000356_0002
A mixture of 1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (693 mg, 3.22 mmol), methyl 3-bromo-5-fluorobenzoate (500 mg, 2.15 mmol), (IR[DF(CF3)PPY]2(DTBPY))PF6 (24 mg, 21.46 µmol), NiCl2.glyme (47 mg, 214.56 µmol) , 4,4'-di-tert-butyl-2,2'-bipyridyl (86 mg, 321.84 µmol) and Cs2CO3 (1.05 g, 3.22 mmol) in DMF (10 mL) in a 20 mL microwave tube was stirred at 20 °C for 16 hours under 72W blue LED Strip Light. The reaction mixture was quenched with NaHCO3 (5 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 15%) to give tert-butyl 2-(3- fluoro-5-(methoxycarbonyl)phenyl)pyrrolidine-1-carboxylate (470 mg). Step 2: methyl 3-fluoro-5-(pyrrolidin-2-yl)benzoate
Figure imgf000357_0001
Figure imgf000357_0002
To a solution of tert-butyl 2-(3-fluoro-5-(methoxycarbonyl)phenyl)pyrrolidine-1- carboxylate (450 mg, 1.39 mmol) in DCM (10 mL) was added TFA (317 mg, 2.78 mmol). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated. Then saturated Na2CO3 was added to adjust pH=9, extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give methyl 3-fluoro-5-(pyrrolidin-2-yl)benzoate (280 mg). Step 3: methyl 3-fluoro-5-(1-methylpyrrolidin-2-yl)benzoate
Figure imgf000357_0003
A mixture of methyl 3-fluoro-5-(pyrrolidin-2-yl)benzoate (250 mg, 1.12 mmol), HCHO (454 mg, 5.60 mmol, 37% in water), Pd/C (100 mg, 10% Pd, 50% water) in MeOH (5 mL) was degassed and purged with H23 times, and then the mixture was stirred at 20 °C for 16 hours under H2 (15 Psi) atmosphere. The reaction mixture was filtered through celite and the filtrate was concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give methyl 3-fluoro-5-(1-methylpyrrolidin-2-yl)benzoate (140 mg). LC-MS (MH+): m/z = 238.1 tR (min, Method c) = 0.525 Step 4: 3-fluoro-5-(1-methylpyrrolidin-2-yl)benzoic acid
Figure imgf000358_0001
To a solution of methyl 3-fluoro-5-(1-methylpyrrolidin-2-yl)benzoate (140 mg, 590.05 µmol) in the mixture of H2O (2 mL) and MeOH (2 mL) was added LiOH.H2O (74 mg, 1.77 mmol). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated to move MeOH, then added HCl (2 M) to adjust pH=2~3 and concentrated to give 3-fluoro- 5-(1-methylpyrrolidin-2-yl)benzoic acid (250 mg, crude, HCl salt). LC-MS (MH+): m/z = 224.1 tR (min, Method c) = 0.290 Step 5: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-methylpyrrolidin-2- yl)benzamido)propanoate
Figure imgf000358_0002
To a solution of 3-fluoro-5-(1-methylpyrrolidin-2-yl)benzoic acid (250 mg, 962.6 µmol, HCl salt) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (281 mg, 770.1 µmol, HCl salt) in DMF (5 mL) was added TBTU (309 mg, 962.6 µmol) and DIPEA (373 mg, 2.89 mmol). The mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) twice to give (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-methylpyrrolidin-2- yl)benzamido)propanoate (180 mg). LC-MS (MH+): m/z = 534.3 tR (min, Method c) = 0.755 Step 6: (2R)-2-amino-3-(3-fluoro-5-(1-methylpyrrolidin-2-yl)benzamido)propanoic acid
Figure imgf000359_0001
A solution of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- methylpyrrolidin-2-yl)benzamido)propanoate (180 mg, 337.3 µmol) in 30% HBr in AcOH (4 mL) was stirred at 20°C for 16 hours. The mixture was concentrated. The residue was purified by preparation HPLC (Method AA) to give (2R)-2-amino-3-(3-fluoro-5-(1- methylpyrrolidin-2-yl)benzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 11.38 (br s, 1H), 9.17-9.10 (m, 1H), 8.57 (br s, 3H), 8.14 (t, J = 7.6 Hz, 1H), 7.89-7.81 (m, 2H), 4.83-4.68 (m, 1H), 4.16-4.07 (m, 1H), 3.86-3.70 (m, 3H), 3.36-3.09 (m, 1H), 2.64 (d, J = 3.6 Hz, 3H), 2.49-2.38 (m, 1H), 2.24-2.07 (m, 3H). LC-MS (MH+): m/z = 310.2 tR (min, Method c) = 2.073 [α]20,D = 0.25 (c = 8 mg/mL, MeOH). Compound 4j (2R)-2-amino-3-(3-(4,4-difluoropyrrolidin-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (2R)-2-amino-3-(3-(4,4- difluoropyrrolidin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000360_0001
Step 1: tert-butyl 4,4-difluoro-2-(3-fluoro-5-(methoxycarbonyl)phenyl)pyrrolidine-1- carboxylate
Figure imgf000360_0002
A mixture of 1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (810 mg, 3.23 mmol), methyl 3-bromo-5-fluorobenzoate (500 mg, 2.15 mmol), NiCl2.glyme (47 mg, 215.0 µmol) , 4,4'-di-tert-butyl-2,2'-bipyridyl (87 mg, 322.5 µmol), (IR[DF(CF3)PPY]2(DTBPY))PF6 (24 mg, 21.50 µmol) and Cs2CO3 (1.05 g, 3.23 mmol) in DMF (10 mL) in a 20 mL microwave tube was stirred at 20 °C for 16 hours under 72W blue LED Strip Light. The reaction mixture was quenched with NaHCO3 (5 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 15%) to give tert-butyl 4,4-difluoro-2-(3-fluoro-5-(methoxycarbonyl)phenyl)pyrrolidine-1- carboxylate (620 mg). LC-MS (M+H-56): m/z = 304.1 tR (min, Method c) = 0.914 Step 2: 3-(1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)-5-fluorobenzoic acid
Figure imgf000361_0001
To a solution of tert-butyl 4,4-difluoro-2-(3-fluoro-5- (methoxycarbonyl)phenyl)pyrrolidine-1-carboxylate (620 mg, 1.73 mmol) in the mixture of H2O (5 mL) and MeOH (5 mL) was added LiOH.H2O (217 mg, 5.18 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated to move MeOH, then added HCl(2 M) to adjust pH=5~6, extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)-5- fluorobenzoic acid (580 mg). LC-MS (MNa+): m/z = 368.1 tR (min, Method c) = 0.923 Step 3: tert-butyl 2-(3-(((R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3- oxopropyl)carbamoyl)-5-fluorophenyl)-4,4-difluoropyrrolidine-1-carboxylate
Figure imgf000361_0002
To a solution of 3-(1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)-5-fluorobenzoic acid (280 mg, 810.9 µmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate hydrochloride (325 mg, 891.9 µmol, HCl salt) in DMF (7 mL) was added TBTU (391 mg, 1.22 mmol) and DIPEA (314 mg, 2.43 mmol). The mixture was stirred at 25 °C for 2 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by preparation HPLC (Method BB) and further purified by Combi Flash on silica gel ( ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) to give 200 mg of crude product, which was further separated by SFC ( Instrument: MG-II; Column: REGIS (s,s) WHELK-O1 (250mm*30mm,5µm); Mobile phase: supercritical CO2/EtOH (0.1% NH3`H2O, v%) = 60/40; Flow Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give tert-butyl 2-(3-(((R)-3- (benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropyl)carbamoyl)-5-fluorophenyl)- 4,4-difluoropyrrolidine-1-carboxylate (120 mg). LC-MS (MH+): m/z = 656.3 tR (min, Method c) = 0.956 Step 4: (2R)-2-amino-3-(3-(1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000362_0001
To a solution of tert-butyl 2-(3-(((R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3- oxopropyl)carbamoyl)-5-fluorophenyl)-4,4-difluoropyrrolidine-1-carboxylate (80 mg, 122.0 µmol) and Pd/C (40 mg, 10% Pd, 50% water) in AcOH (5 mL) was degassed and purged with H23 times, and then the mixture was stirred at 20 °C for 2 hours under H2 atmosphere (15 Psi). The reaction mixture was filtered through celite and the filtrate was concentrated to give (2R)-2-amino-3-(3-(1-(tert-butoxycarbonyl)-4,4- difluoropyrrolidin-2-yl)-5-fluorobenzamido)propanoic acid (45 mg). LC-MS (MH+): m/z = 432.2 tR (min, Method c) = 0.704 Step 5: (2R)-2-amino-3-(3-(4,4-difluoropyrrolidin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000363_0001
A solution of (2R)-2-amino-3-(3-(1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)-5- fluorobenzamido)propanoic acid (45 mg, 104.3 µmol) in HCl/ethyl acetate (4 M, 2 mL) was stirred at 20°C for 2 hours. The mixture was filtered, the solid was washed with ethyl acetate (5 mL) and dried to give (2R)-2-amino-3-(3-(4,4-difluoropyrrolidin-2-yl)-5- fluorobenzamido)propanoic acid (35 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.17-9-13 (m, 1H), 8.58 (br s, 3H), 8.02 (s, 1H), 7.86 (d, J = 9.6 Hz, 1H), 7.79 (d, J = 9.6 Hz, 1H), 5.11-4.99 (m, 1H), 4.16-4.10 (m, 1H), 4.02-3.89 (m, 1H), 3.85-3.79 (m, 3H), 3.14-2.80 (m, 2H). LC-MS (MH+): m/z = 332.1 tR (min, Method c) = 1.202 [α]20,D = -4.6 (c = 1.3 mg/mL, MeOH). Compound 4k (R)-2-amino-3-(1-oxo-1,2-dihydroisoquinoline-7-carboxamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(1-oxo-1,2- dihydroisoquinoline-7-carboxamido)propanoic acid
Figure imgf000364_0001
To a suspension of 7-bromoisoquinolin-1(2H)-one (500 mg, 2.23 mmol) in DMF (10 mL) was added CuCN (400 mg, 4.46 mmol). The reaction was heated to 180°C for 6 hours. The reaction was cooled to room temperature and diluted with water (50 mL). The solution was extracted with ethyl acetate (60 mL × 3), and the combined organic layers were concentrated to give 1-oxo-1,2-dihydroisoquinoline-7-carbonitrile (590 mg, crude). 1H NMR (400 MHz, DMSO-d6) δ 11.62 (br s, 1H), 8.47 (s, 1H), 8.01 (d, J = 8.0 Hz), 7.80 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 6.4 Hz, 1H), 6.61(d, J = 6.4 Hz, 1H).
Figure imgf000364_0002
1-oxo-1,2-dihydroisoquinoline-7-carbonitrile (590 mg, crude) was taken up in ethanol (10 mL) and aqueous NaOH (1 M, 8 mL) was added. The mixture was heated to 100°C and stirred for 2 hours. The reaction was cooled to room temperature and the pH was adjusted to 2 with 1 N aqueous hydrochloric acid. The solid was collected by filtration, rinsed with water (3 mL), and dried to give 1-oxo-1,2-dihydroisoquinoline-7-carboxylic acid (180 mg, crude). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(1-oxo-1,2-dihydroisoquinoline-7- carboxamido)propanoate
Figure imgf000365_0001
To a solution of 1-oxo-1,2-dihydroisoquinoline-7-carboxylic acid (180 mg, crude) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (375 mg, 1.03 mmol, HCl salt) in DMF (10 mL) was added TBTU (458 mg, 1.43 mmol) and DIPEA (369 mg, 2.85 mmol). The reaction mixture was stirred at 10°C for 16 hours. The mixture was diluted with water (30 mL) and extracted with ethyl acetate (40 mL × 3), and the combined organic layers were concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 50% to 100%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(1-oxo-1,2- dihydroisoquinoline-7-carboxamido)propanoate (60 mg). LC-MS (MH+): m/z = 500.2 tR (min, Method c) = 0.846 Steps 4: (R)-2-amino-3-(1-oxo-1,2-dihydroisoquinoline-7-carboxamido)propanoic acid
Figure imgf000365_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(1-oxo-1,2- dihydroisoquinoline-7-carboxamido)propanoate (60 mg, 0.12 mmol) in 30 % HBr in AcOH (2 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(1-oxo- 1,2-dihydroisoquinoline-7-carboxamido)propanoic acid (8.0 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 11.30 (br s, 1H), 8.92-8.85 (m, 1H), 8.71 (d, J = 1.2 Hz, 1H), 8.38 (br s, 2H), 8.13 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.27-7.21 (m, 1H), 6.58 (d, J = 7.2 Hz, 1H), 4.13-4.06 (m, 1H), 3.87-3.68 (m, 2H). LC-MS (MH+): m/z = 276.0 tR (min, WXL-Method F) = 2.826 [α]20,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 4l (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-carboxamido)propanoic acid
Figure imgf000366_0001
Step 1: methyl 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate
Figure imgf000366_0002
To a solution of methyl 3-bromo-5-fluoro-benzoate (1 g, 4.29 mmol) and (2- ethylphenyl)boronic acid (644 mg, 4.29 mmol) in a mixture of dioxane (15 mL) and H2O (1.5 mL) was added Pd(dppf)Cl2 (314 mg, 0.43 mmol) and K2CO3 (1.78 g, 12.87 mmol). The mixture was heated to 90°C for 16 hours. The reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic layer was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (eluent : petroleum) to give methyl 2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-carboxylate (915 mg). 1H NMR (400MHz, CDCl3) δ 7.82 (m, 1H), 7.75-7.70 (m, 1H), 7.38-7.34 (m, 2H), 7.27-7.15 (m, 3H), 3.95 (s, 3H), 2.60 (q, J=7.6 Hz, 2H), 1.12 (t, J=7.6 Hz, 3H). Step 2: 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid
Figure imgf000367_0001
To a solution of methyl 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate (915 mg, 3.54 mmol) in a mixture of MeOH (10 mL), THF (5 mL) and H2O (10 mL) was added LiOH.H2O (446 mg, 10.63 mmol). The mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated, diluted with water (10 mL), adjusted pH = 4~5 with aqueous HCl (2M). The mixture was extracted with ethyl acetate (20 mL × 3), the organic layer was dried over Na2SO4 and concentrated to give 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- carboxylic acid (800 mg). 1H NMR (400MHz, DMSO-d6) δ 7.69-7.65 (m, 2H), 7.50-7.45 (m, 1H), 7.38-7.36 (m, 2H), 7.29-7.25 (m, 1H), 7.20 (d, J=7.6 Hz, 1H), 2.53 (q, J=7.6Hz, 2H), 1.03 (t, J=7.6Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate
Figure imgf000368_0001
To a solution of 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (400 mg, 1.64 mmol) in DMF (10 mL) was added TBTU (789 mg, 2.46 mmol), DIPEA (635 mg, 4.91 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (657 mg, 1.80 mmol, HCl salt). The mixture was stirred at 10 °C for 1 hour. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'- ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (620 mg). 1H NMR (400MHz, CDCl3) δ 7.43-7.26 (m, 15H), 7.20-7.14 (m, 2H), 6.71 (br s, 1H), 5.94- 5.91 (m, 1H), 5.26-5.16 (m, 2H), 5.09 (s, 2H), 4.63-4.54 (m, 1H), 3.99- 3.76 (m, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid
Figure imgf000368_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate (620 mg, 1.12 mmol) in the solution of 30% HBr in AcOH (10 mL) was heated at 50°C for 1 hour. The reaction mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(2'- ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid (310 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.94 (br s, 1H), 7.72-7.64 (m, 2H), 7.41-7.34 (m, 3H), 7.31- 7.25 (m, 1H), 7.19 (d, J = 7.6 Hz, 1H), 3.80-3.75 (m, 1H), 3.65-3.55 (m, 2H), 2.55 (q, J = 7.6 Hz, 2H), 1.03 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 331.1 tR (min, Method D) = 1.832 [α]20D = +10.0 (c = 1.0 mg/mL, CH3OH). Compound 4m (R)-2-amino-3-(3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-fluoro-5-(1- isopropyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000369_0001
Step 1: 5-iodo-1-isopropyl-1H-1,2,3-triazole
Figure imgf000369_0002
To a stirred solution of 1-isopropyl-1H-1,2,3-triazole (300 mg, 2.70 mmol) in THF (10 mL) was added n-BuLi (2.5 M in hexane, 1.30 mL) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (822 mg, 3.24 mmol) (dissolving in 5 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 20°C for 2 hours. The reaction mixture was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give 5-iodo-1-isopropyl- 1H-1,2,3-triazole (480 mg). 1H NMR (400MHz, CDCl3) δ 7.73 (s, 1H), 4.82-4.71 (m, 1H), 1.62 (d, J = 6.8 Hz, 6H). Step 2: methyl 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzoate
Figure imgf000370_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (350 mg, 1.77 mmol), 5-iodo-1- isopropyl-1H-1,2,3-triazole (419 mg, 1.77 mmol), K2CO3 (733 mg, 5.30 mmol) and Pd(dppf)Cl2.CH2Cl2 (144 mg, 0.18 mmol) were taken up into a microwave tube in a mixture of dioxane (8 mL) and H2O (0.8 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give methyl 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5- yl)benzoate (310 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.83 (m, 2H), 7.73 (s, 1H), 7.30-7.27 (m, 1H), 4.69-4.58 (m, 1H), 3.98 (s, 3H), 1.63 (d, J = 6.4 Hz, 6H). Step 3: 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzoic acid
Figure imgf000371_0001
To a solution of methyl 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzoate (310 mg, 1.18 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (124 mg, 2.94 mmol) and the mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzoic acid (290 mg). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-isopropyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate
Figure imgf000371_0002
To a solution of a mixture of 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzoic acid (140 mg, 0.56 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (225 mg, 0.62 mmol, HCl salt) in DMF (8 mL) was added TBTU (271 mg, 0.84 mmol) and DIPEA (218 mg, 1.69 mmol). The reaction mixture was stirred at 25°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-isopropyl-1H- 1,2,3-triazol-5-yl)benzamido)propanoate (300 mg). 1H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.55-7.48 (m, 2H), 7.38-7.29 (m, 10 H), 7.22- 7.14 (m, 2H), 5.94 (br d, J = 6.8 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.65-4.57 (m, 2H), 4.02- 3.93 (m, 1H), 3.87-3.77 (m, 1H), 1.59 (d, J = 6.0 Hz, 6H). Step 5: (R)-2-amino-3-(3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5- yl)benzamido)propanoic acid
Figure imgf000372_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-isopropyl-1H- 1,2,3-triazol-5-yl)benzamido)propanoate (300 mg, 0.54 mmol) in the solution of 30% HBr in AcOH (15 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with MTBE (5 mL × 2). The aqueous layer was lyophilized to give (R)-2-amino-3-(3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5- yl)benzamido)propanoic acid (200 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.01-8.97 (m, 1H), 8.35 (br s, 3H), 7.93 (s, 1H), 7.84-7.79 (m, 2H), 7.68-7.64 (m, 1H), 4.76-4.67 (m, 1H), 4.17-4.09 (m, 1H), 3.85-3.66 (m, 2H), 1.52- 1.46 (m, 6H). LC-MS (MH+): m/z = 336.1 tR (min, Method D) = 1.579 [α]20,D = +1.3 (c = 3 mg/mL,CH3OH). Compound 4n (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-fluoro-5-(1-propyl- 1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000373_0001
To a solution of 2-(trimethylsilyl)-2H-1,2,3-triazole (2 g, 14.16 mmol) and 1-iodopropane (2.65 g, 15.58 mmol) in dioxane (20 mL) was added KOH (794 mg, 14.16 mmol). The resulting solution was stirred at 100°C for 16 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) to give 1-propyl-1H-1,2,3-triazole (1.1 g). 1H NMR (400MHz, DMSO-d6) δ 8.12 (s, 1H), 7.71 (s, 1H), 4.31 (t, J = 7.2 Hz, 2H), 1.87-1.77 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H).
Figure imgf000373_0002
To a stirred solution of 1-propyl-1H-1,2,3-triazole (400 mg, 3.60 mmol) in THF (10 mL) was added n-BuLi (2.5 M in hexane, 1.73 mL) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (1.10 g, 4.32 mmol) (dissolving in 5 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 20°C for 2 hours. The reaction mixture was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give 5-iodo-1-propyl- 1H-1,2,3-triazole (740 mg). 1H NMR (400MHz, CDCl3) δ 7.72 (s, 1H), 4.37 (t, J = 7.6 Hz, 2H), 2.01-1.91 (m, 2H), 0.98 (t, J = 7.6 Hz, 3H). Step 3: methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate
Figure imgf000374_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (400 mg, 2.02 mmol), 5-iodo-1- propyl-1H-1,2,3-triazole (479 mg, 2.02 mmol), K2CO3 (838 mg, 6.06 mmol) and Pd(dppf)Cl2.CH2Cl2 (165 mg, 0.20 mmol) were taken up into a microwave tube in a mixture of dioxane (8 mL) and H2O (0.8 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzoate (470 mg). 1H NMR (400MHz, CDCl3) δ 7.88 (t, J = 1.2 Hz, 1H), 7.86-7.82 (m, 1H), 7.76 (s, 1H), 7.33- 7.29 (m, 1H), 4.35 (t, J = 7.2 Hz, 2H), 3.98 (s, 3H), 1.94 - 1.84 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). Step 4: 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid
Figure imgf000375_0001
To a solution of methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate (470 mg, 1.79 mmol) in a mixture of THF (8 mL) and H2O (4 mL) was added LiOH.H2O (187 mg, 4.46 mmol) and the mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid (420 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate
Figure imgf000375_0002
To a solution of a mixture of 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid (210 mg, 0.84 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (338 mg, 0.93 mmol, HCl salt) in DMF (8 mL) was added TBTU (406 mg, 1.25 mmol) and DIPEA (327 mg, 2.53 mmol). The reaction mixture was stirred at 30°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoate (400 mg). 1H NMR (400 MHz, CDCl3) δ 7.70 (s, 1H), 7.58 (s, 1H), 7.54-7.49 (m, 1H), 7.40-7.28 (m, 11H), 7.23-7.19 (m, 1H), 6.02 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.10 (s, 2H), 4.65-4.58 (m, 1H), 4.31 (t, J = 7.2 Hz, 2H), 4.00-3.80 (m, 2H), 1.89-1.79 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). Step 6: (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000376_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H- 1,2,3-triazol-5-yl)benzamido)propanoate (400 mg, 0.71 mmol) in the solution of 30% HBr in AcOH (15 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with MTBE (5 mL × 2). The aqueous layer was lyophilized to give (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoic acid (250 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.05-8.98 (m, 1H), 8.37 (br s, 3H), 8.00 (s, 1H), 7.88 (s, 1H), 7.84-7.79 (m, 1H), 7.75-7.71 (m, 1H), 4.46-4.39 (m, 2H), 4.18-4.10 (m, 1H), 3.84-3.67 (m, 2H), 1.75-1.64 (m, 2H), 0.77-0.71 (m, 3H). LC-MS (MH+): m/z = 336.0 tR (min, Method D) = 1.249 [α]20,D = +0.6 (c = 7 mg/mL,CH3OH). Compound 4o (R)-2-amino-3-(3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(1-(tert-butyl)-1H- 1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000377_0001
To a solution of 1H-triazole (5.53 g, 80 mmol) and TEA (8.80 g, 87 mmol) in THF (80 mL) was addded TMSCl (9.13 g, 84 mmol) at 0~ 5°C. The mixture was stirred at 0~5°C for 30 minutes. The resulting mixture was heated to 80°C and stirred for 2 hours. The mixture was filtered, the filtration was concentrated. The residue was distilled under reduced pressre to give 2-(trimethylsilyl)-2H-1,2,3-triazole (5.4 g). 1H NMR (400MHz, CDCl3) δ 7.74 (s, 2 H), 0.52 (s, 9H).
Figure imgf000378_0001
To a solution of 2-(trimethylsilyl)-2H-1,2,3-triazole (2.20 g, 15.58 mmol) and 2-bromo-2- methylpropane (2.13 g, 15.58 mmol) in dioxane (20 mL) was added KOH (874 mg, 15.58 mmol). The resulting solution was stirred at 100°C for 16 hours. The reaction mixture was quenched with water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) to give 1-(tert-butyl)-1H- 1,2,3-triazole (400 mg). 1H NMR (400MHz, CDCl3) δ 7.70 (s, 1H), 7.62 (s, 1H), 1.70 (s, 9H).
Figure imgf000378_0002
To a stirred solution of 1-(tert-butyl)-1H-1,2,3-triazole (200 mg, 1.60 mmol) in THF (10 mL) was added n-BuLi (2.5 M in hexane, 0.77 mL) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (487 mg, 1.92 mmol) (dissolving in 5 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 25°C for 2 hours. The reaction mixture was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give 1-(tert-butyl)-5- iodo-1H-1,2,3-triazole (241 mg). 1H NMR (400MHz, CDCl3) δ 7.74 (s, 1H), 1.86 (s, 9H). Step 4: methyl 3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate
Figure imgf000379_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (190 mg, 0.96 mmol), 1-(tert-butyl)- 5-iodo-1H-1,2,3-triazole (241 mg, 0.96 mmol), K2CO3 (398 mg, 2.88 mmol) and Pd(dppf)Cl2.CH2Cl2 (78 mg, 0.10 mmol) were taken up into a microwave tube in a mixture of dioxane (8 mL) and H2O (0.8 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give methyl 3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5- fluorobenzoate (100 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.82 (m, 2H), 7.59 (s, 1H), 7.27-7.24 (m, 1H), 3.96 (s, 3H), 1.59 (s, 9H). Step 5: 3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid
Figure imgf000379_0002
To a solution of methyl 3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate (100 mg, 0.36 mmol) in a mixture of THF (4 mL) and H2O (1 mL) was added LiOH.H2O (38 mg, 0.90 mmol) and the mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid (90 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-(tert-butyl)-1H-1,2,3-triazol- 5-yl)-5-fluorobenzamido)propanoate
Figure imgf000380_0001
To a solution of a mixture of 3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid (110 mg, 0.42 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (168 mg, 0.46 mmol, HCl salt) in DMF (5 mL) was added TBTU (201 mg, 0.63 mmol) and DIPEA (162 mg, 1.25 mmol). The reaction mixture was stirred at 30°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-(tert-butyl)-1H-1,2,3- triazol-5-yl)-5-fluorobenzamido)propanoate (180 mg). 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.51-7.48 (m, 1H), 7.37-7.30 (m, 10H), 7.20-7.16 (m, 1H), 7.12-7.07 (m, 1H), 5.91 (br s, 1H), 5.21 (s, 2H), 5.09 (s, 2H), 4.62-4.55 (m, 1H), 4.00-3.75 (m, 2H), 1.57 (s, 9H). Step 7: (R)-2-amino-3-(3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000381_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-(tert-butyl)-1H-1,2,3- triazol-5-yl)-5-fluorobenzamido)propanoate (85 mg, 0.15 mmol) and Pd/C (40 mg, 10% Pd, 50% water) in AcOH (2 mL) was degassed and purged with H2 for 3 times, and then the mixture was stirred at 30°C for 1 hour under H2 atmosphere (15psi). The reaction mixture was filtered and concentrated. The residue was purified by preparative HPLC (Method DD) to give (R)-2-amino-3-(3-(1-(tert-butyl)-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoic acid (20 mg). 1H NMR (400MHz, DMSO-d6) δ 8.92 (br s, 1H), 7.85-7.80 (m, 1H), 7.74 (s, 1H), 7.71 (s, 1H), 7.64-7.59 (m, 1H), 3.76-3.67 (m, 1H), 3.57-3.55 (m, 2H), 1.50 (s, 9H). LC-MS (MH+): m/z = 294.0 tR (min, Method D) = 1.403 [α]20,D = +18.0 (c = 1 mg/mL,CH3OH). Compound 4p (R)-2-amino-3-(3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1- methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000382_0001
A mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (0.5 g, 2.53 mmol), 5- iodo-1-methyl-1H-pyrazole (525 mg, 2.53 mmol), Pd(dppf)Cl2 (165 mg, 0.25 mmol) and K3PO4 (1.61 g, 7.58 mmol) in the mixture of dioxane (30 mL) and H2O (3 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100°C for 3 hours under N2 atmosphere. The mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL × 2). Dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate: from 0 to 30%) to give methyl 3-fluoro-5-(1-methyl-1H-pyrazol-5-yl)benzoate (550 mg). 1H NMR (400MHz, CDCl3) δ 7.91 (dd, J = 1.6 Hz, 1.2 Hz, 1H), 7.81-7.71 (m, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.37-7.30 (m, 1H), 6.38 (d, J = 2.0 Hz, 1H), 3.96 (s, 3H), 3.93 (s, 3H). Step 2: methyl 3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000383_0001
To a mixture of methyl 3-fluoro-5-(1-methyl-1H-pyrazol-5-yl)benzoate (550 mg, 2.35 mmol) in THF (20 mL) was added NCS (314 mg, 2.35 mmol). The mixture was stirred at 70°C for 16 hours. The mixture was concentrated. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate: from 0 to 30%) to give methyl 3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (540 mg). 1H NMR (400MHz, CDCl3) δ 7.90 (t, J = 1.6 Hz, 1H), 7.84-7.81 (m, 1H), 7.53 (s, 1H), 7.38- 7.35 (m, 1H), 3.96 (s, 3H), 3.84 (s, 3H). Step 3: 3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000383_0002
To a solution of methyl 3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (540 mg, 2.01 mmol) in H2O (20 mL) and MeOH (20 mL) was added LiOH.H2O (169 mg, 4.02 mmol). The mixture was stirred at 25°C for 16 hours. The mixture was adjusted pH=3~4 with KHSO4 (aq.20 mL) and extracted with ethyl acetate (40 mL × 3), dried over Na2SO4 and concentrated to give 3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (445 mg), which was used for the next step without purification. LC-MS (MH+): m/z = 255.1 tR (min, Method c) = 1.472 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-methyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate
Figure imgf000384_0001
To a mixture of 3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.59 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (193 mg, 0.53 mmol, HCl salt) in DMF (5 mL) was added TBTU (284 mg, 0.88 mmol) followed by DIPEA (381 mg, 2.95 mmol) at 25°C. The mixture was stirred at 25°C for 2 hours. The mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) twice to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate(130 mg). 1H NMR (400MHz, CDCl3) δ 7.53 (s, 1H), 7.51 (s, 1H), 7.47 (br, J = 8.0 Hz, 1H), 7.34 - 7.31 (m, 11H), 7.00-6.92 (m, 1H), 5.92 (br d, J = 6.0 Hz, 1H), 5.25-5.20 (m, 2H), 5.11 (s, 2H), 4.62-4.58 (m, 1H), 4.01-3.95 (m, 1H), 3.88-3.80 (m, 4H). Step 5: (R)-2-amino-3-(3-(4-chloro-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000384_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (130 mg, 0.23 mmol) in HBr/AcOH (5 mL, 30% in AcOH) was stirred at 25°C for 16 hours. The mixture was concentrated. The residue was purified by Preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(4-chloro-1- methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid(50 mg) as a HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.21 (br s, 1H), 8.61 (br s, 3H), 7.93-7.90 (m, 2H), 7.70 (s, 1H), 7.66-7.60 (m, 1H), 4.13-4.12 (m, 1H), 3.82-3.79 (m, 5H). LC-MS (MH+): m/z = 341.0 tR (min, Method D) = 1.491 [α]20,D = 4.0 (c = 2.5mg/mL, CH3OH). Compound 4q (R)-2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000385_0001
Step 1: 1-ethyl-5-iodo-1H-pyrazole
Figure imgf000385_0002
To a stirred solution of 1-ethyl-1H-pyrazole (5.0 g, 52.01 mmol) in THF (60 mL) was added n-BuLi (25.0 mL, 2.5M in hexane) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (15.8 g, 62.4 mmol, dissolving in 40 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 30°C for 2 hours. The reaction was quenched with ice water (20 mL) and extracted with ethyl acetate (30 mL × 3). The organic layer was washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give 1-ethyl-5-iodo-1H-pyrazole (11 g). 1H NMR (400MHz, CDCl3) δ 7.51 (d, J = 2.0 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000386_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 1-ethyl-5-iodo- 1H-pyrazole (1.12 g, 5.05 mmol), K2CO3 (2.09 g, 15.15 mmol) and Pd(dppf)Cl2.CH2Cl2 (413 mg, 0.51 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 minutes under microwave. The reaction was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The organic layer was washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 20%) to give methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (1.0 g). 1H NMR (400MHz, CDCl3) δ 7.90 (s, 1H), 7.80-7.76 (m, 1H), 7.58 (s, 1H), 7.34-7.29 (m, 1H), 6.35 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). Step 3: methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000387_0001
To a solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (350 mg, 1.41 mmol) in THF (8 mL) was sadded NIS (1049 mg, 4.65 mmol) and the reaction mixture was stirred at 70°C for 4 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 10%) to give methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg). 1H NMR (400MHz, CDCl3) δ 7.88-7.83 (m, 2H), 7.62 (s, 1H), 7.32-7.29 (m, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H). Step 4: methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000387_0002
A mixture of methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg, 1.34 mmol), methyl 2,2-difluoro-2-fluorosulfonyl-acetate (385 mg, 2.00 mmol), CuI (51 mg,0.27 mmol) in DMF (8 mL) was heated at 120°C for 4 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 15%) to give methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate (240 mg). 1H NMR (400MHz, CDCl3) δ 7.91-7.87 (m, 1H), 7.87-7.85 (m, 1H), 7.80 (s, 1H), 7.32-7.28 (m, 1H), 4.04 (q, J = 6.8 Hz, 2H), 3.97 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). Step 5: 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000388_0001
To a solution of methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate (240 mg, 0.76 mmol) in the mixture of THF (4 mL) and H2O (1 mL) was added LiOH.H2O (48 mg, 1.14 mmol), the mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid (226 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000388_0002
To a mixture of 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid (113 mg, 0.37 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (150 mg, 0.41 mmol, HCl salt) in DMF (6 mL) was added TBTU (180 mg, 0.56 mmol) and DIPEA (145 mg, 1.12 mmol), the mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: ethyl acetate, ethyl acetate from 0% to 30%). The obtained material was further purified by Chiral SFC (Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALCEL OD-H(250mm*30mm,5μm); Mobile phase: supercritical CO2/IPA (0.1% NH3`H2O, v%) = 65/35; low Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (160 mg). 1H NMR (400MHz, CDCl3) δ 7.81 (s, 1H), 7.54-7.48 (m, 2H), 7.38-7.30 (m, 10H), 7.25-7.22 (m, 1H), 6.95 (br s, 1H), 5.89 (br d, J = 6.0 Hz, 1H), 5.26-5.19 (m, 2H), 5.11 (s, 2H), 4.64- 4.57 (m, 1H), 4.09-3.93 (m, 3H), 3.83-3.74 (m, 1H), 1.38 (t, J = 7.6 Hz, 3H). Step 7: (R)-2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000389_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (160 mg, 0.26 mmol) in the solution of 30% HBr in AcOH (5 mL) was stirred at 50°C for 2 hours. The mixture was concentrated. The residue was added HOAc (3 mL), then added MTBE (5 mL). The formed solid was collected by filtration and added water (10 mL) for freeze-drying. The obtained solid was further purified by preparative-HPLC (Method AA) to give (R)-2- amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (28 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.97-8.92 (m, 1H), 8.34-8.30 (m, 3H), 8.04 (s, 1H), 7.90 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.67 (d, J = 9.2 Hz, 1H), 4.16-4.10 (m, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.84-3.65 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 389.0 tR (min, Method D) = 1.756 [α]20,D = -2.7 (c = 4.5 mg/mL, CH3OH). Compound 4r (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(1-propyl- 4-(trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoic acid
Figure imgf000390_0001
A mixture of 5-bromo-1-propyl-pyrazole (0.885 g, 4.68 mmol), (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (772 mg, 3.90 mmol), Pd(dtbpf)Cl2 (254 mg, 0.39 mmol), K3PO4 (2.48 g, 11.70 mmol) in the mixture of dioxane (35 mL) and H2O (3.5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 3 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.90 g). 1H NMR (400MHz, CDCl3) δ 7.88 (t, J=1.2 Hz, 1H), 7.79-7.74 (m, 1H), 7.55 (d, J=2.0 Hz, 1H), 7.33-7.27 (m, 1H), 6.33 (d, J=1.8 Hz, 1H), 4.12 - 4.08 (m, 2H), 3.95 (s, 3H), 1.88-1.79 (m, 2H), 0.82 (t, J=7.6 Hz, 3H). Step 2: methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000391_0001
To a solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (600 mg, 2.29 mmol) in THF (20 mL) was added NIS (1.03 g, 4.58 mmol). The mixture was stirred at 70°C for 2 hours. The reaction mixture was concentrated. The residue was added water (15 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (10 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20 %) to give methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5- yl)benzoate (660 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.81 (m, 2H), 7.61 (s, 1H), 7.32-7.27 (m, 1H), 4.04 (t, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.81-1.71 (m, 2H), 0.80 (t, J = 7.6 Hz, 3H). Step 3: methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoate
Figure imgf000392_0001
To a mixture of methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate (590 mg, 1.52 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (1.46 g, 7.60 mmol) in DMF (15 mL) was added CuI (29 mg, 0.15 mmol). The mixture was stirred at 120°C for 15 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers were washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20%) twice to give methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzoate (280 mg). 1H NMR (400MHz,CDCl3) δ 7.88-7.79 (m, 3H), 7.29-7.26 (m, 1H), 3.96 (s, 3H), 3.95-3.90 (m, 2H), 1.84-1.75 (m, 2H), 0.82 (t, J = 7.6 Hz, 3H). Step 4: 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid
Figure imgf000392_0002
To a solution of methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzoate (280 mg, 0.85 mmol) in a mixture of MeOH (10 mL) and H2O (2 mL) was added LiOH.H2O (90 mg, 2.14 mmol). The mixture was stirred at 30°C for 1 hour. The reaction mixture was concentrated. The residue was added water (10 mL), adjusted pH to 5-6 with 1N HCl and extracted with ethyl acetate (15 mL × 3). The organic layers was dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid (255 mg). 1H NMR (400MHz, DMSO-d6) δ 7.99 (s, 1H), 7.80-7.71 (m, 2H), 7.38 (d, J = 8.8 Hz, 1H), 3.93 (t, J = 7.2 Hz, 2H), 1.72-1.63 (m, 2H), 0.70 (t, J = 7.2 Hz, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate
Figure imgf000393_0001
To a mixture of 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid (100 mg, 0.32 mmol), TBTU (152 mg, 0.47 mmol) and DIPEA (123 mg, 0.95 mmol) in DMF (15 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (138 mg, 0.38 mmol, HCl salt). The mixture was stirred at 35°C for 16 hours. The reaction mixture was added water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoate (110 mg). 1H NMR (400MHz, CDCl3) δ 7.81 (s, 1H), 7.52-7.47 (m, 2H), 7.37-7.31 (m, 10H), 7.25-7.20 (m, 1H), 6.99-6.69 (m, 1H), 5.90 (br d, J = 6.0 Hz, 1H), 5.28-5.17 (m, 2H), 5.11 (s, 2H), 4.60-4.55 (m, 1H), 4.01-3.91 (m, 3H), 3.85-3.74 (m, 1H), 1.85-1.75 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). Step 6: (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid
Figure imgf000394_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate (110 mg, 0.18 mmol) in 30% HBr in AcOH (6 mL) was stirred at 20°C for 16 hours. The reaction mixture was concentrated. The residue was added water and extracted with MTBE (10 ml × 3). The aqueous layer was lyophilized. The product was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid (14 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.09-9.04 (m, 1H), 8.44 (br s, 3H), 8.04 (s, 1H), 7.95-7.92 (m, 1H), 7.79 (s, 1H), 7.66-7.61 (m, 1H), 4.17-4.08 (m, 1H), 3.96-3.92 (m, 2H), 3.85-3.78 (m, 1H), 3.76-3.70 (m, 1H), 1.72-1.63 (m, 2H), 0.71 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 403.2 tR (min, Method D) = 1.891 [α]20,D = 11.3 (c = 1.0 mg/mL, CH3OH). Compound 4s (R)-2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(4-(difluoromethyl)- 1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000395_0001
To a stirred solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg, 2.01 mmol) in POCl3 (7.43 g, 48.42 mmol) was added DMF (1.43 g, 19.50 mmol) and the mixture was heated to 100°C for 16 hours. The mixture was poured into H2O (10 mL) at 0°C, neutralize with sat. aqueous Na2CO3 to pH 7~8 and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, ethyl acetate in petroleum ether) to give methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5- fluorobenzoate (480 mg). 1H NMR (400MHz, CDCl3) δ 9.65 (s, 1H), 8.09 (s, 1H), 7.94-7.88 (m, 2H), 7.38-7.32 (m, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.98 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000396_0001
To a solution of methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5-fluorobenzoate (480 mg, 1.74 mmol) in DCM (10 mL) was added DAST (8.40 g, 52.12 mmol) at 0°C and the resulting mixture was stirred at 25°C for 2 hours. The mixture was poured into sat. aqueous Na2CO3 to pH 7~8 at 0°C and extracted with DCM (30 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 20%, ethyl acetate in petroleum ether) to give methyl 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (410 mg).1H NMR (400MHz, CDCl3) δ 7.90-7.83 (m, 2H), 7.78 (s, 1H), 7.33-7.27 (m, 1H), 6.65 (t, J = 55.6 Hz, 1H), 4.07 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). Step 3: 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000396_0002
To a solution of methyl 3-[4-(difluoromethyl)-2-ethyl-pyrazol-3-yl]-5-fluoro-benzoate (410 mg, 1.37 mmol) in MeOH (8 mL) was added a solution of LiOH.H2O (173 mg, 4.12 mmol) in H2O (4 mL) and the resulting mixture was stirred at 25°C for 2 hours. The mixture was concentrated to move MeOH, then added sat. aqueous KHSO4 to adjust pH = 4~5 and extracted with ethyl acetate (10 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated to give 3-(4-(difluoromethyl)-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (365 mg). 1H NMR (400MHz, DMSO-d6) δ 7.87-7.79 (m, 2H), 7.77 (t, J = 1.6 Hz, 1H), 7.70-7.63 (m, 1H), 7.85 (t, J = 55.2 Hz, 1H), 4.03 (t, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000397_0001
A mixture of 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (180 mg, 0.63 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (277 mg, 0.76 mmol, HCl salt), TBTU (305 mg, 0.95 mmol) and DIPEA (164 mg, 1.27 mmol) in DMF (5 mL) was stirred at 25°C for 16 hours. The mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL × 2). The organic phase was washed with brine (5 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 40%, ethyl acetate in petroleum ether) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (240 mg). 1H NMR (400MHz, CDCl3) δ 7.76 (s, 1H), 7.56-7.48 (m, 2H), 7.39-7.16 (m, 12H), 6.43 (t, J = 55.6 Hz, 1H), 6.15-6.01 (m, 1H), 5.19 (s, 2H), 5.07 (s, 2H), 4.62-4.52 (m, 1H), 4.02 (q, J = 7.2 Hz, 2H), 3.96-3.74 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000398_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (240 mg, 0.40 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 5 hours. The reaction was concentrated. The residue was washed with TBME (2 mL × 3) and the organic layer was decanted, then the residue was submitted to lyophilize to give (R)-2-amino-3-(3-(4-(difluoromethyl)-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (150 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99-8.93 (m, 1H), 8.45-8.25 (m, 3H), 7.89-7.81 (m, 2H), 7.76 (s, 1H), 7.63-7.56 (m, 1H), 6.87 (d, J = 55.2 Hz, 1H), 4.13-4.17 (m, 1H), 4.05-3.99 (m, 2H), 3.85-3.65 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 471.1 tR (min, Method E) = 1.316 [α]20,D = 2.71 (c = 14 mg/mL, CH3OH). Compound 4t (R)-2-amino-3-(3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-fluoro-5-(1-methyl- 1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000399_0001
To a stirred solution of 1-methyl-1H-1,2,3-triazole (300 mg, 3.61 mmol) in THF (10 mL) was added n-BuLi (2.5 M in hexane, 1.73 mL) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (1.10 g, 4.33 mmol) (dissolving in 5 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 20°C for 2 hours. The reaction mixture was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give 5-iodo-1-methyl- 1H-1,2,3-triazole (480 mg). 1H NMR (400MHz, CDCl3) δ 7.74 (s, 1H), 4.11 (s, 3H). Step 2: methyl 3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzoate
Figure imgf000400_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (400 mg, 2.02 mmol), 5-iodo-1- methyl-1H-1,2,3-triazole (422 mg, 2.02 mmol), K2CO3 (838 mg, 6.06 mmol) and Pd(dppf)Cl2.CH2Cl2 (165 mg, 0.20 mmol) were taken up into a microwave tube in a mixture of dioxane (8 mL) and H2O (0.8 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give methyl 3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5- yl)benzoate (400 mg). 1H NMR (400MHz, CDCl3) δ 7.92 (s, 1H), 7.86-7.82 (m, 1H), 7.80 (s, 1H), 7.37-7.33 (m, 1H), 4.13 (s, 3H), 3.98 (s, 3H).
Figure imgf000400_0002
To a solution of methyl 3-fluoro-5-(1-isopropyl-1H-1,2,3-triazol-5-yl)benzoate (400 mg, 1.70 mmol) in a mixture of THF (8 mL) and H2O (4 mL) was added LiOH.H2O (179 mg, 4.25 mmol) and the mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzoic acid (330 mg). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-methyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate
Figure imgf000401_0001
To a solution of a mixture of 3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzoic acid (165 mg, 0.75 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (299 mg, 0.82 mmol, HCl salt) in DMF (8 mL) was added TBTU (359 mg, 1.12 mmol) and DIPEA (289 mg, 2.24 mmol). The reaction mixture was stirred at 20°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) three times to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5- yl)benzamido)propanoate (340 mg). 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.60-7.55 (m, 1H), 7.52-7.48 (m, 1H), 7.38-7.29 (m, 10 H), 7.26-7.24 (m, 1H), 7.18 (br, 1H), 5.96 (br, 1H), 5.23 (s, 2H), 5.11 (s, 2H), 4.65- 4.58 (m, 1H), 4.09 (s, 3H), 4.02-3.95 (m, 1H), 3.85-3.75 (m, 1H). Step 5: (R)-2-amino-3-(3-fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000402_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-methyl-1H- 1,2,3-triazol-5-yl)benzamido)propanoate (340 mg, 0.64 mmol) in the solution of 30% HBr in AcOH (15 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3- fluoro-5-(1-methyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid (135 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.31-9.24 (m, 1H), 8.62 (br s, 3H), 8.08 (s, 1H), 8.05 (s, 1H), 7.86-7.82 (m, 1H), 7.79-7.74 (m, 1H), 4.18-4.11 (m, 4H), 3.84-3.79 (m, 2H). LC-MS (MH+): m/z = 308.1 tR (min, Method F) = 2.792 [α]20,D = +1.7 (c = 7 mg/mL,CH3OH). Compound 4u (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoic acid
Figure imgf000402_0002
Step 1: methyl 3-bromo-5-fluoro-4-hydroxybenzoate
Figure imgf000403_0001
To a solution of methyl 3-fluoro-4-hydroxybenzoate (500 mg, 2.94 mmol) in DMF (5 mL) was added NBS (628 mg, 3.53 mmol) and the reaction mixture was stirred at 30°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give methyl 3-bromo-5-fluoro-4-hydroxybenzoate (700 mg). 1H NMR (400 MHz, CD3Cl) δ 8.02 (dd, J = 2.0 Hz, 1.6 Hz, 1H), 7.74 (dd, J = 10.4 Hz, 1.6 Hz, 1H), 6.17 (s, 1H), 3.91 (s, 3H). Step 2: methyl 3-bromo-5-fluoro-4-methoxybenzoate
Figure imgf000403_0002
To a solution of methyl 3-bromo-5-fluoro-4-hydroxybenzoate (700 mg, 2.81 mmol) in DMF (10 mL) was added K2CO3 (971 mg, 7.03 mmol) and iodomethane (1.20 g, 8.43 mmol), then the mixture was stirred at 60°C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 2%) to give methyl 3-bromo-5-fluoro- 4-methoxybenzoate (700 mg). 1H NMR (400 MHz, CD3Cl) δ 8.02 (dd, J = 2.0 Hz, 2.0 Hz, 1H), 7.74 (dd, J = 11.6 Hz, 2.0 Hz, 1H), 4.05 (d, J = 2.8 Hz, 3H), 3.91 (s, 3H). Step 3: methyl 3-ethyl-5-fluoro-4-methoxybenzoate
Figure imgf000404_0001
A mixture of methyl 3-bromo-5-fluoro-4-methoxybenzoate (700 mg, 2.66 mmol), triethylborane (1 M in THF, 8 mL), Pd(dppf)Cl2 (195 mg, 0.27 mmol) and CsOAc (1.53 g, 7.98 mmol) in THF (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-ethyl-5- fluoro-4-methoxybenzoate (360 mg). 1H NMR (400MHz, CDCl3) δ 7.66 (s, 1H), 7.59 (dd, J = 12.4 Hz, 2.0 Hz, 1H), 4.00 (d, J = 2.8 Hz, 3H), 3.89 (s, 3H), 2.67 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). Step 4: 3-ethyl-5-fluoro-4-methoxybenzoic acid
Figure imgf000404_0002
To a solution of methyl 3-ethyl-5-fluoro-4-methoxybenzoate (360 mg, 1.70 mmol) in a mixture of THF (6 mL) and H2O (3 mL) was added LiOH.H2O (214 mg, 5.09 mmol) and the mixture was stirred at 10°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-ethyl-5-fluoro-4-methoxybenzoic acid (330 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate
Figure imgf000405_0001
To a solution of a mixture of 3-ethyl-5-fluoro-4-methoxybenzoic acid (100 mg, 0.50 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (202 mg, 0.56 mmol, HCl salt) in DMF (5 mL) was added TBTU (243 mg, 0.76 mmol) and DIPEA (196 mg, 1.51 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoate (250 mg). 1H NMR (400MHz, CDCl3) δ 7.35-7.29 (m, 11H), 7.27-7.21 (m, 1H), 6.61 (br s, 1H), 5.98 (br d, J = 6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.62-4.54 (m, 1H), 3.98 (d, J = 2.8 Hz, 3H), 3.94-3.76 (m, 2H), 2.65 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). Step 6: (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid
Figure imgf000405_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate (100 mg, 0.20 mmol) and Pd/C (50 mg, 10% Pd, 50% water) in AcOH (5 mL) was degassed and purged with H2 for 3 times, and then the mixture was stirred at 10°C for 1 hour under H2 atmosphere (15 psi). The reaction mixture was filtered and concentrated. The sample was submitted to lyophilization to give (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid (8 mg). 1H NMR (400MHz, DMSO-d6) δ 8.80 (br s, 1H), 7.60-7.52 (m, 2H), 3.88 (s, 3H), 3.69-3.59 (m, 1H), 3.50-3.40 (m, 2H), 2.62 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 285.0 tR (min, Method E) = 1.443 [α]20,D = +17.0 (c = 2 mg/mL,CH3OH). Compound 4v (R)-2-amino-3-(3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- isopropyl-3-methylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000406_0001
Step 1: 3-isopropyl-5-methylisoxazole and 5-isopropyl-3-methylisoxazole
Figure imgf000407_0001
5-methylhexane-2,4-dione (2.0 g, 15.6 mmol), NH2OH.HCl (1.29 g, 18.61 mmol) were taken up into a microwave tube in EtOH (12 mL). The sealed tube was heated at 130 °C for 5 min under microwave. The reaction was concentrated below 35 °C. The residue was added water (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give a mixture of 3- isopropyl-5-methylisoxazole and 5-isopropyl-3-methylisoxazole (1.9 g, crude, ratio was 5:1). Step 2: 4-bromo-3-isopropyl-5-methylisoxazole and 4-bromo-5-isopropyl-3- methylisoxazole
Figure imgf000407_0002
A mixture of 3-isopropyl-5-methylisoxazole and 5-isopropyl-3-methylisoxazole (1.9 g, crude) in DMF (15 mL) was added NBS (2.97 g, 16.70 mmol) and the reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was added water (15 mL), extracted with ethyl acetate (15mL × 3). The combined organic layers were washed with brine (20 mL × 3) and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 10%) to give a mixture of 4-bromo-3-isopropyl-5-methylisoxazole and 4-bromo-5- isopropyl-3-methylisoxazole (2.00 g). Step 3: 3-fluoro-5-(3-isopropyl-5-methylisoxazol-4-yl)benzoate and 3-fluoro-5-(5- isopropyl-3-methylisoxazol-4-yl)benzoate
Figure imgf000408_0001
The mixture of 4-bromo-3-isopropyl-5-methylisoxazole and 4-bromo-5-isopropyl-3- methylisoxazole (1.80 g, 8.82 mmol) and (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.92 g, 9.70 mmol), Pd(t-Bu3P)2 (451 mg, 0.88 mmol,) and KF (1.28 g, 22.05 mmol) were taken up into a microwave tube in a mixture of dioxane (12 mL) and H2O (1 mL). The sealed tube was heated at 130 °C for 60 min under microwave. The residue was added water (15 mL) and extracted with ethyl acetate (20 mL × 3). Then the combined organic layers concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 15%) to give the crude product in 1.8 g. Further purification by preparative-HPLC (Method CC) gave 1.25 g of product. The product was further separated by SFC (Instrument: Thar200; Column: DAICEL CHIRALCEL OJ-H(250mm*30mm,5 m; Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, v%) = 90/10; Flow Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give methyl 3-fluoro-5-(5-isopropyl-3- methylisoxazol-4-yl)benzoate (330 mg), and methyl 3-fluoro-5-(3-isopropyl-5- methylisoxazol-4-yl)benzoate (140 mg, not pure). methyl 3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzoate: 1H NMR (400MHz,CDCl3) δ 7.76-7.71 (m, 2H), 7.18-7.12 (m, 1H), 3.96 (s, 3H), 3.18-3.05 (m, 1H), 2.24 (s, 3H), 1.31 (d, J = 7.2 Hz, 6H). Step 4: 3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzoic acid
Figure imgf000408_0002
A mixture of methyl 3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzoate (330 mg, 1.19 mmol) in concentrated HCl (10 mL) was stirred at 80 °C for 16 hours . The mixture was adjusted pH to 3~4 with 2N NaOH and extracted with ethyl acetate (20 mL × 5). Then the combined organic layers were dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzoic acid (260 mg). 1H NMR (400MHz, DMSO-d6) δ 7.72-7.66 (m, 2H), 7.57-7.54 (m, 1H), 3.18-3.08 (m, 1H), 2.19 (s, 3H), 1.23 (d, J = 7.2 Hz, 6H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropyl-3- methylisoxazol-4-yl)benzamido)propanoate
Figure imgf000409_0001
To a solution of 3-fluoro-5-(5-isopropyl-3-methyl-isoxazol-4-yl)benzoic acid (130 mg, 0.50 mmol) in DMF (6 mL) was added TBTU (239 mg, 0.74 mmol), DIPEA (192 mg, 1.48 mmol ) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (180 mg, 0.49 mmol, HCl salt). The mixture was stirred at 30 °C for 16 hours. The mixture was added water (15 mL) and extracted with ethyl acetate (20 mL × 3). Then the combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 40%) twice to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropyl-3-methylisoxazol-4- yl)benzamido)propanoate (70 mg). 1H NMR (400MHz, CDCl3) δ 7.89 (br s, 1H), 7.43-7.38 (m, 1H), 7.37-7.30 (m, 10H), 7.09- 7.06 (m, 1H), 6.90 (br s, 1H), 5.93 (br d, J = 7.2 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.63- 4.56 (m, 1H), 3.98-3.94 ( m, 1H), 3.81-3.78 (m, 1H), 3.14-3.07 (m, 1H), 2.24 (s, 3H), 1.30 (d, J = 7.2 Hz, 6H). Step 6: (R)-2-amino-3-(3-fluoro-5-(5-isopropyl-3-methylisoxazol-4- yl)benzamido)propanoic acid
Figure imgf000410_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropyl-3- methylisoxazol-4-yl)benzamido)propanoate (70 mg, 0.12 mmol) in 30% HBr in AcOH (4 mL) was stirred at 30 °C for 16 hours. The reaction mixture was concentrated. The residue was further purified by preparative-HPLC (Method AA) to give (R)-2-amino- 3-(3-fluoro-5-(5-isopropyl-3-methylisoxazol-4-yl)benzamido)propanoic acid (35 mg) as a HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.07-9.00 (m, 1H), 8.46 (br s, 3H), 7.78-7.72 (m, 1H), 7.69 (s, 1H), 7.50-7.44 (m, 1H), 4.15-4.07 (m, 1H), 3.87-3.67 (m, 2H), 3.20-3.09 (m, 1H), 2.20 (s, 3 H), 1.23 (d, J = 6.8 Hz, 6H). LC-MS (MH+): m/z = 350.0 tR (min, Method D) = 1.656 [α]20,D = 2.6 (c = 2.3 mg/mL, CH3OH). Compound 4w (R)-2-amino-3-(3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1- isopropyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000411_0001
Step 1: methyl 3-fluoro-5-(1-isopropyl-1H-pyrazol-5-yl)benzoate
Figure imgf000411_0002
To a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (524 mg, 2.64 mmol), methyl 3-fluoro-5-(1-isopropyl-1H-pyrazol-5-yl)benzoate (0.50 g, 2.64 mmol, 1 eq) and K3PO4 (1.68 g, 7.93 mmol) in the mixture of dioxane (50 mL) and H2O (5 mL) was added Pd(dtbpf)Cl2 (172 mg, 0.26 mmol) under N2. The mixture was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100°C for 3 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-fluoro-5-(1-isopropyl-1H-pyrazol-5-yl)benzoate (308 mg). 1H NMR (400MHz, CDCl3) δ 7.86 (dd, J=1.6 Hz, 1.2 Hz, 1H), 7.80-7.75 (m, 1H), 7.59 (d, J=1.6 Hz, 1H), 7.30-7.26 (m, 1H), 6.29 (d, J=1.6 Hz, 1H), 4.54-4.47 (m, 1H), 3.96 (s, 3H), 1.49 (d, J=6.8 Hz, 6H). Step 2: methyl 3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000412_0001
To a mixture of methyl 3-fluoro-5-(1-isopropyl-1H-pyrazol-5-yl)benzoate (232 mg, 0.89 mmol) in DMF (10 mL) was added NCS (118 mg, 0.88 mmol). The mixture was stirred at 55°C for 16 hours. The mixture was concentrated. The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzoate (190 mg). Step 3: 3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000412_0002
To a mixture of methyl 3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzoate (140 mg, 0.47 mmol) in a mixture of MeOH (10 mL) and H2O (10 mL) was added LiOH.H2O (40 mg, 0.94 mmol) at 25°C. The mixture was stirred at 25°C for 16 hours. The mixture was adjusted pH=4 with KHSO4 (aq.4 mL) and extracted with ethyl acetate (20 mL × 3). The organic phase was concentrated. The mixture was used for next step without further purification to give 3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (130 mg). LC-MS (MH+): m/z = 283.1 tR (min, Method c) = 0.806 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-isopropyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000413_0001
To a mixture of 3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (65.0 mg, 0.23 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (84.0 mg, 0.23 mmol, HCl salt) in DMF (10 mL) was added TBTU (111 mg, 0.34 mmol) followed by addition of DIPEA (149 mg, 1.15 mmol) at 25°C. The mixture was stirred at 25°C for 16 hours. The mixture was diluted with H2O (100 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether : ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (30 mg). 1H NMR (400MHz, CDCl3) δ 7.58 (s, 1H), 7.53-7.43 (m, 2H), 7.39-7.28 (m, 10H), 7.25-7.23 (m,1H), 6.95 (br s, 1H), 5.94 (br d, J=6.8 Hz, 1H), 5.26-5.18 (m, 2H), 5.14-5.06 (m, 2H), 4.65-4.58 (m, 1H), 4.42-4.35 (m, 1H), 4.00-3.79 (m, 2H), 1.45 (d, J=6.4 Hz, 6H). Step 5: (R)-2-amino-3-(3-(4-chloro-1-isopropyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000413_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-isopropyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (30 mg, 0.051 mmol) in HBr/AcOH (5 mL, 30% in AcOH) was stirred at 25°C for 16 hours. The mixture was concentrated. The residue was purified by preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(4-chloro-1- isopropyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.15 (br s, 1H), 8.53 (br s, 3H), 7.95-7.90 (m, 1H), 7.79 (s, 1H), 7.76 (s, 1H), 7.62-7.57 (m, 1H), 4.42-4.36 (m, 1H), 4.23-4.10 (m, 1H), 4.20-4.11 (m, 1H), 3.84-3.76 (m, 1H), 1.38-1.33 (m, 6H). LC-MS (MH+): m/z = 369.0 tR (min, Method D) = 1.664 [α]20,D = 5.0 (c = 1.6 mg/mL, MeOH). Compound 4x (R)-2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1-propyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000414_0001
Step 1: methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000414_0002
A mixture of 5-bromo-1-propyl-pyrazole (0.885 g, 4.68 mmol), (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (772 mg, 3.90 mmol), Pd(dtbpf)Cl2 (254 mg, 0.39 mmol), K3PO4 (2.48 g, 11.70 mmol) in the mixture of dioxane (35 mL) and H2O (3.5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 3 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.9 g). 1H NMR (400MHz, CDCl3) δ 7.88 (t, J = 1.2 Hz, 1H), 7.79-7.74 (m, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.33-7.27 (m, 1H), 6.33 (d, J = 1.8 Hz, 1H), 4.12-4.08 (m, 2H), 3.95 (s, 3H), 1.88-1.79 (m, 2H), 0.82 (t, J = 7.6 Hz, 3H). Step 2: methyl 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000415_0001
To a solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.89 g, 3.39 mmol) in THF (20 mL) was added NCS (680 mg, 5.09 mmol). The mixture was stirred at 70 °C for 2 hours. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (0.9 g). 1H NMR (400MHz, CDCl3) δ 7.87 (t, J=1.6 Hz, 1H), 7.85-7.79 (m, 1H), 7.54 (s, 1H), 7.35- 7.29 (m, 1H), 4.05-3.99 (m, 2H), 3.95 (s, 3H), 1.80-1.74 (m, 2H), 0.80 (t, J=7.6 Hz, 3H). Step 3: 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000415_0002
To a solution of methyl 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (0.90 g, 3.03 mmol) in a mixture of MeOH (10 mL) and H2O (10 mL) was added LiOH.H2O (255 mg, 6.07 mmol). The mixture was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was adjust pH=4 with aqueous KHSO4 (6 mL). The precipitate was filtered to give 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (0.80 g). LC-MS (MH+): m/z = 283.1 tR (min, Method c) = 0.804 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate
Figure imgf000416_0001
A mixture of 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (0.20 g, 0.71 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (258 mg, 0.71 mmol, HCl salt), TBTU (341 mg, 1.06 mmol) and DIPEA (457 mg, 3.54 mmol) in DMF (20 mL) was stirred at 30°C for 16 hours. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 35%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (0.18 g). 1H NMR (400MHz, CDCl3) δ 7.55 (s, 1H), 7.51 (s, 1H), 7.46 (br d, J=8.4 Hz, 1H), 7.37-7.29 (m, 10H), 7.26-7.23 (m, 1H), 6.98 (br, 1H), 5.94 (br d, J=6.4 Hz, 1H), 5.26-5.16 (m, 2H), 5.10 (s, 2H), 4.65-4.54 (m, 1H), 4.00 (t, J=7.2 Hz, 2H), 3.97-3.90 (m, 1H), 3.85-3.76 (m, 1H), 1.82-1.72 (m, 2H), 0.79 (t, J=7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000417_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (0.18 g, 0.3 mmol) and HBr in AcOH (8 mL, 30% in AcOH) were stirred at 30°C for 15 hours. The mixture was concentrated. The residue was diluted with AcOH (3 mL). MTBE (50 mL) was added to the mixture. The precipitate was filtered under N2. The filter cake was lyophilized to give (R)-2-amino-3- (3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99-8.93 (m, 1H), 8.34 (br s, 3H), 7.86 (d, J=8.8 Hz, 1H), 7.79 (s, 1H), 7.75 (s, 1H), 7.63 (d, J=8.8 Hz, 1H), 4.17-4.11 (m, 1H), 4.04 (t, J=6.8 Hz, 2H), 3.85-3.77 (m, 1H), 3.74-3.66 (m, 1H), 1.70-1.58 (m, 2H), 0.70 (t, J=7.8 Hz, 3H). LC-MS (MH+): m/z = 369.0 tR (min, Method D) = 1.663 [α]20,D = 2.0 (c = 2.0 mg/mL, CH3OH). Compound 4y (R)-2-amino-3-(3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3,5- dimethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000418_0001
Step 1: methyl 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoate
Figure imgf000418_0002
To a solution of 4-bromo-3,5-dimethyl-isoxazole (600 mg, 3.41 mmol) and (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (675 mg, 3.41 mmol) in dioxane (15 mL) and H2O (1.5 mL) was added K3PO4 (2.17 g, 10.23 mmol) and Pd(dtbpf)Cl2 (222 mg, 340.89 μmol). The mixture was heated to 100°C for 16 hours. The reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (20 ml × 3). The organic layer was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0~10%) to give methyl 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoate (340 mg). 1H NMR (400MHz, CDCl3) δ 7.75-7.70 (m, 2H), 7.17 (dd, J = 6.8Hz, 2.0 Hz, 1H), 3.96 (s, 3H), 2.44 (s, 3H), 2.30 (s, 3 H). Step 2: 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000418_0003
To a solution of methyl 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoate (330 mg, 1.32 mmol) in a mixture of MeOH (10 mL) and H2O (5 mL) was added LiOH.H2O (167 mg, 3.97 mmol). The mixture was stirred at 35 °C for 2 hours. The mixture was concentrated. The residue was dissolved in water (20 mL) and acidified with 2N HCl to adjust pH to 4 and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-(3,5- dimethylisoxazol-4-yl)-5-fluorobenzoic acid (300 mg). 1H NMR (400MHz, CDCl3) δ 13.45 (br s, 1H), 7.75-7.70 (m, 1H), 7.70-7.64 (m, 1H), 7.62- 7.56 (m, 1H), 2.43 (s, 3H), 2.24 (s, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethylisoxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000419_0001
To a solution of 3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzoic acid (100 mg, 0.43 mmol) in DMF (5 mL) was added TBTU (2056 mg, 0.63 mmol), DIPEA (165 mg, 1.28 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (155 mg, 0.43 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (180 mg). 1H NMR (400MHz, CDCl3) δ 7.41 (s, 1H), 7.38-7.31 (m, 11H), 7.13-7.07 (m, 1H), 6.98 (br s, 1H), 5.92 (br d, J = 6.8 Hz, 1H), 5.23 (s, 2H), 5.11 (s, 2H), 4.64-4.56 (m, 1H), 4.02-3.93 (m, 1H), 3.84-3.74 (m, 1H), 2.43 (s, 3H), 2.29 (s, 3H) Step 4: (R)-2-amino-3-(3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000420_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3,5-dimethylisoxazol-4- yl)-5-fluorobenzamido)propanoate (100 mg, 183.3 μmol) in 30% HBr/AcOH (3 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by Preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(3,5- dimethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.04-9.00 (m, 1H), 8.44 (br s, 3H), 7.76-7.67 (m, 2H), 7.55- 7.50 (m, 1H), 4.15-4.10 (m, 1H), 3.83-3.70 (m, 2H), 2.44 (s, 3H), 2.26 (s, 3H). LC-MS (MH+): m/z = 322.1 tR (min, Method D) = 1.505 [α]20,D = +2.2 (c = 2.1 mg/mL,CH3OH). Compound 4z (R)-2-amino-3-(3-fluoro-5-(5-isopropylisothiazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- isopropylisothiazol-4-yl)benzamido)propanoic acid
Figure imgf000421_0001
Step 1: 5-(prop-1-en-2-yl)isothiazole
Figure imgf000421_0002
To a solution of 5-bromoisothiazole (1.6 g, 9.75 mmol) and 4,4,5,5-tetramethyl-2-(prop- 1-en-2-yl)-1,3,2-dioxaborolane (2.16 g, 11.71 mmol) in a mixture of dioxane (20 mL) and H2O (2 mL) was added Pd(dppf)Cl2 (714 mg, 0.98 mmol) and K2CO3 (3.37 g, 24.39 mmol) under N2 atmosphere. The mixture was stirred at 100 °C for 16 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give 5-(prop-1-en-2-yl)isothiazole (1.1 g). 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 1.6 Hz, 1H), 7.16 (d, J = 1.6 Hz, 1H), 5.54 (s, 1H), 5.21 (s, 1H), 2.18 (s, 3H). Step 2: 5-isopropylisothiazole
Figure imgf000422_0001
To a solution of 5-(prop-1-en-2-yl)isothiazole (1.1 g, 8.79 mmol) in MeOH (30 mL) was added Pd/C (600 mg, 10% Pd, <1% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 16 hours. The mixture was filtered through Celite and the filtrate was concentrated below 30 °C (Note: volatilization) to give 5-isopropylisothiazole (800 mg). 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1 H), 6.98 (s, 1 H), 3.34-3.24 (m, 1H), 1.36 (d, J = 7.2 Hz, 6H). Step 3: 4-bromo-5-isopropylisothiazole
Figure imgf000422_0002
To a solution of 5-isopropylisothiazole (400 mg, 3.14 mmol) in AcOH (8 mL) was added KOAc (463 mg, 4.72 mmol) and Br2 (1.01 g, 6.29 mmol). The mixture was stirred at 100 °C for 3 hours. The mixture was diluted with water (15 mL) and added saturated Na2SO3 solution (5 mL) and was adjusted pH to 7 with 2N NaOH solution. The aqueous solution was extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give 4-bromo-5-isopropylisothiazole (540 mg). 1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 3.41-3.30 (m, 1H), 1.38 (d, J = 6.8 Hz, 6H). Step 4: methyl 3-fluoro-5-(5-isopropylisothiazol-4-yl)benzoate
Figure imgf000423_0001
4-bromo-5-isopropylisothiazole (540 mg, 2.62 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (622 mg, 3.14 mmol), Pd(dppf)Cl2.CH2Cl2 (214 mg, 0.26 mmol) and Na2CO3 (833 mg, 7.86 mmol) were taken up into a microwave tube in a mixture of dioxane (12 mL) and H2O (1.2 mL). The sealed tube was heated at 100 °C for 60 minutes under microwave. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-fluoro-5-(5-isopropylisothiazol-4-yl)benzoate (190 mg). 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 7.87-7.84 (m, 1H), 7.77-7.72 (m, 1H), 7.30-7.26 (m, 1H), 3.96 (s, 3H), 3.46-3.38 (m, 1H), 1.35 (d, J = 6.8 Hz, 6H). Step 5: 3-fluoro-5-(5-isopropylisothiazol-4-yl)benzoic acid
Figure imgf000423_0002
To a solution of methyl 3-fluoro-5-(5-isopropylisothiazol-4-yl)benzoate (190 mg, 0.68 mmol) in MeOH (5 mL) was added LiOH.H2O (86 mg, 2.04 mmol) in H2O (2 mL). The mixture was stirred at 30 °C for 2 hours. The mixture was concentrated. The residue was added water (10 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(5-isopropylisothiazol-4-yl)benzoic acid (170 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.81 (s, 1H), 7.71-7.64 (m, 2H), 3.48-3.41 (m, 1H), 1.29 (d, J = 6.8 Hz, 6H). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropylisothiazol- 4-yl)benzamido)propanoate
Figure imgf000424_0001
To a solution of 3-fluoro-5-(5-isopropylisothiazol-4-yl)benzoic acid (85 mg, 0.32 mmol) in DMF (5 mL) was added TBTU (154 mg, 0.48 mmol), DIPEA (124 mg, 0.96 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (117 mg, 0.32 mmol, HCl salt). The mixture was stirred at 30 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give 120 mg crude product. The crude product was further purified by SFC (Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALCEL OJ-H(250mm*30mm,5μm; Mobile phase: supercritical CO2/i-PrOH (0.1% NH3`H2O, v%) = 75/25; Flow Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-isopropylisothiazol-4- yl)benzamido)propanoate (75 mg). 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 7.51 (s, 1H), 7.39-7.29 (m, 11H), 7.24-7.18 (m, 1H), 6.90 (br s, 1H), 5.93 (br d, J = 6.8 Hz, 1H), 5.27-5.17 (m, 2H), 5.15-5.07 (m, 2H), 4.64- 4.56 (m, 1H), 4.00-3.90 (m, 1H), 3.87-3.77 (m, 1H), 3.46-3.37 (m, 1H), 1.34 (d, J = 6.4 Hz, 6H). Step 7: (R)-2-amino-3-(3-fluoro-5-(5-isopropylisothiazol-4-yl)benzamido)propanoic acid
Figure imgf000425_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5- isopropylisothiazol-4-yl)benzamido)propanoate (75 mg, 0.13 mmol) in30% HBr in AcOH (4 mL) was stirred at 30°C for 15 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with MTBE (5 mL × 2). The aqueous layer was lyophilized to give (R)-2-amino-3-(3-fluoro-5-(5-isopropylisothiazol-4-yl)benzamido)propanoic acid (45 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.95-8.90 (m, 1H), 8.60 (s, 1H), 8.33 (br s, 3H), 7.79 (s, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.58 (d, J = 9.2 Hz, 1H), 4.16-4.08 (m, 1H), 3.86-3.77 (m, 1H), 3.74-3.64 (m, 1H), 3.54-3.43 (m, 1H), 1.28 (d, J = 6.8 Hz, 6H). LC-MS (MH+): m/z = 352.1 tR (min, Method D) = 1.828 [α]20,D = -0.9 (c = 2.2 mg/mL, CH3OH). Compound 5a (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1-ethyl- 3-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000426_0001
To a solution of 3-methyl-1H-pyrazole (4 g, 48.72 mmol) in THF (100 mL) was added NaH (2.92 g, 73.01 mmol, 60% in mineral oil) at 0°C and the reaction was stirred at 0°C for 1 hour. iodoethane (9.12 g, 58.46 mmol) was added at 0°C and the resulting mixture was stirred at 30°C for 16 hours. H2O (100 ml) was added to quench the reaction at 0°C and extracted with DCM (100 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered and concentrated to give 1-ethyl-3-methyl-1H- pyrazole (5 g, crude). Step 2: 1-ethyl-5-iodo-3-methyl-1H-pyrazole
Figure imgf000426_0002
To a solution of 1-ethyl-3-methyl-1H-pyrazole (5 g, crude) in THF (100 mL) was added n- BuLi (20 mL, 2.5 M in hexane) at -78°C and the reaction was stirred at -78°C for 1 hour. I2 (12.1 g, 47.66 mmol) was added and the reaction was stirred at -78°C for 1 hour. H2O (50 ml) was added to quench the reaction and extracted with DCM (100 mL × 3). The combined organic layers were washed with brine (100 mL × 2), dried over Na2SO4, filtered and concentrated to give 1-ethyl-5-iodo-3-methyl-1H-pyrazole (10 g, crude). Step 3: methyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000427_0001
A mixture of 1-ethyl-5-iodo-3-methyl-1H-pyrazole (1.0 g, crude), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (840 mg, 4.24 mmol), Pd(dppf)Cl2 (310 mg, 0.42 mmol) and K2CO3 (1.76 g, 12.73 mmol) in a mixture of dioxane (50 mL) and H2O (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80°C for 2 hours under N2 atmosphere. The mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (100 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~30%) to give methyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (900 mg). 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.78-7.72 (m, 1H), 7.31-7.27 (m, 1H), 6.11 (s, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 2.31 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). Step 4: methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000428_0001
A mixture of methyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (900 mg, 3.43 mmol) and NCS (500 mg, 3.74 mmol) in THF (2 mL) was stirred at 70°C for 2 hours. The mixture was concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~20%) and further purified by preparative HPLC (Method CC) to give methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol- 5-yl)-5-fluorobenzoate (300 mg). 1H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 1.6 Hz, 1.2 Hz, 1H), 7.84-7.79 (m, 1H), 7.36-7.30 (m, 1H), 4.06 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 2.30 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). Step 5: 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000428_0002
A mixture of methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (350 mg, 1.18 mmol) and LiOH.H2O (150 mg, 3.57 mmol) in a mixture of MeOH (8 mL) and H2O (2 mL) was stirred at 30°C for 4 hours. The mixture was concentrated. H2O (2 mL) was added, acidified with satd. aqueous KHSO4 to pH 3~4 and extracted with ethyl acetate (10 mL × 5). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol- 5-yl)-5-fluorobenzoic acid (300 mg). LC-MS (MH+): m/z = 283.0 tR (min, Method D) = 0.761 Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000429_0001
A mixture of 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.53 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (233 mg, 0.64 mmol, HCl salt), TBTU (256 mg, 0.80 mmol) and DIPEA (148 mg, 1.15 mmol) in DMF (5 mL) was stirred at 30°C for 16 hours. The mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (15 mL × 3). The organic phase was washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (270 mg). 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H), 7.48-7.41 (m, 1H), 7.37-7.26 (m, 11H), 6.97-6.84 (m, 1H), 5.91 (d, J = 6.8 Hz, 1H), 5.27-5.17 (m, 2H), 5.15-5.04 (m, 2H), 4.65-4.55 (m, 1H), 4.04 (q, J = 7.2 Hz, 2H), 4.00-3.75 (m, 2H), 2.30 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). Step 7: (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000429_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (270 mg, 0.46 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 3 hours. The solvent was removed under reduced pressure. The solid was suspended in TBME (5 mL), filtered, washed with TBME (1 mL × 2) and lyophilized to give (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoic acid (105 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.02-8.85 (m, 1H), 8.45-8.20 (m, 3H), 7.83 (d, J = 9.2 Hz, 1H), 7.77 (s, 1H), 7.59 (d, J = 9.2 Hz, 1H), 4.15-4.10 (m, 1H), 4.05-4.00 (m, 2H), 3.75-3.65 (m, , 2H), 2.20 (s, 3H), 1.22 (t, J=7.2 Hz, 3H). LC-MS (MH+): m/z = 369.1 tR (min, Method D) = 1.721 [α]20,D = 1.25 (c = 8 mg/mL, MeOH). Compound 5b (R)-2-amino-3-(3-fluoro-5-methylbenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5- methylbenzamido)propanoic acid
Figure imgf000430_0001
Step 1: 3-fluoro-5-methylbenzoyl chloride
Figure imgf000430_0002
3-fluoro-5-methylbenzoic acid (65 mg, 0.422 mmol) was dissolved in DCM (1.5 mL) and oxalyl chloride (130 µl, 1.485 mmol) was added at room temperature. Then DMF (1 drop) was added and the reaction was stirred at room temperature over-night. The reaction was concentrated to afford 3-fluoro-5-methylbenzoyl chloride in 72 mg (crude). The following intermediates was made in a similar way: 3-ethylbenzoyl chloride 3-fluoro-5-methoxybenzoyl chloride Step 2: tert-butyl (R)-2-((tert-butoxycarbonyl)amino)-3-(3-fluoro-5- methylbenzamido)propanoate
Figure imgf000431_0001
(R)-tert-butyl 3-amino-2-((tert-butoxycarbonyl)amino)propanoate (50 mg, 0.192 mmol) was dissolved in THF (2.0 mL) and DIPEA (304 µl, 1.738 mmol) was added under argon atmosphere.3-fluoro-5-methylbenzoyl chloride (60 mg, 0.348 mmol) was suspended in THF (1.0 mL) and added dropwise to the reaction mixture. The mixture was stirred at room temperature for 1 hour. The contents were diluted with water and extracted with ethyl acetate (twice). The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated on vacuum. The residue was purified by Combi flash (silica gel, hepane/ethyl acetate with ethyl acetate from 0~40%) to give tert-butyl (R)-2- ((tert-butoxycarbonyl)amino)-3-(3-fluoro-5-methylbenzamido)propanoate (31 mg). The following intermediates were made in a similar way: tert-butyl (R)-2-((tert-butoxycarbonyl)amino)-3-(3-ethylbenzamido)propanoate tert-butyl (R)-2-((tert-butoxycarbonyl)amino)-3-(3-fluoro-5- methoxybenzamido)propanoate Step 3: (R)-2-amino-3-(3-fluoro-5-methylbenzamido)propanoic acid
Figure imgf000432_0001
(R)-2-((tert-butoxycarbonyl)amino)-3-(3-fluoro-5-methylbenzamido)propanoate (31 mg, 0.078 mmol) was dissolved in DCM (0.83 mL) and TFA ( 0.83 mL) was added at 0 oC. The reaction mixture was stirred over-night at room temperature. Concentration and lyophilizisation afforded (R)-2-amino-3-(3-fluoro-5-methylbenzamido)propanoic acid as TFA salt. 1H NMR (600 MHz, DMSO) δ 8.82-8.80 (m, 1H), 8.41 (br s, 3H), 7.54 (s, 1H), 7.48 – 7.40 (m, 1H), 7.25-7.23 (m, 1H), 4.11-4.09 (m, 1H), 3.79-3.75 (m, 1H), 3.72-3.67 (m, 1H), 2.37 (s, 3H). LC-MS (MH+): m/z = 241.4 tR (min, Method A) = 0.29 The following compounds were synthesized in a similar way as example Compound 5b Compound 5c (R)-2-amino-3-(3-ethylbenzamido)propanoic acid
Figure imgf000432_0002
From 3-ethylbenzoic acid. LC-MS (MH+): m/z = 237.4 tR (min, Method A) = 0.34 Compound 5d (R)-2-amino-3-(3-fluoro-5-methoxybenzamido)propanoic acid
Figure imgf000433_0001
From 3-fluoro-5-methoxybenzoic acid LC-MS (MH+): m/z = 257.4 tR (min, Method A) = 0.28 Compound 5e (R)-2-amino-3-(5-fluoro-2'-(methoxymethyl)-[1,1'-biphenyl]-3- carboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(5-fluoro-2'- (methoxymethyl)-[1,1'-biphenyl]-3-carboxamido)propanoic acid
Figure imgf000433_0002
Step 1: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5- fluorobenzamido)propanoate
Figure imgf000434_0001
3-bromo-5-fluorobenzoic acid (624 mg, 2.851 mmol) was dissolved in anhydrous THF (18 mL), N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine hydrochloride (546 mg, 2.851 mmol) and 1H-benzo[d][1,2,3]triazol-1-ol hydrate (369 mg, 2.412 mmol) were added at room temperature. Then (R)-3-(benzyloxy)-2- (((benzyloxy)carbonyl)amino)-3-oxopropan-1-aminium chloride (800 mg, 2.193 mmol) and DIPEA (1.53 ml, 8.77 mmol) was added and the reaction was stirred at room temperature for 2 hours. The mixture was diluted with H2O and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, heptane/ethyl acetate with ethyl acetate from 0~40%) to give benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5- fluorobenzamido)propanoate in 900 mg. LC-MS (MH+): m/z = 529.5 tR (min, Method A) = 0.82 Step 2: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-(methoxymethyl)-[1,1'- biphenyl]-3-carboxamido)propanoate
Figure imgf000434_0002
(R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-fluorobenzamido)propanoate (100 mg, 0.189 mmol), cesium carbonate (176 mg, 0.541 mmol) and (2- (methoxymethyl)phenyl)boronic acid (44.9 mg, 0.271 mmol) were dissolved in WATER (500 µl) and 1,4-Dioxane (2.0 ml). Flushed with Argon for 5 minutes and then Pd(dppf)Cl2 (20.0 mg, 0.036 mmol) was added. Vial was capped and microwaved at 80° for 15 minuttes. Brine and ethyl acetate was added and the aqueous phase was extracted with ethyl acetate 3 times. The combined organic phase was washed with brine, dried with MgSC , filtrated and evaporated. The residue was purified by Combi flash (silica gel, heptane/ethyl acetate with ethyl acetate from 0~40%) to give benzyl (R)-2- (((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-3- carboxamido)propanoate (80 mg)
LC-MS (MNa+): m/z = 593.7 t« (min, Method A) = 0.85
Step 3: (R)-2-amino-3-(5-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-3- carboxamido)propanoic acid
Figure imgf000435_0001
A solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-(methoxymethyl)-[l,l'- biphenyl]-3-carboxamido)propanoate (80 mg, 0.140 mmol) in MeOH was passed through the H-Cube. The contents were concentrated and purified by preparative HPLC to afford (R)-2-amino-3-(5-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-3- carboxamido)propanoic acid.
XH NMR (600 MHz, DMSO) 6 8.94-8.90 (m, 1H), 8.40 (br s, 3H), 7.74-7.71 (m, 1H), 7.70- 7.65 (m, 1H), 7.54-7.50 (m, 1H), 7.49-7.41 (m, 3H), 7.36-7.32 (m, 1H), 4.30 (s, 2H), 4.11 (br s, 1H), 3.83-3.77 (m, 1H), 3.75-3.67 (m, 1H), 3.23 (s, 3H).
LC-MS (MH+): m/z = 347.4 t« (min, Method A) = 0.45 Compound 5f (R)-2-amino-3-(2'-carbamoyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(2'-carbamoyl-5- fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid
Figure imgf000436_0001
(R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-fluorobenzamido)propanoate (100 mg, 0.189 mmol), cesium carbonate (176 mg, 0.541 mmol) and (2- cyanophenyl)boronic acid (39.8 mg, 0.271 mmol) were dissolved in WATER (500 µl) and 1,4-Dioxane (2.0 ml, 23.4 mmol), under argon atmosphere. Pd(dppf)Cl2 (20.00 mg, 0.036 mmol) was added and the vial was capped and microwaved at 80° 15 minutes. Brine and ethyl acetate was added and the aqueous phase was extracted with ethyl acetate 3 times. The combined organic phase was washed with brine, dried with MgSO4, filtrated and evaporated. The residue was purified by Combi flash (silica gel, heptane/ethyl acetate with ethyl acetate from 0 to 100%) to give benzyl (R)-2- (((benzyloxy)carbonyl)amino)-3-(2'-carbamoyl-5-fluoro-[1,1'-biphenyl]-3- carboxamido)propanoate. The intermediate was stirred in 30% HBr in AcOH (2 mL) at room temperature overnight. The solvent was removed and the product was stirred in heptane:ether (1:1) at room temperature for 30min. The solvent was decanted and the rusty solid was dissolved in DMSO and evaporated on genevac to afford (R)-2-amino-3- (2'-carbamoyl-5-fluoro-[1,1'-biphenyl]-3-carboxamido)propanoic acid as HBr salt. LC-MS (MH+): m/z = 346.4 tR (min, Method A) = 0.30 The following compounds were synthesized in a similar way as example Compound 5f: Compound 5g (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-pyrazol-4-yl)benzamido)propanoic acid
Figure imgf000437_0001
From benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5- fluorobenzamido)propanoate and (1-propyl-1H-pyrazol-4-yl)boronic acid LC-MS (MH+): m/z = 335.4 tR (min, Method A) = 0.39 Compound 5h (R)-2-amino-3-(3-(1,5-dimethyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000437_0002
From benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5- fluorobenzamido)propanoate and (1,5-dimethyl-1H-pyrazol-4-yl)boronic acid LC-MS (MH+): m/z = 321.4 tR (min, Method A) = 0.31 Compound 5i (R)-2-amino-3-(6-fluoro-1,2,3,4-tetrahydroquinoline-8-carboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(6-fluoro-1,2,3,4- tetrahydroquinoline-8-carboxamido)propanoic acid
Figure imgf000438_0001
Step 1: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(6-fluoro-1,2,3,4- tetrahydroquinoline-8-carboxamido)propanoate
Figure imgf000438_0002
6-fluoro-1,2,3,4-tetrahydroquinoline-8-carboxylic acid (50 mg, 0.256 mmol) was dissolved in anhydrous THF (3.0 mL), N1-((ethylimino)methylene)-N3,N3- dimethylpropane-1,3-diamine hydrochloride (73.7 mg, 0.384 mmol) and 1H- benzo[d][1,2,3]triazol-1-ol hydrate (58.8 mg, 0.384 mmol) were added at room temperature. Then (R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropan-1- aminium chloride (140 mg, 0.384 mmol) and DIPEA (224 µl, 1.281 mmol) was added and the reaction was stirred at room temperature overnight. The mixture was diluted with H2O and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, heptane/ethyl acetate with ethyl acetate from 0~50%) to give benzyl (R)-2- (((benzyloxy)carbonyl)amino)-3-(6-fluoro-1,2,3,4-tetrahydroquinoline-8- carboxamido)propanoate (89 mg). LC-MS (MH+): m/z = 506.6 tR (min, Method A) = 0.86 Step 2: (R)-2-amino-3-(6-fluoro-1,2,3,4-tetrahydroquinoline-8-carboxamido)propanoic acid
Figure imgf000439_0001
benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(6-fluoro-1,2,3,4-tetrahydroquinoline-8- carboxamido)propanoate (89 mg, 0.176 mmol) was stirred in 30% HBr in AcOH (1 mL) at room temperature overnight. The solvent was removed and the product was stirred in ether at room temperature for 30 minutes. The solvent was decanted followed by lyophilization to afford (R)-2-amino-3-(6-fluoro-1,2,3,4-tetrahydroquinoline-8- carboxamido)propanoic acid (70 mg) as HBr salt. 1H NMR (600 MHz, DMSO) δ 8.54-8.48 (m, 1H), 8.37-8.21 (br s, 3H), 7.32-7.27 (m, 1H), 7.00-6.95 (m, 1H ), 4.11-404 (m, 1H), 3.73-3.67 (m, 2H), 3.66-3.60 (m, 2H), 3.31-3.24 (m, 2H), 2.76-2.70 (m, 2H), 1.82-1.74 (m, 2H), 1.10-1.06 (m, 1H). LC-MS (MH+): m/z = 282.4 tR (min, Method A) = 0.30 Compound 5j (R)-2-amino-3-(5-fluoro-2'-methoxy-[1,1'-biphenyl]-3-carboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(5-fluoro-2'-methoxy- [1,1'-biphenyl]-3-carboxamido)propanoic acid
Figure imgf000439_0002
Step 1: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-methoxy-[1,1'- biphenyl]-3-carboxamido)propanoate
Figure imgf000440_0001
A solution of 5-fluoro-2'-methoxy-[1,1'-biphenyl]-3-carboxylic acid (200 mg, 0.812 mmol) and (R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropan-1-aminium chloride (356 mg, 0.975 mmol) in DMF (3.0 mL) was treated with HATU (371 mg, 0.975 mmol) and DIPEA (426 μl, 2.437 mmol) and the solution was stirred at room temperature for 72 hours. Ethyl acetate (100 mL), water (50 mL), and brine (50 mL) were added and the phases are separated. The ethyl acetate phase was washed with water (50 mL), and brine (50 mL), and the aqueous phase was then extracted with ethyl acetate. The ethyl acetate phases are combined and concentrated. Purification by combiflash afforded benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-methoxy- [1,1'-biphenyl]-3-carboxamido)propanoate (357 mg). LC-MS (MH+): m/z = 557.5 tR (min, Method A) = 0.89 The following intermediates were made in a similar way: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5- isobutoxybenzamido)propanoate Step 2: (R)-2-amino-3-(5-fluoro-2'-methoxy-[1,1'-biphenyl]-3-carboxamido)propanoic acid
Figure imgf000440_0002
A solution of benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(5-fluoro-2'-methoxy-[1,1'- biphenyl]-3-carboxamido)propanoate (100 mg, 0.180 mmol) in 20 mL methanol:water (9:1) was passed through the H-Cube (1 bar, 1 ml/min) at room temperature. The reslting solution was concentrated to afford (R)-2-amino-3-(5-fluoro-2'-methoxy-[1,1'- biphenyl]-3-carboxamido)propanoic acid (48 mg). 1H NMR (600 MHz, DMSO) δ 8.98-8.90 (m, 1H ), 7.84-7.77 (m, 1H), 7.66-7.56 (m, 1H), 7.52-7.45 (m, 1H), 7.43-7.33 (m, 2H), 7.19-7.11 (m, 1H), 7.09-7.00 (m, 1H), 3.78 (s, 3H), 3.75-3.69 (m, 1H), 3.56-3.44 (m, 2H). LC-MS (MH+): m/z = 333.5 tR (min, Method A) = 0.46 The following compounds were synthesized in a similar way as example Compound 5j: Compound 5k (R)-2-amino-3-(3-fluoro-5-isobutoxybenzamido)propanoic acid
Figure imgf000441_0001
(R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5- isobutoxybenzamido)propanoate. LC-MS (MH+): m/z = 299.4 tR (min, Method A) = 0.46 Compound 5l (R)-2-amino-3-(5,5'-difluoro-2'-methoxy-[1,1'-biphenyl]-3-carboxamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(5,5'-difluoro-2'- methoxy-[1,1'-biphenyl]-3-carboxamido)propanoic acid
Figure imgf000442_0001
Step 1: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(5,5'-difluoro-2'-methoxy-[1,1'- biphenyl]-3-carboxamido)propanoate
Figure imgf000442_0002
To a oven-dried microwave tube, equipped with a magnetic stir bar, was added benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-fluorobenzamido)propanoate (100 mg, 0.189 mmol), (5-fluoro-2-methoxyphenyl)boronic acid (48.2 mg, 0.283 mmol), Pd(dppf)Cl2 (27.6 mg, 0.038 mmol), and K2CO3 (91 mg, 0.661 mmol). The tube was sealed with a microwave cap and evacuated and backfilled with argon (this process was repeated a total of three times). Then degassed dioxane/water (4:1; 5mL) was added and the reaction was heated to 100 °C in a microwave for 1 hour. Ethyl acetate (50 mL) and 1M HCl (50 mL) were added and the phases were separated. The aqueous phase was then extracted with ethyl acetate (2x25 mL), and the ethyl acetate phases were combined, dried over MgSO4, filtered, and concentrated. Purification by combiflash afforded benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(5,5'-difluoro-2'-methoxy-[1,1'- biphenyl]-3-carboxamido)propanoate (56 mg). LC-MS (MH+): m/z = 575.5 tR (min, Method A) = 0.87 Step 2: (R)-2-amino-3-(5,5'-difluoro-2'-methoxy-[1,1'-biphenyl]-3- carboxamido)propanoic acid
Figure imgf000443_0001
A solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-(5,5'-difluoro-2'-methoxy-[1,1'- biphenyl]-3-carboxamido)propanoate (56 mg, 0.097 mmol) in 10 mL methanol:water (9:1) was passed through the H-Cube (1 bar, 1 ml/min) at room temperature. The reslting solution was concentrated to afford (R)-2-amino-3-(5,5'-difluoro-2'-methoxy- [1,1'-biphenyl]-3-carboxamido)propanoic acid (19 mg). 1H NMR (600 MHz, DMSO) δ 9.06-8.99 (m, 1H), 7.86-7.82 (m, 1H), 7.67-7.62 (m, 1H), 7.55-7.50 (m, 1H), 7.28 (dd, J = 9.2, 3.2 Hz, 1H), 7.23 (ddd, J = 9.1, 8.1, 3.2 Hz, 1H), 7.15 (dd, J = 9.1, 4.6 Hz, 1H), 3.81-3.73 (m, 4H), 3.55-3.46 (m, 2H). LC-MS (MH+): m/z = 351.5 tR (min, Method A) = 0.47 Compound 5m (R)-2-amino-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(pentan- 3-yl)benzamido)propanoic acid
Figure imgf000444_0001
Step 1: tert-butyl 3-fluoro-5-(pentan-3-yl)benzoate and tert-butyl 3-fluoro-5-(pentan-2- yl)benzoate
Figure imgf000444_0002
To a microwave tube (which was beforehand oven-dried), equipped with a magnetic stirring bar, was charged with Pd(OAc)2 (16.3 mg, 0.073 mmol) and Cphos (99 mg, 0.025 mmol. The vessel was evacuated and backfilled with argon (this process was repeated a total of 3 times). Then tert-butyl 3-bromo-5-fluorobenzoate (250 mg, 0.909 mmol) and degassed THF (1.8 mL) were added via syringe. The solution was cooled to 0 °C in an ice bath and a THF solution of pentan-3-ylzinc(II) bromide (2.18 mL, 1.090 mmol, 0.5 M in THF) was added dropwise. The ice bath was removed and the resulting solution was stirred at room temperature for 30 minutes. The reaction was diluted with 1M HCl (50 mL) and ethyl acetate (50 mL). The aqueous phase was extracted with ethyl acetate (1x50 mL), combined with the other ethyl acetate fraction, dried over MgSO4, concentrated and purified by combiflash to afford tert-butyl 3-fluoro-5-(pentan-3- yl)benzoate and tert-butyl 3-fluoro-5-(pentan-2-yl)benzoate (137 mg) as a 1.7:1 unseparable mixture. Step 2: 3-fluoro-5-(pentan-3-yl)benzoic acid and 3-fluoro-5-(pentan-2-yl)benzoic acid
Figure imgf000445_0001
The mixture of tert-butyl 3-fluoro-5-(pentan-3-yl)benzoate and tert-butyl 3-fluoro-5- (pentan-2-yl)benzoate (124 mg 0.466 mmol) was dissolved in DCM (3 mL) and TFA (2 mL) and stirred at room temperature for 30 minutes. The contents were concentrated and left on lyophilizised to afford 3-fluoro-5-(pentan-3-yl)benzoic acid and 3-fluoro-5- (pentan-2-yl)benzoic acid (70 mg) as unseparable mixture. Step 3: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(pentan-3- yl)benzamido)propanoate and benzyl (2R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro- 5-(pentan-2-yl)benzamido)propanoate
Figure imgf000445_0002
A solution of 3-fluoro-5-(pentan-3-yl)benzoic acid and 3-fluoro-5-(pentan-2-yl)benzoic acid (70 mg, 0.333 mmol) and (R)-3-3(benzyloxy)carbonyl)amino)-3-oxopropan-1- aminium chloride (146 mg, 0.400 mmol) in DMF (1.33 mL) was treated with HATU (152 mg, 0.400 mmol) and DIPEA (174 μl, 7.82 mmol) and the solution was stirred at room temperature for 2 hours. Ethyl acetate (100 mL) was added and the organics were washed with water (2 x 100 mL), followed by evaporation of the ethyl acetate on rotavap. Purification by Combi Flash afforded the benzyl (R)-2- (((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoate and benzyl (2R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(pentan-2- yl)benzamido)propanoate in 122 mg (70% yield) as an unseparable mixture. Step 4: (R)-2-amino-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoic acid and (2R)-2- amino-3-(3-fluoro-5-(pentan-2-yl)benzamido)propanoic acid
Figure imgf000446_0001
A solution of benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(pentan-3- yl)benzamido)propanoate and benzyl (2R)-2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro- 5-(pentan-2-yl)benzamido)propanoate (120 mg, 0.231 mmol) in 23 mL methanol:water (9:1) was passed through the H-Cube (1 bar, 1 ml/min) at room temperature to afford (R)-2-amino-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoic acid and (2R)-2-amino-3- (3-fluoro-5-(pentan-2-yl)benzamido)propanoic acid (53 mg) as a regioisomeric mixture. The regioisomers were separated by prep HPLC (Method cc). (R)-2-amino-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoic acid: 1H NMR (600 MHz, DMSO) δ 8.96 (s, 1H), 8.28 (s, 1H), 7.58-7.45 (m, 2H), 7.25-7.14 (m, 1H), 3.82-3.74 (m, 1H), 3.59-3.46 (m, 2H), 2.45-2.36 (m, 1H), 1.71-1.59 (m, 2H), 1.58-1.45 (m, 2H), 0.74-0.64 (m, 6H). LC-MS (MH+): m/z = 297.5 tR (min, Method B) = 061 Compound 5n (R)-2-amino-3-(3-fluoro-5-morpholinobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5- morpholinobenzamido)propanoic acid
Figure imgf000447_0001
A solution of 3-fluoro-5-morpholinobenzoic acid hydrochloride (250 mg, 0.955 mmol) and (R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropan-1-aminium chloride (418 mg, 1.146 mmol) in DMF (3.0 mL) was treated with HATU (436 mg, 1.146 mmol) and DIPEA (667 μl, 3.82 mmol) and the solution was stirred at room temperature for 48 hours. Ethyl acetate (100 mL), water (50 mL), and brine (50 mL) were added and the phases were separated. The ethyl acetate phase was washed with water (50 mL) and brine (50 mL), and the aqueous phase was then extracted with ethyl acetate. The ethyl acetate phases are combined and concentrated. Purification by combiflash afforded (R)- 2-amino-3-(3-fluoro-5-morpholinobenzamido)propanoic acid (496 mg). LC-MS (MH+): m/z = 536.7 tR (min, Method A) = 0.77 Step 2: (R)-2-amino-3-(3-fluoro-5-morpholinobenzamido)propanoic acid
Figure imgf000447_0002
A solution of (R)-2-amino-3-(3-fluoro-5-morpholinobenzamido)propanoic acid (100 mg, 0.187 mmol) in 20 mL methanol:water (9:1) was passed through the H-Cube (1 bar, 1 ml/min) at room temperature. The reslting solution was concentrated to afford (R)-2- amino-3-(3-fluoro-5-morpholinobenzamido)propanoic acid (20 mg). 1H NMR (600 MHz, DMSO) δ 8.81-8.74 (m, 1H), 7.98 (br s, 2H), 7.24 (s, 1H), 7.05-7.00 (m, 1H), 6.95-6.90 (m, 1H), 3.78-3.66 (m, 5H), 3.60-3.48 (m, 2H), 3.22-3.14 (m, 4H), LC-MS (MH+): m/z = 312.5 tR (min, Method B) = 0.42 Compound 5o (R)-2-amino-3-(3-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1,3,5-trimethyl- 1H-pyrazol-4-yl)benzamido)propanoic acid
Figure imgf000448_0001
Step 1: methyl 3-bromo-5-iodobenzoate
Figure imgf000448_0002
A round bottom flask was charged with 3-bromo-5-iodo-benzoic acid (8.00 g, 24.5 mmol) and methanol (50 mL). Thionyl chloride (5.36 mL, 73.4 mmol) was slowly added at 0 °C and the reaction mixture was heated at 40 °C for 18 hours. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with aqueous NaHCO3. Removal of the solvent under reduced pressure gave the methyl 3- bromo-5-iodobenzoate. Step 2: methyl 3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate
Figure imgf000449_0001
To a mixture of methyl 3-bromo-5-iodobenzoate (1.05 g, 3.08 mmol), 1,3,5-trimethyl-4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (800 mg, 3.39 mmol), K2CO3 (468 mg, 3.39 mmol), and Pd(dppf)Cl2-CH2Cl2 (151 mg, 0.185 mmol) was added degassed dioxane/water (10:1, 5.0 mL), and the mixture was heated in microwave at 90 °C for 2h under argon atmosphere. Additional 1ml of dioxane/water was added and stirred overnight with conventional heating at 80 °C, under argon atmosphere. After cooling to room temperature the mixture was diluted with water (50 mL) and brine (25 mL) and ethyl acetate (100 mL). The ethyl acetate phase was washed with water (50 mL) and the water phase was extracted with ethyl acetate (2x50 mL), and the combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by combi flash afforded the methyl 3-bromo-5-(1,3,5-trimethyl-1H-pyrazol- 4-yl)benzoate (222 mg). LC-MS (MH+): m/z = 325.4 tR (min, Method A) = 0.72 Step 3: 3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid
Figure imgf000449_0002
A solution of methyl 3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoate (222 mg, 0.687 mmol), LiOH (82 mg, 3.43 mmol) in MeOH (2.0 ml), THF (2.0 ml), and water (2.0 ml) was stirred at room temperature for 1.5 hours. The mixture was adjusted to pH = 1- 2 with HCl (2M) and concentrated in vacuo, to remove MeOH and THF. The residue was diluted with ethyl acetate (25 mL) and water (25 mL) and the phases were separated. The water phase was extracted with ethyl acetate (2x25 mL), and the combined organic phase was washed with brine (1x30 mL), dried over Na2SO4, filtered and concentrated to afford 3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (142 mg) Step 4: benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-(1,3,5-trimethyl-1H- pyrazol-4-yl)benzamido)propanoate
Figure imgf000450_0001
A solution of 3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4-yl)benzoic acid (142 mg, 0.459 mmol) and (R)-3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropan-1-aminium chloride (184 mg, 0.505 mmol) in DMF (2.0 mL) was treated with HATU (192 mg, 0.505 mmol) and DIPEA (241 μl, 1.378 mmol) and the solution was stirred at room temperature for 18 hours. Ethyl acetate (50 mL), water (50 mL) and brine (10 mL) was added and the phases were separated. The ethyl acetate phase was washed with water (2 x 30 mL), dried over Na2SO4, filtered, and concentrated on rotavap. Purification by Combiflash afforded benzyl (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-(1,3,5- trimethyl-1H-pyrazol-4-yl)benzamido)propanoate (286 mg). LC-MS (MH+): m/z = 619.6 tR (min, Method A) = 0.81 Step 5: (R)-2-amino-3-(3-bromo-5-(1,3,5-trimethyl-1H-pyrazol-4- yl)benzamido)propanoic acid
Figure imgf000451_0001
(R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoate (30 mg, 0.051 mmol) in 15 mL THF:water (5:1) was passed through the H-Cube (Pd/c, 10 bar, 1 ml/min) at room temperature. The resulting solution was concentrated to afford (R)-2-amino-3-(3-bromo-5-(l,3,5-trimethyl-lH- pyrazol-4-yl)benzamido)propanoic acid (9 mg).
XH NMR (600 MHz, DMSO) 6 8.73-8.67 (m, 1H), 8.33-8.25 (br s, 2H), 7.79-7.75 (m, 1H), 7.72 (s, 1H), 7.55-7.50 (m, 1H), 7.45-7.41 (m, 1H), 4.14-4.06 (m, 1H), 3.81-3.65 (m, 5H), 2.21 (s, 3H), 2.12 (s, 3H). LC-MS (MH+): m/z = 317.5 tR (min, Method B) = 0.38
Compound 5p
(R)-2-amino-3-(3-bromo-5-(l,3,5-trimethyl-lH-pyrazol-4-yl)benzamido)propanoic acid
Figure imgf000451_0002
A solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5-(l,3,5-trimethyl-lH- pyrazol-4-yl)benzamido)propanoate (257 mg, 0.435 mmol) in HBr/AcOH (10 mL, 33%) was stirred at 50 °C for 20 hours. The solvent was concentrated to remove solvents and to obtain (R)-2-amino-3-(3-bromo-5-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoic acid 230 mg (99% yield) as HBr salt. 1H NMR (600 MHz, DMSO) δ 8.89-8.82 (m, 1H), 8.35-8.26 (m, 3H), 7.97-7.95 (m, 1H), 7.72-7.69 (m, 1H), 7.63-7.60 (m, 1H), 4.14-4.08 (m, 1H), 3.81-3.65 (m, 5H), 2.23 (s, 3H), 2.14 (s, 3H), LC-MS (MH+): m/z = 397.4 tR (min, Method B) = 0.48 Compound 5q (2R)-2-amino-3-(3-fluoro-5-(1-phenoxyethyl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (2R)-2-amino-3-(3-fluoro-5-(1- phenoxyethyl)benzamido)propanoic acid
Figure imgf000452_0001
Step 1: 3-bromo-5-fluorobenzoic acid
Figure imgf000452_0002
To a solution of methyl 3-bromo-5-fluorobenzoate (500 mg, 2.15 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (270 mg, 6.44 mmol). The mixture was stirred at 20 °C for 2 hours. The mixture was concentrated to remove MeOH. Then HCl (2 M) was added to adjust pH=3~4 and the solid was collected and dried to give 3- bromo-5-fluorobenzoic acid (450 mg). 1H NMR (400MHz, DMSO-d6) δ 7.90 - 7.84 (m, 2H), 7.71 - 7.66 (m, 1H). Step 2: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-bromo-5- fluorobenzamido)propanoate
Figure imgf000453_0001
To a solution of 3-bromo-5-fluorobenzoic acid (450 mg, 2.05 mmol) and (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (825 mg, 2.26 mmol, HCl salt) in DMF (10 mL) was added TBTU (990 mg, 3.08 mmol) and DIPEA (797 mg, 6.16 mmol). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-bromo- 5-fluorobenzamido)propanoate (1 g). 1H NMR (400MHz, CDCl3) δ 7.62 (s, 1H), 7.38 - 7.29 (m, 12H), 6.98 (br, 1H), 6.02 (d, J = 6.4 Hz, 1H), 5.24 - 5.15 (m, 2H), 5.15 - 5.06 (m, 2H), 4.66 - 4.50 (m, 1H), 3.95 - 3.75 (m, 2H). Step 3: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- phenoxyethyl)benzamido)propanoate
Figure imgf000453_0002
A mixture of 2-phenoxypropanoic acid (47 mg, 283.36 µmol), (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-bromo-5-fluorobenzamido)propanoate (100 mg, 188.9 µmol) , NiCl2.glyme (4 mg, 18.89 µmol), 4,4'-di-tert-butyl-2,2'-bipyridyl (8 mg, 28.34 µmol), (IR[DF(CF3)PPY]2(DTBPY))PF6 (2.0 mg, 1.89 µmol) and Cs2CO3 (92 mg, 283.36 µmol) in DMF (3 mL) in a tube was stirred at 20 °C for 16 hours under 72W blue LED Strip Light. The reaction mixture was quenched with NaHCO3 (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 10%) to give 60 mg crude product, which was further purified by preparative HPLC (Method BB) to give (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- phenoxyethyl)benzamido)propanoate (40 mg). 1H NMR (400MHz, CDCl3) δ 7.54 (s, 1H), 7.40 - 7.27 (m, 11H), 7.25 - 7.17 (m, 3H), 6.94 - 6.88 (m, 1H), 6.87 - 6.82 (m, 2H), 6.80 - 6.68 (m, 1H), 5.93 (d, J = 6.4 Hz, 1H), 5.32 (q, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.15 – 5.10 (m, 2H), 4.64 - 4.54 (m, 1H), 3.97 - 3.86 (m, 1H), 3.86 - 3.77 (m, 1H), 1.62 (d, J = 6.8 Hz, 3H). Step 4: (2R)-2-amino-3-(3-fluoro-5-(1-phenoxyethyl)benzamido)propanoic acid
Figure imgf000454_0001
A mixture of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1- phenoxyethyl)benzamido)propanoate (37 mg, 64.84 µmol), Pd/C (20 mg, 10% Pd, 50% water) in AcOH (5 mL) was degassed and purged with H23 times, and then the mixture was stirred at 20 °C for 1 hour under H2 atmosphere(15 Psi). The reaction mixture was filtered through celite and the filtrate was concentrated to give (2R)-2-amino-3-(3- fluoro-5-(1-phenoxyethyl)benzamido)propanoic acid (21 mg). 1H NMR (400MHz, DMSO-d6) δ 8.88 (br, 1H), 7.79 (s, 1H), 7.56 (d, J=9.2 Hz, 1H), 7.41 (d, J = 9.2 Hz, 1H), 7.26-7.19 (m, 2H), 6.95-6.83 (m, 3H), 5.57 (q, J = 6.0 Hz, 1H), 3.81-3.59 (m, 1H), 3.51-3.49 (m, 2H), 1.56 (d, J = 6.0 Hz, 3H). LC-MS (MH+): m/z = 347.1 tR (min, Method D) = 1.870 [α]20,D = 10 (c = 1 mg/mL, MeOH). Compound 5r (2R)-2-amino-3-(3-fluoro-5-(methoxy(phenyl)methyl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (2R)-2-amino-3-(3-fluoro-5- (methoxy(phenyl)methyl)benzamido)propanoic acid
Figure imgf000455_0001
Step 1: ethyl 3-fluoro-5-iodobenzoate
Figure imgf000455_0002
To a solution of 3-fluoro-5-iodobenzoic acid (5.0 g, 18.80 mmol) in EtOH (50 mL) was added SOCl2 (4.47 g, 37.59 mmol) at 0°C. The mixture was stirred at 20°C for 16 hours. To the mixture was added SOCl2 (4 mL) and the reaction was stirred at 50°C for another 19 hours. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) twice to give ethyl 3-fluoro-5-iodobenzoate (6 g, crude). 1H NMR (400MHz, CDCl3) δ 8.17 (t, J = 1.6 Hz, 1H), 7.72 - 7.66 (m, 1H), 7.64 - 7.59 (m, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). Step 2: ethyl 3-fluoro-5-(hydroxy(phenyl)methyl)benzoate
Figure imgf000456_0001
To a solution of ethyl 3-fluoro-5-iodobenzoate (500 mg, crude) in THF (9 mL) was added dropwise i-PrMgCl.LiCl (1.3 M in THF, 1.83 mL) at -78 °C. After addition, the mixture was stirred at this temperature for 30 minutes, and then benzaldehyde (180 mg, 1.70 mmol) in THF (1 mL) was added dropwise at -78 °C. The resulting mixture was stirred at 15 °C for 1 hour. The reaction was quenched with saturated solution NH4Cl (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. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 10%) to give ethyl 3-fluoro-5-(hydroxy(phenyl)methyl)benzoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.87 (s, 1H), 7.63 - 7.57 (m, 1H), 7.40 - 7.28 (m, 6H), 5.87 (d, J = 3.2 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 2.52 (d, J = 3.2 Hz, 1H), 1.39 (t, J = 7.2 Hz, 3H). Step 3: ethyl 3-fluoro-5-(methoxy(phenyl)methyl)benzoate
Figure imgf000457_0001
To a solution of ethyl 3-fluoro-5-(hydroxy(phenyl)methyl)benzoate (350 mg, 1.28 mmol) in DMF (8 mL) was added NaH (77 mg, 1.91 mmol, 60% purity in mineral oil) at 0 °C, the mixture was stirred at this temperature for 30 minutes, and then MeI (362 mg, 2.55 mmol) was added at 0 °C. The resulting mixture was stirred at 60 °C for 3 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (10 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 10%) to give ethyl 3-fluoro-5- (methoxy(phenyl)methyl)benzoate (260 mg, crude). Step 4: 3-fluoro-5-(methoxy(phenyl)methyl)benzoic acid
Figure imgf000457_0002
To a solution of ethyl 3-fluoro-5-[methoxy(phenyl)methyl]benzoate (260 mg, 901.8 µmol) in the mixture of H2O (2 mL) and MeOH (2 mL) was added LiOH.H2O (113 mg, 2.71 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated to move MeOH, then added HCl (2 M) to adjust pH=3~4, extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5-(methoxy(phenyl)methyl)benzoic acid (230 mg, crude). Step 5: (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5- (methoxy(phenyl)methyl)benzamido)propanoate
Figure imgf000458_0001
To a solution of 3-fluoro-5-(methoxy(phenyl)methyl)benzoic acid (230 mg, crude) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (355 mg, 972.11 µmol, HCl salt) in DMF (6 mL) was added TBTU (426 mg, 1.33 mmol) and DIPEA (343 mg, 2.65 mmol). The mixture was stirred at 20 °C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) to give 550 mg crude compound, which was purified by preparation HPLC (Method BB) to give (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3- fluoro-5-(methoxy(phenyl)methyl)benzamido)propanoate (280 mg). 1H NMR (400MHz, CDCl3) δ 7.54 (s, 1H), 7.38 - 7.28 (m, 15H), 7.25 - 7.18 (m, 2H), 6.76 (br, 1H), 5.95 (d, J = 6.8 Hz, 1H), 5.24 (s, 1H), 5.20 (s, 2H), 5.11 (s, 2H), 4.62 - 4.54 (m, 1H), 3.95 – 3.75 (m, 2H), 3.38 (s, 3H). Step 6: (2R)-2-amino-3-(3-fluoro-5-(methoxy(phenyl)methyl)benzamido)propanoic acid
Figure imgf000458_0002
A mixture of (2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5- (methoxy(phenyl)methyl)benzamido)propanoate (80 mg, 140.2 µmol) , Pd/C (20 mg, 10% Pd, 50% water) in AcOH (3 mL) was degassed and purged with H23 times, and then the mixture was stirred at 20 °C for 1 hour under H2 atmosphere(15 Psi). The reaction mixture was filtered through celite and the filtrate was concentrated. The residue was purified by preparation HPLC (Method DD) to give (2R)-2-amino-3-(3-fluoro-5- (methoxy(phenyl)methyl)benzamido)propanoic acid (20 mg). 1H NMR (400MHz, DMSO-d6) δ 8.81 (br s, 1H), 7.72 (s, 1H), 7.57-7.53 (m, 1H), 7.41-7.32 (m, 5H), 7.29-7.23 (m, 1H), 5.42 (s, 1H), 3.71-3.60 (m, 1H), 3.53-3.44 (m, 2H), 3.29 (s, 3H). LC-MS (MH+): m/z = 347.2 tR (min, Method D) = 1.811 [α]20,D = 11 (c = 2 mg/mL, MeOH). In vitro and in vivo characterization of compounds of the invention Example 1: Affinity data of the exemplified compounds of the invention for the glycine binding site of the NMDA receptor Scintillation proximity assay (SPA): To determine the affinity of the compounds of the present invention for the glycine binding site of the NMDA receptor a SPA was used. The assay was run in a 384-plate format (OptiPlate-384) where each well contained a mix of 5 µL of test compound, 5 µL NR1s1s2 (ligand binding domains of the NMDA receptor, MW 35.6 kDa, 0.075 ug/well final), 5 µL [3H]-MDL-105,519 (radiolabelled, high affinity N-methyl-D-aspartate (NMDA) glutamate receptor antagonist which binds selectively to the glycine site, final concentration 5 nM, Kd =1.3 nM), 5 µL streptavidin coated imaging beads (Perkin Elmer cat. No.: RPNQ0273, 8 ug/well). The assay buffer contained 100 mM HEPES-NaOH, 150 mM NaCl, 1 mM EDTA, 10% glycerol at pH 7.4 in ultra-pure water. Non-specific binding was defined by inclusion of 10 µM L-689,560 (highly potent NMDA antagonist) and total binding by 1% DMSO. Following 30 minutes incubation in the dark (shaker, Multi- microplate Genie), the SPA beads were allowed to settle for 3 hours after which the signal is read on a Viewlux instrument (Perkin Elmer). Normalized data were used to calculate Ki values. Table 2: SPA Ki (nM) values of the compound of the invention
Figure imgf000460_0002
Figure imgf000460_0001
Figure imgf000460_0003
Figure imgf000461_0001
Figure imgf000461_0002
Figure imgf000461_0003
Table 2 shows that compounds of formula I have affinity to the glycine site of the NMDA receptor.
Example 2: In vivo exposure data
In vivo procedure:
Brain disposition of test compound was evaluated in male Sprague Dawley rats (standard body weight range). Briefly, discrete (nominal dose: 2 mg/kg, 2 ml/kg) or cassetted (nominal dose: 1 mg/kg/compound, 2 ml/kg ) test compounds were administered by intravenous bolus injection (formulated in 10% hydroxypropyl-|3- cyclodextrin or 10-20% Captisol, pH=3) or discrete (nominal dose: 5 mg/kg/compound, 10 ml/kg ) test compounds were administered by p.o. gavage (formulated in 10% hydroxypropyl-p-cyclodextrin, pH=4)
Sample collection:
Serial blood samples were collected from a lateral tail vein at designated time points (n=3 per time point) then rats were put under deep isoflurane induced anesthesia prior to removal of brains (n=3 per time point). Blood was collected into K3-EDTA-coated tubes and the samples were gently turned upside-down to ensure a homogenous sample. The tubes were centrifuged at 3300 x g for 10 minutes at max 4 °C and plasma samples were transferred to Micronic tubes. Brain samples were dissected once the animal had been sacrificed, slightly "dipped" on filter paper to remove blood overflow on the outside and transferred into Covaris AFA tubes. Plasma and brain samples were stored at -80°C until analysis.
Sample preparation:
Seven calibration standards and three QC samples were prepared in plasma and brain homogenate, respectively, in the concentration range 10-10000 ng/mL. Blank samples (control matrix with internal standard) were prepared and treated in the same way as calibration standards. Priorto analysis, the brain samples were homogenized with milliQ water 1:4 (w/v) using a Covaris focused-ultrasonicator. Study samples with expected concentration above upper limit of quantification were diluted with blank matrix. Brain homogenate and plasma from study samples, calibration standards, quality controls and blank samples were subsequently treated with the same extraction procedure, i.e. protein precipitation by adding 150 µL acetonitrile with internal standard (Tolbutamide) to 25 μL of sample. Samples were centrifuged and the supernatant from each sample was diluted 1:1 with water to lower the content of organic solvent. LC-MS/MS: Samples were analyzed using an AB Sciex API4000 triple quadrupole (TQ) mass spectrometer operated in positive and negative electrospray ionization and MS/MS mode (multiple reaction monitoring, MRM). The mass spectrometer was coupled to a Waters Acquity UPLC equipped with a Waters Acquity UPLC HSS C18 SB (1.7 µm, 30 mm x 2.1 mm) analytical column. Chromatographic separation was achieved by a 3-minute gradient starting with 98% mobile phase A (0.1% Formic Acid in water) and 2% mobile phase B (0.1% Formic Acid in Acetonitrile) increasing to 95% mobile phase B. Flow rate was 0.6 mL/min and the column temperature was 40 oC. MRM transitions (m/z) were as follows: 380→248, 350→263, Tolbutamide: 269→106 (neg) and 271→155 (pos). Quantification was performed by linear regression, 1/x2 weighting. The blood brain deposition data is shown in Table 3 below. Table 3: blood brain deposition data for compounds of the invention
Figure imgf000463_0001
*Compounds were administered by iv bolus and B/P’s taken at 0.5 h. **Compound was administered by po gavage and B/P’s taken at 0.33 h. BLOQ: Below Level of Quantification Example 3: Resting state Electroencephalography (rsEEG) in rats On the day of surgery, rats (270-300g) were anesthetized with a 0.25 ml/100g subcutaneous (SC) injection of 1:1 hypnorm/Dormicum and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with blunt ear bars. Marcain (0.2 ml SC) was injected under the scalp, and gel (Neutral Opthta Eye Gel) put on the eyes to prevent the mucous membrane drying out. Holes were burred in the scull to allow for placement of three depth electrodes (E363-series; PlasticsOne, Roanoke, VA, USA) in medial prefrontal cortex (mPFC) (AP: 3.0 mm from the bregma suture, ML: -0.7 mm from the sagittal suture and DV: 3.0 mm from the dura), Nucleus accumbens (NAc): (AP + 1.6 mm from the bregma suture, ML: -1.0 mm from the sagittal suture, DV: -6.7mm from the dura) and thalamus (AP: -2.8 mm from the bregma suture, ML: +0.7 mm from the sagittal suture and DV: 4.4 mm from the dura) and three screw electrodes above the auditory cortex (A1) (AP: -4.8 mm from the bregma suture, ML: +6.4 mm from the sagittal suture), a reference electrode (AP: +8.0 mm and ML: −2.0 mm), and a ground electrode (AP: -5 mm, ML: +5 mm). During surgeries, nails were cut to prevent rats from scratching wounds following surgery. After completion of surgeries, rats were placed under warming lamps until recovery of consciousness (maximum 4 hours). Water soaked food pellets were placed in the home cage, so the rat easily and quickly could start feeding. Extra muesli was supplied to aid the recovery. Rats were treated with Norodyl and Noromox for 5 days in total and closely observed during a 10-14-day post- surgery recovery period. Animal bodyweights were recorded daily. No rats lost more than 10 % of their pre-surgery bodyweight. Sutures were removed after 7-10 days. At the end of experiments electrical lesions were performed in all recording electrodes and brains were cut for visual microscopy inspection of electrode placement. The differences between depth- and screw-electrode impedances were handled by investigating relative power changes and common-mode noise sources were reduced from recording in shielded boxes and excluding power estimates around 50, 100, and 150 Hz from analyses.
Rats were handled daily and habituated to recording box the week before recording sessions. Recordings were performed during the dark phase of the I ight/da rk cycle. At 8 AM, rats (400-500 g) were individually transferred to an acrylic chamber (30 cm wide 45 cm deep 55 cm high) placed within an electrically shielded sound-proof box (90 cm wide 55 cm deep 65 cm high) and were tethered to a six-pin wire suspended from a rotating swivel, allowing free movement within the recording box. There was a 2-hour habituation period followed by 45 minutes of baseline recording, where after rats were injected subcutaneously with 10% 2-Hydroxypropyl-|3-cyclodextrin (HPbetaCD, vehicle), 3 mg/kg compound 3f, 10 mg/kg compound 3f or 30 mg/kg compound 3f and left in the box for two more hours. Rats only went through recording sessions once a week with at least six days between recordings to allow for wash-out of compounds. The analog local field potential and electrocorticogram (LFP and ECoG) signals were amplified and bandpass filtered at 0.01-300 Hz (Precision Model 440; Brownlee, Palo Alto, CA, USA) and converted to a digital signal at a sampling rate of 1 kHz (CED Power 1401, Power 1 (625 kHz, 16 bit) and CED Expansion ADC16; CED, Cambridge, England). Video recordings were processed in EthoVision. The analysis of the locomotor behaviour was based on the recorded accelerometer signal. The accelerometer signal thresholds were validated to detect periods of locomotor activity (Active) and inactivity (Inactive).
The development of the locomotive state-detection algorithm and the state-specific pharmaco-EEG analyses were carried out in MATLAB R2017a (The MathWorks, Inc., Natick, MA, USA) using functions from the sigTOOL toolbox.
As shown in Figure 1, systemic administration of compound 3f at 10 and 30 mg/kg shows a clear effect on High Frequency Oscillations in the range of 130-160 Hz (HFO) in the nucleus accumbens compared to vehicle (10% HPbetaCD). Thus, after a subcutaneous injection of 10 and 30 mg/kg of compound 3f, a significant effect can be observed on the neuronal activity in the nucleus accumbens. Ketamine and d-cycloserine have been shown to exhibit similar effects (I .H. Hansen et. al., Eur J Neurosci., 2019, 50, 1948- 1971).
Example 4- Maximal Electro Shock Threshold
Naive rats were acclimatised to the procedure room in their home cages, with food and water available ad libitum. All rats were weighed at the beginning of the study and randomly assigned to treatment groups. The individual treatment groups were dosed with either 10% hydroxypropyl-|3-cyclodextrin (vehicle) or compound 3f (3, 10, or 30 mg/g). The dosing of the animals was performed by subcutaneous injection 30 minutes before test according to treatment groups. Rats were individually assessed for the production of a tonic hind limb extensor seizure using a Hugo Sachs Electronik stimulator, which delivered an adjustable constant current (1-300 mA) of 0.3 seconds duration via corneal electrodes. The stimulus intensity was varied, from a typical baseline of 25mA, by an 'up and down' method of shock titration. Thus, the first rat within a treatment group was given a shock at the expected or estimated seizure threshold (CC50) current, that is, the current producing tonic hind limb extensor seizure in 50% of animals. For subsequent animals, the stimulus intensity was lowered or raised in log 0.06 :10A(l+x*0.06) mA intervals if the preceding rat did or did not show tonic hind limb extension, respectively. This procedure continued for all rats within a treatment group. Data generated from treatment groups of n=12-16 were used to calculate the CC50 values according to the method of Kimball et al. (Kimball A, Burnett W, Doherty D. Chemical protection against ionizing radiation. I. Sampling methods for screening compounds in radiation protection studies with mice. Radiat Res. 1957;7( 1) :1— 12). Significant differences between drug-treated animals and vehicle were assessed by a one-way ANOVA with Dunnett's post hoc test. Significant differences between drug-treated animals and vehicle were assessed according to Litchfield and Wilcoxon (Litchfield J, Wilcoxon F. A simplified method for evaluating dose-effect experiments. J Pharmacol Exp Ther. 1949; 96(1): 99-113). As shown in Figure 2, administration of compound 3f dosed at 10 mg/kg subcutaneously showed effects on the maximal electro shock threshold as compared to vehicle. Thus, the administration of the compound 3f resulted in a significant change in electrophysiological properties of the brain in rat. NMDA receptor antagonists are known to demonstrate anticonvulsive properties in various rodent seizure/epilepsy models (Parsons CG et.al., Neuropharmacology, (1995), 34, 1239; P. Wlaz et. al., Epilepsia, (1996), 37, 610).

Claims

CLAIMS 1. A compound according to general formula I
Figure imgf000468_0001
wherein R1 is selected from the group consisting of hydrogen and halogen; R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6- membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3- C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1- C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, halo(C1-C4)alkyl, hydroxy(C1- C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1- C4)alkoxy; or R1 and R2, or R2 and R3, together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaromatic ring is optionally substituted with one or more substituents independently selected from (Ci-C4)a Ikyl, and wherein said 5- or 6-membered heterocyclic ring is optionally substituted with one or more substituents independently selected from (Ci-C4)a I kyl and oxo; or a pharmaceutically acceptable salt thereof; with the proviso that at least one of Ri, R2, R3 and R4does not represent hydrogen; with the proviso that when R2 represents halogen or methyl, at least one of Ri, R3 and R4 does not represent hydrogen; with the proviso that when R3 represents halogen or methyl, at least one of Ri, R2 and R4 does not represent hydrogen; with the proviso that when R4 represents halogen or methyl, at least one of Ri, R2 and R3 does not represent hydrogen; and with the proviso that when R3 represents methyl, R2 or R4 does not represent 3-trifluoromethylphenyl. . The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is selected from the group consisting of hydrogen and fluoro. . The compound according to any one of claims 1 - 2, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro and (Ci-Csja Ikyl.
4. The compound according to any one of claims 1 - 3, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen.
5. The compound according to any one of claims 1 - 4, or a pharmaceutically acceptable salt thereof, wherein
R2 is selected from the group consisting of hydrogen, halogen and (Ci-Ce)a Ikyl and wherein
R4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6- membered heteroaryl, phenyl, (Ci-Ce)a Ikyl, 4 - 6 membered heterocyclyl, (C3- Ce)cycloalkyl and (Ci-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (Ci-Ce)alkyl, 4-6 membered heterocyclyl, (Cs-Cejcycloalkyl and (Ci- C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (Ci-C4)a I kyl, (Ci- C4)alkoxy, (Ci-C4)alkoxy(Ci-C4)alkyl, halo(Ci-C4)alkyl, hydroxy(Ci-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (Ci-C4)a Ikyl.
6. The compound according to any one of claims 1 - 5, or a pharmaceutically acceptable salt thereof, wherein R2 and R4 are not hydrogen.
7. The compound according to any one of claims 1 - 6, or a pharmaceutically acceptable salt thereof, wherein
R1 and R3 are hydrogen;
R2 is selected from the group consisting of halogen and (Ci-Ce)a I kyl;
R4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (Ci-Ce)a Ikyl, 4 - 6 membered heterocyclyl, (Cs-Cejcycloalkyl and (Ci-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (Ci- Ce)a Ikyl, 4-6 membered heterocyclyl, (Cs-Cejcycloalkyl and (Ci-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (Ci-C4)a Ikyl, (Ci-C4)alkoxy, (Ci- C4)alkoxy(Ci-C4)alkyl, ha lo(Ci-C4)a Ikyl, hydroxy(Ci-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (Ci-C4)a I kyl .
8. The compound according to any one of claims 1 - 7, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of 5- or 6-membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (Ci-C4)a Ikyl, (Ci-C4)alkoxy, (Ci- C4)alkoxy(Ci-C4)alkyl, ha lo(Ci-C4)a Ikyl, phenyl, phenoxy and -C(O)NH2.
9. The compound according to any one of claims 1 - 8, or a pharmaceutically acceptable salt thereof, wherein R4 is pyrazolyl, wherein said pyrazolyl is optionally substituted with one or more substituents independently selected from the group consisting of fluor, chloro, (Ci-C4)alkyl, (Ci-C4)alkoxy, (Ci- C2)alkoxy(Ci-C2)alkyl, and halo(Ci-C2)alkyl.
10. The compound according to any one of claims 1 - 7, or a pharmaceutically acceptable salt thereof, wherein R4 is phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (Ci-C4)alkyl, (Ci-C4)alkoxy, (Ci-C2)alkoxy(Ci- C2)alkyl, and halo(Ci-C2)alkyl.
11. The compound according to any one of claims 1 - 10, or a pharmaceutically acceptable salt thereof, wherein R2 is fluoro. The compound according to any one of claims 1 - 11, selected from the list consisting of
(R)-2-amino-3-(3-(4-ethyl-l-methyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-Amino-3-(3-(4-ethyl-l-methyl-lH-pyrazol-3-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethylpyridin-2-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethyl-lH-pyrazol-l-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethyl-lH-pyrazol-l-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethyl-lH-imidazol-l-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-ethyl-lH-imidazol-l-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l-ethyl-lH-imidazol-2-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethyl-2-methyloxazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-ethylisoxazol-5-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-ethyl-2-methyloxazol-5-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethyl-l-methyl-lH-pyrazol-4-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l,4-diethyl-lH-pyrazol-3-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(5-ethyl-3-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethyl-5-methylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l-ethyl-4-methyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethyl-l-methyl-lH-pyrazol-4-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(2'-chloro-6'-ethyl-5-fluoro-[l,l'-biphenyl]-3- ylcarboxamido)propanoic acid,
(R)-2-amino-3-(3-(l,4-diethyl-lH-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(2-methoxypropan-2-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-l-methyl-lH-pyrazol-3- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(4-(methoxymethyl)-l-methyl-lH-pyrazol-5- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(3,5-dimethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-methylcyclopropyl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(3,5-dimethyl-lH-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3,5-dimethyl-4H-l,2,4-triazol-4-yl)-5- fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-(3,5-dimethyl-lH-l,2,4-triazol-l-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l-ethylcyclopropyl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l,2-dimethylcyclopropyl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(diethylamino)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethylpyridin-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-chloro-5-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethylmorpholino)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(2-ethylpiperidin-l-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(2'-chloro-5-fluoro-5'-methyl-[l,l'-biphenyl]-3- ylcarboxamido)propanoic acid,
(R)-2-amino-3-(2'-chloro-5-fluoro-4'-methyl-[l,l'-biphenyl]-3- ylcarboxamido)propanoic acid,
(R)-2-amino-3-(3-(4-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethylpyrimidin-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-chloro-6-methylpyridin-2-yl)-5-fluorobenzamido)propanoic acid,
(-)(2R)-2-amino-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoic acid,
(+)(2R)-2-amino-3-(3-(2-ethylcyclohexyl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-methoxycyclobutyl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(4-ethylpyrimidin-5-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(l-methoxycyclopentyl)benzamido)propanoic acid,
(R)-2-amino-3-(5-(5-ethylisothiazol-4-yl)-2-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(3-methoxyoxetan-3-yl)benzamido)propanoic acid,
(R)-2-amino-3-(2-fluoro-3-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoic acid,
(R)-2-amino-3-(2-fluoro-5-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoic acid,
(R)-2-amino-3-(4-fluoro-3-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l-ethyl-4-methyl-lH-pyrazol-5-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid,
(R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-chloro-l-ethyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(+) (2R)-2-amino-3-(3-(l-ethoxyethyl)-5-fluorobenzamido)propanoic acid,
(-) (2R)-2-amino-3-(3-(l-ethoxyethyl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(5-methylisoxazol-4-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(l-ethyl-lH-imidazol-5-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-(tert-butyl)isoxazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-(l-ethyl-lH-l,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(5-isopropylisoxazol-4-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid,
(-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoic acid,
(+)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
(R)-2-amino-3-(3-(4-ethyl-lH-imidazol-5-yl)-5-fluorobenzamido)propanoic acid
(R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(2-ethyl-4-(trifluoromethyl)pyridin-3-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-ethyl-lH-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(2-ethylthiophen-3-yl)-5-fluorobenzamido)propanoic acid
(R)-2-amino-3-(3-(5-ethylthiazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(5-(hydroxymethyl)-l-methyl-lH-pyrazol-4- yl)benzamido)propanoic acid,
(R)-2-amino-3-(5-fluoro-2'-methyl-[l,l'-biphenyl]-3-ylcarboxamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(4-methyl-2-(trifluoromethyl)pyridin-3- yl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(l-propyl-4-(trifluoromethyl)-lH-pyrazol-5- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-butylbenzamido)propanoic acid,
(R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(4-methylquinoline-6-carboxamido)propanoic acid,
(R)-2-amino-3-(lH-benzo[d]imidazole-5-carboxamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-methylpyrrolidin-2-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(4,4-difluoropyrrolidin-2-yl)-5-fluorobenzamido)propanoic aid,
(R)-2-amino-3-(l-oxo-l,2-dihydroisoquinoline-7-carboxamido)propanoic acid,
(R)-2-amino-3-(2'-ethyl-5-fluoro-[l,l'-biphenyl]-3-carboxamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-isopropyl-lH-l,2,3-triazol-5- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-propyl-lH-l,2,3-triazol-5-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(l-(tert-butyl)-lH-l,2,3-triazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-chloro-l-methyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(l-ethyl-4-(trifluoromethyl)-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-propyl-4-(trifluoromethyl)-lH-pyrazol-5- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(4-(difluoromethyl)-l-ethyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(l-methyl-lH-l,2,3-triazol-5-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(5-isopropyl-3-methylisoxazol-4- yl)benzamido)propanoic,
(R)-2-amino-3-(3-(4-chloro-l-isopropyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(4-chloro-l-propyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-(3,5-dimethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(5-isopropylisothiazol-4-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(4-chloro-l-ethyl-3-methyl-lH-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-methylbenzamido)propanoic acid,
(R)-2-amino-3-(3-ethylbenzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-methoxybenzamido)propanoic acid,
(R)-2-amino-3-(5-fluoro-2'-(methoxymethyl)-[l,l'-biphenyl]-3- carboxamido)propanoic acid,
(R)-2-amino-3-(2'-carbamoyl-5-fluoro-[l,l'-biphenyl]-3-carboxamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-propyl-lH-pyrazol-4-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-(l,5-dimethyl-lH-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid, (R)-2-amino-3-(6-fluoro-l,2,3,4-tetrahydroquinoline-8-carboxamido)propanoic acid,
(R)-2-amino-3-(5-fluoro-2'-methoxy-[l,l'-biphenyl]-3-carboxamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-isobutoxybenzamido)propanoic acid,
(R)-2-amino-3-(5,5'-difluoro-2'-methoxy-[l,l'-biphenyl]-3- carboxamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(pentan-3-yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-morpholinobenzamido)propanoic, (R)-2-amino-3-(3-(l,3,5-trimethyl-lH-pyrazol-4-yl)benzamido)propanoic acid, (R)-2-amino-3-(3-bromo-5-(l,3,5-trimethyl-lH-pyrazol-4- yl)benzamido)propanoic acid,
(R)-2-amino-3-(3-fluoro-5-(l-phenoxyethyl)benzamido)propanoic acid, (R)-2-amino-3-(3-fluoro-5-(methoxy(phenyl)methyl)benzamido)propanoic acid, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 12, and one or more pharmaceutically acceptable carriers or diluents. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 for use as a medicament. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 for use in the treatment of depression. The compound or pharmaceutical composition according to claim 15, wherein the depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus. A method for the treatment of depression comprising the administration of a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 to a patient (e.g. a human patient) in need thereof. The method for the treatment of depression according to claim 18, wherein depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
20. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13 for the manufacture of a medicament for use in the treatment of depression. 21. The use of a compound or pharmaceutical composition according to claim 20, wherein the depression is selected from the group consisting of major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
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WO2023247968A2 (en) 2022-06-23 2023-12-28 University Of Strathclyde Modified amino acids and uses thereof

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