EP4630420A1 - Inhibitors of nlrp3 - Google Patents

Inhibitors of nlrp3

Info

Publication number
EP4630420A1
EP4630420A1 EP23818438.6A EP23818438A EP4630420A1 EP 4630420 A1 EP4630420 A1 EP 4630420A1 EP 23818438 A EP23818438 A EP 23818438A EP 4630420 A1 EP4630420 A1 EP 4630420A1
Authority
EP
European Patent Office
Prior art keywords
alkyl
methyl
dihydropyridazino
oxazin
mmol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23818438.6A
Other languages
German (de)
French (fr)
Inventor
Lea Aurelie BOUCHE
Wolfgang Guba
Georg Jaeschke
Stefanie Katharina MESCH
Angélique PATINY-ADAM
Christian Schnider
Jonathan Martin SHANNON
Sandra Steiner
Andreas Michael TOSSTORFF
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of EP4630420A1 publication Critical patent/EP4630420A1/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to compounds that modulate NLRP3 inhibition.
  • the present invention provides novel compounds of formula I wherein,
  • R 1 is H or alkyl
  • R 2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted with 1- to-2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
  • X is -O-, -CH 2 -, -NH-, or -N(CH 3 )-;
  • W is selected from ring systems A and B
  • R 3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R 1 and R 3 is H and the other is not H;
  • R 4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
  • R 5 is H; or R 4 and R 5 , and the atoms to which they are attached, form i) a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii) a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
  • R 6 is halo, haloalkyl or OH
  • R 7 is H or F; and pharmaceutically acceptable salts thereof.
  • the invention includes all racemic mixtures, all their corresponding enantiomers and/or optical isomers.
  • NLR NOD-like receptor
  • NLRP3 pyrin domain-containing protein 3
  • NLRP3 is an intracellular signaling molecule that senses many pathogen-derived, environmental and host-derived factors. Upon activation, NLRP3 binds to apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). ASC then polymerises to form a large aggregate known as an ASC speck. Polymerised ASC in turn interacts with the cysteine protease caspase-1 to form a complex termed the inflammasome. This results in the activation of caspase- 1, which cleaves the precursor forms of the proinflammatory cytokines IL-ip and IL- 18 (termed pro-IL-ip and pro-IL-18 respectively) to thereby activate these cytokines.
  • ASC caspase activation and recruitment domain
  • Caspase-1 also mediates a type of inflammatory cell death known as pyroptosis.
  • the ASC speck can also recruit and activate caspase-8, which can process pro-IL-ip and pro-IL- 18 and trigger apoptotic cell death.
  • Caspase- 1 cleaves pro-IL-ip and pro-IL-18 to their active forms, which are secreted from the cell. Active caspase- 1 also cleaves gasdermin-D to trigger pyroptosis. Through its control of the pyroptotic cell death pathway, caspase- 1 also mediates the release of alarmin molecules such as IL-33 and high mobility group box 1 protein (HMGB1). Caspase-1 also cleaves intracellular IL-1R2 resulting in its degradation and allowing the release of IL-la. In human cells caspase-1 may also control the processing and secretion of IL-37. A number of other caspase-1 substrates such as components of the cytoskeleton and glycolysis pathway may contribute to caspase-1- dependent inflammation.
  • NLRP3 -dependent ASC specks are released into the extracellular environment where they can activate caspase-1, induce processing of caspase-1 substrates and propagate inflammation.
  • cytokines derived from NLRP3 inflammasome activation are important drivers of inflammation and interact with other cytokine pathways to shape the immune response to infection and injury.
  • IL-ip signalling induces the secretion of the pro-inflammatory cytokines IL-6 and TNF.
  • IL-ip and IL- 18 synergise with IL-23 to induce IL- 17 production by memory CD4 Th 17 cells and by y5 T cells in the absence of T cell receptor engagement.
  • IL- 18 and IL-12 also synergise to induce IFN-y production from memory T cells and NK cells driving a Thl response.
  • NLRP3 The inherited CAPS diseases Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS) and neonatal -onset multisystem inflammatory disease (NOMID) are caused by gain-of-function mutations in NLRP3, thus defining NLRP3 as a critical component of the inflammatory process.
  • NLRP3 has also been implicated in the pathogenesis of a number of complex diseases, notably including metabolic disorders such as type 2 diabetes, atherosclerosis, obesity and gout.
  • NLRP3 has also been suggested to have a role in a number of central nervous system conditions, including Parkinson's disease (PD), Alzheimer's disease (AD), dementia, Huntington's disease, cerebral malaria, brain injury from pneumococcal meningitis (Walsh et al., Nature Reviews, 15: 84-97, 2014, and Dempsey et al. Brain. Behav. Immun. 201761 : 306-316).
  • Parkinson's disease PD
  • AD Alzheimer's disease
  • dementia Huntington's disease
  • cerebral malaria brain injury from pneumococcal meningitis
  • NLRP3 has also been shown to play a role in a number of lung diseases including chronic obstructive pulmonary disorder (COPD), asthma (including steroid-resistant asthma), asbestosis, and silicosis (De Nardo et al., Am. J. Pathol., 184: 42-54, 2014 and Kim et al. Am J Respir Crit Care Med. 2017 196(3): 283-97). Furthermore, NLRP3 has a role in the development of liver disease, kidney disease and aging. Many of these associations were defined using Nlrp3 ⁇ ⁇ mice, but there have also been insights into the specific activation of NLRP3 in these diseases. In type 2 diabetes mellitus (T2D), the deposition of islet amyloid polypeptide in the pancreas activates NLRP3 and IL-ip signalling, resulting in cell death and inflammation.
  • COPD chronic obstructive pulmonary disorder
  • asthma including steroid-resistant asthma
  • asbestosis asbestosis
  • Glyburide inhibits IL-ip production at micromolar concentrations in response to the activation of NLRP3 but not NLRC4 or NLRP1.
  • Other previously characterised weak NLRP3 inhibitors include parthenolide, 3,4-methylenedioxy-P-nitrostyrene and dimethyl sulfoxide (DMSO), although these agents have limited potency and are nonspecific.
  • NLRP3-related diseases include biologic agents that target IL-1. These are the recombinant IL-1 receptor antagonist anakinra, the neutralizing IL-ip antibody canakinumab and the soluble decoy IL-1 receptor rilonacept. These approaches have proven successful in the treatment of CAPS, and these biologic agents have been used in clinical trials for other IL-ip-associated diseases.
  • the present invention provides novel compounds of formula I wherein,
  • R 1 is H or alkyl
  • R 2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted with 1- to-2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
  • X is -O-, -CH2-, -NH-, or -N(CH 3 )-;
  • W is selected from ring systems A and B
  • R 3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R 1 and R 3 is H and the other is not H;
  • R 4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
  • R 5 is H; or R 4 and R 5 , and the atoms to which they are attached, form i) a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii) a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
  • R 6 is halo, haloalkyl or OH
  • R 7 is H or F; and pharmaceutically acceptable salts thereof.
  • alkyl denotes a monovalent linear or branched saturated hydrocarbon group of 1 to 6 carbon atoms. In some embodiments, if not otherwise described, alkyl comprises 1 to 6 carbon atoms (Ci-6-alkyl), or 1 to 4 carbon atoms (Ci-4-alkyl).
  • Ci-6-alkyl include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl.
  • Particular alkyl groups include methyl and ethyl.
  • alkoxy denotes a group of the formula -O-R’, wherein R’ is a Ci-6-alkyl group.
  • Ci-6-alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and tert-butoxy.
  • alkoxyalkyl denotes an alkyl group wherein one of the hydrogen atoms of the alkyl group have been replaced by an alkoxy group. Examples of alkoxyalkyl are methoxymethyl and meth oxy ethyl.
  • cycloalkyl denotes monocyclic or polycyclic saturated or partially unsaturated, non-aromatic hydrocarbon. In some embodiments, unless otherwise described, cycloalkyl comprises 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. In some embodiments, cycloalkyl is a saturated monocyclic or polycyclic hydrocarbon. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, and the like. Particular example is cyclobutyl.
  • halogen halide and halo are used interchangeably herein and denote fluoro, chloro, bromo or iodo.
  • haloalkyl denotes a Ci-6-alkyl group wherein at least one of the hydrogen atoms of the Ci-6-alkyl group has been replaced by the same or different halogen atoms.
  • Example of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.
  • haloalkoxy denotes a Ci-6-alkoxy group wherein at least one of the hydrogen atoms of the Ci-6-alkoxy group has been replaced by the same or different halogen atoms.
  • haloalkoxy are difluoromethoxy, trifluoromethoxy, difluoroethoxy and tri fluoroethoxy.
  • heterocycle ring denotes a monovalent saturated or partly unsaturated mono- or bicyclic ring system of 4 to 9 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
  • Examples for monocyclic saturated heterocycle rings are azetidinyl, diazepanyl, pyrrolidinyl, tetrahydrofuranyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, and piperazinyl.
  • polycyclic saturated heterocycle rings examples include azaspiroheptanyl, diazaspiroheptanyl, azaspirooctanyl, diazospirooctanyl, diazaspirononanyl, oxaazaspirooctanyl, and oxadiazaspirononanyl.
  • a particular example of a heterocycle ring is piperidinyl.
  • hydroxy denotes a -OH group.
  • hydroxyalkyl denotes an alkyl group wherein at least one of the hydrogen atoms of the alkyl group has been replaced by a hydroxy group.
  • examples of hydroxyalkyl include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxymethylethyl, hydroxymethylpropyl and dihydroxypropyl.
  • salts refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.
  • the salts are formed with inorganic acids such as trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein.
  • salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts.
  • Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.
  • the compound of formula I can also be present in the form of zwitterions.
  • Particularly preferred pharmaceutically acceptable salts of compounds of formula I are the salts formed with formic acid and the salts formed with hydrochloric acid yielding a hydrochloride, dihydrochloride or trihydrochloride salt.
  • the abbreviation uM means microMolar and is equivalent to the symbol pM.
  • the abbreviation uL means microliter and is equivalent to the symbol pL.
  • the abbreviation ug means microgram and is equivalent to the symbol pg.
  • the compounds of formula I can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • the asymmetric carbon atom can be of the "R” or "S” configuration.
  • an embodiment of the present invention provides compounds according to formula I as described herein and pharmaceutically acceptable salts or esters thereof, in particular compounds according to formula I as described herein and pharmaceutically acceptable salts thereof, more particularly compounds according to formula I as described herein.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 1 is H or alkyl and R 3 is H, alkyl or alkoxyalkyl, wherein one of R 1 or R 3 is H and the other is not H.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 1 is H or alkyl and R 3 is H or alkyl, wherein one of R 1 or R 3 is H and the other is alkyl.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 1 is H and R 3 is alkyl.
  • R 2 is selected from i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl.
  • R 2 is selected from i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and ii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH.
  • R 2 is a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH.
  • R 2 is selected from i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-membered cycloalkyl substituted with alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl.
  • R 2 is selected from i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein X is X is O or -CH2-.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 7 is H.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein W is ring system A
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 5 is H, or R 4 and R 5 , and the atoms to which they are attached, form i. a 5-membered cycloalkyl ring, or ii. a 5-membered heterocycle ring comprising a single O heteroatom.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 5 is H, or R 4 and R 5 , and the atoms to which they are attached, form i. a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 5 is H.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein W is selected from ring systems A, C and D
  • R 3 is H or alkyl, wherein one of R 1 and R 3 is H and the other is not H;
  • R 4 is alkyl, cyano, haloalkyl, or haloalkoxy
  • R 5 is H
  • R 6 is OH
  • Y is CH 2 or O.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 4 is halo, cyano, haloalkyl or haloalkoxy.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 4 is alkyl, cyano, haloalkyl or haloalkoxy.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 4 is cyano.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein R 6 is OH.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein,
  • R 1 is H or alkyl
  • R 2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and
  • a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH;
  • X is -O- or -CH2-;
  • W is selected from ring systems A, C and D wherein
  • R 3 is H or alkyl, wherein one of R 1 and R 3 is H and the other is not H;
  • R 4 is alkyl, cyano, haloalkyl, or haloalkoxy
  • R 5 is H
  • R 6 is OH
  • Y is CH 2 or O; and pharmaceutically acceptable salts thereof.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein
  • R 1 is H or alkyl
  • R 2 is a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and
  • X is -O- or -CH2-
  • W is selected from ring systems C and D wherein
  • R 3 is H or alkyl, wherein one of R 1 and R 3 is H and the other is not H;
  • R 4 is alkyl, cyano, haloalkyl, or haloalkoxy
  • R 5 is H
  • R 6 is OH
  • Y is CH 2 or O; and pharmaceutically acceptable salts thereof.
  • R 1 is H or alkyl
  • R 2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
  • X is -O-, -CH 2 -, -NH-, or -N(CH 3 )-;
  • W is selected from ring systems A and B
  • R 3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R 1 and R 3 is H and the other is not H;
  • R 4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
  • R 5 is H; or R 4 and R 5 , and the atoms to which they are attached, form i. a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
  • R 6 is halo, haloalkyl or OH
  • R 7 is H or F; and pharmaceutically acceptable salts thereof.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein
  • R 1 is H or alkyl and R 3 is H or alkyl, wherein one of R 1 or R 3 is H and the other is alkyl;
  • R 2 is selected from: i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH;
  • X is O or -CH2-
  • W is ring system A:
  • R 4 is cyano
  • R 5 is H
  • R 6 is OH; and pharmaceutically acceptable salts thereof.
  • An embodiment of the present invention provides compounds according to formula I as described herein, wherein
  • R 1 is H and R 3 is alkyl
  • R 2 is selected from: i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH;
  • X is O or -CH2-
  • W is ring system A:
  • R 4 is cyano;
  • R 5 is H;
  • R 6 is OH; and pharmaceutically acceptable salts thereof.
  • Particular examples of compounds of formula I as described herein are selected from 4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hydroxy-5-methyl-benzonitrile;formic acid;
  • Preferred examples of compounds of formula I as described herein are selected from 5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;
  • the compound of formula I may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
  • physiologically acceptable carriers i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
  • the pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8.
  • a compound of formula I is formulated in an acetate buffer, at pH 5.
  • the compound of formula I is sterile.
  • the compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
  • compositions are formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc.
  • Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
  • a typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient.
  • Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.
  • the formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
  • buffers stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing
  • the compounds of formula I and their pharmaceutically acceptable salts can be processed with pharmaceutically inert, inorganic or organic adjuvants for the production of tablets, coated tablets, dragees, hard gelatin capsules, injection solutions or topical formulations Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used, for example, as such adjuvants for tablets, dragees and hard gelatin capsules.
  • Suitable adjuvants for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid substances and liquid polyols, etc.
  • Suitable adjuvants for the production of solutions and syrups are, for example, water, polyols, saccharose, invert sugar, glucose, etc.
  • Suitable adjuvants for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils, etc.
  • Suitable adjuvants for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols, etc.
  • Suitable adjuvants for topical ocular formulations are, for example, cyclodextrins, mannitol or many other carriers and excipients known in the art.
  • the pharmaceutical preparations can contain preservatives, solubilizers, viscosity-increasing substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
  • the dosage can vary in wide limits and will, of course, be fitted to the individual requirements in each particular case.
  • the formulation can contain 0.001% to 15% by weight of medicament and the required dose, which can be between 0.1 and 25 mg in can be administered either by single dose per day or per week, or by multiple doses (2 to 4) per day, or by multiple doses per week It will, however, be clear that the upper or lower limit given herein can be exceeded when this is shown to be indicated.
  • An embodiment of the present invention is a compound according to formula I as described herein for use as a therapeutically active substance.
  • An embodiment of the present invention is a compound according to formula I as described herein for use in the treatment or prevention of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.
  • An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition, wherein the disorder or condition is responsive to NLRP3 inhibition.
  • NLRP3 inhibition refers to the complete or partial reduction in the level of activity of NLRP3 and includes, for example, the inhibition of active NLRP3 and/or the inhibition of activation of NLRP3.
  • NLRP3 -induced IL-1 and IL- 18 There is evidence for a role of NLRP3 -induced IL-1 and IL- 18 in the inflammatory responses occurring in connection with, or as a result of, a multitude of different disorders (Menu et al., Clinical and Experimental Immunology, 166: 1-15, 2011; Strowig et al., Nature, 481 : 278- 286, 2012).
  • the disease, disorder or condition is selected from:
  • the disease, disorder or condition is selected from:
  • the disease, disorder or condition is inflammation.
  • inflammation examples include inflammatory responses occurring in connection with, or as a result of:
  • a skin condition such as contact hypersensitivity, bullous pemphigoid, sunburn, psoriasis, atopical dermatitis, contact dermatitis, allergic contact dermatitis, seborrhoetic dermatitis, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, erythemas, or alopecia;
  • a joint condition such as osteoarthritis, systemic juvenile idiopathic arthritis, adult-onset Still’s disease, relapsing polychondritis, rheumatoid arthritisjuvenile chronic arthritis, gout, or a seronegative spondyloarthropathy (e.g. ankylosing spondylitis, psoriatic arthritis or Reiter’s disease);
  • a muscular condition such as polymyositis or myasthenia gravis
  • a gastrointestinal tract condition such as inflammatory bowel disease (including Crohn’s disease and ulcerative colitis), colitis, gastric ulcer, Coeliac disease, proctitis, pancreatitis, eosinopilic gastro-enteritis, mastocytosis, antiphospholipid syndrome, or a food-related allergy which may have effects remote from the gut (e.g., migraine, rhinitis or eczema);
  • a respiratory system condition such as chronic obstructive pulmonary disease (COPD), asthma (including eosinophilic, bronchial, allergic, intrinsic, extrinsic or dust asthma, and particularly chronic or inveterate asthma, such as late asthma and airways hyper-responsiveness), bronchitis, rhinitis (including acute rhinitis, allergic rhinitis, atrophic rhinitis, chronic rhinitis, rhinitis caseosa, hypertrophic rhinitis, rhinitis pumlenta, rhinitis sicca, rhinitis medicamentosa, membranous rhinitis, seasonal rhinitis e.g.
  • COPD chronic obstructive pulmonary disease
  • asthma including eosinophilic, bronchial, allergic, intrinsic, extrinsic or dust asthma, and particularly chronic or inveterate asthma, such as late asthma and airways hyper-responsiveness
  • bronchitis
  • hay fever, and vasomotor rhinitis sinusitis, idiopathic pulmonary fibrosis (IPF), sarcoidosis, farmer’s lung, silicosis, asbestosis, volcanic ash induced inflammation, adult respiratory distress syndrome, hypersensitivity pneumonitis, or idiopathic interstitial pneumonia;
  • IPF idiopathic pulmonary fibrosis
  • sarcoidosis farmer’s lung, silicosis, asbestosis, volcanic ash induced inflammation, adult respiratory distress syndrome, hypersensitivity pneumonitis, or idiopathic interstitial pneumonia
  • vascular condition such as atherosclerosis, Behcet’s disease, vasculitides, or Wegener’s granulomatosis;
  • an autoimmune condition such as systemic lupus erythematosus, Sjogren’s syndrome, systemic sclerosis, Hashimoto’s thyroiditis, type I diabetes, idiopathic thrombocytopenia purpura, or Graves disease;
  • an ocular condition such as uveitis, allergic conjunctivitis, or vernal conjunctivitis;
  • a nervous condition such as multiple sclerosis or encephalomyelitis
  • x an infection or infection-related condition, such as Acquired Immunodeficiency Syndrome (AIDS), acute or chronic bacterial infection, acute or chronic parasitic infection, acute or chronic viral infection, acute or chronic fungal infection, meningitis, hepatitis (A, B or C, or other viral hepatitis), peritonitis, pneumonia, epiglottitis, malaria, dengue hemorrhagic fever, leishmaniasis, streptococcal myositis, mycobacterium tuberculosis (including mycobacterium tuberculosis and HIV co-infection), mycobacterium avium intracellulare, pneumocystis carinii pneumonia, orchitis/epidydimitis, legionella, Lyme disease, influenza A, Epstein-Barr virus infection, viral encephalitis/aseptic meningitis, or pelvic inflammatory disease;
  • AIDS Acquired Immunodeficiency Syndrome
  • acute or chronic bacterial infection such as acute or
  • a renal condition such as mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis, glomerular nephritis, obesity related glomerulopathy, acute renal failure, acute kidney injury, uremia, nephritic syndrome, kidney fibrosis including chronic crystal nephropathy, or renal hypertension;
  • xiii a condition of, or involving, the immune system, such as hyper IgE syndrome, lepromatous leprosy, familial hemophagocytic lymphohistiocytosis, or graft versus host disease;
  • a hepatic condition such as chronic active hepatitis, non-alcoholic steatohepatitis (NASH), alcohol-induced hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), primary biliary cirrhosis, fulminant hepatitis, liver fibrosis, or liver failure;
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • AFLD alcoholic fatty liver disease
  • ASH alcoholic steatohepatitis
  • primary biliary cirrhosis fulminant hepatitis
  • liver fibrosis or liver failure
  • a metabolic disease such as type 2 diabetes (T2D), atherosclerosis, obesity, gout or pseudo-gout; and/or
  • An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition selected from: inflammation; an auto-immune disease; cancer; an infection; a central nervous system disease; a metabolic disease; a cardiovascular disease; a respiratory disease; a liver disease; a renal disease; an ocular disease; a skin disease; a lymphatic condition; a psychological disorder; graft versus host disease; allodynia; a condition associated with diabetes; and any disease where an individual has been determined to carry a germline or somatic non- silent mutation in NLRP3.
  • a disease, disorder or condition selected from: inflammation; an auto-immune disease; cancer; an infection; a central nervous system disease; a metabolic disease; a cardiovascular disease; a respiratory disease; a liver disease; a renal disease; an ocular disease; a skin disease; a lymphatic condition; a psychological disorder; graft versus host disease; allodynia; a condition associated
  • An embodiment of the present invention is the use of a compound according to formula I as described herein in the treatment or prophylaxis of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.
  • An embodiment of the present invention is the use of a compound according to formula I as described herein in the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease.
  • An embodiment of the present invention is the use a compound according to formula I as described herein for use in the treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD.
  • An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease.
  • An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD.
  • An embodiment of the present invention is the use of a compound according to formula I as described herein for preparation of a medicament for the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease.
  • An embodiment of the present invention is the use of a compound according to formula I as described herein for the preparation of a medicament for the treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD.
  • An embodiment of the present invention is a method of treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease, which method comprises administering an effective amount of a compound according to formula I as described herein.
  • An embodiment of the present invention is a method of treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD, which method comprises administering an effective amount of a compound according to formula I as described herein.
  • An embodiment of the present invention relates to a method of inhibiting NLRP3, which method comprises administering an effective amount of a compound according to formula I as described herein.
  • An embodiment of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to formula I as described herein and a therapeutically inert carrier.
  • THP-1 cells (ATCC # TIB-202) were grown in RPMI containing L-glutamine (Gibco #11835) supplemented with ImM sodium pyruvate (Sigma # S8636) and penicillin (lOOunits/ml) / streptomycin (O.lmg/ml) (Sigma # P4333) in 10% Fetal Bovine Serum (FBS) (Sigma # F0804). The cells were routinely passaged and grown to confluency ( ⁇ 10 6 cells/ml). On the day of the experiment, THP-1 cells were harvested and resuspended into RPMI medium (without FBS). The cells were then counted and viability (>90%) checked by Trypan blue (Sigma # T8154).
  • IL-ip was measured according to the manufacturer protocol (Perkin Elmer- AlphaLisa IL-1 Kit AL220F-5000)
  • IC50 data is fitted to a non-linear regression equation (log inhibitor vs response-variable slope 4- parameters)
  • the CHO crelox hERG cell line (ATCC reference Nr. PTA-6812, female Chinese hamster cells) was generated and validated at Roche. Ready-to-use frozen instant CHO-hERG cells were cryopreserved at Evotec (Germany) and used directly in the experiments.
  • the extracellular solution contains (in mM): NaCl 150; KC1 4; CaCh 1; MgCh 1; HEPES 10; pH 7.2-7.4 with NaOH, osmolarity 290-330 mOsm.
  • the hERG test is performed using automated patch clamp system SynchroPatch® 384 (Nanion Technologies GmbH, Germany). K+ currents are measured with the patch-voltage-clamp technique in the whole-cell configuration at 35-37°C.
  • the amplitudes of IKhERG were recorded in each concentration of drug and they were compared to the vehicle control values (taken as 100%) to define fractional blocks.
  • the concentration-response data were fitted with the following relationship:
  • the general assay uses transfected LLC-PK1 cells (porcine kidney epithelial cells) overexpressing human or mouse P-gp, cultured on 96 well semi-permeable filter membrane plates, where they form a polarized monolayer with tight junctions, and act as a barrier between the apical and basolateral compartment.
  • P-gp is expressed in the apical-facing membrane of the monolayer.
  • the tightness of the cell monolayer and functional activity of P-gp are confirmed by addition of a cell-impermeable marker, Lucifer yellow, and a reference P-gp substrate, edoxaban, respectively.
  • PAMPA Parallel Artificial Membrane Permeability Assay
  • the PAMPA assay mimics the transcellular absorption conditions using an artificial phospholipid membrane. This assay determines a permeability value that can be used for compound optimization and ranking purposes as well as input parameters for in silico models to predict intestinal absorption.
  • the donor concentration is measured at t-start (reference) and compared with the donor and acceptor concentration after a certain time (t-end) to calculate the extent of passage of the compound through the membrane.
  • Incubations of test compounds at 1 pM in microsomes (0.5 mg/mL) plus cofactor NADPH are performed in 96 well plates at 37°C on a TEC AN (Tecan Group Ltd, Switzerland) automated liquid handling system. After a 10 minutes pre-incubation step of the test compound with the microsomes, the enzymatic reaction is started by the addition of cofactors. At 1, 3, 6, 9, 15, 25, 35 and 45 minutes, aliquots of the incubations are removed and quenched with 1 :3 (v/v) acetonitrile containing internal standard. Samples are then cooled and centrifuged before analysis of the supernatant by LC-MS/MS 2.
  • Figure 1 illustrates the voltage pattern that was used for cells that were held at a resting voltage of -80 mV (pulse pattern used to elicit outward K+ current at 35-37°C) to activate hERG channels and conduct outward IKhERG current, at a stimulation frequency of 0.1 Hz (6 bpm).
  • aqueous phase was extracted with dichloromethane (3 x 400 mL), and the combined extracts were dried over Na2SC>4, fdtered and concentrated under reduced pressure, and purified by reversed-phase flash (CombiElash 0.1% TEA aqueous-ACN condition) and followed by lyophilization to afford the title compound (4.20 g, 56% yield) as a colorless oil.
  • LCMS m/z 226.9 [M+H] + , ESI pos.
  • Step F 3-Methyl-4-(4,4.5.5-tetramethyl-1.3.2-dioxaborolan-2-yl)-5-( (2 -(trimethylsilyl) ethoxy)methoxy)benzonitrile
  • Step B 2-(3.6-Dichloropyridazin-4-yl)oxyacetaldehyde
  • Step C ( 1 /?.2/?)-2-
  • Step D / -Biityl -I ( I /?.2/?)-2-(3-chloro-6.7 -dihydropyridazino[4,3 -bl [ 1 ,41oxazin-8-yl)cyclohexoxyl - dimethyl-silane
  • Step B 2-(3,6-Dichloropyridazin-4-yl)oxyethyl ethanesulfonate
  • Step C 2-(3,6-Dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate
  • reaction mixture was purified by reversed phase flash (column: Xtimate Cis, 250mm*50mm* 10pm; mobile phase: [water (0.1% ammonia hydroxide, v/v)-ACN]; B%: 5%-70%, 10 minutes) to afford the title compound (15.0 mg, 1% yield) as a white solid.
  • LCMS m/z 398.2 [M+H] + , ESI pos.
  • Step A (3S)-3-12-(3.6-Dichloropyridazin-4-yl)oxyethylamino]piperidine-l-carboxylic acid tert-butyl ester
  • 3,6-dichloro-4-(2-iodoethoxy)pyridazine (CAS # 2490352- 76-4, prepared following to W02020190793, 300 mg, 893.6 umol, 1.00 eq) and (37?)-3-aminopiperidine- 1 -carboxylic acid tert-butyl ester (CAS # 188111-79-7, 196.7 mg, 189.3 uL, 982.9 umol, 1.10 eq) in DMSO (5 mL) was added N, A-Diisopropylcthylaminc (312 uL, 1.79 mmol, 2.00 eq).
  • Step C (3/?)-3-
  • Step D 3-Hydroxy-5-methyl-4-[8-[(3R)-3-piperidyll-6.7-dihydropyridazino[4,3-bl [1,41 oxazin-3 - yllbenzonitrile 1 : 1 hydrogen chloride
  • Step A (/?)-3-Chloro-8-( 1 -ethylpiperidin-3-yl)-5.6.7.8-tetrahydropyrido[2,3-c1pyridazine
  • Step B -4-(8-( l -Ethylpipcridin-3-yl)-5.6.7.8-tctrahvdropyrido
  • Step C 4-[8-[(3R)-l-Ethyl-3-piperidyl1-6,7-dihydro-5FI-pyridor2.3-c1pyridazin-3-yl1-3-hydroxy-5- methyl -benzonitrile
  • Step A 3-[2-(3.6-Dichloropyridazin-4-yl)oxyethylaminol-l-methyl-cyclobutanol
  • 3, 6-dichloro-4-(2 -iodoethoxy )pyridazine (CAS # 2490352-76-4, 0.54 g, 1.69 mmol, 1.0 eq)
  • 3-amino-l-methyl-cyclobutanol CAS # 1523606-23-6, 188.4 mg, 1.86 mmol, 1.1 eq)
  • DIPEA (1.18 m , 6.77 mmol, 4.0 eq) were dissolved in DMSO (20 m ) and stirred at 50 °C for 48 h.
  • Step B 3 -(3 -Chloro-6,7 -dihydropyridazino [4, 3 -b] 11 ,4] oxazin-8 -yl) - 1 -methyl -cyclobutanol
  • Pd-176 (CAS 879689-47-1, 55.2 mg, 0.07 mmol, 0.2 eq), aforementioned 3-[2-(3,6-dichloropyridazin-4- yl)oxyethylamino]-l -methyl -cyclobutanol (100 mg, 0.34 mmol, 1.0 eq) and caesium carbonate (334.6 mg, 1.03 mmol, 3.0 eq) were dissolved in t-BuOH (4 mb) and the mixture degassed (N2, 5 min), then stirred at 90 °C for 3 h. The mixture was filtered through a plug of celite, and the celite washed with EtOAc (30 mb).
  • Step C 3-Hvdroxy-4-[8-(3-hvdroxy-3-methyl-cvclobutyl)-6.7-dihvdropyridazino[4.3-b1 [1.41oxazin-3-yl1- 5 -methyl -benzonitrile
  • reaction mixture was heated to 80 °C and stirred for 3 h.
  • the reaction mixture was filtered through a plug of celite and the filtrate dry -loaded onto silica gel.
  • the product was purified by flash chromatography (silica gel, 4 g column, 0-10% (0.7 N ammonia in MeOH/DCM)) to afford the title compound (8.95 mg, 19%) as a light brown solid.
  • Step A 3-Hydroxy-5-methyl-4-[8-[(17?,27?)-2-[terZ-butyl(dimethyl)silyl1oxycyclohexyl1-6,7- dihvdropyridazino[4,3-b1[L41oxazin-3-yl1benzonitrile
  • Step B 3-Hvdroxy-4-[8-[ -2-hvdroxycvclohexyl]-6.7-dihvdropyridazino[4.3- Z>iri,41oxazin-3-yl1-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid
  • Step A 5-r8-r lA,2A)-2-rterZ-butyl(dimethyl)silyl1oxycyclohexyl1-6,7-dihydropyridazino[4,3- b1[E41oxazin-3-yl1-6-methyl-indan-4-ol
  • Step B 5-(8-((lR,2R)-2-hvdroxycvclohexyl)-7,8-dihvdro-6H-Dyridazino[4,3-b1[L41oxazin-3- yl )-6-methyl-2.3-dihydro- l H-inden-4-ol; 2,2,2-trifluoroacetic acid
  • Step B 5-[8-[(lA,2A)-2-hvdroxycvclohexyl]-4-rnethyl-6,7-dihvdropyridazino[4,3-b][L4]oxazin- 3-yl]-2,3-dihvdrobenzofuran-4-ol;2,2,2-trifluoroacetic acid
  • Step A terLButyl (3A,5A)-3-amino-5-rterLbutyl(dimethyl)silyl1oxy-piperidine-l-carboxylate
  • tert-butyl (3A,55)-3-amino-5-hydroxy-piperidine-l -carboxylate CAS # 1932513- 59-1, 550.0 mg, 2.54 mmol, 1.0 eq.
  • triethylamine (0.47 mL, 3.36 mmol, 1.32 eq) in DCM (10 mL) was added tert-butyl dimethyl silyl chloride (421.6 mg, 2.8 mmol, 1.1 eq).
  • Step B tert-butyl (3£,5A)-3-[terZ-Butyl(dimethyl)silyl]oxy-5-[2-(3,6-dichloropyridazin-4- yl)oxyethylamino]piperidine-l -carboxylate
  • Step C tert-butyl (3A5A)-3-rterZ-Butyl(dimethyl)silyl1oxy-5-(3-chloro-6,7- dihydropyridazino[4,3-birL41oxazin-8-yl)piperidine-l-carboxylate
  • Step D tert-butyl (3X5A)-3-rterZ-Butyl(dimethyl)silyl1oxy-5-[3-r4-cvano-2-methyl-6-(2- trimethylsilylethoxymethoxy)phenyl]-6,7-dihvdropyridazino[4,3-Z>][L4]oxazin-8-yl]piperidine- 1 -carboxylate
  • Step E 3-Hvdroxy-4- 5-hvdroxy-3-piperidyl1-6,7-dihvdropyridazinol4,3- Z>irL41oxazin-3-yl1-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid
  • Step F 4-r8-r(3R,5S)-l-ethyl-5-hydroxy-3-piperidyl1-6,7-dihydropyridazinor4,3-birL41oxazin- 3-yl]-3-hydroxy-5-methyl-benzonitrile
  • Step B (lA,2A)-2-[2-(3,6-Dichloropyridazin-4-yl)oxyethylamino1cvclohexanol
  • Step C (lA,2A)-2-(3-Chloro-6,7-dihvdropyridazino[4,3-Z>irL41oxazin-8-yl)cvclohexanol
  • Pd-176 360.0 mg, 0.45 mmol, 0.17 eq
  • cesium carbonate 2.6 g, 7.98 mmol, 2.96 eq
  • 7.98 mmol, 2.96 eq cesium carbonate
  • Step D (17?,27?)-2-[3-(4-Benzyloxy-6-methyl-2,3-dihvdrobenzofuran-5-yl)-6,7- dihvdropyridazino[4,3-Z>irL41oxazin-8-yl1cvclohexanol 2-(4-Benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (CAS # 2923540-31-0, 82.0 mg, 0.22 mmol, 1.59 eq), (17?,27?)-2-(3-chloro-6,7- dihydropyridazino[4,3-b][l,4]oxazin-8-yl)cyclohexanol (38.0 mg, 0.14 mmol, 1.0 eq) and cesium carbonate (136.0 mg, 0.42 mmol, 2.96 eq) in water (0.400 mL
  • At-column dilution pump gives 2 mL min-1 methanol over the entire method, which is included in the following MeCN percentages.
  • Gradient information 0.0-0.5 min, 12.5% MeCN; 0.5-5.5 min, ramped from 12.5% MeCN to 42.5% MeCN; 5.5-5.6 min, ramped from 42.5% MeCN to 100% MeCN; 5.6-8.5 min, held at 100% MeCN.
  • the clean fractions were evaporated in a Genevac. The dried fractions were transferred to a vial and freeze-dried to provide the title compound (16.0 mg, 53% yield) as an off- white amorphous freeze-dried solid.
  • LCMS m/z 384.2 [M+H] + , ESIpos.
  • XPhos Pd G3 (18.0 mg, 0.02 mmol, 0.15 eq) was added and the reaction mixture was heated to 80 °C and stirred for 3 h. The reaction mixture was dry-loaded onto silica gel. The product was purified by flash chromatography on silica gel (0-60% (10% MeOH [0.7 M NH3] in DCM) : DCM) eluting at 45% to afford the product with some minor impurities as a yellow gum. The residue was triturated with TBME and PE 40:60. The resulting precipitate was filtered and desiccated for 24 h to provide the title compound (24.0 mg, 39% yield) as a yellow solid. LCMS: m/z 410.2 [M+H] + , ESI pos.
  • Step A tert-butyl (3A)-3-[2-(3,6-Dichloropyridazin-4-yl)oxyethylamino1pyrrolidine-l- carboxylate
  • Step B tert-butyl (3A)-3-(3-Chloro-6,7-dihydropyridazino[4,3-Z>1[L41oxazin-8-yl)pyrrolidine-l- carboxylate
  • tert-butyl (3R)-3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]pyrrolidine-l- carboxylate 2.0 g, 5.3 mmol, 1.0 eq
  • BINAP (429.1 mg, 0.69 mmol, 0.13 eq)
  • CS2CO3 3281.7 mg, 10.07 mmol, 1.9 eq
  • Pd(OAc)2 95.2 mg, 0.42 mmol, 0.08 eq
  • Step C 3-Chloro-8-[ -pyrrolidin-3-yl1-6,7-dihvdropyridazino[4,3-Z>1[L41oxazine
  • Step D 3-Chloro-8-[(3/?)-l-ethylpyrrolidin-3-yl1-6,7-dihydropyridazino[4,3-Z>1[L41oxazine
  • Step E 4-[8-[(3/?)-l-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-Z>][L4]oxazin-3-yl]-3- methyl-5-(2 -trimethylsilylethoxymethoxy )benzonitrile
  • Step F 4-[8-[(3A)-l-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-Z>][L4]oxazin-3-yl]-3- hvdroxy-5-methyl-benzonitrile;2.2.2-trifluoroacetic acid
  • Step B 2-(2-Benzyloxy-4,6-dimethyl-phenyl)-4A5,5-tetramethyl-L3,2-dioxaborolane
  • Step C 3,5-Dimethyl-2-(4,4,5,5-tetramethyl-L3,2-dioxaborolan-2-yl)phenol
  • Step D 2-r8-r(lA,2A)-2-rterLButyl(dimethyl)silyl1oxycyclohexyl1-6,7-dihydropyridazino[4,3- b1[L41oxazin-3-yl1-3,5-dimethyl-phenol
  • Step A 3-Methyl-5-(trifluoromethoxy)aniline
  • 3-bromo-5-(trifluoromethoxy)aniline (10.0 g, 39.1 mmol, 1.0 eq)
  • trimethylboroxine (16.7 mL, 58.6 mmol, 1.5 eq) in 1,4-Dioxane (100 mL) and Water (20 mL)
  • K2CO3 (10.8 g, 78.1 mmol, 2.0 eq) and the mixture was degassed and purged with N2 for three times.
  • Pd(dppf)C12 (1.43 g, 1.95 mmol, 0.05 eq) was added to the above mixture.
  • Step E 2-[2-Benzyloxy-6-methyl-4-('trifluoromethoxy)phenyl]-4A5.5-tetramethyl- l .3.2- dioxaborolane
  • Step F [(77?,27?)-2-[3-[2-Benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-6,7- dihydropyridazino[4,3-Z>][L4]oxazin-8-yl]cyclohexoxy]-tert-butyl-dimethyl-silane
  • Step G 2- -2-rtert-Butyl(dimethyl)silyl1oxycvclohexyl1-6,7-dihvdropyridazinol4,3- birL41oxazin-3-yl1-3-methyl-5-(tri fluoromethoxylphenol
  • Step H 2-[8-[(7A,2A)-2-Hydroxycyclohexyl1-6,7-dihydropyridazino[4,3-Z>1[L41oxazin-3-yl1-3- methyl-5-(trifluoromethoxy)phenol;2,2,2-trifluoroacetic acid
  • Step A 3-Chloro-8-[(3A)-l-ethyl-3-DiDeridyl1-4-methyl-6,7-dihydroDyridazino[4,3- blUAIoxazine
  • Step B 4-[8-[(3A)-l-Ethyl-3-piperidyl1-4-methyl-6,7-dihydropyridazino[4,3-b1[L41oxazin-3-yl1- 3-(2-trimethylsilylethoxymethoxy)benzonitrile
  • reaction mixture was stirred at 95 °C for 4 h under nitrogen.
  • the above reaction mixture was cooled to room temperature.
  • the mixture was concentrated in vacuum.
  • the residue was purified by reversed phase flash (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% ammonia hydroxide v/v)-ACN]; B%: 5%-35%, 10 minutes) and followed by lyophilization to give the title compound (11.0 mg, 34% yield) as a yellow solid.
  • LCMS m/z 510.5 [M+H] + , ESI pos.
  • Step C 4-[8-[(3A)-l-Ethyl-3-piperidyl1-4-methyl-6,7-dihydropyridazino[4,3-Z>1[L41oxazin-3-yl1-
  • Step A 5-[8-[(77?,27?)-2-[terLbutyl(dimethyl)silyl1oxycvclohexyl1-4-methyl-6,7- dihydropyridazino[4,3-birE41oxazin-3-yl1indan-4-ol
  • Step B 5-[8-[(77?,27?)-2-hydroxycyclohexyl1-4-methyl-6,7-dihydropyridazino[4,3-Z>1[L41oxazin- 3-yl1indan-4-ol;2,2,2-trifluoroacetic acid
  • Step B terLButyl-r(7A,2A)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-birL41oxazin-8- yDcyclohexoxyl-dimethyl-silane
  • Step C [ -2-[3-(2 -benzyl oxy-3-bicvclo[4.2, 0]octa-L3,5-tri enyl)-4-m ethyl-6,7- dihydropyridazinol4,3-birE41oxazin-8-yl1cyclohexoxy1-terZ-butyl-dimethyl-silane
  • Step D 3-[8-[(17?,27?)-2-[terLbutyl(dimethyl)silyl1oxycvclohexyl1-4-methyl-6,7- dihydropyridazinor4,3-birL41oxazin-3-yl1bicyclol4.2.01octa-L3,5-trien-2-ol
  • Step E 3-[8-[(77?,27G-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-Z>][L4]oxazin- 3-yl1bicyclor4.2.01octa-L3,5-trien-2-ol;2,2,2-trifluoroacetic acid
  • Step A Butyl (dimethyl )silyl1oxy-A f -r2-('3.6-dichloropyridazin-4- yl )oxy ethyl 1 cy cl ohexanamine
  • Step B terLButyl-r4-(3-chloro-6,7-dihvdropyridazinor4,3-Z>irL41oxazin-8-yl)cvclohexoxy1- dimethyl -silane
  • Step C 5-r8-r4-rferZ-Butyl(dimethyl)silyl1oxycyclohexyl1-6,7-dihydropyridazinol4,3- Z>11E41oxazin-3-yl1-6-methyl-2,3-dihydrobenzofuran-4-ol
  • Step D 5-r8-(4-Hvdroxycvclohexyl)-6,7-dihvdropyridazinol4,3-Z>irL41oxazin-3-yl1-6-methyl- 2.3-dihydrobenzofuran-4-ol
  • Step A (7A,35 f )-3 butyl(dimethyl)silyl1oxy-A-r2-(3,6-dichloropyridazin-4-yl)oxyethyl1- cyclohexanamine
  • Step B terLButyl-r -3-(3-chloro-6,7-dihydropyridazinol4,3-Z>irL41oxazin-8- vDcyclohexoxyI-dimethyl-silane
  • Step D 5-[8-[(77?,3M-3-Hydroxycyclohexyl]-6 J-dihydropyridazino[4,3-Z>][L4]oxazin-3-yl]-6- methyl-2.3-dihydrobenzofuran-4-ol
  • Step B terLButyl-r(3 ,4A)-4-(3-chloro-6,7-dihydropyridazinol4,3-Z>irL41oxazin-8- yl)tetrahydropyran-3-yl1oxy-dimethyl-silane
  • Step C 5-r8 -3-lterLButyl(dimethyl)silyl1oxytetrahydropyran-4-yl1-6,7- dihydropyridazinol4,3-Z>irL41oxazin-3-yl1-6-methyl-2,3-dihydrobenzofuran-4-ol
  • Step D 5-18-1(3 , 4A)-3-Hvdroxytetrahvdropyran-4-yl1-6,7-dihvdropyridazinol4,3-Z>irL41oxazin- 3-yl1-6-methyl-2,3-dihvdrobenzofuran-4-ol
  • a compound of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
  • a compound of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:

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Abstract

The invention relates to novel compounds having the general formula (I), (I) wherein R1, R2, X and W are as described herein, composition including the compounds and methods of using the compounds.

Description

INHIBITORS OF NLRP3
Field of the Invention
The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to compounds that modulate NLRP3 inhibition.
The present invention provides novel compounds of formula I wherein,
R1 is H or alkyl;
R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted with 1- to-2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
X is -O-, -CH2-, -NH-, or -N(CH3)-;
W is selected from ring systems A and B
R3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
R5 is H; or R4 and R5, and the atoms to which they are attached, form i) a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii) a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
R6 is halo, haloalkyl or OH;
R7 is H or F; and pharmaceutically acceptable salts thereof.
Furthermore, the invention includes all racemic mixtures, all their corresponding enantiomers and/or optical isomers.
Background of the Invention
The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activity is pathogenic in inherited disorders such as cryopyrin-associated periodic syndromes (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer’s disease and atherosclerosis.
NLRP3 is an intracellular signaling molecule that senses many pathogen-derived, environmental and host-derived factors. Upon activation, NLRP3 binds to apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). ASC then polymerises to form a large aggregate known as an ASC speck. Polymerised ASC in turn interacts with the cysteine protease caspase-1 to form a complex termed the inflammasome. This results in the activation of caspase- 1, which cleaves the precursor forms of the proinflammatory cytokines IL-ip and IL- 18 (termed pro-IL-ip and pro-IL-18 respectively) to thereby activate these cytokines. Caspase-1 also mediates a type of inflammatory cell death known as pyroptosis. The ASC speck can also recruit and activate caspase-8, which can process pro-IL-ip and pro-IL- 18 and trigger apoptotic cell death.
Caspase- 1 cleaves pro-IL-ip and pro-IL-18 to their active forms, which are secreted from the cell. Active caspase- 1 also cleaves gasdermin-D to trigger pyroptosis. Through its control of the pyroptotic cell death pathway, caspase- 1 also mediates the release of alarmin molecules such as IL-33 and high mobility group box 1 protein (HMGB1). Caspase-1 also cleaves intracellular IL-1R2 resulting in its degradation and allowing the release of IL-la. In human cells caspase-1 may also control the processing and secretion of IL-37. A number of other caspase-1 substrates such as components of the cytoskeleton and glycolysis pathway may contribute to caspase-1- dependent inflammation.
NLRP3 -dependent ASC specks are released into the extracellular environment where they can activate caspase-1, induce processing of caspase-1 substrates and propagate inflammation.
Active cytokines derived from NLRP3 inflammasome activation are important drivers of inflammation and interact with other cytokine pathways to shape the immune response to infection and injury. For example, IL-ip signalling induces the secretion of the pro-inflammatory cytokines IL-6 and TNF. IL-ip and IL- 18 synergise with IL-23 to induce IL- 17 production by memory CD4 Th 17 cells and by y5 T cells in the absence of T cell receptor engagement. IL- 18 and IL-12 also synergise to induce IFN-y production from memory T cells and NK cells driving a Thl response.
The inherited CAPS diseases Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS) and neonatal -onset multisystem inflammatory disease (NOMID) are caused by gain-of-function mutations in NLRP3, thus defining NLRP3 as a critical component of the inflammatory process. NLRP3 has also been implicated in the pathogenesis of a number of complex diseases, notably including metabolic disorders such as type 2 diabetes, atherosclerosis, obesity and gout.
A role for NLRP3 in diseases of the central nervous system is emerging, and lung diseases have also been shown to be influenced by NLRP3. NLRP3 has also been suggested to have a role in a number of central nervous system conditions, including Parkinson's disease (PD), Alzheimer's disease (AD), dementia, Huntington's disease, cerebral malaria, brain injury from pneumococcal meningitis (Walsh et al., Nature Reviews, 15: 84-97, 2014, and Dempsey et al. Brain. Behav. Immun. 201761 : 306-316). NLRP3 has also been shown to play a role in a number of lung diseases including chronic obstructive pulmonary disorder (COPD), asthma (including steroid-resistant asthma), asbestosis, and silicosis (De Nardo et al., Am. J. Pathol., 184: 42-54, 2014 and Kim et al. Am J Respir Crit Care Med. 2017 196(3): 283-97). Furthermore, NLRP3 has a role in the development of liver disease, kidney disease and aging. Many of these associations were defined using Nlrp3~ ~ mice, but there have also been insights into the specific activation of NLRP3 in these diseases. In type 2 diabetes mellitus (T2D), the deposition of islet amyloid polypeptide in the pancreas activates NLRP3 and IL-ip signalling, resulting in cell death and inflammation.
Several small molecules have been shown to inhibit the NLRP3 inflammasome. Glyburide inhibits IL-ip production at micromolar concentrations in response to the activation of NLRP3 but not NLRC4 or NLRP1. Other previously characterised weak NLRP3 inhibitors include parthenolide, 3,4-methylenedioxy-P-nitrostyrene and dimethyl sulfoxide (DMSO), although these agents have limited potency and are nonspecific.
Current treatments for NLRP3-related diseases include biologic agents that target IL-1. These are the recombinant IL-1 receptor antagonist anakinra, the neutralizing IL-ip antibody canakinumab and the soluble decoy IL-1 receptor rilonacept. These approaches have proven successful in the treatment of CAPS, and these biologic agents have been used in clinical trials for other IL-ip-associated diseases.
There is a need to provide compounds with improved pharmacological and/or physiological and/or physicochemical properties and/or those that provide a useful alternative to known compounds.
Summary of the Invention
The present invention provides novel compounds of formula I wherein,
R1 is H or alkyl;
R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted with 1- to-2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
X is -O-, -CH2-, -NH-, or -N(CH3)-;
W is selected from ring systems A and B
R3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
R5 is H; or R4 and R5, and the atoms to which they are attached, form i) a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii) a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
R6 is halo, haloalkyl or OH;
R7 is H or F; and pharmaceutically acceptable salts thereof.
The term “alkyl” denotes a monovalent linear or branched saturated hydrocarbon group of 1 to 6 carbon atoms. In some embodiments, if not otherwise described, alkyl comprises 1 to 6 carbon atoms (Ci-6-alkyl), or 1 to 4 carbon atoms (Ci-4-alkyl). Examples of Ci-6-alkyl include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl. Particular alkyl groups include methyl and ethyl.
The term “alkoxy” denotes a group of the formula -O-R’, wherein R’ is a Ci-6-alkyl group. Examples of Ci-6-alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and tert-butoxy.
The term “alkoxyalkyl” denotes an alkyl group wherein one of the hydrogen atoms of the alkyl group have been replaced by an alkoxy group. Examples of alkoxyalkyl are methoxymethyl and meth oxy ethyl. The term “cycloalkyl” denotes monocyclic or polycyclic saturated or partially unsaturated, non-aromatic hydrocarbon. In some embodiments, unless otherwise described, cycloalkyl comprises 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. In some embodiments, cycloalkyl is a saturated monocyclic or polycyclic hydrocarbon. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, and the like. Particular example is cyclobutyl.
The term “halogen”, “halide” and “halo” are used interchangeably herein and denote fluoro, chloro, bromo or iodo.
The term “haloalkyl” denotes a Ci-6-alkyl group wherein at least one of the hydrogen atoms of the Ci-6-alkyl group has been replaced by the same or different halogen atoms. Example of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.
The term “haloalkoxy” denotes a Ci-6-alkoxy group wherein at least one of the hydrogen atoms of the Ci-6-alkoxy group has been replaced by the same or different halogen atoms. Examples of haloalkoxy are difluoromethoxy, trifluoromethoxy, difluoroethoxy and tri fluoroethoxy.
The term “heterocycle ring” denotes a monovalent saturated or partly unsaturated mono- or bicyclic ring system of 4 to 9 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon. Examples for monocyclic saturated heterocycle rings are azetidinyl, diazepanyl, pyrrolidinyl, tetrahydrofuranyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, and piperazinyl. Examples of polycyclic saturated heterocycle rings are azaspiroheptanyl, diazaspiroheptanyl, azaspirooctanyl, diazospirooctanyl, diazaspirononanyl, oxaazaspirooctanyl, and oxadiazaspirononanyl. A particular example of a heterocycle ring is piperidinyl.
The term “hydroxy” denotes a -OH group.
The term “hydroxyalkyl” denotes an alkyl group wherein at least one of the hydrogen atoms of the alkyl group has been replaced by a hydroxy group. Examples of hydroxyalkyl include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxymethylethyl, hydroxymethylpropyl and dihydroxypropyl. The term “cyano” denotes a -C=N group.
The term “oxo” denotes a divalent oxygen atom =0.
The term “pharmaceutically acceptable salts" refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein. In addition these salts may be prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The compound of formula I can also be present in the form of zwitterions. Particularly preferred pharmaceutically acceptable salts of compounds of formula I are the salts formed with formic acid and the salts formed with hydrochloric acid yielding a hydrochloride, dihydrochloride or trihydrochloride salt.
The abbreviation uM means microMolar and is equivalent to the symbol pM. The abbreviation uL means microliter and is equivalent to the symbol pL. The abbreviation ug means microgram and is equivalent to the symbol pg.
The compounds of formula I can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
According to the Cahn-Ingold-Prelog Convention the asymmetric carbon atom can be of the "R" or "S" configuration.
Also an embodiment of the present invention provides compounds according to formula I as described herein and pharmaceutically acceptable salts or esters thereof, in particular compounds according to formula I as described herein and pharmaceutically acceptable salts thereof, more particularly compounds according to formula I as described herein.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R1 is H or alkyl and R3 is H, alkyl or alkoxyalkyl, wherein one of R1 or R3 is H and the other is not H. An embodiment of the present invention provides compounds according to formula I as described herein, wherein R1 is H or alkyl and R3 is H or alkyl, wherein one of R1 or R3 is H and the other is alkyl.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R1 is H and R3 is alkyl.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R2 is selected from i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R2 is selected from i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and ii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R2 is a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R2 is selected from i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-membered cycloalkyl substituted with alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R2 is selected from i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein X is X is O or -CH2-.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R7 is H.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein W is ring system A
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R5 is H, or R4 and R5, and the atoms to which they are attached, form i. a 5-membered cycloalkyl ring, or ii. a 5-membered heterocycle ring comprising a single O heteroatom.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R5 is H, or R4 and R5, and the atoms to which they are attached, form i. a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom. An embodiment of the present invention provides compounds according to formula I as described herein, wherein R5 is H.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein W is selected from ring systems A, C and D
R3 is H or alkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is alkyl, cyano, haloalkyl, or haloalkoxy;
R5 is H;
R6 is OH; and
Y is CH2 or O.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R4 is halo, cyano, haloalkyl or haloalkoxy.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R4 is alkyl, cyano, haloalkyl or haloalkoxy.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R4 is cyano.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein R6 is OH.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein,
R1 is H or alkyl;
R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and
11. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; X is -O- or -CH2-;
W is selected from ring systems A, C and D wherein
R3 is H or alkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is alkyl, cyano, haloalkyl, or haloalkoxy;
R5 is H;
R6 is OH;
Y is CH2 or O; and pharmaceutically acceptable salts thereof.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein
R1 is H or alkyl;
R2 is a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and
X is -O- or -CH2-;
W is selected from ring systems C and D wherein
R3 is H or alkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is alkyl, cyano, haloalkyl, or haloalkoxy;
R5 is H;
R6 is OH;
Y is CH2 or O; and pharmaceutically acceptable salts thereof. An embodiment of the present invention provides compounds according to formula I as described herein, wherein,
R1 is H or alkyl;
R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
X is -O-, -CH2-, -NH-, or -N(CH3)-;
W is selected from ring systems A and B
R3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
R5 is H; or R4 and R5, and the atoms to which they are attached, form i. a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
R6 is halo, haloalkyl or OH;
R7 is H or F; and pharmaceutically acceptable salts thereof. An embodiment of the present invention provides compounds according to formula I as described herein, wherein
R1 is H or alkyl and R3 is H or alkyl, wherein one of R1 or R3 is H and the other is alkyl;
R2 is selected from: i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH;
X is O or -CH2-;
W is ring system A:
R4 is cyano;
R5 is H;
R6 is OH; and pharmaceutically acceptable salts thereof.
An embodiment of the present invention provides compounds according to formula I as described herein, wherein
R1 is H and R3 is alkyl;
R2 is selected from: i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH;
X is O or -CH2-;
W is ring system A:
R4 is cyano; R5 is H;
R6 is OH; and pharmaceutically acceptable salts thereof.
Particular examples of compounds of formula I as described herein are selected from 4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hydroxy-5-methyl-benzonitrile;formic acid;
4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hydroxy-5-methyl -benzonitrile;
4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-3-yl]-3- hydroxy-5-methyl -benzonitrile;
3-Hydroxy-4-[8-(3 -hydroxy-3 -methyl-cyclobutyl)-6,7-dihydropyridazino[4, 3- b][l,4]oxazin-3-yl]-5-methyl -benzonitrile; and pharmaceutically acceptable salts thereof.
Other particular examples of compounds of formula I as described herein are selected from
3-hydroxy-4-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid;
3-hydroxy-4-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-5-methyl -benzonitrile;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-indan-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-indan-4-ol;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol;
4-[8-[(3R,5S)-l-ethyl-5-hydroxy-3-piperidyl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-3-hydroxy-5-methyl -benzonitrile;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; 2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-3-methyl-5-(trifluoromethyl)phenol;
4-[8-[(3R)-l-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-
3-hydroxy-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid;
4-[8-[(3R)-l-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-
3 -hydroxy- 5 -methyl -b enzonitril e;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-3,5-dimethyl-phenol;2,2,2-trifluoroacetic acid
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl] -3 , 5 -dimethyl -phenol ;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-3-methyl-5-(trifluoromethoxy)phenol;2,2,2-trifluoroacetic acid;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-3-methyl-5-(trifluoromethoxy)phenol;
4-[8-[(3R)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-3-hydroxy-benzonitrile;2,2,2-trifluoroacetic acid;
4-[8-[(3R)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3- b] [ 1 ,4]oxazin-3 -yl ] -3 -hydroxy-benzonitrile;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]indan-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3- b] [ 1 ,4]oxazin-3 -yl]indan-4-ol;
3-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]bicyclo[4.2.0]octa-l,3,5-trien-2-ol;2,2,2-trifluoroacetic acid;
3-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]bicyclo[4.2.0]octa-l,3,5-trien-2-ol;
5-[8-[(lR,3S)-3-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;
5-[8-[(3S,4R)-3-hydroxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; and pharmaceutically acceptable salts thereof. Other particular examples of compounds of formula I as described herein are selected from
5-[8-(4-hydroxycyclohexyl)-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl-2,3-dihydrobenzofuran-4-ol; and pharmaceutically acceptable salts thereof.
Preferred examples of compounds of formula I as described herein are selected from 5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-indan-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-6-methyl-indan-4-ol; and pharmaceutically acceptable salts thereof.
Another embodiment of the invention provides a pharmaceutical composition or medicament containing a compound of the invention and a therapeutically inert carrier, diluent or excipient, as well as a method of using the compounds of the invention to prepare such composition and medicament. In one example, the compound of formula I may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula I is formulated in an acetate buffer, at pH 5. In another embodiment, the compound of formula I is sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
A typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
The compounds of formula I and their pharmaceutically acceptable salts can be processed with pharmaceutically inert, inorganic or organic adjuvants for the production of tablets, coated tablets, dragees, hard gelatin capsules, injection solutions or topical formulations Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used, for example, as such adjuvants for tablets, dragees and hard gelatin capsules.
Suitable adjuvants for soft gelatin capsules, are, for example, vegetable oils, waxes, fats, semi-solid substances and liquid polyols, etc.
Suitable adjuvants for the production of solutions and syrups are, for example, water, polyols, saccharose, invert sugar, glucose, etc. Suitable adjuvants for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils, etc.
Suitable adjuvants for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols, etc.
Suitable adjuvants for topical ocular formulations are, for example, cyclodextrins, mannitol or many other carriers and excipients known in the art.
Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, viscosity-increasing substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
The dosage can vary in wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of oral administration a daily dosage of about 0.1 mg to 20 mg per kg body weight, preferably about 0.5 mg to 4 mg per kg body weight (e.g. about 300 mg per person), divided into preferably 1-3 individual doses, which can consist, for example, of the same amounts, should it be appropriate. In the case of topical administration, the formulation can contain 0.001% to 15% by weight of medicament and the required dose, which can be between 0.1 and 25 mg in can be administered either by single dose per day or per week, or by multiple doses (2 to 4) per day, or by multiple doses per week It will, however, be clear that the upper or lower limit given herein can be exceeded when this is shown to be indicated.
An embodiment of the present invention is a compound according to formula I as described herein for use as a therapeutically active substance.
An embodiment of the present invention is a compound according to formula I as described herein for use in the treatment or prevention of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.
An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition, wherein the disorder or condition is responsive to NLRP3 inhibition.
As used herein, the term “NLRP3 inhibition” refers to the complete or partial reduction in the level of activity of NLRP3 and includes, for example, the inhibition of active NLRP3 and/or the inhibition of activation of NLRP3. There is evidence for a role of NLRP3 -induced IL-1 and IL- 18 in the inflammatory responses occurring in connection with, or as a result of, a multitude of different disorders (Menu et al., Clinical and Experimental Immunology, 166: 1-15, 2011; Strowig et al., Nature, 481 : 278- 286, 2012).
In one embodiment, the disease, disorder or condition is selected from:
(i) inflammation;
(ii) an auto-immune disease;
(iii) cancer;
(iv) an infection;
(v) a central nervous system disease;
(vi) a metabolic disease;
(vii) a cardiovascular disease;
(viii) a respiratory disease;
(ix) a liver disease;
(x) a renal disease;
(xi) an ocular disease;
(xii) a skin disease;
(xiii) a lymphatic condition;
(xiv) a psychological disorder;
(xv) graft versus host disease;
(xvi) allodynia;
(xvii) a condition associated with diabetes; and
(xviii) any disease where an individual has been determined to carry a germline or somatic non-silent mutation in NLRP3
In another embodiment, the disease, disorder or condition is selected from:
(i) cancer;
(ii) an infection;
(iii) a central nervous system disease;
(iv) a cardiovascular disease;
(v) a liver disease;
(vi) an ocular disease; and
(vii) a skin disease. In a further typical embodiment of the invention, the disease, disorder or condition is inflammation. Examples of inflammation that may be treated or prevented include inflammatory responses occurring in connection with, or as a result of:
(i) a skin condition such as contact hypersensitivity, bullous pemphigoid, sunburn, psoriasis, atopical dermatitis, contact dermatitis, allergic contact dermatitis, seborrhoetic dermatitis, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, erythemas, or alopecia;
(ii) a joint condition such as osteoarthritis, systemic juvenile idiopathic arthritis, adult-onset Still’s disease, relapsing polychondritis, rheumatoid arthritisjuvenile chronic arthritis, gout, or a seronegative spondyloarthropathy (e.g. ankylosing spondylitis, psoriatic arthritis or Reiter’s disease);
(iii) a muscular condition such as polymyositis or myasthenia gravis;
(iv) a gastrointestinal tract condition such as inflammatory bowel disease (including Crohn’s disease and ulcerative colitis), colitis, gastric ulcer, Coeliac disease, proctitis, pancreatitis, eosinopilic gastro-enteritis, mastocytosis, antiphospholipid syndrome, or a food-related allergy which may have effects remote from the gut (e.g., migraine, rhinitis or eczema);
(v) a respiratory system condition such as chronic obstructive pulmonary disease (COPD), asthma (including eosinophilic, bronchial, allergic, intrinsic, extrinsic or dust asthma, and particularly chronic or inveterate asthma, such as late asthma and airways hyper-responsiveness), bronchitis, rhinitis (including acute rhinitis, allergic rhinitis, atrophic rhinitis, chronic rhinitis, rhinitis caseosa, hypertrophic rhinitis, rhinitis pumlenta, rhinitis sicca, rhinitis medicamentosa, membranous rhinitis, seasonal rhinitis e.g. hay fever, and vasomotor rhinitis), sinusitis, idiopathic pulmonary fibrosis (IPF), sarcoidosis, farmer’s lung, silicosis, asbestosis, volcanic ash induced inflammation, adult respiratory distress syndrome, hypersensitivity pneumonitis, or idiopathic interstitial pneumonia;
(vi) a vascular condition such as atherosclerosis, Behcet’s disease, vasculitides, or Wegener’s granulomatosis;
(vii) an autoimmune condition such as systemic lupus erythematosus, Sjogren’s syndrome, systemic sclerosis, Hashimoto’s thyroiditis, type I diabetes, idiopathic thrombocytopenia purpura, or Graves disease; (viii) an ocular condition such as uveitis, allergic conjunctivitis, or vernal conjunctivitis;
(ix) a nervous condition such as multiple sclerosis or encephalomyelitis;
(x) an infection or infection-related condition, such as Acquired Immunodeficiency Syndrome (AIDS), acute or chronic bacterial infection, acute or chronic parasitic infection, acute or chronic viral infection, acute or chronic fungal infection, meningitis, hepatitis (A, B or C, or other viral hepatitis), peritonitis, pneumonia, epiglottitis, malaria, dengue hemorrhagic fever, leishmaniasis, streptococcal myositis, mycobacterium tuberculosis (including mycobacterium tuberculosis and HIV co-infection), mycobacterium avium intracellulare, pneumocystis carinii pneumonia, orchitis/epidydimitis, legionella, Lyme disease, influenza A, Epstein-Barr virus infection, viral encephalitis/aseptic meningitis, or pelvic inflammatory disease;
(xi) a renal condition such as mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis, glomerular nephritis, obesity related glomerulopathy, acute renal failure, acute kidney injury, uremia, nephritic syndrome, kidney fibrosis including chronic crystal nephropathy, or renal hypertension;
(xii) a lymphatic condition such as Castleman’s disease;
(xiii) a condition of, or involving, the immune system, such as hyper IgE syndrome, lepromatous leprosy, familial hemophagocytic lymphohistiocytosis, or graft versus host disease;
(xiv) a hepatic condition such as chronic active hepatitis, non-alcoholic steatohepatitis (NASH), alcohol-induced hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), primary biliary cirrhosis, fulminant hepatitis, liver fibrosis, or liver failure;
(xv) a cancer, including those cancers listed above;
(xvi) a bum, wound, trauma, haemorrhage or stroke;
(xvii) radiation exposure;
(xviii) a metabolic disease such as type 2 diabetes (T2D), atherosclerosis, obesity, gout or pseudo-gout; and/or
(xix) pain such as inflammatory hyperalgesia, pelvic pain, allodynia, neuropathic pain, or cancer-induced bone pain. An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition selected from: inflammation; an auto-immune disease; cancer; an infection; a central nervous system disease; a metabolic disease; a cardiovascular disease; a respiratory disease; a liver disease; a renal disease; an ocular disease; a skin disease; a lymphatic condition; a psychological disorder; graft versus host disease; allodynia; a condition associated with diabetes; and any disease where an individual has been determined to carry a germline or somatic non- silent mutation in NLRP3.
An embodiment of the present invention is the use of a compound according to formula I as described herein in the treatment or prophylaxis of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.
An embodiment of the present invention is the use of a compound according to formula I as described herein in the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease.
An embodiment of the present invention is the use a compound according to formula I as described herein for use in the treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD. An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease.
An embodiment of the present invention is a compound according to formula I as described herein for the treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD.
An embodiment of the present invention is the use of a compound according to formula I as described herein for preparation of a medicament for the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease.
An embodiment of the present invention is the use of a compound according to formula I as described herein for the preparation of a medicament for the treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD.
An embodiment of the present invention is a method of treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease, which method comprises administering an effective amount of a compound according to formula I as described herein.
An embodiment of the present invention is a method of treatment or prophylaxis of a disease, disorder or condition selected from Asthma and COPD, which method comprises administering an effective amount of a compound according to formula I as described herein.
An embodiment of the present invention relates to a method of inhibiting NLRP3, which method comprises administering an effective amount of a compound according to formula I as described herein.
Also an embodiment of the present invention are compounds of formula I as described herein, when manufactured according to any one of the described processes.
An embodiment of the present invention is a pharmaceutical composition comprising a compound according to formula I as described herein and a therapeutically inert carrier.
Assay Procedures
NLRP3 and Pyroptosis
It is well established that the activation of NLRP3 leads to cell pyroptosis and this feature plays an important part in the manifestation of clinical disease (Yan-gang Liu et al., Cell Death & Disease, 2017, 8(2), e2579; Alexander Wree et al., Hepatology, 2014, 59(3), 898-910; Alex Baldwin etal., Journal of Medicinal Chemistry, 2016, 59(5), 1691-1710; Ema Ozaki et al., Journal of Inflammation Research, 2015, 8, 15-27; Zhen Xie & Gang Zhao, Neuroimmunology Neuroinflammation, 2014, 1(2), 60-65; Mattia Cocco et al., Journal of Medicinal Chemistry, 2014, 57(24), 10366-10382; T. Satoh etal., Cell Death & Disease, 2013, 4, e644). Therefore, it is anticipated that inhibitors of NLRP3 will block pyroptosis, as well as the release of pro- inflammatory cytokines (e.g. IL-ip) from the cell.
THP-1 Cells: Culture and Preparation
THP-1 cells (ATCC # TIB-202) were grown in RPMI containing L-glutamine (Gibco #11835) supplemented with ImM sodium pyruvate (Sigma # S8636) and penicillin (lOOunits/ml) / streptomycin (O.lmg/ml) (Sigma # P4333) in 10% Fetal Bovine Serum (FBS) (Sigma # F0804). The cells were routinely passaged and grown to confluency (~106cells/ml). On the day of the experiment, THP-1 cells were harvested and resuspended into RPMI medium (without FBS). The cells were then counted and viability (>90%) checked by Trypan blue (Sigma # T8154). Appropriate dilutions were made to give a concentration of 625,000cells/ml. To this diluted cell solution was added LPS (Sigma # L4524) to give a Ipg/ml Final Assay Concentration (FAC). 40pl of the final preparation was aliquoted into each well of a 96-well plate. The plate thus prepared was used for compound screening.
THP-1 Cells Pyroptosis Assay
The following method step-by-step assay was followed for compound screening.
Seed THP-1 cells (25,000cells/well) containing l.Opg/ml LPS in 40pl of RPMI medium (without FBS) in 96-well, black walled, clear bottom cell culture plates coated with poly-D-lysine (VWR # 734-0317)
Add 5 pl compound (8 points half-log dilution, with lOpM top dose) or vehicle (DMSO 0.1% FAC) to the appropriate wells
Incubate for 3 hours at 37 °C, 5% CO2
Add 5 l nigericin (Sigma # N7143) (FAC 5pM) to all wells
Incubate for Ihr at 37°C, 5% CO2
At the end of the incubation period, spin plates at 300xg for 3mins and remove supernatant Then add 50 pl of resazurin (Sigma # R7017) (FAC 100 pM resazurin in RPMI medium without FBS) and incubate plates for a further 1-2 hours at 37 °C and 5% CO2 Plates were read in an Envision reader at Ex 560nm and Em 590nm IC50 data is fitted to a non-linear regression equation (log inhibitor vs response-variable slope 4- parameters)
The results of the pyroptosis assay are summarised in Table 1 below as THP IC50.
Human Whole Blood IL- lb Release Assay
For systemic delivery, the ability to inhibit NLRP3 when the compounds are present within the bloodstream is of great importance. For this reason, the NLRP3 inhibitory activity of a number of compounds in human whole blood was investigated in accordance with the following protocol.
Human whole blood in Li-heparin tubes was obtained from healthy donors from a volunteer donor panel.
Plate out 80pl of whole blood containing Ipg/ml of LPS in 96-well, clear bottom cell culture plate (Coming # 3585)
Add lOpl compound (8 points half-log dilution with lOpM top dose) or vehicle (DMSO 0.1% FAC) to the appropriate wells
Incubate for 3 hours at 37 °C, 5% CO2
Add 10 pl nigericin (Sigma # N7143) (lOpM FAC) to all wells
Incubate for Ihr at 37°C, 5% CO2
At the end of the incubation period, spin plates at 300xg for 5mins to pellet cells and remove 20pl of supernatant and add to 96-well v-bottom plates for IL-ip analysis (note: these plates containing the supernatants can be stored at -80°C to be analysed at a later date)
IL-ip was measured according to the manufacturer protocol (Perkin Elmer- AlphaLisa IL-1 Kit AL220F-5000)
IC50 data is fitted to a non-linear regression equation (log inhibitor vs response-variable slope 4- parameters)
The results of the human whole blood assay are summarised in Table 1 below as HWB IC50. hERG screening assay
In the drug development process of small molecules, one of the most frequent adverse side effects, leading to the failure of drugs, is the cardiac arrhythmias. Such failure is often related to the capacity of the drug to inhibit the human ether-a-go-go-related gene (hERG) cardiac potassium channel. Having no or low inhibition of the hERG cardiac potassium channel is therefore considered as beneficial. Cells
The CHO crelox hERG cell line (ATCC reference Nr. PTA-6812, female Chinese hamster cells) was generated and validated at Roche. Ready-to-use frozen instant CHO-hERG cells were cryopreserved at Evotec (Germany) and used directly in the experiments.
Experimental solutions
The extracellular solution contains (in mM): NaCl 150; KC1 4; CaCh 1; MgCh 1; HEPES 10; pH 7.2-7.4 with NaOH, osmolarity 290-330 mOsm. The internal solution contains (in mM): KC1, 10; KF, 100; NaCl, 10; HEPES, 10; EGTA, 20; pH = 7.0-7.4 with KOH, osmolarity 260-300 mOsm.
Electrophysiology
The effects of a compound on hERG K+-currents parameters will be evaluated at 2 concentrations in at least 4 cells.
The hERG test is performed using automated patch clamp system SynchroPatch® 384 (Nanion Technologies GmbH, Germany). K+ currents are measured with the patch-voltage-clamp technique in the whole-cell configuration at 35-37°C.
Cells were held at a resting voltage of -80 mV and they were stimulated by a voltage pattern shown in Figure 1 (pulse pattern used to elicit outward K+ current at 35-37°C) to activate hERG channels and conduct outward IKhERG current, at a stimulation frequency of 0.1 Hz (6 bpm) Data analysis
The amplitudes of IKhERG were recorded in each concentration of drug and they were compared to the vehicle control values (taken as 100%) to define fractional blocks. The concentration-response data were fitted with the following relationship:
Concentration-response curves were fitted by non-linear regression analysis using EworkBook suite (ID Business Solutions Ltd, UK). Data fit was done with the 4 Parameter Logistic Model (fit = (A+(B/(l+((x/C)AD)))), where A=0 and B=100). The results of the hERG assay are summarised in Table 2 below as hERG IC20.
Transcellular P-gp Assay:
The general assay uses transfected LLC-PK1 cells (porcine kidney epithelial cells) overexpressing human or mouse P-gp, cultured on 96 well semi-permeable filter membrane plates, where they form a polarized monolayer with tight junctions, and act as a barrier between the apical and basolateral compartment.
P-gp is expressed in the apical-facing membrane of the monolayer.
The tightness of the cell monolayer and functional activity of P-gp are confirmed by addition of a cell-impermeable marker, Lucifer yellow, and a reference P-gp substrate, edoxaban, respectively.
PAMPA:
PAMPA (Parallel Artificial Membrane Permeability Assay) is a first line permeability screen for drug candidates. The PAMPA assay mimics the transcellular absorption conditions using an artificial phospholipid membrane. This assay determines a permeability value that can be used for compound optimization and ranking purposes as well as input parameters for in silico models to predict intestinal absorption.
The donor concentration is measured at t-start (reference) and compared with the donor and acceptor concentration after a certain time (t-end) to calculate the extent of passage of the compound through the membrane.
Microsomal Stability:
Incubations of test compounds at 1 pM in microsomes (0.5 mg/mL) plus cofactor NADPH are performed in 96 well plates at 37°C on a TEC AN (Tecan Group Ltd, Switzerland) automated liquid handling system. After a 10 minutes pre-incubation step of the test compound with the microsomes, the enzymatic reaction is started by the addition of cofactors. At 1, 3, 6, 9, 15, 25, 35 and 45 minutes, aliquots of the incubations are removed and quenched with 1 :3 (v/v) acetonitrile containing internal standard. Samples are then cooled and centrifuged before analysis of the supernatant by LC-MS/MS 2.
Metabolic Stability in Hepatocytes:
Assay descriptions: Biological materials. Cryopreserved hepatocytes [mouse, rat, rabbit, monkey and human (male and female; mixed)] are obtained. Viability of hepatocytes after reconstitution is at least 80% throughout the study. Ready-to-use rat/human HepatoPac® cultures [long-term hepatocyte cocultures; pooled (n=5 for male and n=5 for female for human)] with stromal mouse fibroblasts (negative control; pooled) with the plates for incubations, application medium and maintenance medium are acquired.
Metabolism by suspended hepatocytes. Primary pooled cryopreserved hepatocytes are reconstituted in pre-warmed William’s E media containing 10% FCS, 0.05 mg/mL streptomycin and 50 U/mL penicillin and 0.4 mM L-glutamine; and 0.01 mg/mL gentamicin, 0.048 mg/mL hydrocortisone and 0.004 mg/mL insulin, to a final suspension density of 1 x 106 cells/mL. The incubation was performed fully automatically with Liquid Handling System (Tecan) equipped with a CO2 incubator with an orbital shaker. After the addition of a test compound at e.g. 1 pM to the wells (1 x 105 cells/well), the 96-well hepatocyte suspension culture plates are incubated in a 5% CO2 at 37°C. Samples are quenched by addition of acetonitrile (including an internal standard) to the incubation well at the designated time points up to 2 h.
Metabolism by HepatoPac®. Incubations for a test article (at e.g. 1 pM, 0.1% v/v DMSO) as conducted in suspension assays are performed in 96-well plates containing either a co-culture of adherent hepatocytes with mouse fibroblast control cells or control cells alone (5% CO2 atmosphere and 37°C). The incubation media in human HepatoPac® is identical with that in suspended hepatocytes. At defined time points (2, 18, 26, 48, 72 and 96 h), whole wells are quenched with ice-cold acetonitrile containing an internal standard.
Samples are then centrifuged appropriately and the supernatant analyzed by LC-MS/MS. The incubation is conducted in n=l or 2.
Table 1: NLRP3 inhibitory activity
Table 2: hERG inhibition assay
Brief description of the figures
Figure 1 illustrates the voltage pattern that was used for cells that were held at a resting voltage of -80 mV (pulse pattern used to elicit outward K+ current at 35-37°C) to activate hERG channels and conduct outward IKhERG current, at a stimulation frequency of 0.1 Hz (6 bpm).
The invention will now be illustrated by the following examples which have no limiting character. In case the preparative examples are obtained as a mixture of enantiomers or diastereoisomers, the pure enantiomers or diastereomers can be obtained by methods described herein or by methods known to those skilled in the art, such as e.g. chiral chromatography or crystallization.
Experimental Methods
Abbreviations:
All examples and intermediates were prepared under nitrogen atmosphere if not specified otherwise.
Intermediates:
Intermediate 1: (4-Cyano-2-hydroxy-6-methyl-phenyl)boronic acid Step A: 4-Amino-3-methoxy-5-methyl-benzonitrile
Two Batches were carried out in parallel.
To a solution of commercially available 4-bromo-2-methoxy-6-methylbenzenamine (CAS # 348169-39-1, 25.0 g, 115 mmol, 1.00 eq) in DMF (250 mL) was added Zn(CN)2 (13.5 g, 115 mmol, 7.34 mb, 1.00 eq) and Pd(PPhs)4 (66.8 g, 57.8 mmol, 0.50 eq). The reaction mixture was stirred at 100 °C for 12 hrs. The reaction mixture was poured into water (1.50 L) and extracted with ethyl acetate (1 L x 3). The organic phase was washed with brine (I L x 3), dried over Na2SC>4, fdtered and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiC>2, petroleum ether/ethyl acetate = 100/1 to 0/1) to give the title compound (28.0 g, 75% yield) as a yellow solid. ’H NMR (DMSO-de) 5 7.05 (s, 2H), 5.47 (bs, 2H), 3.81 (s, 3H), 2.09 (s, 3H).
Step B: 4-Bromo-3-methoxy-5-methyl-benzonitrile
To a solution of CuBr (46.4 g, 323 mmol, 9.86 mL, 1.50 eq) in MeCN (180 mL) was added t-BuONO (33.3 g, 323 mmol, 38.5 mL, 1.50 eq) and stirred at 65 °C. Then solution of aforementioned Intermediate 2B 4- bromo-3-methoxy-5 -methyl -benzonitrile (35.0 g, 215 mmol, 1.00 eq) in MeCN (180 mL) was added at 65 °C. The mixture was stirred at 65 °C for 3.5 hrs. After completion, sat. aq. Na2SOs (400 mL) and sat. aq. NH4CI (200 mL) was added to the mixture and extracted with ethyl acetate (500 mL x 3). The organic phase was washed with brine (500 mL x 2), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCL, petroleum ether/ethyl acetate = 100/1 to 0/1, Rf = 0.75) to give the title compound (20.7 g, 42% yield) as a white solid. ' H NMR (DMSO-de) 5 7.43, 7.40 (2s, IH each), 3.90 (s, 3H), 2.37 (s, 3H).
Step C: 3-Methoxy-5-methyl-4-(4.4.5.5-tetramethyl-1.3.2-dioxaborolan-2-yl)benzonitrile
To a solution of aformentioned 4-bromo-3-methoxy-5-methyl -benzonitrile (18.0 g, 79.6 mmol, 1.00 eq) in DMF (180 mL) was added B2Pin2 (30.3 g, 119 mmol, 1.50 eq) and AcOK (35.1 g, 358 mmol, 4.50 eq). The mixture was stirred at 20 °C for 0.5 hr and Pd^ppQCL’CFLCL (13.0 g, 15.9 mmol, 0.20 eq) was added. The mixture was stirred at 100 °C for 12 hrs. The mixture was filtered with diatomite and dilutered with H2O (500 mL) and extracted with ethyl acetate (800 mL x 3). The organic phase was washed with brine (800 mL x 3), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCL, petroleum ether/ethyl acetate = 100/1 to 1/1, Rf = 0.30) to give the title compound (18.0 g, 83% yield) as a white solid. ’H NMR (DMSO-de) 5 7.22, 7.21 (2s, 1H each), 3.75 (s, 3H), 2.27 (s, 3H), 1.30 (s, 12H).
Step D: (4-Cyano-2-hydroxy-6-methyl-phenyl)boronic acid
A solution of aforementioned 3-methoxy-5-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)benzonitrile (17.0 g, 96.0 mmol, 1.00 eq) in dichloromethane (170 mL) was cooled to 0 °C and BBrs (38.9 g, 155 mmol, 2.50 eq) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 0.5 hr. The mixture was poured into H2O (200 mL), fdtered, and the cake was collected and triturated with EtOAc (20 mL) to give the title compound (4.67 g, 42% yield) as a gray solid. LCMS: m/z 178.1 [M+H]+, ESI pos.
Intermediate 2: 3,6-Dichloro-4-(3-chloropropyl)r)yridazine
To a stirred suspension of 3,6-dichloropyridazine (CAS: # 141-30-0, 5.0 g, 33.6 mmol, 1.0 eq) in water (125 mL) was added H2SO4 (6.6 g, 67. 1 mmol, 2.0 eq) . The mixture was heated to 70 °C before the addition of 4-chlorobutyric acid (CAS # 627-00-9, 4.50 g, 36.9 mmol, 1.1 eq) followed by a solution of silver nitrate (1.30 g, 7.65 mmol, 0.23 eq) in water (2.5 mL) over 1 min. At this point, the mixture became milky in appearance, and a solution of ammonium persulfate (25.8 g, 113.1 mmol, 3.40 eq.) in water (65 mL) was added slowly over 20-30 min, which caused the product to form a sticky precipitate. The reaction mixture was stirred for 1 hour at 70 °C. Upon reaction completion, the mixture was cooled to room temperature, poured into ice, and basified with concentrated aqueous ammonia, adjusting pH to about 8, keeping the temperature below 5 °C. The aqueous phase was extracted with dichloromethane (3 x 400 mL), and the combined extracts were dried over Na2SC>4, fdtered and concentrated under reduced pressure, and purified by reversed-phase flash (CombiElash 0.1% TEA aqueous-ACN condition) and followed by lyophilization to afford the title compound (4.20 g, 56% yield) as a colorless oil. LCMS: m/z 226.9 [M+H]+, ESI pos.
Intermediate 3: 3-Methyl-4-(4.4.5.5-tetramethyl-1.3.2-dioxaborolan-2-yl)-5-((2-(trimethylsilyl)ethoxy) methoxylbenzonitrile
Step A: 4-Bromo-2-methoxy-6-methylaniline
To a solution of 2-methoxy-6-methylaniline (CAS # 50868-73-0, 50.0 g, 364.5 mmol, 1.0 eq) in methanol (150 mL) and acetic acid (50.0 mL, 874.2 mmol, 2.4 eq) was dropped B (22.4 mL, 437.4 mmol, 1.2 eq) slowly, under 0 °C, then stirred at 25 °C for 2 hours. Upon reaction completion, the above reaction solution was diluted with water (300 mL), and extracted with ethyl acetate (100 mL*3). The combined organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, fdtered and the filtrate was concentrated under reduced pressure and purified over column chromatography (hexane/EtOAc, 1:0 to 10: 1), then slurried with petroleum ether (30 mL), filtered and the cake was collected to afford the title compound (30.3 g, 39% yield) as a brown solid. ’H NMR (400 MHz, DMSO-de) 5 6.82 (d, 1H), 6.78 (d, 1H), 4.57 (m, 2H), 3.77 (s, 3H), 2.06 (s, 3H).
Step B: 4-Amino-3-methoxy-5-methylbenzonitrile
To a solution of aforementioned 4-bromo-2-methoxy-6-methyl -aniline (30.3 g, 140.2 mmol, 1.0 eq) in DMF (260 mL) was added Zn(CN)2 (16.5 g, 140.5 mmol, 1.0 eq) and Pd(PPhs)4 (32.4 g, 28.1 mmol, 0.2 eq). The mixture was stirred at 100 °C for 12 hours under nitrogen atmosphere. Upon reaction completion, the mixture was cooled to 20 °C, poured into H2O (500 mL) and extracted with ethyl acetate (300 mL * 3). The organic phase was washed with brine (200 mL * 3), dried over Na2SC>4, filtered, then the filtrate was concentrated under reduced pressure and purified over column chromatography (hexane/EtOAc, 1 : 0 to 5 : 1 ) to afford the title compound (22.0 g, 97% yield) as a pink solid. ’H NMR (400 MHz, DMSO-de) 5 7.05 (s, 2H), 5.48 (s, 2H), 3.81 (s, 3H), 2.09 (s, 3H).
Step C: 4-Bromo-3-methoxy-5-methylbenzonitrile
To a solution of aforementioned 4-amino-3-methoxy-5-methyl-benzonitrile (22.0 g, 135.6 mmol, 1.0 eq), CuBr (29.2 g, 203.5 mmol, 1.5 eq) in ACN (220 mL) was added butyl nitrite (20.98 mL, 203.47 mmol, 1.5 eq), The mixture was stirred at 65 °C for 2 hours. Upon reaction completion, the mixture was cooled to 20 °C, diluted with ACN (150 mL), filtered and the filtrate was concentrated under reduced pressure and purified over column chromatography (hexane/EtOAc, 1:0 to 5: 1), then slurried with the mixture of ethyl acetate (30 mL) and petroleum ether (3 mL), filtered and the cake was collected to afford the title compound (18.0 g, 59% yield) as a white solid. ’H NMR (400 MHz, DMSO-de) 5 7.46 (s, 1H), 7.43 (s, 1H), 3.91 (s, 3H), 2.39 (s, 3H).
Step D: 4-Bromo-3-hydroxy-5-methylbenzonitrile
To a solution of aforementioned 4-bromo-3-methoxy-5-methyl-benzonitrile (6.0 g, 26.5 mmol, 1.0 eq) in DCM (30 mL) was added BBr, (30.0 mL, 316.1 mmol, 11.9 eq) at 0 °C, then stirred at 25 °C for 1 hour. Upon reaction completion, combined with another batch (10 g), then diluted with water (500 mL), and extracted with ethyl acetate (150 mL*3). The combined organic phase was washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure and purified over column chromatography (hexane/EtOAc, 1:0 to 10: 1) to afford the title compound (13.3 g, 89% yield) as a yellow solid. ' H NMR (400 MHz, DMSO-de) 5 10.15 (s, 1H), 6.42 (d, 1H), 6.24 (d, 1H), 1.51 (s, 3H). Step E: 4-Bromo-3-methyl-5-((2-(trimethylsilyl)ethoxy)methoxy)benzonitrile
To a solution of aforementioned 4-bromo-3-hydroxy-5-methyl-benzonitrile (13.3 g, 62.7 mmol, 1.0 eq) in DMF (60 mL) was added CS2CO3 (33.3 g, 102.1 mmol, 1.63 eq) at 0 °C , then stirred at 20 °C for 0.5 hour, then 2-(trimethylsilyl)ethoxymethyl chloride (15.5 mL, 87.8 mmol, 1.4 eq) was dropped at 0 °C, then stirred at 20 °C for 1 hour. Upon reaction completion, diluted with water (200 mL), and extracted with MTBE (150 mL*3). The combined organic phase was washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated reduced pressure and purified over column chromatography (hexane/EtOAc, 1:0 to 10: 1) to get the title compound (16.3 g, 76% yield) as a white solid ’H NMR (400 MHz, CDCh) 5 7.27 (d, 1H), 7.18 (d, 1H), 5.30 (s, 2H), 3.77 (t, 2H), 2.44 (s, 3H), 0.95 (t, 2H), 0 (s, 9H).
Step F: 3-Methyl-4-(4,4.5.5-tetramethyl-1.3.2-dioxaborolan-2-yl)-5-( (2 -(trimethylsilyl) ethoxy)methoxy)benzonitrile
To a solution of aforementioned 4-bromo-3-methyl-5-(2-trimethylsilylethoxymethoxy)benzonitrile (10.0 g, 29.2 mmol, 1.0 eq.) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(l,3,2-dioxaborolane) (14.8 g, 58.4 mmol, 2.0 eq) in 1,4-dioxane (100 mL) was added CS2CO3 (19.0 g, 58.4 mmol, 2.0 eq), tris(4-methoxy-3,5- dimethylphenyl)phosphane (CAS # 121898-64-4, 1.28 g, 2.92 mmol, 0.1 eq). Pd(OAc)2 (0.66 g, 2.92 mmol, 0.1 eq), under N2, then stirred at 95 °C for 5 hours. Upon reaction completion, the mixture was cooled to room temperature, diluted with water (150 mL) and ethyl acetate (50 mL), filtered and the filtrate was extracted with ethyl acetate (100 mL*3). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and purified over column chromatography (hexane/EtOAc, 1 :0 to 10: 1) (17 g), then triturated with MeOH (51 mL), filtered and the cake was collected to afford the title compound (8.38 g, 74% yield) as white solid. ’H NMR (400 MHz, CDCh) 5 7.13 (s, 1H), 7.07 (s, 1H), 5.18 (s, 2H), 3.72 (t, 2H), 2.36 (s, 3H), 1.38 (s, 12H), 0.94 (t, 2H), 0.00 (s, 9H).
Intermediate 4: ( -2-Uert-Butyl(dimethyl)silylloxycvclohexanamine
To a solution of 2-aminocyclohexanol (10.0 g, 86.83 mmol, 1.0 eq) in DCM (100 mL) was added TEA (14.5 mL, 104.2 mmol, 1.2 eq) and tert-butyldimethylchlorosilane (15.7 g, 104.2 mmol, 1.2 eq), then the mixture was stirred at 20 °C for 12 h. Upon the reaction was completion, the above reaction solution was diluted with water (50 mL), extracted with ethyl acetate (60 mL- 3). The combined organic phases were washed with brine (100 ml 3), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by silica gel column (petroleum ether: ethyl acetate = 1 :0 to 3: 1) to afford the title compound (20.5 g, 82% yield) as yellow liquid. 'HNMR (400 MHz, CDC13) d [ppm]: 3.20 - 3.14 (m, 1H), 2.54 - 2.48 (m, 1H), 1.86 - 1.82 (m, 2H), 1.70 - 1.50 (m, 2H), 1.28 - 1.21 (m, 3H), 1.14 - 1.04 (m, 1H), 0.90 (s, 9H), 0.09 - 0.07 (d, 6H).
Intermediate 5: terf-Butyl-r(U?.2j?)-2-(3-chloro-6.7-dihvdropyridazinol4.3-biri.41oxazin-8- vDcyclohexoxyl -dimethyl -silane
Step A: Methyl 2-(3,6-dichloropyridazin-4-yl)oxyacetate
To a solution of methyl glycolate (CAS # 96-35-5, 16.4 mL, 212.6 mmol, 1.0 eq) in THF (100 mL) was added NaH (12.8 g, 318.9 mmol, 1.5 eq, purity: 60% in mineral oil) in portions at 25 °C under N2 and the resulting mixture was stirred at 25 °C for 10 minutes. Then 3,4,6-trichloropyridazine (CAS # 6082-66-2, 39.0 g, 212.6 mmol, 1.0 eq) was added to the above mixture, the reaction mixture was stirred at 25 °C for 1 h under N2. Upon the reaction completion, the mixture was quenched with H2O (100 mL), and extracted with EtOAc (150 mLx 3). The combined organic phases were washed with brine (100 mLx 2), dried over anhydrous Na2SC>4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 3: 1 to 2: 1) to afford the title compound (20.3 g, 40% yield) as a yellow solid. LCMS: 236.9 [M+H]+, ESI pos.
Step B: 2-(3.6-Dichloropyridazin-4-yl)oxyacetaldehyde
A solution of methyl 2-(3,6-dichloropyridazin-4-yl)oxyacetate (0.18 g, 0.76 mmol, 1 eq) in THF (4 mL) was cooled to -65 °C, then DIBAL-H (IM in Toluene, 2.1 mL, 2.1 mmol, 2.77 eq) was added dropwise into above mixture under N2. Then the reaction mixture was stirred at -65 °C for 2 h under N2. Upon the reaction was completion, the reaction mixture was quenched with ice water (5 mL) at 0 °C, and extracted with EtOAc (15 mLx 2). The combined phases were washed with brine (10 mLx 2), filtered and the filtrate was dried over Na2SO4, filtered, and the filtrate was concentrated in vacuum to give the crude product (180 mg, 57% yield) as yellow oil. The crude was used directly in the next step without further purification step. Step C: ( 1 /?.2/?)-2-|/fc77-Biityl(dimcthyl)silyl |oxy-A-| 2-(3.6-dichloropyridazin-4-yl)oxycthyl |- cyclohexanamine
To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (180 mg, crude from above step.) in DCE (4 mL) was added trans-( 1 R,2R)-2-[tert-butyl(dimethyl)silyl ] oxy cyclohexanamine (199.5 mg, 0.87 mmol, 1.0 eq). The mixture was stirred at 20 °C for 15 minutes, then NaBH(OAc)3 (460.7 mg, 2.17 mmol, 2.5 eq) was added to the above mixture. The reaction mixture was stirred at 20 °C for 15 minutes. Upon the reaction was completion, the reaction mixture was diluted with water (10 mL), and extracted with EtOAc (10 mLx 2). The combined extracts were washed with brine (10 mL), filtered and the filtrate was dried over Na2SC>4, filtered, and the filtrate was concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 1: 1) to afford the title compound (50.0 mg, 12% yield over 2 steps) as yellow oil. LCMS: m/z 420.2 [M+H]+, ESI pos.
Step D: / -Biityl -I ( I /?.2/?)-2-(3-chloro-6.7 -dihydropyridazino[4,3 -bl [ 1 ,41oxazin-8-yl)cyclohexoxyl - dimethyl-silane
To a solution of ( lR,2R)-2-[tert-butyl(dimethyl)silyl |oxy-A-|2-(3.6-dichloropyridazin-4-yl)oxycthyl ]- cyclohexanamine (100.0 mg, 0.24 mmol, 1.0 eq), CS2CO3 (147.2 mg, 0.45 mmol, 1.9 eq) in 1,4-Dioxane (2 mL) was added Pd-PEPPSI-IHEPTCl (19.9 mg, 0.02 mmol, 0.1 eq) under N2. Then the reaction mixture was stirred at 100°C for 4 hours under N2. Upon the reaction completion, the above reaction mixture was cooled to room temperature, and poured into ice (20 mL), extracted with ethyl acetate (50 mL x 2). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 5: 1, Rf = 0.6) to afford the title compound (15.0 mg, 12% yield) as yellow oil. LCMS: m/z 384.2, [M+H]+, ESI pos.
Intermediate 6: tert-butyl-K 1 /?.2/?)-2-(3-Chloro-4-mcthyl-6.7-dihvdropyridazino|4.3-b I 11,41 oxazin-8 - yDcyclohexoxyl -dimethyl-silane
Step A: 2-(3,6-Dichloropyridazin-4-yl)oxyethanol
To a solution of methyl 2-(3,6-dichloropyridazin-4-yl)oxyacetate (16.5 g, 69.6 mmol, 1.0 eq) in methanol (180 mL) and THF (90 mL) was added NaBELj (5266.6 mg, 139.2 mmol, 2.0 eq) at 0 °C, then the reaction mixture was stirred at 25 °C for 1 h. Upon the reaction was completion, the mixture was quenched by saturated aqueous NH4CI solution (100 mL), and washed with EtOAc (100 mL x 2). The organic phases were washed with brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 2: 1 to 1 : 1) to afford the title compound (9.8 g, 67% yield) as a yellow solid. 'HNMR (400 MHz, DMSO-t/6) 3 [ppm]: 7.75 (s, 1H), 5.03 (t, 1H), 4.32 (t, 2H), 3.80 - 3.70 (m, 2H).
Step B: 2-(3,6-Dichloropyridazin-4-yl)oxyethyl ethanesulfonate
To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyethanol (1.0 g, 4.78 mmol, 1.0 eq) in DCM (10 mL) was added DIEA (2.37 mL, 14.35 mmol, 3.0 eq), then the solution of ethanesulfonyl chloride (CAS # 594-44-5, 0.68 mL, 7.18 mmol, 1.5 eq) in DCM (2 mL) was added dropwise to the above mixture, the reaction mixture was stirred at 20 °C for 1 h. Upon the reaction was completion, the above reaction mixture was diluted with water (50 mL), extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with brine (80 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate =1 :0 to 1 : 1) to afford the title compound (1.0 g, 63% yield) as yellow solid. ‘H NMR (400 MHz, DMSO-t/6) d [ppm]: 7.80 (s, 1H), 4.65 - 4.52 (m, 4H), 3.44 - 3.37 (m, 2H), 1.26 (t, 3H).
Step C: 2-(3,6-Dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate
To a stirred suspension of 2-(3,6-dichloropyridazin-4-yl)oxyethyl ethanesulfonate (4.0 g, 13.3 mmol, 1.0 eq) and silver nitrate (1.53 g, 9.01 mmol, 0.68 eq) in water (30 mL) was added CH3COOH (2.28 mL, 39.9 mmol, 3.0 eq). The mixture was heated to 50 °C, and then a solution of sulfuric acid (2.17 mL, 39.9 mmol, 3.0 eq) in water (15 mL) was added dropwise to the above mixture. The temperature was then raised to 70 °C and a solution of ammonium persulfate (9093.1 mg, 39.85 mmol, 3.0 eq) in water (15 mL) was added dropwise to the above mixture over 30 minutes. Then the reaction mixture was heated at 70 °C and stirred for a further 30 minutes. Upon the reaction completion, the reaction mixture was cooled to room temperature. EtOAc (150 mL) was added to the above mixture and the precipitate was filtered off. The filtrate was washed with brine (150 mL), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 3: 1) to afford the title compound (1200.0 mg, 26% yield) as colorless oil. LCMS: m/z 315.0 [M+H]+, ESI pos. Step D: /c/V-butyl-lCI >.2 >)-2-('3-Chloro-4-methyl-6.7-dihvdropyridazino[4.3-b1[ l .41oxazin-8- yDcyclohexoxyl-dimethyl-silane
To a solution of 2-(3,6-dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate (1000.0 mg, 3.17 mmol, 1.0 eq), trans-(lR,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexanamine (1000.0 mg, 4.36 mmol, 1.37 eq) in DMF (5 mL) was added DIEA (736.8 mg, 5.71 mmol, 1.8 eq), then the reaction mixture was stirred at 80 °C for 12 h under N2. Upon the reaction completion, the reaction mixture was purified by reversed phase flash (column: Xtimate Cis, 250mm*50mm* 10pm; mobile phase: [water (0.1% ammonia hydroxide, v/v)-ACN]; B%: 5%-70%, 10 minutes) to afford the title compound (15.0 mg, 1% yield) as a white solid. LCMS: m/z 398.2 [M+H]+, ESI pos.
Intermediate 7 : (3X4A)-3-[terZ-Butyl(dimethyl)silyl1oxytetrahydropyran-4-amine
To a soluton of /raw -4-aminocyclohexanol (3.0 g, 26.05 mmol, 1.0 eq) in DCM (30 mL) was added TEA (4.36 mL, 31.26 mmol, 1.2 eq) and tert-butyldimethylchlorosilane (4.71 g, 31.26 mmol, 1.2 eq) , then was stirred at 20 °C for 12 hours. The above reaction mixture was filtered and the filtrate was concentrated under reduced pressure to get the crude product. The crude product was purified by silica gel column (ethyl acetate : MeOH= 1 :0 to 0:1) to get the title compound as a yellow oil (2.1 g, 32% yield) was obtained as a yellow oil.
Intermediate -3-[/c/7-Butyl (dimethyl isilylloxycyclohexanamine
To a soluton of (IS, 3R)-3 -aminocyclohexanol (0.9 g, 7.81 mmol, 1.0 eq) in DCM (10 mL) was added TEA (3.27 mL, 23.44 mmol, 3.0 eq) and /c/V-butyldimethylchlorosilane (1.41 g, 9.38 mmol, 1.2 eq), and the reaction was stirred at 20 °C for 12 hours. Then the above reaction mixture was filtered and the filtrate was concentrated under reduced pressure to get the crude product. The crude product was combined with another batch and the combined crude was purified by silica gel column (PE : EA= 1 :0 to 0: 1) to get the title compound as a colorless oil (1.2 g, 60% yield). Intermediate 4-[/cte/-Butyl(dimeth yl )sil ylloxycyclohexanamine
To a soluton of Zraw -4-aminocyclohexanol (3.0 g, 26.05 mmol, 1.0 eq) in DCM (30 mL) was added TEA (4.36 mL, 31.26 mmol, 1.2 eq) and tert-butyldimethylchlorosilane (4.71 g, 31.26 mmol, 1.2 eq) and the mixture was stirred at 20 °C for 12 hours. The above reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (ethyl acetate : MeOH= 1 :0 to 0: 1) to get the title compound as a yellow oil (2.1 g, 32% yield).
Examples
Example 1:
4- [8- [(37?)- l-Ethyl-3-piperidyl] -6,7-dihydropyridazino [4,3-b] [1,4] oxazin-3-yl] -3-hydroxy-5-methyl- benzonitrile;formic acid
Step A: (3S)-3-12-(3.6-Dichloropyridazin-4-yl)oxyethylamino]piperidine-l-carboxylic acid tert-butyl ester According to W02020190793, to a solution of 3,6-dichloro-4-(2-iodoethoxy)pyridazine (CAS # 2490352- 76-4, prepared following to W02020190793, 300 mg, 893.6 umol, 1.00 eq) and (37?)-3-aminopiperidine- 1 -carboxylic acid tert-butyl ester (CAS # 188111-79-7, 196.7 mg, 189.3 uL, 982.9 umol, 1.10 eq) in DMSO (5 mL) was added N, A-Diisopropylcthylaminc (312 uL, 1.79 mmol, 2.00 eq). The mixture was stirred at 50 °C for 4 h and then overnight. The reaction was quenched with water (50 mL), extracted with ethyl acetate (2 x 50 mL), the combined organic layers combined, dried over anhydrous sodium sulfate, fdtered, and concentrated. The residue was purified by silica gel flash chromatography with gradient elution (solid deposit, 30-80% ethyl acetate/heptane) to give the title compound (202 mg, 58%) as yellow oil. LCMS: m/z 391.3 [M+H]+, ESI pos. Step B: (3/?)-3-(3-Chloro-6.7-dihvdropyridazino|4.3-/? || 1.4|oxazin-8-yl)pipcridinc-l -carboxylic acid tertbutyl ester
According to W02020190793, a mixture of aforementioned (3.S')-3-|2-(3.6-dichloropyridazin-4- yl)oxyethylamino]piperidine-l -carboxylic acid tert-butyl ester (202 mg, 516.2 umol, 1.00 eq), palladium(ii) acetate (CAS # 3375-31-3, 9.27 mg, 41.3 umol, 0.08 eq), (R)-(+)-binap (CAS # 76189-55-4, 41.79 mg, 67.1 umol, 0.130 eq) and cesium carbonate (319.6 mg, 980.9 umol, 1.90 eq) in toluene (4 mL) was degassed with N2 three times and stirred at 110 °C for 3 h. After cooling to room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate (2x2 OmL). The crude material was purified by flash chromatography on silica gel (24 g, EtOAc in Heptane 0 to 50% ) to the title compound (54 mg, 29%) as white powder. LCMS: m/z 355.3 [M+H]+, ESI pos.
Step C: (3/?)-3-| 3-(4-Cvano-2-hvdroxy-6-incthyl-phcnyl)-6.7-dihvdropyridazino|4.3-/? 11 1 .4 |oxazin-8- yllpiperidine-1 -carboxylic acid tert-butyl ester
In a sealed tube, a mixture of aforementioned (3/?)-3-(3-chloro-6.7-dihydropyridazino|4.3-b|| l,4]oxazin- 8 -yl)piperidine-l -carboxylic acid tert-butyl ester (54 mg, 0.152 mmol, 1.00 eq), (4-cyano-2-hydroxy-6- methyl-phenyl)boronic acid Intermediate 1 (45.8 mg, 258.7 umol, 1.70 eq), and caesium carbonate (148.8 mg, 456.6 umol, 3.00 eq) in 1,4-dioxane (2 mL) and water (400 uL) was stirred and argon was bubbled through the mixture for 2 min, then the catalyst (XPhos Pd G3) (19.3 mg, 22.8 umol, 0.15 eq) was added finally. The sealed tube was stirred at 100 °C for 3 h. The reaction mixture was extracted with ethyl acetate (2x 80 mL) and half-saturated NH4CI -solution (20 mL). The organic layers were washed with water (20 mL) and brine (20 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography with gradient elution (solid deposit, 0-50% ethyl acetate/heptane) to give the title compound (17 mg, 25%) as light yellow powder. LCMS: m/z 452.4 [M+H]+, ESI pos.
Step D: 3-Hydroxy-5-methyl-4-[8-[(3R)-3-piperidyll-6.7-dihydropyridazino[4,3-bl [1,41 oxazin-3 - yllbenzonitrile 1 : 1 hydrogen chloride
To a solution of aforementioned (3R)-3-[3-(4-cyano-2-hydroxy-6-methyl-phenyl)-6,7- dihydropyridazino[4,3-b][l,4]oxazin-8-yl]piperidine-l-carboxylic acid tert-butyl ester (17 mg, 37.7 umol, 1.00 eq) in dichloromethane (0.2 mL) and methanol (0.1 mL) was added at room temperature dropwise hydrochloric acid (4M in Dioxane, 75.3 uL, 301.2 umol, 8.00 eq). The reaction mixture was stirred at 23 °C for 2 hours. The reaction mixture was concentrated in vacuo to give the title compound (1: 1 hydrochloride chloride, 18 mg) as light yellow viscous oil, which was used directly in the next step without further purification step. LCMS: m/z 348.3 ([{35C1}M-H]-), 350.3 ([{37C1 }M-H]-), ESI neg. Step E: 4-[8-[(3R)-l-Ethyl-3-piperidyl1-6.7-dihydropyridazino[4,3-b1[1.4]oxazin-3-yl]-3-hydroxy-5- methyl-benzonitrile;formic acid
To a suspension of aforementioned 3-hydroxy-5-methyl-4-[8-[(3R)-3-piperidyl]-6,7- dihydropyridazino[4,3-b][l,4]oxazin-3-yl]benzonitrile;hydrochloride (18 mg, 46.4 nmol, 1.00 eq) in dichloromethane (0.1 mL) was added triethylamine (9.65 uL, 69.6 nmol, 1.50 eq). Then, under ice cooling, acetaldehyde (6.51 uL, 116.02 umol, 2.50 eq) was added, following by the addition of sodium triacetoxyborohydride (17.7 mg, 83.5 umol, 1.80 eq). The reaction mixture was stirred at room temperature and under nitrogen atmosphere for 45 min. After reaction completion, the reaction mixture was extracted with DCM, ammonium chloride and water. The aqueous layers were back -extracted twice with DCM. The combined organic layers were washed with water and brine, then dried with sodium sulfate, filtered and concentrated in vacuo and finally purified using RP HPLC (Gemini NX, 12 nm, 5 pm, 100x30 mm; ACN/water+0.1% HCOOH with gradient 5-50 CAN) to afford the title compound as a 1: 1 formic acid (9 mg, 41%) as light brown powder. LCMS: m/z 378.3 [M+H]+, ESI pos. 1H NMR (600 MHz, DMSO-de) 5 ppm 10.31 (br s, 1 H), 9.56 - 9.85 (m, 1 H), 7.19 - 7.27 (m, 1 H), 7.12 (d, 1 H), 6.82 - 6.89 (m, 1 H), 5.15 - 5.19 (m, 1 H), 4.23 - 4.42 (m, 2 H), 3.52 - 3.63 (m, 2 H), 3.40 - 3.52 (m, 1 H), 3.03 - 3.24 (m, 2 H), 2.70 - 2.97 (m, 1 H), 2.10 (s, 3 H), 1.60 - 2.06 (m, 5 H), 1.23 - 1.25 (m, 2 H).
Example 2: 4-[8-[(37?)-l-Ethyl-3-piperidyl]-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin-3-yl]-3-hydroxy-5-methyl- benzonitrile
Step A: (/?)-3-Chloro-8-( 1 -ethylpiperidin-3-yl)-5.6.7.8-tetrahydropyrido[2,3-c1pyridazine
To a mixture of 3,6-dichloro-4-(3-chloropropyl)pyridazine Intermediate 2 (500.0 mg, 2.22 mmol, 1.0 eq), (3R)-l-ethylpiperidin-3-amine (611.8 mg, 4.77 mmol, 2.15 eq) in DMF (4 mL) was added TEA (725 mg, 6.09 mmol, 2.75 eq), stirred for 2 hours at 150 °C. The above reaction mixture was cooled to room temperature, which was purified by reversed-phase flash (CombiFlash 0.1% NH3H2O aqueous-ACN condition) and followed by lyophilization to give the title compound (111 mg, 18% yield) as a white solid. LCMS: m/z 281.0 [M+H]+, ESI pos. Step B: -4-(8-( l -Ethylpipcridin-3-yl)-5.6.7.8-tctrahvdropyrido|2.3-c|pyridazin-3-yl)-3-nicthyl-5-((2- (trimethylsilyl)ethoxy)methoxy)benzonitrile
To a solution of aforementioned 3-chloro-8-|(3/?)- 1 -ethyl-3 -piperidyl ]-6,7-dihydro-5H-pyrido[2,3- c]pyridazine (140 mg, 0.50 mmol, 1.0 eq) and 3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)- 5-(2-trimethylsilylethoxymethoxy)benzonitrile Intermediate 3 (291 mg, 0.75 mmol, 1.5 eq) in 1,4- dioxane (4 mL) and water (0.8 mL) was added CsF (227.2 mg, 1.5 mmol, 3.0 eq) and Xphos Pd G3 (42.3 mg, 0.05 mmol, 0.1 eq), then stirred at 100 °C for 4 hours under nitrogen. The above reaction mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure to get the crude product which was purified by reversed-phase flash (CombiFlash 0.1% NH3H2O aqueous-ACN condition) and followed by lyophilisation to give the title compound (25.0 mg, 9% yield) as a yellow solid. LCMS: m/z 508.4 [M+H]+, ESI pos.
Step C: 4-[8-[(3R)-l-Ethyl-3-piperidyl1-6,7-dihydro-5FI-pyridor2.3-c1pyridazin-3-yl1-3-hydroxy-5- methyl -benzonitrile
To a solution of aforementioned 4-[8-[(3R)-l -ethyl-3 -piperidyl ]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin- 3-yl]-3-methyl-5-(2-trimethylsilylethoxymethoxy)benzonitrile (20.0 mg, 0.04 mmol, 1.0 eq.) in DCM (1 mL) was added TFA (0.5 mL), then stirred at 25 °C for 1 h under nitrogen. The above reaction mixture was cooled to room temperature, filtered and the filtrate was concentrated under reduced pressure to get the crude product which was purified by reversed-phase flash (CombiFlash 0. 1% NH3-H2O aqueous-ACN condition) and followed by lyophilization to give the title compound (13.1 mg, 84% yield) as a yellow solid. LCMS: m/z 378.3 [M+H]+, ESI pos. ’H NMR (400 MHz, CD3OD) 5 7.15 (s, 1H), 7.09(s, 1H), 7.04 (s, 1H), 5.12-5.09 (m, 1H), 3.55-3.49 (m,2H), 3.11-3.08 (m, 1H), 2.97-2.94 (m, 1H), 2.81-2.78 (m, 2H), 2.53-2.46 (m, 2H), 2.22-2.20 (m, 1H),2.17 (s, 3H), 2.00-1.92 (m, 5H), 1.78-1.71 (m, 2H), 1.15 (t, 3H).
Example 3: 3-Hydroxy-4-[8-(3-hydroxy-3-methyl-cyclobutyl)-6,7-dihydropyridazino[4,3-b] [l,4] oxazin-3-yl]-5- methyl-benzonitrile
Step A: 3-[2-(3.6-Dichloropyridazin-4-yl)oxyethylaminol-l-methyl-cyclobutanol Commercially available 3, 6-dichloro-4-(2 -iodoethoxy )pyridazine (CAS # 2490352-76-4, 0.54 g, 1.69 mmol, 1.0 eq), 3-amino-l-methyl-cyclobutanol (CAS # 1523606-23-6, 188.4 mg, 1.86 mmol, 1.1 eq), DIPEA (1.18 m , 6.77 mmol, 4.0 eq) were dissolved in DMSO (20 m ) and stirred at 50 °C for 48 h. The mixture was diluted with EtOAc (100 mb) and washed with brine (100 mb) and 10 wt% aqueous EiCl (3 x 50 mb), dried using a phase separator and concentrated in vacuo. The resulting residue was purified by flash chromatography (silica gel, 24 g, 0-10% MeOH in DCM) to afford the title compound (119.0 mg, 23%) as a light brown solid. ECMS: m/z 292.1 ([35C1] [M+H]+, ESI pos.
Step B : 3 -(3 -Chloro-6,7 -dihydropyridazino [4, 3 -b] 11 ,4] oxazin-8 -yl) - 1 -methyl -cyclobutanol
Pd-176 (CAS 879689-47-1, 55.2 mg, 0.07 mmol, 0.2 eq), aforementioned 3-[2-(3,6-dichloropyridazin-4- yl)oxyethylamino]-l -methyl -cyclobutanol (100 mg, 0.34 mmol, 1.0 eq) and caesium carbonate (334.6 mg, 1.03 mmol, 3.0 eq) were dissolved in t-BuOH (4 mb) and the mixture degassed (N2, 5 min), then stirred at 90 °C for 3 h. The mixture was filtered through a plug of celite, and the celite washed with EtOAc (30 mb). The filtrate was dry loaded onto silica gel and purified by flash chromatography (silica gel, 12 g, 0-100% EtOAc, followed by isocratic 10% MeOH in DCM) to afford the title compound (32.0 mg, 31%) as a lightyellow solid. ECMS m/z 256.1 [M+H]+, ESI pos.
Step C: 3-Hvdroxy-4-[8-(3-hvdroxy-3-methyl-cvclobutyl)-6.7-dihvdropyridazino[4.3-b1 [1.41oxazin-3-yl1- 5 -methyl -benzonitrile
Aforementioned (4-cyano-2-hydroxy-6-methyl-phenyl)boronic acid (33.2 mg, 0.19 mmol, 1.5 eq), 3-(3- chloro-6,7-dihydropyridazino[4,3-b][l,4]oxazin-8-yl)-l-methyl-cyclobutanol (32.0 mg, 0.13 mmol, 1.0 eq), Xphos Pd G3 (15.91 mg, 0.02 mmol, 0.15 eq) and caesium carbonate (110.09 mg, 0.34 mmol, 2.7 eq) were suspended in 1,4-dioxane (3 mb) and water (0.75 mb) and degassed with N2 (5 min). The reaction mixture was heated to 80 °C and stirred for 3 h. The reaction mixture was filtered through a plug of celite and the filtrate dry -loaded onto silica gel. The product was purified by flash chromatography (silica gel, 4 g column, 0-10% (0.7 N ammonia in MeOH/DCM)) to afford the title compound (8.95 mg, 19%) as a light brown solid. ECMS m/z 352.8 [M+H]+, ESI pos.
Example 4:
3-Hydroxy-4- [8- [(77?,27?)-2-hydr oxy cyclohexyl] -6, 7-dihydr opyridazino [4 ,3- Z>] [1,4] oxazin-3-yl] -5- methyl-benzonitrile;2,2,2-trifluoroacetic acid
Step A: 3-Hydroxy-5-methyl-4-[8-[(17?,27?)-2-[terZ-butyl(dimethyl)silyl1oxycyclohexyl1-6,7- dihvdropyridazino[4,3-b1[L41oxazin-3-yl1benzonitrile
To a solution of 3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5-((2-(trimethylsilyl)ethoxy) methoxy)benzonitrile (106.5 mg, 0.27 mmol, 1.5 eq; Intermediate 3), ISfeCCL (57.9 mg, 0.55 mmol, 3.0 eq), tert-butyl-[(77?,27?)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][l,4]oxazin-8- yl)cyclohexoxy]-dimethyl-silane (70.0 mg, 0.18 mmol, 1.0 eq) in 1,4-dioxane (1 mL) and water (0.2 mL) was added XphosPdGs (15.5 mg, 0.02 mmol, 0.1 eq). Then the reaction mixture was added at 95 °C for 2 h under N2. Upon the reaction completion, the above reaction mixture was cooled to room temperature and diluted with water (20 mL), extracted with ethyl acetate (20 mL x3). The combined organic phases were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 5: 1) to afford the title compound (20.0 mg, 12% yield) as orange oil. LCMS: m/z 611.5 [M+H]+, ESI pos.
Step B: 3-Hvdroxy-4-[8-[ -2-hvdroxycvclohexyl]-6.7-dihvdropyridazino[4.3- Z>iri,41oxazin-3-yl1-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid
A solution of 3-hydroxy-5-methyl-4-[8-[(17?,27?)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]- 6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]benzonitrile (20.0 mg, 0.03 mmol, 1.0 eq) and HCl/dioxane (0.5 mL, 1.0 mmol, 2M in dioxane) was stirred at 25°C for 10 mins. The mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Phenomenex Gemini, 150mm*30mm*25um; mobile phase: [water (0.1% TFA, V/V)-ACN]; B%: 22%-56%, 13 mins) to afford the title compound (8.6 mg, 70% yield) as a white solid. LCMS: m/z 367.1 [M+H]+, ESI pos.
Example 5:
5- [8- [( 1?,21?)-2-hydroxy cyclohexyl] -6,7-dihydropyridazino [4,3-6] [1,4] oxazin-3-yl] -6- methyl-indan-4-ol;2,2,2-trifluoroacetic acid
Step A: 5-r8-r lA,2A)-2-rterZ-butyl(dimethyl)silyl1oxycyclohexyl1-6,7-dihydropyridazino[4,3- b1[E41oxazin-3-yl1-6-methyl-indan-4-ol
To a solution of 6-methyl-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)indan-4-ol (5.4 mg, 0.02 mmol, 1.0 eq), tert-butyl-[(lR,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][l,4]oxazin-8- yl)cyclohexoxy]-dimethyl-silane (10.0 mg, 0.02 mmol, 1.0 eq) and CsF (9.0 mg, 0.06 mmol, 3.0 eq) in 1,4-dioxane (0.5 mL) and water (0.1 mL) was added XPhosPdGs (3.4 mg, 0.01 mmol, 0.2 eq) under N2, then the reaction vessel was sealed and heated in microwave at 95 °C for 1 h under N2. Upon the reaction was completion, the reaction mixture was cooled to 25 °C and concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 2: 1) to afford the title compound (7.0 mg, 71% yield) as yellow oil. LCMS: m/z 496.3 [M+H]+, ESI pos.
Step B: 5-(8-((lR,2R)-2-hvdroxycvclohexyl)-7,8-dihvdro-6H-Dyridazino[4,3-b1[L41oxazin-3- yl )-6-methyl-2.3-dihydro- l H-inden-4-ol; 2,2,2-trifluoroacetic acid
A solution of aforementioned 5-[8-[( l A>,2A>)-2-[/c/7-butyl(dimethyl)silyl]oxycyclohexyl]-6,7- dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6-methyl-indan-4-ol (10.0 mg, 0.02 mmol, 1.0 eq) and HCl/dioxane (0.1 mL, 0.2 mmol, 2M in dioxane) was stirred at 20 °C for 10 mins. Upon the reaction was completion, the reaction mixture was concentrated in vacuum, the residue was purified by prep-HPLC (column: Xtimate Cis, 250 mm*50mm*10pm; mobile phase: [water (0.1% TFA, v/v)-ACN]; B%: 5%-55%, 10 minutes) to afford the title compound (2.7 mg, 35 % yield) as a white solid. LCMS: m/z 382.2 [M+H]+, ESI pos.
Example 6:
5-[8-[(ll?,21?)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3- yl]-2,3-dihydrobenzofuran-4-ol;2,2,2-trifluoroacetic acid Step A: 5-[8-[(lA,2A)-2-[terLButyl(dimethyl)silyl1oxycvclohexyl1-4-methyl-6,7- dihydropyridazino[4,3-birL41oxazin-3-yl1-2,3-dihydrobenzofuran-4-ol
To a solution of 5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-4-ol (11.9 mg, 0.05 mmol, 1.2 eq; CAS # 2923540-32-1) tert-butyl-[(lA,2A)-2-(3-chloro-4-methyl-6,7- dihydropyridazino[4,3-b][l,4]oxazin-8-yl)cyclohexoxy]-dimethyl-silane (15.0 mg, 0.04 mmol, 1.0 eq) and CsF (17.2 mg, 0.11 mmol, 3.0 eq) in 1,4-dioxane (1 mL) and water (0.1 mL) was added XphosPdGs (6.4 mg, 0.01 mmol, 0.2 eq) under N2. The reaction vessel was sealed and heated in microwave at 95 °C for 0.5 h under N2. Upon the reaction completion, the reaction mixture was cooled to 20 °C and concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 5: 1) to afford the title compound (15.0 mg, 74% yield) as yellow oil. LCMS: m/z 498.2 [M+H]+, ESI pos.
Step B: 5-[8-[(lA,2A)-2-hvdroxycvclohexyl]-4-rnethyl-6,7-dihvdropyridazino[4,3-b][L4]oxazin- 3-yl]-2,3-dihvdrobenzofuran-4-ol;2,2,2-trifluoroacetic acid
A mixture of aforementioned 5-[8-[(lR,2R)-2-[te/7-butyl(dimethyl)silyl]oxycyclohexyl]-4- methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol (15.0 mg, 0.03 mmol, 1.0 eq) and HCl/dioxane (1.51 mL, 3.01 mmol, 2M in dioxane) was stirred at 20 °C for 1 h. Upon the reaction completion, the reaction mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% TFA, v/v)-ACN]; B%: 5%-60%, 18 min) to afford the title compound (7.2 mg, 48% yield) as a white solid. LCMS: m/z 383.45 [M+H]+, ESI pos.
Example 7:
4-[8-[(51?,55)-l-ethyl-5-hydroxy-3-piperidyl]-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-
3-hydroxy-5-methyl-benzonitrile
Step A: terLButyl (3A,5A)-3-amino-5-rterLbutyl(dimethyl)silyl1oxy-piperidine-l-carboxylate To a mixture of tert-butyl (3A,55)-3-amino-5-hydroxy-piperidine-l -carboxylate (CAS # 1932513- 59-1, 550.0 mg, 2.54 mmol, 1.0 eq.), triethylamine (0.47 mL, 3.36 mmol, 1.32 eq) in DCM (10 mL) was added tert-butyl dimethyl silyl chloride (421.6 mg, 2.8 mmol, 1.1 eq). The reaction mixture was stirred at 25 °C for 20 h. Upon the reaction completion, the reaction mixture was treated with ethyl acetate (15 mL) and a mixture of water/brine (1 : 1, V/V) (30 mL). The aqueous layer was extracted with ethyl acetate (50 mL x 3). The organic layers were washed with brine (60 mL), dried over sodium sulfate, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 :5 to 0:1) to afford the title compound (380.0 mg, 45% yield) as yellow oil. 'H NMR (400 MHz, CD3OD) 3 [ppm]: 3.89 - 3.77 (m, 2H), 3.77 - 3.70 (m, 1H), 2.96 - 2.74 (m, 3H), 2.16 - 2.06 (m, 1H), 1.46 (s, 9H), 1.44 - 1.38 (m, 1H), 0.92 (s, 9H), 0.12 (d, 6H).
Step B: tert-butyl (3£,5A)-3-[terZ-Butyl(dimethyl)silyl]oxy-5-[2-(3,6-dichloropyridazin-4- yl)oxyethylamino]piperidine-l -carboxylate
To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (540.0 mg, 1.3 mmol, 1.0 eq) in DCE (11 mL) was added tert-butyl (3R,5S)-3-amino-5-[tert-butyl(dimethyl)silyl]oxy-piperidine- 1-carboxylate (340.0 mg, 1.03 mmol, 0.79 eq). Then the mixture was stirred at 20 °C for 0.5 h, then NaBH(OAc)s (691.1 mg, 3.26 mmol, 2.5 eq) into above mixture in portions and the resulting mixture was stirred at 20 °C for 0.5 h. Upon the reaction completion, the reaction mixture was cooled to room temperature, and quenched with water (30 mL). The aqueous phase was extracted with DCM (30 mL x 2). The combined phases were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 : 1 to 1 :3) to afford the title compound (200.0 mg, 29% yield) as yellow oil. LCMS: m/z 521.2 [M+H]+, ESIpos.
Step C: tert-butyl (3A5A)-3-rterZ-Butyl(dimethyl)silyl1oxy-5-(3-chloro-6,7- dihydropyridazino[4,3-birL41oxazin-8-yl)piperidine-l-carboxylate
To a solution of tert-butyl (3S,5R)-3-[tert-butyl(dimethyl)silyl]oxy-5-[2-(3,6-dichloropyridazin-4- yl)oxyethylamino]piperidine-l -carboxylate (350.0 mg, 0.67 mmol, 1.0 eq) and CS2CO3 (437.3 mg, 1.34 mmol, 2.0 eq) in 1,4-dioxane (6 mL) was added Pd-PEPPSI-IHEPTCl (CAS # 1814936-54-3, 56.4 mg, 0.07 mmol, 0.1 eq). Then the mixture was stirred at 80 °C for 12 h under N2. Upon the reaction completion, the reaction mixture was cooled to 20 °C. Then the mixture was concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 : 1 to 0: 1) to afford the title compound (140.0 mg, 22% yield) as yellow oil. LCMS: m/z 485.1 [M+H]+, ESI pos.
Step D: tert-butyl (3X5A)-3-rterZ-Butyl(dimethyl)silyl1oxy-5-[3-r4-cvano-2-methyl-6-(2- trimethylsilylethoxymethoxy)phenyl]-6,7-dihvdropyridazino[4,3-Z>][L4]oxazin-8-yl]piperidine- 1 -carboxylate
To a solution of 3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5-(2- trimethylsilylethoxymethoxy)benzonitrile (60.1 mg, 0.15 mmol, 1.2 eq), tert-butyl (3S,5R)-3- [tert-butyl(dimethyl)silyl]oxy-5-(3-chl oro-6, 7-dihydropyridazino[4,3-b][l,4]oxazin-8 yl)piperidine-l -carboxylate (120.0 mg, 0.13 mmol, 1.0 eq) and CsF (78.2 mg, 0.51 mmol, 4.0 eq) in 1,4-dioxane (2 mL) and water (0.4 mL) was added XphosPdGs (12.0 mg, 0.01 mmol, 0.11 eq) under N2. The reaction vessel was sealed and heated in microwave at 95 °C for 30 min. Upon the reaction completion, the reaction mixture was cooled to 20 °C and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 : 1 to 0: 1) to afford the title compound (60.0 mg, 47% yield) as yellow oil. LCMS: m/z 712.4 [M+H]+, ESI pos.
Step E: 3-Hvdroxy-4- 5-hvdroxy-3-piperidyl1-6,7-dihvdropyridazinol4,3- Z>irL41oxazin-3-yl1-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid
A solution of tert-butyl (3S,5R)-3-[tert-butyl(dimethyl)silyl]oxy-5-[3-[4-cyano-2-methyl-6-(2- trimethyl silyl ethoxymethoxy )phenyl]-6,7-dihydropyridazino[4, 3 -b][l, 4]oxazin-8-yl]piperidine- 1-carboxylate (50.0 mg, 0.05 mmol, 1.0 eq) in TFA (2.03 mL, 27.31 mmol) was stirred at 40°C for 16 hours. Upon the reaction completion, the reaction mixture was concentrated in vacuum. The residue was purified by reversed phase flash (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% TFA, v/v)-ACN]; B%: 5%-40%, 13 min) to the title compound (20.0 mg, 82% yield) as a yellow solid. LCMS: m/z 368.2 [M+H]+, ESI pos.
Step F: 4-r8-r(3R,5S)-l-ethyl-5-hydroxy-3-piperidyl1-6,7-dihydropyridazinor4,3-birL41oxazin- 3-yl]-3-hydroxy-5-methyl-benzonitrile
To a solution of 3-hydroxy-4-[8-[(3R,5S)-5-hydroxy-3-piperidyl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid (15.0 mg, 0.03 mmol, 1.0 eq) and DIEA (10.1 mg, 0.08 mmol, 2.5 eq) in DMF (1 mL) was added iodoethane (5.8 mg, 0.04 mmol, 1.2 eq). Then the reaction mixture was stirred at 20°C for 1 hour. Upon the reaction completion, water (O.lmL) was added, then the mixture was purified by prep-HPLC (column: Phenomenex Gemini, 150mm*25mm*10um; mobile phase: [water (0.1% NH4 HCO3, V7V)- ACN]; B%: 22%-52%, 12 mins) to afford the title compound (5.47 mg, 45% yield) as a white solid. LCMS: m/z 396.2 [M+H]+, ESI pos.
Example 8:
5-[8-[(71?,21?)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-6- methyl-2,3-dihydrobenzofuran-4-ol
Step A: 3,6-Dichloro-4-(2-iodoethoxy)pyridazine
2-Iodoethanol (107.0 pL, 1.37 mmol, 1.01 eq) was dissolved in anhydrous THF (7 mL) and sodium hydride (60% in mineral oil) (82.0 mg, 2.05 mmol, 1.5 eq) was added portion-wise at 0 °C. The mixture was stirred at room temperature for 15 min, then 3,4,6-trichloropyridazine (250.0 mg, 1.36 mmol, 1.0 eq) was added and the mixture stirred at 40 °C for overnight (18 h). The reaction was left to stir an additional day. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic extracts were dried (ISfeSCh), and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel (40 g column, 0-40% EtOAc in heptane), eluting at 30% to afford the title compound (88.0 mg, 20% yield) as a light yellow solid. LCMS: m/z 318.9 2x 35C1 [M+H]+, ESI pos.
Step B: (lA,2A)-2-[2-(3,6-Dichloropyridazin-4-yl)oxyethylamino1cvclohexanol
3,6-Dichloro-4-(2-iodoethoxy)pyridazine (418.0 mg, 1.31 mmol, 1.0 eq), (lR,2R)-2- aminocyclohexanol (227.0 mg, 1.97 mmol, 1.5 eq) and A,A-diisopropylethylamine (0.9 mL, 5.17 mmol, 3.94 eq) were dissolved in DMSO (8 mL) and stirred at 50 °C for 2 h. The reaction was let to react overnight (16 h). Afterwards, the reaction mixture was diluted with EtOAc (50 mL) and 50 w% brine (50 mL) and the layers separated. The aqueous layer was extracted with EtOAc (2 x 50 mL) after which the combined organics were dried (Na2SO4), filtered and concentrated in vacuo. The crude product was combined with another batch and purified by silica gel chromatography (40 g cartridge, 0-10% (MeOH : EtOAc) eluting at 10% to give the title compound (124.0 mg, 28% yield) as a light yellow solid. LCMS: m/z 306.1 [M+H]+, ESI pos.
Step C: (lA,2A)-2-(3-Chloro-6,7-dihvdropyridazino[4,3-Z>irL41oxazin-8-yl)cvclohexanol Pd-176 (360.0 mg, 0.45 mmol, 0.17 eq) and cesium carbonate (2.6 g, 7.98 mmol, 2.96 eq) were added to a flask containing (7A,2A)-2-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]- cyclohexanol (825.0 mg, 2.69 mmol, 1.0 eq) in tert-butanol (35 mL) which had been sparged (bubbling N2 while sonicating for 5 min). The reaction mixture was heated to 80 °C and left to stir for 2 h. The mixture was combined with other batches and filtered through a plug of celite, and the celite washed with EtOAc (30 mL). The concentrated filtrate (1.69 g) was dry loaded onto celite and purified by reverse-phase flash chromatography (Cis, 43 g column, 10-100% MeCN [0.1% formic acid]: 0.1% aq. formic acid) eluting at 25% to give after extraction of the appropriate fractions with DCM (3 x 50 mL), drying (Na2SO4), filtration and concentration to give the title compound (76.0 mg, 10% yield) as a light yellow solid. LCMS: m/z 270.1/272.1 [M+H]+, ESI pos.
Step D: (17?,27?)-2-[3-(4-Benzyloxy-6-methyl-2,3-dihvdrobenzofuran-5-yl)-6,7- dihvdropyridazino[4,3-Z>irL41oxazin-8-yl1cvclohexanol 2-(4-Benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (CAS # 2923540-31-0, 82.0 mg, 0.22 mmol, 1.59 eq), (17?,27?)-2-(3-chloro-6,7- dihydropyridazino[4,3-b][l,4]oxazin-8-yl)cyclohexanol (38.0 mg, 0.14 mmol, 1.0 eq) and cesium carbonate (136.0 mg, 0.42 mmol, 2.96 eq) in water (0.400 mL) and 1,4-dioxane (2 mL) were degassed with N2 (sonication, 5 min). XPhos Pd G3 (18.0 mg, 0.02 mmol, 0.15 eq) was added and the reaction mixture was heated to 80 °C and stirred for 4 h. The reaction was cooled and left to stir over the weekend wherein no change was seen. Additional 2-(4-benzyloxy-6-methyl-2,3- dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (27.0 mg, 0.07 mmol, 0.52 eq) and cesium carbonate (49.0 mg, 0.15 mmol, 1.07 eq) was added, the reaction mixture was sparged as before and then XPhos Pd G3 (12.0 mg, 0.01 mmol, 0.1 eq) was added and the reaction mixture heated as before for 2 h. The reaction mixture was dry -loaded onto silica gel. The product was purified by flash chromatography on silica gel (24 g column, 0-10% MeOH : EtOAc eluting at 5% to afford the title compound (13.0 mg, 18% yield) was obtained as a light yellow solid. LCMS: m/z 474.3 [M+H]+, ESI pos. Step E: 5-r8- -2-Hvdroxycvclohexyl1-6,7-dihydroDyridazinor4,3-Z>iri,41oxazin-3-yl1-6- methyl -2.3-dihydrobenzofuran-4-ol
Pd/C (Type 87) (16.0 mg, 0.01 mmol, 0.1 eq) was added to a stirred solution of (77?,27?)-2-[3-(4- benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-6,7-dihydropyridazino[4,3-Z>][l,4]oxazin-8- yl] cyclohexanol (36.0 mg, 0.08 mmol, 1.0 eq) in ethanol (3 mL). The hydrogenation vessel was placed under an atmosphere of hydrogen (2 bar) at r.t and vigorously stirred for 2 h. The reaction mixture was filtered through celite, rinsed with EtOH and concentrated under reduced pressure to give the crude residue (34 mg). The crude was dissolved in 1.58 mL with DMSO, filtered and purified by reversed phase preparative HPLC (Waters 2767 Sample Manager, Waters 2545 Binary Gradient Module, Waters Systems Fluidics Organiser, Waters 515 ACD pump, Waters 515 Makeup pump, Waters 2998 Photodiode Array Detector, Waters QDa) on a Waters X-Select CSH Cis ODB prep column, 130A, 5 pm, 30 mm X 100 mm, flow rate 40 mL min-1 eluting with a 0.1% formic acid in water-MeCN gradient over 8.5 mins using UV across all wavelengths with PDA as well as a QDA and ELS detector. At-column dilution pump gives 2 mL min-1 methanol over the entire method, which is included in the following MeCN percentages. Gradient information: 0.0-0.5 min, 12.5% MeCN; 0.5-5.5 min, ramped from 12.5% MeCN to 42.5% MeCN; 5.5-5.6 min, ramped from 42.5% MeCN to 100% MeCN; 5.6-8.5 min, held at 100% MeCN. The clean fractions were evaporated in a Genevac. The dried fractions were transferred to a vial and freeze-dried to provide the title compound (16.0 mg, 53% yield) as an off- white amorphous freeze-dried solid. LCMS: m/z 384.2 [M+H]+, ESIpos.
Example 9:
2-[8-[(71?,21?)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-3- methyl-5-(trifluoromethyl)phenol
3-Methyl-2-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (CAS # 2557358-38-8, 66.0 mg, 0.22 mmol, 1.55 eq), (77?,27?)-2-(3-chloro-6,7-dihydropyridazino[4,3- Z>][l,4]oxazin-8-yl)cyclohexanol (38.0 mg, 0.14 mmol, 1.0 eq; Example 8, step C) and caesium carbonate (131.0 mg, 0.4 mmol, 2.85 eq) in water (0.400 mL) and 1,4-dioxane (2 mL) were degassed with N2. XPhos Pd G3 (18.0 mg, 0.02 mmol, 0.15 eq) was added and the reaction mixture was heated to 80 °C and stirred for 3 h. The reaction mixture was dry-loaded onto silica gel. The product was purified by flash chromatography on silica gel (0-60% (10% MeOH [0.7 M NH3] in DCM) : DCM) eluting at 45% to afford the product with some minor impurities as a yellow gum. The residue was triturated with TBME and PE 40:60. The resulting precipitate was filtered and desiccated for 24 h to provide the title compound (24.0 mg, 39% yield) as a yellow solid. LCMS: m/z 410.2 [M+H]+, ESI pos.
Example 10:
4-[8-[(31?)-l-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-3- hydroxy-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid
Step A: tert-butyl (3A)-3-[2-(3,6-Dichloropyridazin-4-yl)oxyethylamino1pyrrolidine-l- carboxylate
To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyethyl ethanesulfonate (2600.0 mg, 8.63 mmol, 1.0 eq) and (A)-(+)-l-BOC-3-aminopyrrolidine (3216.1 mg, 17.27 mmol, 2.0 eq) in DMSO (13 mL) was added DIEA (1670.6 mg, 12.95 mmol, 1.5 eq). Then the reaction mixture was stirred at 80 °C for 12 h. Upon the reaction completion, the mixture was poured into water (30 mL), and extracted with EtOAc (200 mL x 2). The combined organic phases were washed with brine (100 mL x 2), dried over anhydrous Na2SC>4, filtered and concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 0: 1 to 1 : 1) to afford the title compound (2.0 g, 61% yield) as a yellow solid. 'H NMR (400 MHz, CDCI3) <5 [ppm]: 7.06 (s, 1H), 4.45 - 4.32 (m, 1H), 3.78 - 3.65 (m, 3H), 3.63 - 3.51 (m, 3H), 3.39 - 3.23 (m, 2H), 2.26 - 2.13 (m, 1H), 2.05 - 1.95 (m, 1H), 1.48 (s, 9H).
Step B: tert-butyl (3A)-3-(3-Chloro-6,7-dihydropyridazino[4,3-Z>1[L41oxazin-8-yl)pyrrolidine-l- carboxylate To a slution of tert-butyl (3R)-3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]pyrrolidine-l- carboxylate (2.0 g, 5.3 mmol, 1.0 eq), BINAP (429.1 mg, 0.69 mmol, 0.13 eq), CS2CO3 (3281.7 mg, 10.07 mmol, 1.9 eq) in toluene (20 mL) was added Pd(OAc)2 (95.2 mg, 0.42 mmol, 0.08 eq) under N2 at 25°C. Then the reaction mixture was stirred at 110 °C for 2 h. The mixture was poured into water (20 mL) and extracted with EtOAc (50 mL*3). The combined organic layers were washed with brine (20 mL*2), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1 to 5: 1) to afford the title compound (160.0 mg, 9% yield) as a yellow solid. LCMS: m/z 341.1 [M+H]+, ESI pos.
Step C: 3-Chloro-8-[ -pyrrolidin-3-yl1-6,7-dihvdropyridazino[4,3-Z>1[L41oxazine
A solution of tert-butyl (37?)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][l,4]oxazin-8- yl)pyrrolidine-l-carboxylate (160.0 mg, 0.47 mmol, 1.0 eq) in DCM (1 mL) and TFA (0.5 mL) was stirred at 25 °C for 0.5 h. Upon the reaction completion, the reaction mixture was concentrated in vacuum. The residue was purified by reversed phase flash (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% ammonia hydroxide v/v)-ACN]; B%: 5%- 35%, 10 minutes) and followed by lyophilization to afford the title compound (30.0 mg, 27% yield) as yellow oil. LCMS: m/z 241.0 [M+H]+, ESI pos.
Step D: 3-Chloro-8-[(3/?)-l-ethylpyrrolidin-3-yl1-6,7-dihydropyridazino[4,3-Z>1[L41oxazine
To a solution of 3-chloro-8-[(37?)-pyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazine (30.0 mg, 0.12 mmol, 1.0 eq), DIEA (32.16 mg, 0.25 mmol, 2.0 eq) in DMF (1 mL) was added iodoethane (0.01 mL, 0.19 mmol, 1.5 eq). Then the mixture was stirred at 20°C for 1 hour. Upon the reaction completion, the reaction mixture was concentrated in vacuum. The residue was purified by reversed phase flash (column: Xtimate Cis, 250mm*40mm*10pm; mobile phase: [water (0.1% ammonia hydroxide v/v)-MeCN]; B%: 5%-38%, 15 min) and followed by lyophilization to afford the title compound (22.0 mg, 66% yield) as a yellow solid. LCMS: m/z 269.1 [M+H]+, ESI pos.
Step E: 4-[8-[(3/?)-l-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-Z>][L4]oxazin-3-yl]-3- methyl-5-(2 -trimethylsilylethoxymethoxy )benzonitrile
To a mixture of CsF (37.3 mg, 0.25 mmol, 3.0 eq), 3 -methyl -4-(4, 4, 5, 5 -tetramethyl -1,3,2- dioxaborolan-2-yl)-5-((2-(trimethylsilyl)ethoxy) methoxy)benzonitrile (47.8 mg, 0.12 mmol, 1.5 eq, Intermediate 3) and 3-chloro-8-[(3A)-l-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3- Z>][ 1,4] oxazine (22.0 mg, 0.08 mmol, 1.0 eq) in 1,4-dioxane (1 mL) and water (0.2 mL) was added XPhos Pd G3 (6.9 mg, 0.01 mmol, 0.1 eq) under N2 at 25 °C. The mixture was stirred at 95 °C for 2.5 h. Upon the reaction completion, the reaction mixture was cooled to room temperature. EtOAc (30 mL) and brine (20 mL) were added into the mixture and the layers were separated. The aqueous phase was extracted with EtOAc (30 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 1 : 1 to 0: 1) to afford the title compound (21.0 mg, 42% yield) as a yellow solid. JH NMR (400 MHz, CD3OD) 6 [ppm]: 7.54 (s, 1H), 7.42 (s, 1H), 7.09 (s, 1H), 5.36 (s, 2H), 5.01 - 5.00 (m, 1H), 4.78 - 4.75 (m, 2H), 4.05 - 4.00 (m, 1H), 3.82 - 3.80 (m, 2H), 3.67 (t, 2H), 3.35 - 3.34 (m, 3H), 3.27 - 3.18 (m, 1H), 2.85 - 2.70 (m, 2H), 2.22 (s, 3H), 1.39 (t, 3H), 0.91 (t, 2H), 0.00 (s, 9H).
Step F: 4-[8-[(3A)-l-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-Z>][L4]oxazin-3-yl]-3- hvdroxy-5-methyl-benzonitrile;2.2.2-trifluoroacetic acid
A solution of 4-[8-[(3A)-l-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- methyl-5-(2 -trimethylsilylethoxymethoxy )benzonitrile (21.0 mg, 0.03 mmol, 1.0 eq) in DCM (1 mL) and TFA (0.5 mL) was stirred at 25 °C for 1 h. Upon the reaction completion, the reaction mixture was concentrated in vacuum and dissolved in MeOH (2mL), basified to pH = 7 by NH3 H2O. Then the mixture was filtered, and the filtrate was concentrated in vacuum. The residue was purified by prep-HPLC (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% TFA, v/v)-ACN]; B%: 5%-35%, 10 minutes) and followed by lyophilization to afford the title compound (10.98 mg, 62% yield) as a light yellow solid. LCMS: m/z 366.1 [M+H]+, ESI pos.
Example 11:
2-[8-[(71?,21?)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-3,5- dimethyl-phenol;2,2,2-trifluoroacetic acid Step A : l-Benzyloxy-2-bromo-3,5-dimethyl-benzene
To a solution of 2-bromo-3,5-dimethyl-phenol (5.0 g, 24.9 mmol, 1.0 eq), bromomethylbenzene (5.1 g, 29.8 mmol, 1.2 eq) in DMF (50 mL) was added CS2CO3 (16.2 g, 49.7 mmol, 2.0 eq). The reaction mixture was stirred at 25°C for 1 hour under nitrogen atmosphere. The above reaction mixture was diluted with water (200 mL), extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with brine (300 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether) to afford the title compound (7.0 g, 87% yield) as colorless oil. JH NMR (400 MHz, DMSO-ifc) 3 [ppm]: 7.54 - 7.28 (m, 5H), 6.90 - 6.83 (m, 1H), 6.80 - 6.74 (m, 1H), 5.16 (s, 2H), 2.30 (s, 3H), 2.24 (s, 3H).
Step B : 2-(2-Benzyloxy-4,6-dimethyl-phenyl)-4A5,5-tetramethyl-L3,2-dioxaborolane
To a solution of l-benzyloxy-2-bromo-3,5-dimethyl-benzene (7.0 g, 24.04 mmol, 1.0 eq) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(l,3,2-dioxaborolane) (12.21 g, 48.08 mmol, 2.0 eq), CS2CO3 (15.67 g, 48.08 mmol, 2.0 eq), tris(4-methoxy-3,5-dimethylphenyl)phosphane (1.05 g, 2.4 mmol, 0.1 eq) in 1,4-dioxane (70 mL) was added Pd(OAc)2 (0.54 g, 2.4 mmol, 0.1 eq). Then the mixture was stirred at 95°C for 2 hours under N2. The above reaction mixture was cooled to room temperature, then diluted with water (300 mL) and ethyl acetate (200 mL), filtered and the filtrate was extracted with ethyl acetate (200 mL x3). The combined organic phases were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 :0 to 0: 1) to afford the title compound (3.5 g, 39% yield) as colorless oil. 'H NMR (400 MHz, DMSO-ifc) 3 [ppm]: 7.66 - 7.20 (m, 5H), 6.68 (s, 1H), 6.55 (s, 1H), 5.01 (s, 2H), 2.25 (s, 3H), 2.22 (s, 3H), 1.22 (s, 12H).
Step C : 3,5-Dimethyl-2-(4,4,5,5-tetramethyl-L3,2-dioxaborolan-2-yl)phenol
Pd/C (0.26 g, 0.22 mmol, 0.11 eq purity: 10%) in EtOAc (4 mL) was added a solution of 2-(2- benzyloxy-4,6-dimethyl-phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (0.65 g, 1.92 mmol, 1.0 eq) in EtOAc (1 mL). Then the reaction mixture was stirred at 25°C for 2 hours under H2 (1100 mmHg). The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure to afford the title compound (420.0 mg, 79% yield) as orange oil. 1 H NMR (400 MHz, CD3OD) 3 [ppm]: 6.49 (s, 1H), 6.43 (s, 1H), 2.38 (s, 3H), 2.21 (s, 3H), 1.38 (s, 12H). Step D: 2-r8-r(lA,2A)-2-rterLButyl(dimethyl)silyl1oxycyclohexyl1-6,7-dihydropyridazino[4,3- b1[L41oxazin-3-yl1-3,5-dimethyl-phenol
To a mixture of tert-butyl-[(7A,2A)-2-(3-chloro-6,7-dihydropyridazino[4,3-Z>][l,4]oxazin-8- yl)cyclohexoxy]-dimethyl-silane (50.0 mg, 0.13 mmol, 1.0 eq), Na2COs (41.4 mg, 0.39 mmol, 3.0 eq), 3,5-dimethyl-2-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenol (48.5 mg, 0.2 mmol, 1.5 eq) in 1,4-dioxane (2.5 mL) and water (0.5 mL) was added and XphosPdGs (22.1 mg, 0.03 mmol, 0.2 eq). Then the reaction mixture was stirred at 95 °C for 1.5 h under N2. The above reaction mixture was cooled to room temperature. The reaction mixture was filtered off and the filtrate was concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 3: 1) to afford the title compound (40.0 mg, 31% yield) as orange oil. LCMS: m/z 470.4 [M+H]+, ESI pos.
Ste^E^ 2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3,5- dimethyl-phenol;2,2,2-trifluoroacetic acid
A solution of 2-[8-[(lR,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7- dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3,5-dimethyl-phenol (20.0 mg, 0.04 mmol, 1.0 eq) and HCl/dioxane (0.65 mL, 1.3 mmol, 2M in dioxane) was stirred at 25°C for 10 mins. The mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Phenomenex Gemini, 150mm*30mm*15um; mobile phase: [water (0.1% TFA, V/V)-ACN]; B%: l%-55%, 12 mins) to afford the title compound (19.4 mg, 49% yield) as a white solid. LCMS: m/z 356.1 [M+H]+, ESI pos.
Example 12:
2-[8-[(71?,21?)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-3- methyl-5-(trifluoromethoxy)phenol;2,2,2-trifluoroacetic acid
Step A: 3-Methyl-5-(trifluoromethoxy)aniline To a solution mixture of 3-bromo-5-(trifluoromethoxy)aniline (10.0 g, 39.1 mmol, 1.0 eq), trimethylboroxine (16.7 mL, 58.6 mmol, 1.5 eq) in 1,4-Dioxane (100 mL) and Water (20 mL) was added K2CO3 (10.8 g, 78.1 mmol, 2.0 eq) and the mixture was degassed and purged with N2 for three times. Then Pd(dppf)C12 (1.43 g, 1.95 mmol, 0.05 eq) was added to the above mixture. The mixture was stirred at 100 °C for 12 h under N2. Upon then reaction completion, the reaction mixture was cooled to room temperature. EtOAc (150 mL) and water (50 mL) were added to the above mixtyure and layers were separated. The aqueous phase was extracted with EtOAc (200 mL x 2). The combined extracts were washed with brine (100 mL x 2), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1 to 5: 1) to afford the title compound (6.2 g, 56% yield) as yellow oil. LCMS: m/z 192.2 [M+H]+, ESI pos
Step B: 3-Methyl-5-(trifluoromethoxy)phenol
To a mixture of 3-methyl-5-(trifluoromethoxy)aniline (7.2 g, 37.7 mmol, 1.0 eq) in sulfuric acid (35.0 mL)/water (35 mL) was added NabTCh (5.2 g, 75.33 mmol, 2.0 eq) and purged with N2 for three times The mixture was stirred at 80 °C for 3 h. Upon the reaction completion, the mixture was poured into water (50 mL) at 0 °C. EtOAc (100 mL) was added to the above mixyture and the layers were separated. The aqueous phase was extracted with EtOAc (100 mL x 2). The combined phases were washed with brine (50 mL x 2), dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 10/0 to 5/1) to afford the title compound (1.3 g, 16% yield) as brown oil. LCMS: m/z 191.2 [M- H]’, ESI neg.
Step C: 2-Iodo-3-methyl-5-(trifluoromethoxy)phenol
To a mixture of 3-methyl-5-(trifluoromethoxy)phenol (3.2 g, 16.65 mmol, 1.0 eq) in toluene (60 mL) was added NaH (1.33 g, 33.3 mmol, 2.0 eq 60% in mineral oil ) under N2, then the reaction mixture was stirred for 30 mins. I2 (4.23 g, 16.65 mmol, 1.0 eq) was added to the above mixture. The mixture was stirred at 25 °C for 16 h. Upon the reaction completion, the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (20 mL). The aqueous phase was extracted with EtOAc (30 mL x 2). The combined phases were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 :0 to 100: 1) to afford the title compound (2.3 g, 39% yield) as yellow oil. LCMS: m/z 317.0 [M+H]+, ESI pos. Step D: l-Benzyloxy-2-iodo-3-methyl-5-(trifluoromethoxy)benzene
To a mixture of 2-iodo-3-methyl-5 -(trifluorometh oxy)phenol (1.27 g, 3.59 mmol, 1.0 eq) in DMF (10 mL) was added K2CO3 (0.99 g, 7.19 mmol, 2.0 eq) and BnBr (0.85 mL, 7.16 mmol, 1.1 eq). The reaction mixture was stirred at 25 °C for 16 h. Upon the reaction completion, the mixture was quenched by IN HC1 (10 mL). EtOAc (40 mL) and water (40 mL) were added to the above mixture and the layers were separated. The aqueous phase was extracted with EtOAc (30 mL x 2). The combined phases were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 :0 to 10: 1) to afford the title compound (1.2 g, 78% yield) as colorless oil. JH NMR (400 MHz, CD3OD) 3 [ppm]: 7.52 (d, 2H), 7.41 - 7.35 (m, 2H), 7.33 - 7.29 (m, 1H), 6.88 (s, 1H), 6.75 (d, 1H), 5.17 (s, 2H), 2.49 (s, 3H).
Step E: 2-[2-Benzyloxy-6-methyl-4-('trifluoromethoxy)phenyl]-4A5.5-tetramethyl- l .3.2- dioxaborolane
To a solution of l-benzyloxy-2-iodo-3-methyl-5-(trifluoromethoxy)benzene (1.10 g, 2.7 mmol, 1.0 eq) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(l,3,2-dioxaborolane) (1.37 g, 5.39 mmol, 2.0 eq), CS2CO3 (1.76 g, 5.39 mmol, 2.0 eq), tris(4-methoxy-3,5-dimethylphenyl)phosphane (235.3 mg, 0.54 mmol, 0.2 eq) in 1,4-dioxane (20 mL) was added Pd(OAc)2 (121.0 mg, 0.54 mmol, 0.2 eq) under N2 at 25°C. Then the mixture was stirred at 95 °C for 4 h under N2. Upon the reaction completion, the reaction mixture was cooled to roon temperature. EtOAc (50 mL) and water (20 mL) were added to the above mixture and the layers were separated. The aqueous phase was extracted with EtOAc (50 mL x 2). The combined phases were washed with brine (20 mL x 2), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum ether: ethyl acetate = 1 :0 to 10: 1) to afford the title compound (620.0 mg, 54% yield) as a white solid. 'H NMR (400 MHz, CD3OD ) 3 [ppm]: 7.48 (d, 2H), 7.39 - 7.34 (m, 2H), 7.33 - 7.29 (m, 1H), 6.71 (s, 1H), 6.68 (s, 1H), 5.03 (s, 2H), 2.35 (s, 3H), 1.28 (s, 12H).
Step F: [(77?,27?)-2-[3-[2-Benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-6,7- dihydropyridazino[4,3-Z>][L4]oxazin-8-yl]cyclohexoxy]-tert-butyl-dimethyl-silane
To a solution of 2-[2-benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-4,4,5,5-tetramethyl-l,3,2- dioxaborolane (31.9 mg, 0.08 mmol, 1.5 eq), Na2COs (16.6 mg, 0.16 mmol, 3.0 eq), /c/7-butyl- [(77?,27?)-2-(3-chloro-6,7-dihydropyridazino[4,3-Z>][l,4]oxazin-8-yl)cyclohexoxy]-dimethyl- silane (20.0 mg, 0.05 mmol, 1.0 eq) in 1,4-dioxane (1 mL)/water (0.20 mL) was added XphosPdGs (8.8 mg, 0.01 mmol, 0.2 eq) under N2 at 25 °C. The mixture was stirred at 95 °C for 16 h. Upon the reaction completion, the reaction was cooled to room temperature. EtOAc (10 mL) and water (5 mL) were added to the above mixture and the layers were separated. The aqueous phase was extracted with EtOAc (10 mL x 2). The combined phases were washed with brine (10 mL x 2), dried over Na2SO4, filtered, and concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate 5: 1) to afford the title compound (12.0 mg, 35% yield) as a yellow solid. LCMS: m/z 630.4 [M+H]+, ESI pos.
Step G: 2- -2-rtert-Butyl(dimethyl)silyl1oxycvclohexyl1-6,7-dihvdropyridazinol4,3- birL41oxazin-3-yl1-3-methyl-5-(tri fluoromethoxylphenol
To a solution of Pd/C (10.0 mg) in EtOAc (1 mL) and methanol (0.1 mL) was added [(lR,2R)-2- [3-[2-benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-6,7-dihydropyridazino[4,3b][l,4]oxazin- 8-yl]cyclohexoxy]-tert-butyl-dimethyl-silane (70.0 mg, 0.11 mmol, 1.0 eq) under N2. The reaction mixture was stirred at 25 °C for 1 h under H2 (1100 mmHg). Upon the reaction completion, the suspension was filtered through a pad of Celite and the filter cake was washed with EtOAc (20 mL><4). The combined filtrates were concentrated in vacuum to afford the title compound (60.0 mg, 85% yield) as yellow oil. LCMS: m/z 540.3 [M+H]+, ESI pos.
Step H: 2-[8-[(7A,2A)-2-Hydroxycyclohexyl1-6,7-dihydropyridazino[4,3-Z>1[L41oxazin-3-yl1-3- methyl-5-(trifluoromethoxy)phenol;2,2,2-trifluoroacetic acid
A solution of 2-[8-[(lR,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7- dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol (50.0 mg, 0.09 mmol, 1.0 eq) and HCl/dioxane (0.5 mL, 1.0 mmol, 2M in dioxane) was stirred at 25°C for 10 minutes. Upon the reaction completion, the reaction mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Phenomenex Gemini, 150mm*30mm*15um; mobile phase: [water (0.1% TFA, V/V)-ACN]; B%: 3%-53%, 12 mins) to afford the title compound (33.1 mg, 65% yield) as a white solid. LCMS: m/z 426.2 [M+H]+, ESI pos.
Example 13:
4-[8-[(51?)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3-yl]-3- hydroxy-benzonitrile;2,2,2-trifluoroacetic acid
Step A: 3-Chloro-8-[(3A)-l-ethyl-3-DiDeridyl1-4-methyl-6,7-dihydroDyridazino[4,3- blUAIoxazine
To a solution of 2-(3,6-dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate (220.0 mg, 0.7 mmol, 1.0 eq), (3R)-l-ethylpiperi din-3 -amine (116.3 mg, 0.91 mmol, 1.3 eq) in DMSO (1 mL) was added DIEA (162.1 mg, 1.26 mmol, 1.8 eq) under N2. Then the reaction mixture was stirred at 80 °C for 16 h. Upon the reaction completion, the above reaction mixture was cooled to 25 °C. The solution was purified by reversed phase flash (column: Xtimate C18, 250mm*50mm*10pm; mobile phase: [water (0.1% FA, v/v)-ACN]; B%: 5%-35%, 10 minutes) and followed by lyophilization to give the title compound (18.0 mg, 8.32% yield) as a yellow solid. LCMS: m/z 297.2 [M+H]+, ESIpos.
Step B: 4-[8-[(3A)-l-Ethyl-3-piperidyl1-4-methyl-6,7-dihydropyridazino[4,3-b1[L41oxazin-3-yl1- 3-(2-trimethylsilylethoxymethoxy)benzonitrile
To a solution of 3-chloro-8-[(3A)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazine (18.0 mg, 0.06 mmol, 1.0 eq), 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-3- (2 -trimethylsilylethoxymethoxy )benzonitrile (34.1 mg, 0.09 mmol, 1.5 eq), CsF (27.5 mg, 0.18 mmol, 3.0 eq) in dioxane (1.0 mL) and water (0.2 mL) was added XphosPdGs (5.1 mg, 0.01 mmol, 0.1 eq). Then the reaction mixture was stirred at 95 °C for 4 h under nitrogen. The above reaction mixture was cooled to room temperature. The mixture was concentrated in vacuum. The residue was purified by reversed phase flash (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% ammonia hydroxide v/v)-ACN]; B%: 5%-35%, 10 minutes) and followed by lyophilization to give the title compound (11.0 mg, 34% yield) as a yellow solid. LCMS: m/z 510.5 [M+H]+, ESI pos.
Step C: 4-[8-[(3A)-l-Ethyl-3-piperidyl1-4-methyl-6,7-dihydropyridazino[4,3-Z>1[L41oxazin-3-yl1-
3 -hydroxy-benzoni tri le;2.2.2-tri fluoroacetic acid A solution of 4-[8-[(3R)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]-3-(2-trimethylsilylethoxymethoxy)benzonitrile (11.0 mg, 0.02 mmol, 1.0 eq), TFA (104.7 mg, 1.08 mmol, 50.0 eq) in DCM (1 mL) was stirred at 25°C for 1 hour under nitrogen. Upon the reaction completion, the reaction mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Xtimate Cis, 250mm*50mm*10pm; mobile phase: [water (0.1% TFA, v/v)-ACN]; B%: 10%-100%, 15 minutes) and followed by lyophilization to give the title compound (5.3 mg, 50% yield) as a yellow solid. LCMS: m/z 380.2 [M+H]+, ESI pos.
Example 14:
5-[8-[(71?,21?)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3- yl]indan-4-ol;2,2,2-trifluoroacetic acid
Step A: 5-[8-[(77?,27?)-2-[terLbutyl(dimethyl)silyl1oxycvclohexyl1-4-methyl-6,7- dihydropyridazino[4,3-birE41oxazin-3-yl1indan-4-ol
To a solution of Intermediate 6 tert-butyl-[(77?,27?)-2-(3-chloro-4-methyl-6,7- dihydropyridazino[4,3-b][l,4]oxazin-8-yl)cyclohexoxy]-dimethyl-silane (10.0 mg, 0.03 mmol, 1.0 eq) in 1,4-dioxane (1 mL) / water (0.200 mL) was added ISfeCCL (7.99 mg, 0.08 mmol, 3.0 eq), 5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)indan-4-ol (9.8 mg, 0.04 mmol, 1.5 eq) and Xphos Pd g3 (4.26 mg, 0.01 mmol, 0.2 eq) and the mixture was heated at 95 °C for 1.5 hours. Then the reaction mixture was was cooled to room temperature, combined with another batch and concentrated under reduced pressure. The crude product was purified by prep-TLC (petroleum ether : ethyl acetate = 5: 1, Rf = 0.5 ) to get the title compound as an orange oil (10.0 mg, 71% yield). LCMS m/z: 496.2 [M+H]+, ESI pos.
Step B: 5-[8-[(77?,27?)-2-hydroxycyclohexyl1-4-methyl-6,7-dihydropyridazino[4,3-Z>1[L41oxazin- 3-yl1indan-4-ol;2,2,2-trifluoroacetic acid
To a solution of 5-[8-[(77?,27?)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7- dihydropyridazino[4,3-b][l,4]oxazin-3-yl]indan-4-ol (5.0 mg, 0.01 mmol, 1.0 eq) in 1,4-dioxane (0.5 mL) was added dioxane/HCl (0.5 mL, 1.0 mmol, 99 eq) and the mixture was stirred at 25 °C for 0.08 hours. The mixture was then concentrated in vacuo and the crude product was purified by preparative HPLC ( Column Phenomenex Luna C18 150*25mm*10um Condition water (TFA)-ACN Begin B 20 End B 50 Gradient Time (min) 9 100%B Hold Time(min) 2 FlowRate (ml/min) 25) to obtain the title compound as a white solid. The product was combined with another batch. (4.2 mg, 83% yield). LCMS m/z: 382.2 [M+H]+, ESI pos.
Example 15:
3-[8-[(71?,21?)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-6][l,4]oxazin-3- yl]bicyclo[4.2.0]octa-l,3,5-trien-2-ol;2,2,2-trifluoroacetic acid
Step A: 3,6-Dichloro-4-(2-iodoethoxy)-5-methyl-pyridazine
To a solution of 2-(3,6-dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate (1.45 g, 4.6 mmol, 1.0 eq) in MeCN (15 mL) was added Nal (1.03 g, 6.9 mmol, 1.5 eq.), then the reaction mixture was stirred at 80°C for 1 hour. The above reaction mixture was cooled to room temperature, and then diluted with water (100 mL), and extracted with ethyl acetate (100 mL x 3). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1 :0 to 2: 1) to afford the title compound (1.0 g, 59% yield) as yellow oil. 'H NMR (400 MHz, CD3OD) 3 [ppm]: 4.46 (t, 2H), 3.57 (t, 2H), 2.47 (s, 3H).
Step B: terLButyl-r(7A,2A)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-birL41oxazin-8- yDcyclohexoxyl-dimethyl-silane
To a solution of 3,6-dichloro-4-(2-iodoethoxy)-5-methyl-pyridazine (500.0 mg, 1.5 mmol, 1.0 eq.) in DMF (10 mL) was added DIEA (0.49 mL, 3.0 mmol, 2.0 eq.) and trans-(77?,27?)-2-[tert- butyl(dimethyl)silyl]oxycyclohexanamine (516.8 mg, 2.25 mmol, 1.5 eq.), then the mixture was stirred at 50°C for 12 hours. The above reaction mixture was cooled to room temperature and diluted with water (50 mL), extracted with ethyl acetate (50 mL*3). The combined organic phases were washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by prep-TLC (PE: EA = 5: 1) to the title compound (15.0 mg, 2% yield) as yellow oil. LCMS: m/z 398.2 [M+H]+, ESI pos.
Step C: [ -2-[3-(2 -benzyl oxy-3-bicvclo[4.2, 0]octa-L3,5-tri enyl)-4-m ethyl-6,7- dihydropyridazinol4,3-birE41oxazin-8-yl1cyclohexoxy1-terZ-butyl-dimethyl-silane
To a solution of tert-butyl-[(17?,27?)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-8-yl)cyclohexoxy]-dimethyl-silane (15.0 mg, 0.02 mmol, 1.0 eq) in 1,4-dioxane (2 mL) /water (0.4 mL) was added ISfeCCL (5.9 mg, 0.06 mmol, 3.0 eq.), 2-(2 -benzyl oxy-3 - bicyclo[4.2.0]octa-l,3,5-trienyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (19.0 mg, 0.03 mmol, 1.5 eq.) and XphosPdGs (6.4 mg, 0.01 mmol, 0.2 eq.), then was added at 95°C for 1.5 hours under N2 atmosphere. The above reaction mixture was cooled to room temperature and diluted with water (20 mL), extracted with ethyl acetate (50 mL*3). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated in vacuum. The residue was purified by prep-TLC (petroleum ether: ethyl acetate = 3 : 1) to afford the title compound (20.0 mg, 38% yield) as orange oil. LCMS: m/z 572.3 [M+H]+, ESI pos.
Step D: 3-[8-[(17?,27?)-2-[terLbutyl(dimethyl)silyl1oxycvclohexyl1-4-methyl-6,7- dihydropyridazinor4,3-birL41oxazin-3-yl1bicyclol4.2.01octa-L3,5-trien-2-ol
To Pd/C (60.0 mg, purity: 10%) in EtOAc (2 mL) / methanol (0.20 mL) [(17?,27?)-2-[3-(2- benzyloxy-3-bicyclo[4.2.0]octa-l,3,5-trienyl)-4-methyl-6,7-dihydropyridazino[4,3- b][l,4]oxazin-8-yl]cyclohexoxy]-tert-butyl-dimethyl-silane (20.0 mg, 0.03 mmol, 1.0 eq) was added and the mixture was stirred at 25°C for 1 hour under H2 atmosphere. The mixture was filtered through celite and the filtrate was concentrated in vacuum to afford the title compound (15.0 mg, 26% yield) as orange oil. LCMS: m/z 482.2 [M+H]+, ESI pos.
Step E: 3-[8-[(77?,27G-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-Z>][L4]oxazin- 3-yl1bicyclor4.2.01octa-L3,5-trien-2-ol;2,2,2-trifluoroacetic acid
A solution of 3-[8-[(77?,27?)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7- dihydropyridazino[4,3-Z>][l,4]oxazin-3-yl]bicyclo[4.2.0]octa-l,3,5-trien-2-ol (15.0 mg, 0.03 mmol, 1.0 eq) in HCl/dioxane (0.5 mL, 2M in dioxane) was stirred at 25°C for 0.08 hour. The mixture was concentrated in vacuum. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150mm*25mm*10um; mobile phase: [water (0.1% TFA, v/v)-MeCN]; B%: 12%-42%, 12 mins) to afford the title compound (3.7 mg, 24% yield) as a white solid. LCMS: m/z 368.2 [M+H]+, ESI pos.
Example 16:
5-[8-(4-Hydroxycyclohexyl)-6,7-dihydropyridazino[4,3-^][l,4]oxazin-3-yl]-6-methyl-2,3- dihydrobenzofuran-4-ol
Step A: Butyl (dimethyl )silyl1oxy-Af-r2-('3.6-dichloropyridazin-4- yl )oxy ethyl 1 cy cl ohexanamine
A solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (600.0 mg, 2.9 mmol, 1.0 eq) and traw -4-[te/7-butyl(dimethyl)silyl]oxycyclohexanamine (665.01 mg, 2.9 mmol, 1.0 eq) in DCE (12 mL) was stirred at 25 °C for 10 mins. Then NaBH(OAc)3 (1225.97 mg, 5.8 mmol, 2.0 eq) was added in portions at 25 °C. The reaction mixture was stirred at 25 °C for 30 mins. Upon the reaction completion, the mixture was quenched with water (20 mL), then EtOAc (40 mL) was added and layers were separated. The aqueous phase was extracted with EtOAc (30 mL x 3). Combined extracts were washed with brine (30 mL x 2), dried over Na2SO4, filtered, and concentrated under vacuum. The crude was purified by column chromatography on silica gel (EA: MeOH = 1 :0 to 5: 1) to yield the title compound (490.0 mg, 40% yield) as a white solid. LCMS m/z: 420.2 [M+H]+, ESI pos.
Step B: terLButyl-r4-(3-chloro-6,7-dihvdropyridazinor4,3-Z>irL41oxazin-8-yl)cvclohexoxy1- dimethyl -silane
To a mixture of /ra//.s-4-[/c77-butyl(dimethyl)silyl]oxy-A-[2-(3,6-dichloropyridazin-4- yl)oxyethyl]cyclohexanamine (490.0 mg, 1.17 mmol, 1.0 eq), CS2CO3 (1.14 g, 3.5 mmol, 3.0 eq) in 1,4-dioxane (10 mL) was added Xphos Pd G4 (0.2 g, 0.23 mmol, 0.2 eq) under N2, and the mixturewas stirred for 2 hours at 90 °C. Upon reaction completion, the mixture was cooled to room temperature. Then, EtOAc (20 mL) and water (10 mL) were added and layers were separated. The aqueous phase was extracted with EtOAc (20 mL x 3). Combined extracts were washed with brine (10 mL x 2), dried over Na2SO4, filtered, and concentrated under vacuum. The crude was purified by column chromatography on silica gel (petroleum ether : ethyl acetate = 1 :2 to 2: 1) to give the title compound (80.0 mg, 16% yield) as a yellow solid. LCMS m/z: 384.1 [M+H]+, ESI pos.
The isomer /c/7-butyl-[4-(3-chl oro-6, 7-dihydropyridazino[3,4-b][l,4]oxazin-5-yl)cyclohex oxy]- dimethyl -silane (60.0 mg, 9% yield) was obtained as yellow solid.
Step C: 5-r8-r4-rferZ-Butyl(dimethyl)silyl1oxycyclohexyl1-6,7-dihydropyridazinol4,3- Z>11E41oxazin-3-yl1-6-methyl-2,3-dihydrobenzofuran-4-ol
To a solution of te/7-butyl-[4-(3-chloro-6,7-dihydropyridazino[4,3-Z>][l,4]oxazin-8-yl)- cyclohexoxy]-dimethyl-silane (70.0 mg, 0.18 mmol, 1.01 eq) and 6-methyl-5-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-4-ol (50.0 mg, 0.18 mmol, 1.0 eq) in 1,4-dioxane (1 mL) / water (0.2 mL) was added ISfeCCL (57.57 mg, 0.54 mmol, 3.0 eq), and XPhos Pd g3 (30.69 mg, 0.04 mmol, 0.2 eq) under N2. The reaction mixture was heated under microwave irradiation at 95 °C for 2 hours under N2. Upon reaction completion, the reaction mixture was cooled to room temperature. Then EtOAc (10 mL) and water (5 mL) were added and layers were separated. The aqueous phase was extracted with EtOAc (10 mL x 3). Combined extracts were washed with brine (10 mL x 3), dried over Na2SO4, filtered, and concentrated under vacuum and the crude was combined with another batch. The residue was purified by preparative TLC (PEZEA 4: 1, Rf = 0.3) to give the title compound (35.0 mg, 34% yield) as yellow solid. LCMS m/z: 498.3 [M+H]+, ESI pos.
Step D: 5-r8-(4-Hvdroxycvclohexyl)-6,7-dihvdropyridazinol4,3-Z>irL41oxazin-3-yl1-6-methyl- 2.3-dihydrobenzofuran-4-ol
To a solution of 5-[8-[4-[terLbutyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3- Z>][l,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol (25.0 mg, 0.05 mmol, 1.0 eq) in 1,4- dioxane (0.500 mL) was added HCl/dioxane (0.05 mL, 0.05 mmol, 1.0 eq) and the mixture was stirred at 25 °C for 10 minutes. Upon reaction completion, the mixture was concentrated under vacuum. The residue was purified by Cis column chromatography (0.1% TFA in water/MeCN, MeCN:20%-30%, 4 minutes) to afford the title compound by LCMS m/z: 384.1 [M+H]+, ESI pos.
Example 17:
5- [8- 1 ( / /?,.?.S)-3- II y d roxy cyclohexyl] -6,7-dihydropyridazino [4 ,3-b] [1,4] oxazin-3-yl] -6- methyl-2,3-dihydrobenzofuran-4-ol
Step A: (7A,35f)-3 butyl(dimethyl)silyl1oxy-A-r2-(3,6-dichloropyridazin-4-yl)oxyethyl1- cyclohexanamine
To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (300.0 mg, 1.45 mmol, 1.0 eq) in DCE (6 mL) was added (77?,35)-3-[terLbutyl(dimethyl)silyl]oxycyclohexanamine (332.51 mg, 1.45 mmol, 1.0 eq) followed by NaBH(OAc)3 (614.46 mg, 2.90 mmol, 2.0 eq) and the mixture was stirred at 25 °C for 0.5 hour. The above reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to get the crude product, which was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1 :0 to 0: 1) to yield the title compound as a white solid (90.0 mg, 12% yield).
Step B: terLButyl-r -3-(3-chloro-6,7-dihydropyridazinol4,3-Z>irL41oxazin-8- vDcyclohexoxyI-dimethyl-silane
To a solution of (77?,35)-3-[terLbutyl(dimethyl)silyl]oxy-A-[2-(3,6-dichloropyridazin-4- yl)oxyethyl]cyclohexanamine (90.0 mg, 0.21 mmol, 1.0 eq) in 1,4-dioxane (3 mL) was added CS2CO3 (208.7 mg, 0.64 mmol, 3.0 eq) and Xphos Pd G4 (36.84 mg, 0.04 mmol, 0.2 eq) under N2 and the reaction was stirred at 90 °C for 2 hours under N2. The reaction mixture was cooled to room temperature and diluted with water (20 mL), extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to get the crude product and combined with another batch. The combined crude was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 1 :0 to 0: 1) to yield the title compound as a yellow oil (50.0 mg, 61% yield). LCMS m/z: 384.2 [M+H]+, ESI pos. The isomer terLbutyl-[(75,37?)-3-(3- chl oro-6, 7-dihydropyridazino[3,4-Z>][l,4]oxazin-5-yl)cyclohexoxy]-dimethyl-silane (40.0 mg, 46% yield) was obtained as a yellow oil. Step C: 5-[8-[ -3-[terLButyl(dimethyl)silyl1oxycvclohexyl1-6,7-dihvdropyridazino[4,3- Z>iri,41oxazin-3-yl1-6-methyl-2,3-dihydrobenzofuran-4-ol
To a solution of /c77-butyl-[(//?,3A')-3-(3-chloro-6,7-dihydropyridazino[4,3-/i][ l ,4]oxazin-8- yl)cyclohexoxy]-dimethyl-silane (40.0 mg, 0.1 mmol, 1.0 eq) in 1,4-dioxane (2 mL) / water (0.200 mL) was added ISfeCCL (22.08 mg, 0.21 mmol, 2.0 eq), 6-methyl-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)-2,3-dihydrobenzofuran-4-ol (34.52 mg, 0.13 mmol, 1.2 eq) and Xphos Pd g3 (17.66 mg, 0.02 mmol, 0.2 eq) . The reaction mixture was then stirred at 95 °C for 2 hours under N2. The above reaction mixture was combined with another batch, then diluted with water (20 mL), extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to get the crude product. The crude product was purified by prep-TLC (petroleum ether : ethyl acetate = 4: 1, Rf = 0.4 ) to yield the title compound as a yellow solid (30.0 mg, 34% yield). LCMS m/z: 498.3 [M+H]+, ESI pos.
Step D: 5-[8-[(77?,3M-3-Hydroxycyclohexyl]-6 J-dihydropyridazino[4,3-Z>][L4]oxazin-3-yl]-6- methyl-2.3-dihydrobenzofuran-4-ol
To a solution of 5-[8-[(77?,35)-3-[terLbutyl(dimethyl)silyl]oxycyclohexyl]-6,7- dihydropyridazino[4,3-Z>][l,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol (20.0 mg, 0.04 mmol, 1.0 eq) in 1,4-dioxane (2 mL) was added dioxane/HCl (1.0 mL, 2.0 mmol, 49.77 eq) and the mixture was stirred at 25 °C for 0.08 hour. The mixture was then concentrated in vacuo and was combined with another batch. The pH was adjusted to pH ~ 8 with aq. NaHCCh (0.4 mL), then was filtered and the filtrate was concentrated under reduced pressure to get the crude product. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150mm*25mm*5um; mobile phase: [water (0.1% NHi’^O, V/V)-ACN]; B%: 10%-40%, 10 mins) to yield the title compound (10.3 mg, 66% yield) as a white solid. LCMS m/z: 384.2 [M+H]+,ESI pos.
Example 18:
5- [8- [(35, 41?)-3-Hydroxytetrahydropyran-4-yl] -6,7-dihydropyridazino [4,3-6] [1,4] oxazin-3- yl]-6-methyl-2,3-dihydrobenzofuran-4-ol Step A: butyl(dimethyl)silyl1oxy-7V-r2-(3,6-dichloroDyridazin-4- yl)oxyethyl1tetrahydropyran-4-amine
To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (320.0 mg, 1.55 mmol, 1.0 eq) in DCE (5 mL) was added (35,4A)-3-[tert-butyl(dimethyl)silyl]oxytetrahydropyran-4-amine (429.26 mg, 1.85 mmol, 1.2 eq) and the mixture was stirred at 20 °C for 5 min. Then NaBH(OAc)3 (655.24 mg, 3.09 mmol, 2.0 eq) was added into above mixture in portions. Then the reaction mixture was stirred at 20 °C for 5 minutes. Upon the reaction completion, the reaction mixture was cooled to room temperature. EtOAc (40 mL) and water (40 mL) were added and layers were separated. The aqueous phase was extracted with EtOAc (30 mL x 2). Combined extracts were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography on silica gel (petroleum ether : ethyl acetate = 3 : 1 to 2: 1), to afford the title compound (400.0 mg, 55% yield) as yellow oil. LCMS m/z: 422.2 [M+H]+, ESI pos.
Step B: terLButyl-r(3 ,4A)-4-(3-chloro-6,7-dihydropyridazinol4,3-Z>irL41oxazin-8- yl)tetrahydropyran-3-yl1oxy-dimethyl-silane
To a mixture of aforementioned (3A-// )-3-[/c/7-butyl(dimethyl)silyl]oxy-A-[2-(3,6- dichloropyridazin-4-yl)oxyethyl]tetrahydropyran-4-amine (350.0 mg, 0.83 mmol, 1.0 eq), CS2CO3 (807.9 mg, 2.49 mmol, 3.0 eq) in 1,4-dioxane (20 mL) was added Xphos Pd G4 (142.58 mg, 0.17 mmol, 0.2 eq) under N2, then stirred for 2 hours at 90 °C under N2. Upon the reaction completion, the reaction mixture was cooled to room temperature. EtOAc (50 mL) and water (40 mL) were added and layers were separated. The aqueous phase was extracted with EtOAc (50 mL x 2). Combined extracts were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography on silica gel (petroleum ether : ethyl acetate = 5: 1 to 3: 1) to afford the title compound (90.0 mg, 24% yield) as yellow oil. LCMS m/z: 386.2 [M+H]+, ESI pos.
Step C: 5-r8 -3-lterLButyl(dimethyl)silyl1oxytetrahydropyran-4-yl1-6,7- dihydropyridazinol4,3-Z>irL41oxazin-3-yl1-6-methyl-2,3-dihydrobenzofuran-4-ol
To a solution of tert-butyl-[(35',4A)-4-(3-chloro-6,7-dihydropyridazino[4,3-Z>][l,4]oxazin-8- yl)tetrahydropyran-3-yl]oxy-dimethyl-silane (60.0 mg, 0.13 mmol, 1.0 eq) in 1,4-dioxane (3 mL) and water (0.30 mL) was added ISfeCCL (28.3 mg, 0.27 mmol, 2.0 eq), 6-methyl-5-(4, 4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-4-ol (36.9 mg, 0.13 mmol, 1.0 eq) and Xphos Pd g3 (22.66 mg, 0.03 mmol, 0.2 eq), then was stirred at 95 °C for 2 hours under N2. Upon the reaction completion, the reaction mixture was cooled to room temperature. EtOAc (40 mL) and water (30 mL) were added and layers were separated. The aqueous phase was extracted with EtOAc (30 mL x 2). Combined extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give a residue. The residue was purified by preparative TLC (PE:EA= 2.5: 1, Rf= 0.2) to afford the title compound (50.0 mg, 77% yield) as a yellow oil. LCMS m/z: 500.3 [M+H]+, ESI pos.
Step D: 5-18-1(3 , 4A)-3-Hvdroxytetrahvdropyran-4-yl1-6,7-dihvdropyridazinol4,3-Z>irL41oxazin- 3-yl1-6-methyl-2,3-dihvdrobenzofuran-4-ol
To a solution of 5-[8-[(LS',-/ >)-3-[/c/7-butyl(dirnethyl)silyl]oxytetrahydropyran-4-yl]-6,7- dihydropyridazino[4,3-Z>][l,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol (40.0 mg, 0.08 mmol, 1.0 eq) in 1,4-dioxane (3 mL) was added HCl/dioxane (2.0 mL, 2 M in dioxane) . Then the reaction mixture was stirred at 20 °C for 30 minutes. The reaction mixture was concentrated in vacuum at 30 °C. Then the residue was dissolved with MeOH (3 mL) and pH was adjusted to pH ~7 with NaHCCL, and then purified by reversed phase flash (CombiFlash, mobile phase: (water (0.1% ammonia hydroxide v/v)-ACN); B%: 23%-25%) to afford the title compound (20.3 mg, 64% yield) as white solid. LCMS m/z: 386.1 [M+H]+, ESI pos.
Example A
A compound of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
Per tablet
Active ingredient 200 mg
Microcrystalline cellulose 155 mg
Com starch 25 mg
Talc 25 mg
Hydroxypropylmethylcellulose 20 mg
425 mg Example B
A compound of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:
Per capsule
Active ingredient 100.0 mg
Com starch 20.0 mg
Lactose 95.0 mg
Talc 4.5 mg
Magnesium stearate 0.5 mg
220.0 mg

Claims

Claims ompound of formula I wherein,
R1 is H or alkyl;
R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted with l-to-2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo; ii. is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
X is -O-, -CH2-, -NH-, or -N(CH3)-;
W is selected from ring systems A and B
R3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
R5 is H; or R4 and R5, and the atoms to which they are attached, form i. a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
R6 is halo, haloalkyl or OH;
R7 is H or F; and pharmaceutically acceptable salts thereof. A compound according to claim 1, wherein R1 is H or alkyl and R3 is H, alkyl or alkoxyalkyl, wherein one of R1 or R3 is H and the other is not H. A compound according to claim 1 or claim 2, wherein R1 is H or alkyl and R3 is H or alkyl, wherein one of R1 or R3 is H and the other is alkyl. A compound according to any of claims 1 to 3, wherein R2 is selected from i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-membered cycloalkyl substituted with alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl. A compound according to any of claims 1 to 4, wherein R2 is selected from a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH. A compound according to any of claims 1 to 5 wherein X is X is O or -CH2-. A compound according to any of claims 1 to 6, wherein W is W is selected from ring systems
A, C and D wherein Y is CH2 or -O-. A compound according to any of claims 1 to 7, wherein W is ring system A A compound according to any of claims 1 to 8, wherein R4 is alkyl, cyano, haloalkyl or haloalkoxy. A compound according to any of claims 1 to 9, wherein R4 is cyano. A compound according to any of claims 1 to 10, wherein R5 is H, or R4 and R5, and the atoms to which they are attached, form i. 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom. A compound according to any of claims 1 to 10, wherein R5 is H, or R4 and R5, and the atoms to which they are attached, form either a 5-membered cycloalkyl ring, or a 5-membered heterocycle ring comprising a single O heteroatom. A compound according to any of claims 1 to 11, wherein R5 is H. A compound according to any of claims 1 to 12, wherein R6 is OH. A compound according to claim 1, wherein
R1 is H or alkyl; R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl, halo, haloalkyl, hydroxyalkyl or oxo; ii. a -CH2-heterocycle, wherein the heterocycle is a 5-6 membered heterocycle comprising a single O heteroatom; iii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle comprising a single N heteroatom optionally substituted with alkyl;
X is -O-, -CH2-, -NH-, or -N(CH3)-;
W is selected from ring systems A and B
R3 is H, halo, alkyl, alkoxy, or alkoxyalkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is halo, alkyl, alkoxy, cyano, haloalkyl, or haloalkoxy;
R5 is H; or R4 and R5, and the atoms to which they are attached, form i. a 4-5 membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle ring comprising a single O heteroatom optionally substituted with l-to-2 substituents independently selected from alkyl and halo;
R6 is halo, haloalkyl or OH;
R7 is H or F; and pharmaceutically acceptable salts thereof. compound according to any of claims 1 to 13, wherein
R1 is H or alkyl and R3 is H or alkyl, wherein one of R1 or R3 is H and the other is alkyl; R2 is selected from: i. a 6-membered heterocycle comprising a single N heteroatom substituted with alkyl, and ii. a 4-membered cycloalkyl substituted with alkyl and OH;
X is O or -CH2-;
W is ring system A:
R4 is cyano;
R5 is H;
R6 is OH; and pharmaceutically acceptable salts thereof. ompound according to claim 1, wherein
R1 is H or alkyl;
R2 is selected from: i. a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and ii. a 4-6 membered cycloalkyl optionally substituted with l-to-2 substituents independently selected from alkyl and OH;
X is -O- or -CH2-;
W is selected from ring systems A, C and D
R3 is H or alkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is alkyl, cyano, haloalkyl, or haloalkoxy;
R5 is H;
R6 is OH;
Y is CH2 or O; and pharmaceutically acceptable salts thereof. ompound according to claim 1, wherein
R1 is H or alkyl;
R2 is a 5-6 membered heterocycle comprising a single O or a single N heteroatom atom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH;
X is -O- or -CH2-;
W is selected from ring systems C and D
R3 is H or alkyl, wherein one of R1 and R3 is H and the other is not H;
R4 is alkyl, cyano, haloalkyl, or haloalkoxy;
R5 is H;
R6 is OH;
Y is CH2 or O; and pharmaceutically acceptable salts thereof. ompound according to any one of claims 1 to 14, wherein the compound is selected from 4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hydroxy-5-methyl-benzonitrile;formic acid;
4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hy droxy- 5 -methyl -b enzonitril e;
4-[8-[(3R)-l-Ethyl-3-piperidyl]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-3-yl]-3-hydroxy- 5 -methyl -b enzonitril e;
3-Hydroxy-4-[8-(3 -hydroxy-3 -methyl-cyclobutyl)-6,7-dihydropyridazino[4, 3- b][l,4]oxazin-3-yl]-5-methyl -benzonitrile; and pharmaceutically acceptable salts thereof. ompound according to any one of claims 1 to 14, wherein the compound is selected from 3-hydroxy-4-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid;
3-hydroxy-4-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3- b][l,4]oxazin-3-yl]-5-methyl -benzonitrile;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl-indan-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl -indan -4-ol;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]-2,3-dihydrobenzofuran-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin-
3-yl]-2,3-dihydrobenzofuran-4-ol;
4-[8-[(3R, 5 S)- 1 -ethyl-5-hydroxy-3 -piperidyl]-6,7-dihydropyridazino[4,3 -b] [ 1 ,4]oxazin-
3-yl]-3-hydroxy-5-methyl-benzonitrile;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl-2,3-dihydrobenzofuran-4-ol;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- methyl-5-(trifluoromethyl)phenol;
4-[8-[(3R)-l-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hydroxy-5-methyl-benzonitrile;2,2,2-trifluoroacetic acid;
4-[8-[(3R)-l-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- hy droxy- 5 -methyl -b enzonitril e;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3,5- dimethyl-phenol;2,2,2-trifluoroacetic acid 2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3,5- dimethyl -phenol;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- methyl-5-(trifluoromethoxy)phenol;2,2,2-trifluoroacetic acid;
2-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-3- methyl-5-(trifluoromethoxy)phenol;
4-[8-[(3R)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-
3-hydroxy-benzonitrile;2,2,2-trifluoroacetic acid; 4-[8-[(3R)-l-ethyl-3-piperidyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]- 3 -hydroxy-benzonitrile;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]indan-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]indan-4-ol;
3-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]bicyclo[4.2.0]octa-l,3,5-trien-2-ol;2,2,2-trifluoroacetic acid;
3-[8-[(lR,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]bicyclo[4.2.0]octa-l,3,5-trien-2-ol;
5-[8-[(lR,3S)-3-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl-2,3-dihydrobenzofuran-4-ol;
5-[8-[(3S,4R)-3-hydroxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin- 3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; and pharmaceutically acceptable salts thereof. A compound according to any one of claims 1 to 14, wherein the compound is selected from
5-[8-(4-hydroxycyclohexyl)-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6-methyl-2,3- dihydrobenzofuran-4-ol; and pharmaceutically acceptable salts thereof. A compound according to any one of claims 1 to 14, wherein the compound is selected from
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl-2,3-dihydrobenzofuran-4-ol;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl-indan-4-ol;2,2,2-trifluoroacetic acid;
5-[8-[(lR,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][l,4]oxazin-3-yl]-6- methyl -indan -4-ol; and pharmaceutically acceptable salts thereof. A compound according to any one of claims 1 to 22 for use as a therapeutically active substance. A compound according to any one of claims 1 to 22 for use in the treatment or prevention of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition. A pharmaceutical composition comprising a compound according to any one of claims 1 to 22 and a therapeutically inert carrier. The use of a compound according to any one of claims 1 to 22 for the treatment or prophylaxis of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition. A compound according to any one of claims 1 to 22 for use in the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease. The use of a compound according to any one of claims 1 to 22 in the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease. The use of a compound according to any one of claims 1 to 22 for the preparation of a medicament for the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer’s disease and Parkinson’s disease. A method of inhibiting NLRP3, which method comprises administering an effective amount of a compound as claimed in any one of claims 1 to 22 to inhibit NLRP3. A method for the treatment or prophylaxis of a disease, disorder or condition, which method comprises administering an effective amount of a compound according to any one of claims 1 to 22, wherein the disease, disorder or condition is selected from Alzheimer’s disease and Parkinson’s disease. The invention as hereinbefore described.
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