Inhibitors of NLRP3

Abstract

The present invention relates to novel compounds having the general formula (I) # imgabs0 # wherein R1, R2, X and W are as described herein; comprising the compound; and methods of using the compounds.

Description

Technical Field

[0001] The present invention relates to organic compounds that can be used for the treatment and / or prevention in mammals, and particularly relates to compounds that modulate NLRP3 inhibition.

[0002] The present invention provides novel compounds of formula I,

[0003]

[0004] wherein,

[0005] R 1 is H or alkyl;

[0006] R 2 is selected from:

[0007] i. a 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted with 1 to 2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo;

[0008] ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom;

[0009] iii. a 4- to 6-membered cycloalkyl optionally substituted with 1 to 2 substituents independently selected from alkyl and OH; and

[0010] iv. a 9-membered bicyclic heterocycle containing a single N heteroatom optionally substituted with alkyl;

[0011] X is –O-, –CH2-, -NH- or –N(CH3)-;

[0012] W is selected from ring systems A and B

[0013]

[0014] 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;

[0015] R 4 is halo, alkyl, alkoxy, cyano, haloalkyl or haloalkoxy;

[0016] R 5 is H;

[0017] Or R 4 and R 5 together with the atoms to which they are attached form

[0018] i) An optionally oxo-substituted 4- to 5-membered cycloalkyl ring, or

[0019] ii) A 5-membered heterocycle containing a single O heteroatom optionally substituted with 1 to 2 substituents independently selected from alkyl and halo;

[0020] R 6 is halo, haloalkyl or OH;

[0021] R 7 is H or F;

[0022] and pharmaceutically acceptable salts thereof.

[0023] In addition, the present invention includes all racemic mixtures, all their corresponding enantiomers and / or optical isomers. Background Art

[0024] The NOD-like receptor (NLR) family (the pyrin domain-containing protein 3 (NLRP3) inflammasome) is a component of the inflammatory process, and its abnormal activity is pathogenic in genetic disorders such as cryopyrin-associated periodic syndromes (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis.

[0025] 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). Then, ASC polymerizes to form large aggregates called ASC specks. The polymeric ASC then interacts with the cysteine protease caspase-1 to form a complex called the inflammasome. This leads to the activation of caspase-1, which cleaves the precursor forms of the pro-inflammatory cytokines IL-1β and IL-18 (called pro-IL-1β and pro-IL-18, respectively), thus activating these cytokines. Caspase-1 also mediates a type of inflammatory cell death called pyroptosis. ASC specks can also recruit and activate caspase-8, which can process pro-IL-1β and pro-IL-18 and trigger apoptotic cell death.

[0026] Caspase-1 cleaves pro-IL-1β and pro-IL-18 into their active forms, which are secreted by cells. Active caspase-1 also cleaves gasdermin-D to trigger pyroptosis. Caspase-1 can also mediate the release of alarm protein molecules such as IL-33 and high-mobility group box 1 (HMGB1) by controlling the pyroptotic cell death pathway. Caspase-1 also cleaves intracellular IL-1R2, leading to its degradation and the release of IL-1α. In human cells, caspase-1 can also control the processing and secretion of IL-37. Many other substrates of caspase-1, such as components of the cytoskeleton and glycolytic pathways, may contribute to caspase-1-dependent inflammation.

[0027] NLRP3-dependent ASC specks are released into the extracellular environment, where they can activate caspase-1, induce the processing of caspase-1 substrates, and propagate inflammation.

[0028] The active cytokines derived from NLRP3 inflammasome activation are important drivers of inflammation and interact with other cytokine pathways to form an immune response to infection and injury. For example, IL-1β signaling induces the secretion of the pro-inflammatory cytokines IL-6 and TNF. IL-1β and IL-18 act in concert with IL-23 to induce memory CD4 Th17 cells and γδ T cells to produce IL-17 in the absence of T cell receptor engagement. IL-18 and IL-12 also act in concert to induce IFN-γ production from memory T cells and NK cells, driving a Th1 response.

[0029] The hereditary 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 key component of the inflammatory process. NLRP3 is also associated with the pathogenesis of many complex diseases, which particularly include metabolic disorders such as type 2 diabetes, atherosclerosis, obesity, and gout.

[0030] The role of NLRP3 in central nervous system diseases is emerging, and lung diseases have also been shown to be affected by NLRP3. NLRP3 has also been implicated in a number of central nervous system disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), dementia, Huntington's disease, cerebral malaria, and brain injury caused by pneumococcal meningitis (Walsh et al., Nature Reviews, 15:84-97, 2014, and Dempsey et al. Brain. Behav. Immun. 2017 61:306-316). NLRP3 has also been shown to play a role in a number of lung diseases, including chronic obstructive pulmonary disease (COPD), asthma (including steroid-resistant asthma), asbestosis, and silicosis (DeNardo et al., Am. J. Pathol., 184:42-54, 2014 and Kim et al. Am J Respir Crit Care Med. 2017 196(3):283-97). In addition, NLRP3 has a role in the development of liver disease, kidney disease, and aging. Many of these associations have been defined using Nlrp3 - / - mice, but there are also insights into the specific activation of NLRP3 in these diseases. In type 2 diabetes (T2D), the deposition of islet amyloid polypeptide in the pancreas activates NLRP3 and IL-1β signaling, leading to cell death and inflammation.

[0031] Several small molecules have been shown to inhibit the NLRP3 inflammasome. Glibenclamide inhibits IL-1β production at micromolar concentrations in response to the activation of NLRP3 but not NLRC4 or NLRP1. Other previously characterized weak NLRP3 inhibitors include parthenolide, 3,4-methylenedioxy-β-nitrostyrene, and dimethyl sulfoxide (DMSO), although these agents have limited potency and are non-specific.

[0032] 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-1β antibody canakinumab, and the soluble decoy IL-1 receptor rilonacept. These approaches have been shown to successfully treat CAPS, and these biologic agents have been used in clinical trials for other IL-1β-related diseases.

[0033] There is a need to provide compounds having improved pharmacological and / or physiological and / or physicochemical properties, and / or compounds that provide useful alternatives to known compounds. SUMMARY OF THE INVENTION

[0034] The present invention provides novel compounds of formula I,

[0035]

[0036] wherein,

[0037] R 1 is H or alkyl;

[0038] R 2 is selected from:

[0039] i. a 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted with 1 to 2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo;

[0040] ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom;

[0041] iii. a 4- to 6-membered cycloalkyl optionally substituted with 1 to 2 substituents independently selected from alkyl and OH; and

[0042] iv. a 9-membered bicyclic heterocycle containing a single N heteroatom optionally substituted with alkyl;

[0043] X is –O-, –CH2-, -NH- or –N(CH3)-;

[0044] W is selected from ring systems A and B

[0045]

[0046] R 3 is H, halo, alkyl, alkoxy or alkoxyalkyl, wherein R 1 and R 3 one of them is H and the other is not H;

[0047] R 4 is halo, alkyl, alkoxy, cyano, haloalkyl or haloalkoxy;

[0048] R 5 is H;

[0049] or R 4 and R 5 and the atoms to which they are attached form

[0050] i) a 4- to 5-membered cycloalkyl ring optionally substituted with oxo, or

[0051] ii) a 5-membered heterocycle containing a single O heteroatom optionally substituted with 1 to 2 substituents independently selected from alkyl and halo;

[0052] R 6is halogen, haloalkyl or OH;

[0053] R 7 is H or F;

[0054] and its pharmaceutically acceptable salts.

[0055] The term "alkyl" means a monovalent straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, unless otherwise specified, alkyl contains 1 to 6 carbon atoms (C 1-6 -alkyl) or 1 to 4 carbon atoms (C 1-4 -alkyl). Examples of C 1-6 -alkyl include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and pentyl. Particular alkyls include methyl and ethyl.

[0056] The term "alkoxy" means a group of the formula -O-R', where R' is a C 1-6 -alkyl group. Examples of C 1-6 -alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and tert-butoxy.

[0057] The term "alkoxyalkyl" means an alkyl group in which one of the hydrogen atoms of the alkyl group has been replaced by an alkoxy group. Examples of alkoxyalkyl are methoxymethyl and methoxyethyl.

[0058] The term "cycloalkyl" means a monocyclic or polycyclic saturated or partially unsaturated non-aromatic hydrocarbon. In some embodiments, unless otherwise described, cycloalkyl contains 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, etc. A particular example is cyclobutyl.

[0059] The terms "halogen", "halide" and "halo" are used interchangeably herein and mean fluorine, chlorine, bromine or iodine.

[0060] The term "haloalkyl" means a C 1-6 -alkyl group in which at least one hydrogen atom of the C 1-6 -alkyl group has been replaced by the same or different halogen atoms. Examples of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.

[0061] The term "haloalkoxy" means a C 1-6 -alkoxy group in which at least one hydrogen atom of the C 1-6 -alkoxy group has been replaced by the same or different halogen atoms. Examples of haloalkoxy are difluoromethoxy, trifluoromethoxy, difluoroethoxy and trifluoroethoxy.

[0062] The term "heterocycle" means a monocyclic or bicyclic system of 4 to 9 ring atoms, which is monovalent saturated or partially unsaturated and contains 1, 2 or 3 ring heteroatoms selected from N, O and S, and the remaining ring atoms are carbon. Examples of monocyclic saturated heterocycles are azetidinyl, diazepanyl, pyrrolidinyl, tetrahydrofuranyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl and piperazinyl. Examples of polycyclic saturated heterocycles are azaspiroheptyl, diazaspiroheptyl, azaspirooctyl, diazospirooctyl, diazaspirononyl, oxaazaspirooctyl and oxadiazaspirononyl. A specific example of a heterocycle is piperidinyl.

[0063] The term "hydroxy" means the -OH group.

[0064] The term "hydroxyalkyl" means an alkyl group in which at least one hydrogen atom 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" means the –C≡N group.

[0065] The term "oxo" means the divalent oxygen atom =O.

[0066] The term "pharmaceutically acceptable salt" refers to those salts that retain the biological effects and properties of the free base or free acid and are not undesirable biologically or otherwise. These salts are formed with inorganic acids such as trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (especially 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-acetylcysteine. Additionally, these salts can be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines and tertiary amines, including substituted amines of 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 compounds of formula I can also exist in zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the compounds of formula I are salts formed with formic acid and salts formed with hydrochloric acid, yielding hydrochloride, dihydrochloride or trihydrochloride.

[0067] The abbreviation uM means micromole and is equivalent to the symbol μM.

[0068] The abbreviation uL means microliter and is equivalent to the symbol μL.

[0069] The abbreviation ug means microgram and is equivalent to the symbol μg.

[0070] The compounds of formula I may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers (e.g., racemates), optically pure diastereomers, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

[0071] According to the Cahn-Ingold-Prelog convention, an asymmetric carbon atom can be of the "R" or "S" configuration.

[0072] Another embodiment of the present invention provides a compound according to formula I as described herein and its pharmaceutically acceptable salts or esters, particularly a compound according to formula I as described herein and its pharmaceutically acceptable salts, and more particularly a compound according to formula I as described herein.

[0073] One embodiment of the present invention provides a compound 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.

[0074] One embodiment of the present invention provides a compound 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.

[0075] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 1 is H and R 3 is alkyl.

[0076] One embodiment of the present invention provides a compound according to formula (I) as described herein, wherein R 2 is selected from

[0077] i. a 5- to 6-membered heterocycle containing a single O or a single N heteroatom, wherein the heterocycle is optionally substituted by alkyl, halogen, haloalkyl, hydroxyalkyl or oxo;

[0078] ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom;

[0079] iii. a 4- to 6-membered cycloalkyl optionally substituted by 1 to 2 substituents independently selected from alkyl and OH; and

[0080] iv. Optionally alkyl-substituted 9-membered bicyclic heterocycle containing a single N heteroatom.

[0081] One embodiment of the invention provides a compound according to formula (I) as described herein, wherein R 2 is selected from

[0082] i. A 5- to 6-membered heterocycle containing a single O or a single N heteroatom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH; and

[0083] ii. A 4- to 6-membered cycloalkyl optionally substituted by 1 to 2 substituents independently selected from alkyl and OH.

[0084] One embodiment of the invention provides a compound according to formula I as described herein, wherein R 2 is a 5- to 6-membered heterocycle containing a single O or a single N heteroatom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH.

[0085] One embodiment of the invention provides a compound according to formula (I) as described herein, wherein R 2 is selected from

[0086] i. A 6-membered heterocycle containing a single N heteroatom substituted by alkyl;

[0087] ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom;

[0088] iii. A 4-membered cycloalkyl substituted by alkyl and OH; and

[0089] iv. Optionally alkyl-substituted 9-membered bicyclic heterocycle containing a single N heteroatom.

[0090] One embodiment of the invention provides a compound according to formula (I) as described herein, wherein R 2 is selected from

[0091] i. A 6-membered heterocycle containing a single N heteroatom substituted by alkyl, and

[0092] ii. A 4-membered cycloalkyl substituted by alkyl and OH.

[0093] One embodiment of the invention provides a compound according to formula I as described herein, wherein X is O or –CH2-.

[0094] One embodiment of the invention provides a compound according to formula I as described herein, wherein R 7 is H.

[0095] One embodiment of the invention provides a compound according to formula I as described herein, wherein W is ring system A

[0096]

[0097] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 5 is H, or R 4 and R 5 together with the atoms to which they are attached form

[0098] i. a 5-membered cycloalkyl ring, or

[0099] ii. a 5-membered heterocycle containing a single O heteroatom.

[0100] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 5 is H, or R 4 and R 5 together with the atoms to which they are attached form

[0101] i. an optionally oxo-substituted 4- to 5-membered cycloalkyl ring, or

[0102] ii. a 5-membered heterocycle containing a single O heteroatom.

[0103] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 5 is H.

[0104] One embodiment of the present invention provides a compound according to formula I as described herein, wherein W is selected from ring systems A, C, and D

[0105] wherein

[0106] R 3 is H or alkyl, wherein one of R 1 and R 3 is H and the other is not H;

[0107] R 4 is alkyl, cyano, haloalkyl, or haloalkoxy;

[0108] R 5 is H;

[0109] R 6 is OH; and

[0110] Y is CH2 or O.

[0111] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 4 is halo, cyano, haloalkyl, or haloalkoxy.

[0112] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 4 is alkyl, cyano, haloalkyl or haloalkoxy.

[0113] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 4 is cyano.

[0114] One embodiment of the present invention provides a compound according to formula I as described herein, wherein R 6 is OH.

[0115] One embodiment of the present invention provides a compound according to formula I as described herein, wherein,

[0116] R 1 is H or alkyl;

[0117] R 2 is selected from:

[0118] i. a 5- to 6-membered heterocycle containing a single O or a single N heteroatom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH; and

[0119] ii. a 4- to 6-membered cycloalkyl optionally substituted by 1 to 2 substituents independently selected from alkyl and OH;

[0120] X is –O- or –CH2-;

[0121] W is selected from ring systems A, C and D

[0122] wherein

[0123] R 3 is H or alkyl, wherein one of R 1 and R 3 is H and the other is not H;

[0124] R 4 is alkyl, cyano, haloalkyl or haloalkoxy;

[0125] R 5 is H;

[0126] R 6 is OH;

[0127] Y is CH2 or O;

[0128] and its pharmaceutically acceptable salts.

[0129] One embodiment of the present invention provides a compound according to formula I as described herein, wherein

[0130] R 1 is H or alkyl;

[0131] R 2 is a 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted by alkyl or by both alkyl and OH, and

[0132] X is –O- or –CH2-;

[0133] W is selected from ring systems C and D

[0134] wherein

[0135] R 3 is H or alkyl, wherein 1 and R 3 one of them is H and the other is not H;

[0136] R 4 is alkyl, cyano, haloalkyl or haloalkoxy;

[0137] R 5 is H;

[0138] R 6 is OH;

[0139] Y is CH2 or O;

[0140] and pharmaceutically acceptable salts thereof.

[0141] One embodiment of the present invention provides a compound according to formula I as described herein, wherein,

[0142] R 1 is H or alkyl;

[0143] R 2 is selected from:

[0144] i. a 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted by alkyl, halo, haloalkyl, hydroxyalkyl or oxo;

[0145] ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom;

[0146] iii. a 4- to 6-membered cycloalkyl optionally substituted by 1 to 2 substituents independently selected from alkyl and OH; and

[0147] iv. a 9-membered bicyclic heterocycle containing a single N heteroatom optionally substituted by alkyl;

[0148] X is –O-, –CH2-, -NH- or –N(CH3)-;

[0149] W is selected from ring systems A and B

[0150]

[0151] R 3 is H, halogen, alkyl, alkoxy or alkoxyalkyl, wherein R 1 and R 3 one of them is H and the other is not H;

[0152] R 4 is halogen, alkyl, alkoxy, cyano, haloalkyl or haloalkoxy;

[0153] R 5 is H;

[0154] or R 4 and R 5 and the atoms to which they are attached form

[0155] i. an optionally oxo-substituted 4- to 5-membered cycloalkyl ring, or

[0156] ii. a 5-membered heterocycle containing a single O heteroatom optionally substituted with 1 to 2 substituents independently selected from alkyl and halogen;

[0157] R 6 is halogen, haloalkyl or OH;

[0158] R 7 is H or F;

[0159] and its pharmaceutically acceptable salts.

[0160] One embodiment of the present invention provides a compound according to formula I as described herein, wherein

[0161] R 1 is H or alkyl and R 3 is H or alkyl, wherein R 1 or R 3 one of them is H and the other is alkyl;

[0162] R 2 is selected from:

[0163] i. a 6-membered heterocycle containing a single N heteroatom substituted with alkyl, and

[0164] ii. a 4-membered cycloalkyl substituted with alkyl and OH;

[0165] X is O or –CH2-;

[0166] W is ring system A:

[0167]

[0168] R4 is cyano;

[0169] R 5 is H;

[0170] R 6 is OH;

[0171] and its pharmaceutically acceptable salts.

[0172] One embodiment of the present invention provides a compound according to formula I as described herein, wherein

[0173] R 1 is H and R 3 is alkyl;

[0174] R 2 is selected from:

[0175] i. a 6-membered heterocycle containing a single N heteroatom substituted with alkyl, and

[0176] ii. a 4-membered cycloalkyl substituted with alkyl and OH;

[0177] X is O or –CH2-;

[0178] W is ring system A:

[0179]

[0180] R 4 is cyano;

[0181] R 5 is H;

[0182] R 6 is OH;

[0183] and its pharmaceutically acceptable salts.

[0184] Particular examples of the compounds of formula I as described herein are selected from

[0185] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; formic acid;

[0186] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile;

[0187] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-3-yl]-3-hydroxy-5-methyl-benzonitrile;

[0188] 3-Hydroxy-4-[8-(3-hydroxy-3-methyl-cyclobutyl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile;

[0189] and its pharmaceutically acceptable salts.

[0190] Other specific examples of the compounds of formula I as described herein are selected from

[0191] 3-Hydroxy-4-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid;

[0192] 3-Hydroxy-4-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile;

[0193] 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol; 2,2,2-trifluoroacetic acid;

[0194] 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol;

[0195] 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol; 2,2,2-trifluoroacetic acid;

[0196] 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol;

[0197] 4-[8-[(3R,5S)-1-ethyl-5-hydroxy-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile;

[0198] 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;

[0199] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethyl)phenol;

[0200] 4-[8-[(3R)-1-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; 2,2,2-Trifluoroacetic acid;

[0201] 4-[8-[(3R)-1-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile;

[0202] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-dimethyl-phenol; 2,2,2-Trifluoroacetic acid

[0203] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-dimethyl-phenol;

[0204] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol; 2,2,2-Trifluoroacetic acid;

[0205] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol;

[0206] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-benzonitrile; 2,2,2-Trifluoroacetic acid;

[0207] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-benzonitrile;

[0208] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]indan-4-ol; 2,2,2-Trifluoroacetic acid;

[0209] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]indan-4-ol;

[0210] 3-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol; 2,2,2-trifluoroacetic acid;

[0211] 3-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol;

[0212] 5-[8-[(1R,3S)-3-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;

[0213] 5-[8-[(3S,4R)-3-Hydroxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;

[0214] and their pharmaceutically acceptable salts.

[0215] Other particular examples of the compounds of formula I as described herein are selected from

[0216] 5-[8-(4-Hydroxycyclohexyl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;

[0217] and their pharmaceutically acceptable salts.

[0218] Preferred examples of the compounds of formula I as described herein are selected from

[0219] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol;

[0220] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol; 2,2,2-trifluoroacetic acid;

[0221] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-inden-4-ol;

[0222] and its pharmaceutically acceptable salts.

[0223] Another embodiment of the present invention provides a pharmaceutical composition or a medicament containing the compound of the present invention and a therapeutically inert carrier, diluent or excipient, and a method for using the compound of the present invention to prepare such compositions and medicaments. In one example, the compound of formula I can be formulated into a galenical administration form by mixing with a physiologically acceptable carrier (i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used) at ambient temperature at an appropriate pH and desired purity. The pH of the formulation depends mainly on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the 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 can be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.

[0224] The compositions are formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this case 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 to which the agent is to be delivered, the method of administration, the timing of administration, and other factors known to the practicing physician.

[0225] The compounds of the present invention can be administered by any suitable means, including orally, topically (including buccal and sublingual), rectally, vaginally, transdermally, parenterally, subcutaneously, intraperitoneally, intralungally, intradermally, intrathecally and epidurally and intranasally, and (if needed for local treatment) intralesionally. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. The compounds of the present invention can be administered in any convenient form of administration, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions can contain conventional components in pharmaceutical formulations, for example, diluents, carriers, pH regulators, sweeteners, fillers and other active agents.

[0226] Conventional formulations are prepared by mixing the compounds of the present invention with carriers or excipients. Suitable carriers and excipients are well-known to those skilled in the art and are described in detail, for example, in Ansel, Howard C. et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams and Wilkins, 2004; Gennaro, Alfonso R. et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams and Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also contain one or more buffering agents, stabilizers, surfactants, wetting agents, lubricants, emulsifying agents, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, flavoring agents, diluents, and other known additives to provide an aesthetic presentation of the medicament (e.g., the compound of the present invention or its pharmaceutical composition) or to facilitate the preparation of the pharmaceutical product (e.g., the drug).

[0227] The compounds of formula I and their pharmaceutically acceptable salts can be processed with pharmaceutically inert inorganic or organic auxiliaries for the production of tablets, coated tablets, dragees, hard gelatin capsules, injections, or topical preparations. For example, lactose, corn starch or its derivatives, talc, stearic acid or its salts, etc. can be used as such auxiliaries for tablets, dragees, and hard gelatin capsules.

[0228] Suitable auxiliaries for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid substances, and liquid polyols, etc.

[0229] Suitable auxiliaries for the preparation of solutions and syrups are, for example, water, polyols, sucrose, invert sugar, glucose, etc.

[0230] Suitable auxiliaries for injections are, for example, water, alcohols, polyols, glycerol, vegetable oils, etc.

[0231] Suitable auxiliaries for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols, etc.

[0232] Suitable auxiliaries for topical ophthalmic preparations are, for example, cyclodextrins, mannitol, or many other carriers and excipients known in the art.

[0233] In addition, the pharmaceutical preparation may contain preservatives, solubilizers, thickening substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, fragrances, salts for altering the osmotic pressure, buffering agents, masking agents or antioxidants. They may also contain other therapeutically valuable substances.

[0234] The dosage can vary within a wide range and will of course be suitable for the various requirements in each particular case. Generally speaking, 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), should be appropriate, which is preferably divided into 1 to 3 separate dosages (which may consist of, for example, the same amount). In the case of topical administration, the formulation may contain 0.001% to 15% by weight of the drug, and the required dosage can be 0.1 mg to 25 mg, administered once a day or once a week, or several times a day (2 to 4 times), or several times a week. However, it is obvious that the upper or lower limits given herein can be exceeded when shown to be applicable.

[0235] One embodiment of the present invention is a compound according to formula I as described herein, which is used as a therapeutically active substance.

[0236] One embodiment of the present invention is a compound according to formula I as described herein, which is used for treating or preventing a disease, disorder or condition, wherein the disease, disorder or condition responds to NLRP3 inhibition.

[0237] One embodiment of the present invention is a compound according to formula I as described herein, which is used for treating or preventing a disease, disorder or condition, wherein the disorder or condition responds to NLRP3 inhibition.

[0238] As used herein, the term "NLRP3 inhibition" refers to a complete or partial reduction in the level of NLRP3 activity and includes, for example, inhibiting active NLRP3 and / or inhibiting the activation of NLRP3.

[0239] There is evidence that NLRP3-induced IL-1 and IL-18 play a role in the inflammatory responses associated with or caused by a variety of different disorders (Menu et al., Clinical and Experimental Immunology, 166:1-15, 2011; Strowig et al., Nature, 481:278-286, 2012).

[0240] In one embodiment, the disease, disorder or condition is selected from:

[0241] (i) Inflammation;

[0242] (ii) Autoimmune diseases;

[0243] (iii) Cancer;

[0244] (iv) Infection;

[0245] (v) Central nervous system diseases;

[0246] (vi) Metabolic diseases;

[0247] (vii) Cardiovascular diseases;

[0248] (viii) Respiratory diseases;

[0249] (ix) Liver diseases;

[0250] (x) Kidney diseases;

[0251] (xi) Eye diseases;

[0252] (xii) Skin diseases;

[0253] (xiii) Lymphatic disorders;

[0254] (xiv) Psychological disorders;

[0255] (xv) Graft-versus-host disease;

[0256] (xvi) Abnormal pain;

[0257] (xvii) Conditions associated with diabetes; and

[0258] (xviii) Any disease in which an individual has been determined to carry a germline or somatic non-silent mutation in NLRP3

[0259] In another embodiment, the disease, disorder or condition is selected from:

[0260] (i) Cancer;

[0261] (ii) Infection;

[0262] (iii) Central nervous system diseases;

[0263] (iv) Cardiovascular diseases;

[0264] (v) Liver diseases;

[0265] (vi) Eye diseases; and

[0266] (vii) Skin diseases.

[0267] In yet another exemplary embodiment of the invention, the disease, disorder or condition is inflammation. Examples of inflammation that can be treated or prevented include inflammatory responses associated with or caused by the following diseases:

[0268] (i) Skin diseases such as contact hypersensitivity, bullous pemphigoid, sunburn, psoriasis, atopic dermatitis, contact dermatitis, allergic contact dermatitis, seborrheic dermatitis, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, erythema or alopecia;

[0269] (ii) Arthropathies such as osteoarthritis, systemic juvenile idiopathic arthritis, adult-onset Still's disease, relapsing polychondritis, rheumatoid arthritis, juvenile chronic arthritis, gout or seronegative spondyloarthropathies (such as ankylosing spondylitis, psoriatic arthritis or Reiter's disease);

[0270] (iii) Muscle diseases such as polymyositis or myasthenia gravis;

[0271] (iv) Gastrointestinal diseases such as inflammatory bowel disease (including Crohn's disease and ulcerative colitis), colitis, gastric ulcer, celiac disease, proctitis, pancreatitis, eosinophilic gastroenteritis, mastocytosis, antiphospholipid syndrome, or food-related allergies that may have effects outside the gut (such as migraine, rhinitis or eczema);

[0272] (v) Respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma (including eosinophilic asthma, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma or dust asthma, and especially chronic or refractory asthma such as late asthma and airway hyperreactivity), bronchitis, rhinitis (including acute rhinitis, allergic rhinitis, atrophic rhinitis, chronic rhinitis, caseous rhinitis, hypertrophic rhinitis, pustular rhinitis, dry rhinitis, drug-induced rhinitis, membranous rhinitis, seasonal rhinitis such as hay fever and vasomotor rhinitis), sinusitis, idiopathic pulmonary fibrosis (IPF), sarcoidosis, farmer's lung, silicosis, asbestosis, inflammation caused by volcanic ash, adult respiratory distress syndrome, allergic pneumonia or idiopathic interstitial pneumonia;

[0273] (vi) Vascular diseases such as atherosclerosis, Behcet's disease, vasculitis or Wegener's granulomatosis;

[0274] (vii) Autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, Hashimoto's thyroiditis, type I diabetes, idiopathic thrombocytopenic purpura or Graves' disease;

[0275] (viii) Ocular diseases such as uveitis, allergic conjunctivitis or vernal conjunctivitis;

[0276] (ix) Neurological diseases such as multiple sclerosis or encephalomyelitis;

[0277] (x) Infections or infection-related diseases, such as acquired immunodeficiency syndrome (AIDS), acute or chronic bacterial infections, acute or chronic parasitic infections, acute or chronic viral infections, acute or chronic fungal infections, meningitis, hepatitis (hepatitis A, B, C or other viral hepatitis), peritonitis, pneumonia, epiglottitis, malaria, dengue hemorrhagic fever, leishmaniasis, streptococcal myositis, Mycobacterium tuberculosis (including co-infection with Mycobacterium tuberculosis and HIV), Mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, orchitis / epididymitis, Legionella, Lyme disease, influenza A, Epstein-Barr virus infection, viral encephalitis / aseptic meningitis or pelvic inflammatory disease;

[0278] (xi) Kidney diseases, such as mesangial proliferative glomerulonephritis, nephrotic syndrome, nephritis, glomerulonephritis, obesity-related glomerulopathy, acute renal failure, acute kidney injury, uremia, nephrotic syndrome, renal fibrosis (including chronic crystal nephropathy) or renal hypertension;

[0279] (xii) Lymphatic diseases, such as Castleman disease;

[0280] (xiii) Diseases of the immune system or diseases involving the immune system, such as hyper-IgE syndrome, leprosy, familial hemophagocytic lymphohistiocytosis or graft-versus-host disease;

[0281] (xiv) Liver diseases, such as chronic active hepatitis, non-alcoholic fatty liver disease (NASH), alcoholic 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;

[0282] (xv) Cancers, including those listed above;

[0283] (xvi) Burns, trauma, injuries, bleeding or stroke;

[0284] (xvii) Radiation exposure;

[0285] (xviii) Metabolic diseases, such as type 2 diabetes (T2D), atherosclerosis, obesity, gout or pseudogout; and / or

[0286] (xix) Pain, such as inflammatory hyperalgesia, pelvic pain, allodynia, neuropathic pain or bone pain caused by cancer.

[0287] One embodiment of the present invention is a compound according to Formula I as described herein, which is used for treating or preventing a disease, disorder or condition selected from the following:

[0288] Inflammation;

[0289] Autoimmune diseases;

[0290] Cancer;

[0291] Infection;

[0292] Central nervous system diseases;

[0293] Metabolic diseases;

[0294] Cardiovascular diseases;

[0295] Respiratory diseases;

[0296] Liver diseases;

[0297] Kidney diseases;

[0298] Eye diseases;

[0299] Skin diseases;

[0300] Lymphatic disorders;

[0301] Psychological disorders;

[0302] Graft-versus-host disease;

[0303] Allodynia;

[0304] Conditions associated with diabetes; and

[0305] Any disease in which it has been determined that the individual carries a germline or somatic non-silent mutation in NLRP3.

[0306] One embodiment of the invention is the use of a compound according to formula I as described herein in the treatment or prevention of a disease, disorder or condition that responds to NLRP3 inhibition.

[0307] One embodiment of the invention is the use of a compound according to formula I as described herein in the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

[0308] One embodiment of the invention is the use of a compound according to formula I as described herein for the treatment or prevention of a disease, disorder or condition selected from asthma and COPD.

[0309] One embodiment of the invention is a compound according to formula I as described herein for the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

[0310] One embodiment of the invention is a compound according to formula I as described herein for the treatment or prevention of a disease, disorder or condition selected from asthma and COPD.

[0311] One 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 prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

[0312] One 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 prevention of a disease, disorder or condition selected from asthma and COPD.

[0313] One embodiment of the present invention is a method for the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease, the method comprising administering an effective amount of a compound according to formula I as described herein.

[0314] One embodiment of the present invention is a method for the treatment or prevention of a disease, disorder or condition selected from asthma and COPD, the method comprising administering an effective amount of a compound according to formula I as described herein.

[0315] One embodiment of the present invention relates to a method for inhibiting NLRP3, the method comprising administering an effective amount of a compound according to formula I as described herein.

[0316] Another embodiment of the present invention is a compound of formula I as described herein, which is manufactured according to any one of the methods.

[0317] One embodiment of the present invention is a pharmaceutical composition comprising a compound according to formula I as described herein and a therapeutically inert carrier.

[0318] Determination Procedure

[0319] NLRP3 and Pyroptosis

[0320] It is well known that the activation of NLRP3 leads to pyroptosis, and this feature plays an important role in the manifestation of clinical diseases (Yang-gang Liu et al., Cell Death & Disease, 2017, 8(2), e2579; Alexander Wree et al., Hepatology, 2014, 59(3), 898-910; Alex Baldwin et al., Journal of Medicinal Chemistry, 2016, 59(5), 1691-1710; Ema Ozaki et al., Journal of Inflammation Research, 2015, 8, 15-27; Zhen Xie and Gang Zhao, Neuroimmunology Neuroinflammation, 2014, 1(2), 60-65; Mattia Cocco et al., Journal of Medicinal Chemistry, 2014, 57(24), 10366-10382; T. Satoh et al., Cell Death & Disease, 2013, 4, e644). Therefore, inhibitors of NLRP3 are expected to prevent pyroptosis and the release of pro-inflammatory cytokines (such as IL-1β) from cells.

[0321] THP-1 Cells: Culture and Preparation

[0322] THP-1 cells (ATCC#TIB-202) were grown in RPMI containing L-glutamine (Gibco#11835) and supplemented with 1 mM sodium pyruvate (Sigma#S8636) and penicillin (100 units / ml) / streptomycin (0.1 mg / ml) (Sigma#P4333) in 10% fetal bovine serum (FBS) (Sigma#F0804). Cells were passaged routinely and grown to confluence (about 10 6 cells / ml). On the day of the experiment, THP-1 cells were harvested and resuspended in RPMI medium (without FBS). The cells were then counted and viability was checked by trypan blue (Sigma#T8154) (>90%). Appropriate dilutions were made to obtain a concentration of 625,000 cells / ml. LPS (Sigma#L4524) was added to the diluted cell solution to obtain a final assay concentration (FAC) of 1 μg / ml. 40 μl of the final preparation was aliquoted into each well of a 96-well plate. The plates thus prepared were used for compound screening.

[0323] THP-1 Cell Pyroptosis Assay

[0324] Compound screening was performed step by step according to the following method.

[0325] THP-1 cells (25,000 cells / well) containing 1.0 μg / ml LPS were seeded in 40 μl of RPMI medium (without FBS) in a 96-well, black-walled, clear-bottom cell culture plate coated with poly-D-lysine (VWR #734-0317).

[0326] 5 μl of the compound (8-point half-log dilution, 10 μM highest dose) or vehicle (DMSO 0.1% FAC) was added to the appropriate wells.

[0327] Incubate at 37 °C, 5% CO2 for 3 hours.

[0328] 5 μl of nigericin (Sigma #N7143) (FAC 5 μM) was added to all wells.

[0329] Incubate at 37 °C, 5% CO2 for 1 hr.

[0330] At the end of the incubation period, spin the plate at 300 x g for 3 minutes and remove the supernatant.

[0331] Then 50 μl of resazurin (Sigma #R7017) (FAC 100 μM resazurin, dissolved in RPMI medium without FBS) was added, and the plate was incubated for an additional 1 - 2 hours at 37 °C and 5% CO2. Read the plate in an Envision reader at Ex 560 nm and Em 590 nm.

[0332] IC 50 The data fit a non-linear regression equation (log inhibitor vs. response variable slope 4-parameter).

[0333] The results of the pyroptosis assay are summarized in Table 1 below as THP IC 50 .

[0334] Human Whole Blood IL-1β Release Assay

[0335] For systemic delivery, the ability to inhibit NLRP3 when the compound is present in the bloodstream is very important. For this reason, the NLRP3 inhibitory activity of various compounds in human whole blood was investigated according to the following protocol.

[0336] Human whole blood in lithium heparin tubes was obtained from healthy donors from a volunteer donor panel.

[0337] 80 μl of whole blood containing 1 μg / ml LPS was placed in a 96-well clear-bottom cell culture plate (Corning #3585).

[0338] Add 10 μl of the compound (8-point half-log dilution, 10 μM highest dose) or vehicle (DMSO 0.1% FAC) to the appropriate wells

[0339] Incubate at 37 °C, 5% CO2 for 3 hours

[0340] Add 10 μl of nigericin (Sigma #N7143) (10 μM FAC) to all wells and incubate at 37 °C, 5% CO2 for 1 hr

[0341] At the end of the incubation period, spin the plate at 300 x g for 5 minutes to pellet the cells and remove 20 μl of the supernatant, and add it to a 96-well V-bottom plate for IL-1β analysis (Note: These plates containing the supernatant can be stored at -80 °C for later analysis)

[0342] IL-1β was measured according to the manufacturer's protocol (Perkin Elmer - AlphaLisa IL-1 Kit AL220F - 5000)

[0343] IC 50 The data fit a non-linear regression equation (log inhibitor vs. response variable slope 4-parameter)

[0344] The results of the human whole blood assay are summarized in Table 1 below as HWB IC 50 。

[0345] hERG Screening Assay

[0346] During the development of small molecule drugs, one of the most common adverse side effects leading to drug failure is arrhythmia. Such failures are often related to the ability of the drug to inhibit the human ether-à-go-go related gene (hERG) cardiac potassium channel. Therefore, no inhibition or low inhibition of the hERG cardiac potassium channel is considered beneficial.

[0347] Cells

[0348] The CHO crelox hERG cell line (ATCC reference number PTA-6812, female Chinese hamster cells) was generated and validated at Roche. The ready-to-use frozen CHO-hERG cells were cryopreserved at Evotec (Germany) and used directly in the experiments.

[0349] Experimental solutions

[0350] The extracellular solution contains (in mM): NaCl 150; KCl 4; CaCl2 1; MgCl2 1; HEPES 10; pH 7.2 to 7.4 (with NaOH), and osmotic pressure 290 mOsm to 330 mOsm. The internal solution contains (in mM): KCl, 10; KF, 100; NaCl, 10; HEPES, 10; EGTA, 20; pH = 7.0 to 7.4 (with KOH), and osmotic pressure 260 mOsm to 300 mOsm.

[0351] Electrophysiology

[0352] The effects of compounds on hERG K+ current parameters will be evaluated at two concentrations in at least 4 cells.

[0353] Using an automated patch clamp system 384 (Nanion Technologies GmbH, Germany) for hERG testing. K+ currents were measured using the patch-voltage-clamp technique in the whole-cell configuration at 35°C to 37°C.

[0354] The cells were held at a resting voltage of -80 mV and stimulated by Figure 1 the voltage pattern shown in (pulse pattern for eliciting outward K + currents at 35°C to 37°C) to activate the hERG channels and conduct IKhERG currents outwardly, with a stimulation frequency of 0.1 Hz (6 bpm)

[0355] Data analysis

[0356] The amplitudes of IKhERG were recorded at each drug concentration and compared with the vehicle control value (taken as 100%) to define fractional block. Concentration-response data were fitted with the following relationship:

[0357]

[0358] Using the EworkBook suite (ID Business Solutions Ltd, UK), concentration-response curves were fitted by non-linear regression analysis. A four-parameter logistic model (fit = (A + (B / (1 + ((x / C)^D)))), where A = 0 and B = 100) was used to complete the data fitting.

[0359] The results of the hERG assay are summarized as hERG IC 20 in Table 2 below.

[0360] Transcellular P-gp Assay:

[0361] For general assays, transfected LLC-PK1 cells (porcine kidney epithelial cells) overexpressing human or mouse P-gp are used and cultured on 96-well semipermeable filter plates, where they form a polarized monolayer with tight junctions and act as a barrier between the apical and basolateral compartments.

[0362] P-gp is expressed in the apical membrane of the monolayer.

[0363] The tightness of the cell monolayer and the functional activity of P-gp are confirmed by adding the cell-impermeable tracer fluorescein and the reference P-gp substrate edoxaban, respectively.

[0364] PAMPA :

[0365] PAMPA (Parallel Artificial Membrane Permeability Assay) is the first-line permeability screening method for candidate drugs. The PAMPA assay uses an artificial phospholipid membrane to mimic transcellular absorption conditions. The assay determines permeability values that can be used for compound optimization and ranking purposes, as well as input parameters for computer simulation models to predict intestinal absorption.

[0366] The donor concentration is measured at t-start (reference) and compared with the donor and acceptor concentrations after a certain time (t-end) to calculate the extent of compound passage through the membrane.

[0367] Microsomal Stability :

[0368] Incubation is carried out at 37 °C in a 96-well plate on a TECAN (Tecan Group Ltd, Switzerland) automated liquid handling system with 1 μM test compound (0.5 mg / mL) in microsomes plus the cofactor NADPH. After pre-incubating the test compound with microsomes for 10 minutes, the enzyme reaction is started by adding the cofactor. At 1, 3, 6, 9, 15, 25, 35, and 45 minutes, aliquots of the incubation are taken and quenched with 1:3 (v / v) acetonitrile containing an internal standard. The samples are then cooled and centrifuged, and the supernatant is analyzed by LC-MS / MS2.

[0369] Metabolic Stability in Hepatocytes :

[0370] Assay description:

[0371] Biomaterials. Cryopreserved hepatocytes [mouse, rat, rabbit, monkey, and human (male and female; mixed)] are obtained. Throughout the study, the viability after hepatocyte reconstitution is at least 80%. Ready-to-use rat / human Cultures [long-term hepatocyte co-cultures; pooled (for humans, n = 5 males, n = 5 females)] with stromal mouse fibroblasts (negative control; pooled), as well as plates for incubation, application media, and maintenance media.

[0372] Metabolism by suspended hepatocytes. Primary pooled cryopreserved hepatocytes were reconstituted in pre-warmed William's E medium containing 10% FCS, 0.05 mg / mL streptomycin, 50 U / mL penicillin, 0.4 mM L-glutamine, 0.01 mg / mL gentamicin, 0.048 mg / mL hydrocortisone, and 0.004 mg / mL insulin to a final suspension density of 1×106 cells / mL. Incubation was performed fully automatically using a liquid handling system (Tecan) equipped with a CO2 incubator with an orbital shaker. After adding the test compound, for example, at 1 μM to the wells (1×105 cells / well), the 96-well hepatocyte suspension culture plates were incubated at 37 °C in 5% CO2. Samples were quenched by adding acetonitrile (including internal standard) to the incubation wells at specified time points up to 2 hours.

[0373] Metabolism by was performed. Incubation of the test article (at, for example, 1 μM, 0.1% v / v DMSO) was carried out as in the suspension assay in 96-well plates containing co-cultures of adherent hepatocytes and mouse fibroblast control cells or individual control cells (5% CO2 atmosphere and 37 °C). The incubation medium for humans was the same as that for suspended hepatocytes. At defined time points (2, 18, 26, 48, 72, and 96 hours), all wells were quenched with ice-cold acetonitrile containing internal standard.

[0374] The samples were then centrifuged appropriately, and the supernatant was analyzed by LC-MS / MS. Incubation was performed with n = 1 or 2.

[0375] Table 1: NLRP3 inhibitory activity

[0376]

[0377]

[0378] Table 2: hERG inhibition assay

[0379] Description of the drawings

[0380] Figure 1A voltage mode is shown for maintaining cells at a resting voltage of -80 mV, activating the hERG channel at a stimulation frequency of 0.1 Hz (6 bpm), and conducting the IKhERG current outward (a pulse mode for inducing outward K+ current at 35 °C to 37 °C).

[0381] The present invention will now be illustrated by the following examples, which are not limiting.

[0382] In the case where the preparation example is obtained as a mixture of enantiomers or diastereomers, the pure enantiomers or diastereomers can be obtained by the methods described herein or methods known to those skilled in the art, such as chiral chromatography or crystallization.

[0383] Experimental Methods

[0384] Abbreviations:

[0385]

[0386]

[0387] All examples and intermediates were prepared under a nitrogen atmosphere unless otherwise stated.

[0388] Intermediate:

[0389] Intermediate 1: (4-Cyano-2-hydroxy-6-methyl-phenyl)boronic acid

[0390]

[0391] Step A: 4-Amino-3-methoxy-5-methyl-benzonitrile

[0392] Two batches were carried out in parallel.

[0393] To a solution of commercially available 4-bromo-2-methoxy-6-methylaniline (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 mL, 1.00 eq) and Pd(PPh3)4 (66.8 g, 57.8 mmol, 0.50 eq). The reaction mixture was stirred at 100 °C for 12 hours. The reaction mixture was poured into water (1.50 L) and extracted with ethyl acetate (1 L × 3). The organic phase was washed with brine (1 L × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give the title compound as a yellow solid (28.0 g, 75% yield). 11H NMR (DMSO-d6) δ 7.05 (s, 2H), 5.47 (bs, 2H), 3.81 (s, 3H), 2.09 (s, 3H).

[0394] Step B: 4-Bromo-3-methoxy-5-methyl-benzonitrile

[0395] 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 the mixture was stirred at 65 °C. Then, a solution of the above 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 h. After completion, saturated aqueous Na2SO3 solution (400 mL) and saturated aqueous NH4Cl solution (200 mL) were added to the mixture, and the mixture was extracted with ethyl acetate (500 mL × 3). The organic phase was washed with brine (500 mL × 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1, Rf = 0.75) to give the title compound as a white solid (20.7 g, 42% yield). 1 1H NMR (DMSO-d6) δ 7.43, 7.40 (2s, 1H each), 3.90 (s, 3H), 2.37 (s, 3H).

[0396] Step C: 3-Methoxy-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene Nitrile

[0397] To a solution of the above-mentioned 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 h and Pd(dppf)Cl2·CH2Cl2 (13.0 g, 15.9 mmol, 0.20 eq) was added. The mixture was stirred at 100 °C for 12 h. The mixture was filtered through celite, diluted with H2O (500 mL), and extracted with ethyl acetate (800 mL × 3). The organic phase was washed with brine (800 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 1 / 1, Rf = 0.30) to give the title compound as a white solid (18.0 g, 83% yield). 1 1H NMR (DMSO-d6) δ 7.22, 7.21 (2 s, 1H each), 3.75 (s, 3H), 2.27 (s, 3H), 1.30 (s, 12H).

[0398] Step D: (4-Cyano-2-hydroxy-6-methyl-phenyl)boronic acid

[0399] A solution of the above-mentioned 3-methoxy-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (17.0 g, 96.0 mmol, 1.00 eq) in dichloromethane (170 mL) was cooled to 0 °C, and BBr3 (38.9 g, 155 mmol, 2.50 eq) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 0.5 h. The mixture was poured into H2O (200 mL), filtered, and then the filter cake was collected and triturated with EtOAc (20 mL) to give the title compound as a gray solid (4.67 g, 42% yield). LCMS: m / z 178.1 [M+H] + , ESIpos.

[0400] Intermediate 2: 3,6-Dichloro-4-(3-chloropropyl)pyridazine

[0401]

[0402] 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, after which 4-chlorobutyric acid (CAS#627-00-9, 4.50 g, 36.9 mmol, 1.1 eq) was added, followed by addition of a solution of silver nitrate (1.30 g, 7.65 mmol, 0.23 eq) in water (2.5 mL) over 1 minute. At this point, the appearance of the mixture became milky, and a solution of ammonium persulfate (25.8 g, 113.1 mmol, 3.40 eq.) in water (65 mL) was added slowly over 20 to 30 minutes, which resulted in the formation of a viscous precipitate. The reaction mixture was stirred at 70 °C for 1 hour. After completion of the reaction, the mixture was cooled to room temperature, poured into ice, basified with concentrated ammonia, adjusted to pH ~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 Na2SO4, filtered and concentrated under reduced pressure, and purified by reverse-phase flash chromatography (CombiFlash 0.1% TFA aqueous solution - ACN conditions), and then lyophilized to give the title compound as a colorless oil (4.20 g, 56% yield). LCMS: m / z 226.9 [M+H] + , ESI pos.

[0403] Intermediate 3: 3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((2-(trimethylsilyl)ethoxy)methoxy)benzonitrile Step A: 4-Bromo-2-methoxy-6-methylaniline

[0404]

[0405] Step B: 4-Amino-3-methoxy-5-methylbenzonitrile

[0406] 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) at 0 °C was slowly added dropwise Br2 (22.4 mL, 437.4 mmol, 1.2 eq), and then the mixture was stirred at 25 °C for 2 hours. After completion of the reaction, the above reaction solution was diluted with water (300 mL) and extracted with ethyl acetate (100 mL * 3). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane / EtOAc, 1:0 to 10:1), then slurried with petroleum ether (30 mL), filtered and the cake was collected to give the title compound as a brown solid (30.3 g, 39% yield). 11H NMR (400 MHz, DMSO-d6) δ 6.82 (d, 1H), 6.78 (d, 1H), 4.57 (m, 2H), 3.77 (s, 3H), 2.06 (s, 3H).

[0407] Step C: 4-Bromo-3-methoxy-5-methylbenzonitrile

[0408] To a solution of the above-mentioned 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(PPh3)4 (32.4 g, 28.1 mmol, 0.2 eq). Under a nitrogen atmosphere, the mixture was stirred at 100 °C for 12 h. After completion of the reaction, 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 Na2SO4, filtered, and then the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane / EtOAc, 1:0 to 5:1) to obtain the title compound as a pink solid (22.0 g, 97% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 7.05 (s, 2H), 5.48 (s, 2H), 3.81 (s, 3H), 2.09 (s, 3H).

[0409] Step D: 4-Bromo-3-hydroxy-5-methylbenzonitrile

[0410] To a solution of the above-mentioned 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 h. After completion of the reaction, the mixture was cooled to 20 °C, diluted with ACN (150 mL), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane / EtOAc, 1:0 to 5:1), then slurried with a mixture of ethyl acetate (30 mL) and petroleum ether (3 mL), filtered and the cake was collected to obtain the title compound as a white solid (18.0 g, 59% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 7.46 (s, 1H), 7.43 (s, 1H), 3.91 (s, 3H), 2.39 (s, 3H).

[0411] Step E: 4-Bromo-3-methyl-5-((2-(trimethylsilyl)ethoxy)methoxy)benzonitrile

[0412] At 0 °C, BBr3 (30.0 mL, 316.1 mmol, 11.9 eq) was added to a solution of the above 4-bromo-3-methoxy-5-methyl-benzonitrile (6.0 g, 26.5 mmol, 1.0 eq) in DCM (30 mL), and then stirred at 25 °C for 1 hour. After the reaction was completed, it was combined with another batch (10 g), then diluted with water (500 mL), and extracted with ethyl acetate (150 mL * 3). The combined organic phases were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane / EtOAc, 1:0 to 10:1) to obtain the title compound as a yellow solid (13.3 g, 89% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 6.42 (d, 1H), 6.24 (d, 1H), 1.51 (s, 3H).

[0413] Step F: 3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((2-(trimethylsilyl)ethoxy)methoxy)benzonitrile

[0414] At 0 °C, Cs2CO3 (33.3 g, 102.1 mmol, 1.63 eq) was added to a solution of the above 4-bromo-3-hydroxy-5-methyl-benzonitrile (13.3 g, 62.7 mmol, 1.0 eq) in DMF (60 mL), then stirred at 20 °C for 0.5 hour, then 2-(trimethylsilyl)ethoxymethyl chloride (15.5 mL, 87.8 mmol, 1.4 eq) was added dropwise at 0 °C, and then stirred at 20 °C for 1 hour. After the reaction was completed, it was diluted with water (200 mL), and extracted with MTBE (150 mL * 3). The combined organic phases were washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane / EtOAc, 1:0 to 10:1) to obtain the title compound as a white solid (16.3 g, 76% yield). 1 1H NMR (400 MHz, CDCl3) δ 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).

[0415] Intermediate 4: (1R,2R)-2-[tert-Butyl(dimethyl)silyl]oxycyclohexanamine Intermediate 5: tert-Butyl-[(1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane

[0416] To a solution of the above-mentioned 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'-bis(1,3,2-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)phosphine (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, the mixture was then stirred at 95 °C for 5 h. After completion of the reaction, 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 phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (hexane / EtOAc, 1:0 to 10:1) (17 g), then triturated with MeOH (51 mL), filtered, and the filter cake was collected to give the title compound as a white solid (8.38 g, 74% yield). 1 1H NMR (400 MHz, CDCl3) δ 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).

[0417] Step A:

[0418]

[0419] 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), and then the mixture was stirred at 20 °C for 12 h. After completion of the reaction, the above reaction solution was diluted with water (50 mL) and 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 vacuo. The residue was purified by silica gel column (petroleum ether:ethyl acetate = 1:0 to 3:1) to give the title compound as a yellow liquid (20.5 g, 82% yield). 11H NMR (400 MHz, CDCl3) δ [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).

[0420] Step B: Step C: (1R,2R)-2-[tert-Butyl(dimethyl)silyl]oxy-N-[2-(3,6-dichloropyridazin-4-yl)oxyethyl]-cyclohexanamine

[0421]

[0422] Step D: tert-Butyl-[(1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane Methyl 2-(3,6-dichloropyridazin-4-yl)oxyacetate

[0423] Under N2 at 25 °C, NaH (12.8 g, 318.9 mmol, 1.5 eq, purity: 60%, in mineral oil) was added portionwise to a solution of methyl glycolate (CAS# 96 - 35 - 5, 16.4 mL, 212.6 mmol, 1.0 eq) in THF (100 mL), 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, and the reaction mixture was stirred at 25 °C under N2 for 1 hour. After completion of the reaction, the mixture was quenched with H2O (100 mL) and extracted with EtOAc (150 mL × 3). The combined organic phases were washed with brine (100 mL × 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 3:1 to 2:1) to give the title compound as a yellow solid (20.3 g, 40% yield). LCMS: 236.9 [M + H] + , ESIpos.

[0424] Intermediate 6: tert-Butyl-[(1R,2R)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane 2-(3,6-Dichloropyridazin-4-yl)oxyacetaldehyde

[0425] 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, and then DIBAL-H (1 M in toluene, 2.1 mL, 2.1 mmol, 2.77 eq) was added dropwise to the above mixture under N2. Then the reaction mixture was stirred at -65 °C for 2 h under N2. After completion of the reaction, the reaction mixture was quenched with ice water (5 mL) at 0 °C and extracted with EtOAc (15 mL × 2). The combined organic phases were washed with brine (10 mL × 2), filtered, and the filtrate was dried over Na2SO4, filtered and the filtrate was concentrated in vacuo to give the crude product as a yellow oil (180 mg, 57% yield). The crude product was used directly in the next step without further purification steps.

[0426] Step A: 2-(3,6-Dichloropyridazin-4-yl)oxyethanol Step B: 2-(3,6-Dichloropyridazin-4-yl)oxyethyl ethanesulfonate

[0427] To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (180 mg, crude from the above step) in DCE (4 mL) was added trans-(1R,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 min, 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 min. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (10 mL × 2). The combined extracts were washed with brine (10 mL), filtered, and the filtrate was dried over Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 1:1) to give the title compound as a yellow oil (50.0 mg, 12% yield, over 2 steps). LCMS: m / z 420.2 [M+H] + ,ESI pos.

[0428] Step C: 2-(3,6-Dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate Step D: tert-Butyl-[(1R,2R)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane

[0429] Under N2, Pd-PEPPSI-IHEPTCl (19.9 mg, 0.02 mmol, 0.1 eq) was added to a solution of (1R,2R)-2-[tert-butyl(dimethyl)silyl]oxy-N-[2-(3,6-dichloropyridazin-4-yl)oxyethyl]-cyclohexanamine (100.0 mg, 0.24 mmol, 1.0 eq) and Cs2CO3 (147.2 mg, 0.45 mmol, 1.9 eq) in 1,4-dioxane (2 mL). Then the reaction mixture was stirred at 100 °C for 4 h under N2. After completion of the reaction, the above reaction mixture was cooled to room temperature and poured into ice water (20 mL), and extracted with ethyl acetate (50 mL x 2). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 5:1, Rf = 0.6) to give the title compound as a yellow oil (15.0 mg, 12% yield). LCMS: m / z 384.2, [M+H] + , ESIpos.

[0430] (3S,4R)-3-[tert-Butyl(dimethyl)silyl]oxytetrahydropyran-4-amine ​

[0431]

[0432] ​

[0433] At 0 °C, NaBH4 (5266.6 mg, 139.2 mmol, 2.0 eq) was added 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), and then the reaction mixture was stirred at 25 °C for 1 h. After completion of the reaction, the mixture was quenched with saturated aqueous NH4Cl solution (100 mL) and washed with EtOAc (100 mL x 2). The organic phase was washed with brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 2:1 to 1:1) to give the title compound as a yellow solid (9.8 g, 67% yield). 1 1H NMR (400 MHz, DMSO-d6) δ [ppm]: 7.75 (s, 1H), 5.03 (t, 1H), 4.32 (t, 2H), 3.80 - 3.70 (m, 2H).

[0434] ​

[0435] 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), and then a solution of ethylsulfonyl 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. After completion of the reaction, the above reaction mixture was diluted with water (50 mL) and 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 vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:0 to 1:1) to afford the title compound as a yellow solid (1.0 g, 63% yield). 1 H NMR (400 MHz, DMSO-d6) δ [ppm]: 7.80 (s, 1H), 4.65 - 4.52 (m, 4H), 3.44 - 3.37 (m, 2H), 1.26 (t, 3H).

[0436] ​

[0437] To a stirred suspension of 2-(3,6-dichloropyridazin-4-yl)oxyethyl ethylsulfonate (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. Then the temperature was 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 min. Then the reaction mixture was heated at 70 °C and further stirred for 30 min. After completion of the reaction, 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 vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 3:1) to afford the title compound as a colorless oil (1200.0 mg, 26% yield). LCMS: m / z 315.0 [M+H] + , ESI pos.

[0438] ​ ​

[0439] 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-(1R,2R)-2-[(tert-butyl(dimethyl)silyl)oxy]cyclohexanamine (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), and then the reaction mixture was stirred at 80 °C under N2 for 12 h. After completion of the reaction, the reaction mixture was purified by reversed-phase flash chromatography (column: Xtimate C 18 , 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% ammonium hydroxide, v / v)-ACN]; B%: 5% - 70%, 10 min) to give the title compound as a white solid (15.0 mg, 1% yield). LCMS: m / z 398.2 [M+H] + , ESIpos.

[0440] Intermediate 7: ​

[0441]

[0442] To a solution of trans-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 then the mixture was stirred at 20 °C for 12 h. The above reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel column (ethyl acetate:MeOH = 1:0 to 0:1) to give the title compound as a yellow oil (2.1 g, 32% yield).

[0443] Intermediate 8: (1R,3S)-3-[(tert-Butyl(dimethyl)silyl)oxy]cyclohexanamine

[0444]

[0445] To a solution of (1S,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 tert-butyldimethylchlorosilane (1.41 g, 9.38 mmol, 1.2 eq), and the reaction was stirred at 20 °C for 12 h. Then the above reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give 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 give the title compound as a colorless oil (1.2 g, 60% yield).

[0446] Intermediate 9: trans-4-[(tert-Butyl(dimethyl)silyl)oxy]cyclohexanamine

[0447]

[0448] To a solution of trans-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 h. The above reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate:MeOH = 1:0 to 0:1) to give the title compound as a buttery solid (2.1 g, 32% yield).

[0449] Example

[0450] Example 1:

[0451] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; formic acid

[0452]

[0453] Step A: tert-Butyl (3S)-3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]piperidine-1-carboxylate

[0454] According to WO2020190793, N,N-diisopropylethylamine (312 μL, 1.79 mmol, 2.00 eq) was added to a solution of 3,6-dichloro-4-(2-iodoethoxy)pyridazine (CAS#2490352-76-4, prepared according to WO2020190793, 300 mg, 893.6 μmol, 1.00 eq) and tert-butyl (3R)-3-aminopiperidine-1-carboxylate (CAS#188111-79-7, 196.7 mg, 189.3 μL, 982.9 μmol, 1.10 eq) in DMSO (5 mL). 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 were dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography on silica gel with gradient elution (solid deposition, 30%-80% ethyl acetate / heptane) to give the title compound as a yellow oil (202 mg, 58%). LCMS: m / z 391.3 [M+H] + , ESI pos.

[0455] Step B: tert-Butyl (3R)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)piperidine-1-carboxylate Step C: tert-Butyl (3R)-3-[3-(4-cyano-2-hydroxy-6-methylphenyl)-6,7-dihydropyridazino[4,3-b][1,

[0456] According to WO2020190793, a mixture of the above tert-butyl (3S)-3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]piperidine-1-carboxylate (202 mg, 516.2 μmol, 1.00 eq), palladium(II) acetate (CAS#3375-31-3, 9.27 mg, 41.3 μmol, 0.08 eq), (R)-(+)-binap (CAS#76189-55-4, 41.79 mg, 67.1 μmol, 0.130 eq) and cesium carbonate (319.6 mg, 980.9 μmol, 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 (2 x 20 mL). The crude material was purified by flash chromatography on silica gel (24 g, EtOAc in heptane, 0% to 50%) to give the title compound as a white powder (54 mg, 29%). LCMS: m / z 355.3 [M+H] + , ESI pos.

[0457] 4]oxazin-8-yl]piperidine-1-carboxylate Step D: 3-Hydroxy-5-methyl-4-[8-[(3R)-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxa

[0458] In a sealed tube, the above-mentioned tert-butyl (3R)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)piperidine-1-carboxylate (54 mg, 0.152 mmol, 1.00 eq), (4-cyano-2-hydroxy-6-methyl-phenyl)boronic acid intermediate 1 (45.8 mg, 258.7 μmol, 1.70 eq), and cesium carbonate (148.8 mg, 456.6 μmol, 3.00 eq) were stirred in a mixture of 1,4-dioxane (2 mL) and water (400 μL), and argon was bubbled through the mixture for 2 minutes. Then, the catalyst (XPhos Pd G3) (19.3 mg, 22.8 μmol, 0.15 eq) was added last. The sealed tube was stirred at 100 °C for 3 hours. The reaction mixture was extracted with ethyl acetate (2 x 80 mL) and semi-saturated NH4Cl solution (20 mL). The organic layer was 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 flash chromatography on silica gel with gradient elution (solid deposition, 0%-50% ethyl acetate / heptane) to give the title compound as a light yellow powder (17 mg, 25%). LCMS: m / z 452.4 [M+H] + , ESI pos.

[0459] zin-3-yl]benzonitrile 1:1 hydrogen chloride Step E: 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-

[0460] At room temperature, hydrochloric acid (4 M in dioxane, 75.3 μL, 301.2 μmol, 8.00 eq) was added dropwise to a solution of the above-mentioned tert-butyl (3R)-3-[3-(4-cyano-2-hydroxy-6-methyl-phenyl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl]piperidine-1-carboxylate (17 mg, 37.7 μmol, 1.00 eq) in dichloromethane (0.2 mL) and methanol (0.1 mL). The reaction mixture was stirred at 23 °C for 2 hours. The reaction mixture was concentrated in vacuo to give the title compound as a light yellow viscous oil (1:1 hydrochloride, 18 mg), which was used directly in the next step without further purification. LCMS: m / z 348.3 ([{35Cl}M-H]-), 350.3 ([{37Cl}M-H]-), ESI neg.

[0461] yl]-3-hydroxy-5-methyl-benzonitrile; formic acid Example 2:

[0462] To a suspension of 3-hydroxy-5-methyl-4-[8-[(3R)-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]benzonitrile; hydrochloride (18 mg, 46.4 μmol, 1.00 eq) in dichloromethane (0.1 mL) was added triethylamine (9.65 μL, 69.6 μmol, 1.50 eq). Then, under ice-cooling, acetaldehyde (6.51 μL, 116.02 μmol, 2.50 eq) was added, followed by sodium triacetoxyborohydride (17.7 mg, 83.5 μmol, 1.80 eq). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 45 minutes. After completion of the reaction, the reaction mixture was extracted with DCM, ammonium chloride, and water. The aqueous layer was back-extracted twice with DCM. The combined organic layers were washed with water and brine, then dried over sodium sulfate, filtered, and concentrated in vacuo, and finally purified using RP HPLC (Gemini NX, 12 nm, 5 μm, 100x30 mm; ACN / water + 0.1% HCOOH, gradient 5-50 CAN) to give the title compound as a light brown powder, which was 1:1 formic acid (9 mg, 41%). LCMS: m / z 378.3 [M+H] + , ESI pos. 1 H NMR (600 MHz, DMSO-d6) δ ppm 10.31 (br s, 1H), 9.56 - 9.85 (m, 1H), 7.19 - 7.27 (m, 1H), 7.12 (d, 1H), 6.82 - 6.89 (m, 1H), 5.15 - 5.19 (m, 1H), 4.23 - 4.42 (m, 2H), 3.52 - 3.63 (m, 2H), 3.40 - 3.52 (m, 1H), 3.03 - 3.24 (m, 2H), 2.70 - 2.97 (m, 1H), 2.10 (s, 3H), 1.60 - 2.06 (m, 5H), 1.23 - 1.25 (m, 2H).

[0463] Step A: (R)-3-Chloro-8-(1-ethylpiperidin-3-yl)-5,6,7,8-tetrahydropyrido[2,3-c]pyridazine

[0464] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-3-yl]-3-hydroxy-5-methyl-benzonitrile

[0465]

[0466] Step B: (R)-4-(8-(1-ethylpiperidin-3-yl)-5,6,7,8-tetrahydropyrido[2,3-c]pyridazin-3-yl)-

[0467] To a mixture of 3,6-dichloro-4-(3-chloropropyl)pyridazine intermediate 2 (500.0 mg, 2.22 mmol, 1.0 eq), (3R)-1-ethylpiperidin-3-amine (611.8 mg, 4.77 mmol, 2.15 eq) in DMF (4 mL) solution was added TEA (725 mg, 6.09 mmol, 2.75 eq), and the mixture was stirred at 150 °C for 2 h. The above reaction mixture was cooled to room temperature, purified by reverse-phase flash chromatography (CombiFlash 0.1% aqueous NH3H2O - ACN condition), and then lyophilized to obtain the title compound as a white solid (111 mg, 18% yield). LCMS: m / z 281.0 [M+H]+, ESI pos.

[0468] 3-methyl-5-((2-(trimethylsilyl)ethoxy)methoxy)benzonitrile Step C: 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-3-

[0469] To a solution of the above 3-chloro-8-[(3R)-1-ethyl-3-piperidinyl]-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-1,3,2-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), and then the mixture was stirred at 100 °C under nitrogen for 4 h. The above reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by reverse-phase flash chromatography (CombiFlash 0.1% aqueous NH3H2O - ACN condition) and then freeze-dried to obtain the title compound as a yellow solid (25.0 mg, 9% yield). LCMS: m / z 508.4 [M+H] + ,ESIpos.

[0470] yl]-3-hydroxy-5-methyl-benzonitrile Example 3:

[0471] To a solution of 4-[8-[(3R)-1-ethyl-3-piperidinyl]-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), and then the mixture was stirred at 25 °C under nitrogen for 1 h. The above reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was purified by reverse-phase flash chromatography (CombiFlash 0.1% aqueous NH3-H2O - ACN conditions) and then lyophilized to give the title compound as a yellow solid (13.1 mg, 84% yield). LCMS: m / z 378.3 [M+H]+, ESI pos. 1 H NMR (400 MHz, CD3OD) δ 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).

[0472] Step A: 3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]-1-methyl-cyclobutanol

[0473] 3-Hydroxy-4-[8-(3-hydroxy-3-methyl-cyclobutyl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile

[0474]

[0475] Step B: 3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)-1-methyl-cyclobutanol

[0476] Commercially available 3,6-dichloro-4-(2-iodoethoxy)pyridazine (CAS# 2490352-76-4, 0.54 g, 1.69 mmol, 1.0 eq), 3-amino-1-methyl-cyclobutanol (CAS# 1523606-23-6, 188.4 mg, 1.86 mmol, 1.1 eq), and DIPEA (1.18 mL, 6.77 mmol, 4.0 eq) were dissolved in DMSO (20 mL) and stirred at 50 °C for 48 h. The mixture was diluted with EtOAc (100 mL) and washed with brine (100 mL) and 10 wt% aqueous LiCl solution (3 x 50 mL), 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 as a light brown solid (119.0 mg, 23%). LCMS: m / z 292.1 ([35Cl][M+H]+, ESIpos).

[0477] Step C: 3-Hydroxy-4-[8-(3-hydroxy-3-methyl-cyclobutyl)-6,7-dihydropyridazino[4,3-b][1,4]oxa

[0478] Pd-176 (CAS 879689-47-1, 55.2 mg, 0.07 mmol, 0.2 eq), the above 3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]-1-methyl-cyclobutanol (100 mg, 0.34 mmol, 1.0 eq), and cesium carbonate (334.6 mg, 1.03 mmol, 3.0 eq) were dissolved in t-BuOH (4 mL) and the mixture was degassed (N2, 5 min), then stirred at 90 °C for 3 h. The mixture was filtered through a Celite plug and the Celite was washed with EtOAc (30 mL). The filtrate was dried and loaded onto silica gel and purified by flash chromatography (silica gel, 12 g, 0%-100% EtOAc, then isocratic 10% MeOH in DCM) to afford the title compound as a light yellow solid (32.0 mg, 31%). LCMS m / z 256.1 [M+H]+, ESIpos.

[0479] zin-3-yl]-5-methyl-benzonitrile Example 4:

[0480] (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][1,4]oxazin-8-yl)-1-methyl-cyclobutanol (32.0 mg, 0.13 mmol, 1.0 eq), XphosPd G3 (15.91 mg, 0.02 mmol, 0.15 eq) and cesium carbonate (110.09 mg, 0.34 mmol, 2.7 eq) were suspended in 1,4-dioxane (3 mL) and water (0.75 mL) 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 Celite plug and the filtrate was dried and 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 give the title compound as a light brown solid (8.95 mg, 19%). LCMS m / z 352.8 [M+H]+, ESI pos.

[0481] Step A: 3-Hydroxy-5-methyl-4-[8-[(1R,2R)-2-[(tert-butyl(dimethyl)silyl)oxy]cyclohexyl]-6,

[0482] 3-Hydroxy-4-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid

[0483]

[0484] 7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-benzonitrile Step B: 3-Hydroxy-4-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxa

[0485] To a solution of 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((2-(trimethylsilyl)ethoxy)methoxy)benzonitrile (106.5 mg, 0.27 mmol, 1.5 eq; Intermediate 3), Na2CO3 (57.9 mg, 0.55 mmol, 3.0 eq), tert-butyl-[(1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane (70.0 mg, 0.18 mmol, 1.0 eq) in 1,4-dioxane (1 mL) and water (0.2 mL) was added XphosPdG3 (15.5 mg, 0.02 mmol, 0.1 eq). The reaction mixture was then heated at 95 °C under N2 for 2 h. After completion of the reaction, the above reaction mixture was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 5:1) to give the title compound as an orange oil (20.0 mg, 12% yield). LCMS: m / z 611.5 [M+H] + , ESI pos.

[0486] zin-3-yl]-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid Example 5:

[0487] A solution of 3-hydroxy-5-methyl-4-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-benzonitrile (20.0 mg, 0.03 mmol, 1.0 eq) and HCl / dioxane (0.5 mL, 1.0 mmol, 2 M in dioxane) was stirred at 25 °C for 10 min. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150 mm * 30 mm * 25 um; mobile phase: [water (0.1% TFA, V / V)-ACN]; B%: 22% - 56%, 13 min) to give the title compound as a white solid (8.6 mg, 70% yield). LCMS: m / z 367.1 [M+H] + , ESI pos.

[0488] Step A: 5-[8-[(1R,2R)-2-[(tert-butyl(dimethyl)silyl)oxy]cyclohexyl]-6,7-dihydropyridazino[4,

[0489] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol; 2,2,2-trifluoroacetic acid

[0490]

[0491] 3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol ​

[0492] Under N2, XPhosPdG3 (3.4 mg, 0.01 mmol, 0.2 eq) was added to a solution of 6-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indan-4-ol (5.4 mg, 0.02 mmol, 1.0 eq), tert-butyl-[(1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-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). Then the reaction vessel was sealed and heated in a microwave at 95 °C for 1 h under N2. After completion of the reaction, the reaction mixture was cooled to 25 °C and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 2:1) to give the title compound as a yellow oil (7.0 mg, 71% yield). LCMS: m / z 496.3 [M+H] + , ESIpos.

[0493] Step B: 5-(8-((1R,2R)-2-Hydroxycyclohexyl)-7,8-dihydro-6H-pyridazino[4,3-b][1,4]oxazin- 3-yl)-6-methyl-2,3-dihydro-1H-inden-4-ol; 2,2,2-Trifluoroacetic acid

[0494] A solution of the above 5-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]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, 2 M in dioxane) was stirred at 20 °C for 10 min. After completion of the reaction, the reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC (column: Xtimate C 18 , 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% TFA, v / v)-ACN]; B%: 5%-55%, 10 min) to give the title compound as a white solid (2.7 mg, 35% yield). LCMS: m / z 382.2 [M+H] + , ESIpos.

[0495] Example 6:

[0496] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol; 2,2,2-trifluoroacetic acid

[0497]

[0498] Step A: 5-[8-[(1R,2R)-2-[(tert-Butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7-dihydropyrido [4,3-b][1,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol

[0499] Under N2, XphosPdG3 (6.4 mg, 0.01 mmol, 0.2 eq) was added to a solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-4-ol (11.9 mg, 0.05 mmol, 1.2 eq; CAS# 2923540-32-1), tert-butyl-[(1R,2R)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-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). The reaction vessel was sealed and heated in a microwave at 95 °C for 0.5 h under N2. After completion of the reaction, the reaction mixture was cooled to 20 °C and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 5:1) to give the title compound as a yellow oil (15.0 mg, 74% yield). LCMS: m / z 498.2 [M+H] + , ESI pos.

[0500] Step B: 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxa zin-3-yl]-2,3-dihydrobenzofuran-4-ol; 2,2,2-Trifluoroacetic acid

[0501] A mixture of the above 5-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]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, 2 M in dioxane) was stirred at 20 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Xtimate C 18, 250 mm * 50 mm * 10 μm; Mobile phase: [Water (0.1% TFA, v / v) - ACN]; B%: 5% - 60%, 18 minutes) for purification to obtain the title compound as a white solid (7.2 mg, 48% yield). LCMS: m / z 383.45 [M+H] + , ESI pos.

[0502] Example 7:

[0503] 4-[8-[(3R,5S)-1-Ethyl-5-hydroxy-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile

[0504]

[0505] Step A: (3R,5S)-3-Amino-5-[(tert-butyl(dimethyl)silyl]oxy-piperidine-1-carboxylic acid tert-butyl ester

[0506] To a mixture of tert-butyl (3R,5S)-3-amino-5-hydroxypiperidine-1-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), tert-butyldimethylsilyl chloride (421.6 mg, 2.8 mmol, 1.1 eq) was added. The reaction mixture was stirred at 25 °C for 20 h. After completion of the reaction, the reaction mixture was treated with a mixture of ethyl acetate (15 mL) and water / brine (1:1, V / V) (30 mL). The aqueous layer was extracted with ethyl acetate (50 mL × 3). The organic layer was washed with brine (60 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:5 to 0:1) to obtain the title compound as a yellow oil (380.0 mg, 45% yield). 1 HNMR (400 MHz, CD3OD) δ [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).

[0507] Step B: (3S,5R)-3-[tert-Butyl(dimethyl)silyl]oxy-5-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]piperidine-1-carboxylic acid tert-butyl ester

[0508] 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 (3R,5S)-3-amino-5-[tert-butyl(dimethyl)silyl]oxy-piperidine-1-carboxylic acid tert-butyl ester (340.0 mg, 1.03 mmol, 0.79 eq). The mixture was then stirred at 20 °C for 0.5 h, then NaBH(OAc)3 (691.1 mg, 3.26 mmol, 2.5 eq) was added portionwise to the above mixture, and the resulting mixture was stirred at 20 °C for 0.5 h. After completion of the reaction, the reaction mixture was cooled to room temperature and quenched with water (30 mL). The aqueous phase was extracted with DCM (30 mL × 2). The combined phases were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:1 to 1:3) to afford the title compound as a yellow oil (200.0 mg, 29% yield). LCMS: m / z 521.2 [M+H] + , ESI pos.

[0509] Step C: (3S,5R)-3-[(tert-Butyl(dimethyl)silyl]oxy-5-(3-chloro-6,7-dihydropyridazino[4,3-b] [1,4]oxazin-8-yl)piperidine-1-carboxylic acid tert-butyl ester

[0510] To a solution of (3S,5R)-3-[tert-butyl(dimethyl)silyl]oxy-5-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]piperidine-1-carboxylic acid tert-butyl ester (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). The mixture was then stirred under N2 at 80 °C for 12 h. After completion of the reaction, the reaction mixture was cooled to 20 °C. The mixture was then concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:1 to 0:1) to afford the title compound as a yellow oil (140.0 mg, 22% yield). LCMS: m / z 485.1 [M+H] + , ESI pos.

[0511] Step D: (3S,5R)-3-[(tert-Butyl(dimethyl)silyl]oxy-5-[3-[4-cyano-2-methyl-6-(2-trimethyl silylethoxymethoxy)phenyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl]piperidine-1-carboxylic acid tert-but yl ester

[0512] Under N2, XphosPdG3 (12.0 mg, 0.01 mmol, 0.11 eq) was added to a solution of 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-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-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)piperidine-1-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). The reaction vessel was sealed and heated in a microwave at 95 °C for 30 minutes. After completion of the reaction, the reaction mixture was cooled to 20 °C and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:1 to 0:1) to afford the title compound as a yellow oil (60.0 mg, 47% yield). LCMS: m / z 712.4 [M+H] + , ESIpos.

[0513] Step E: 3-Hydroxy-4-[8-[(3R,5S)-5-hydroxy-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1, 4]oxazin-3-yl]-5-methyl-benzonitrile; 2,2,2-Trifluoroacetic acid

[0514] A solution of tert-butyl (3S,5R)-3-[(tert-butyl(dimethyl)silyl)oxy]-5-[3-[4-cyano-2-methyl-6-(2-trimethylsilylethoxymethoxy)phenyl]-6,7-dihydropyridazino[4,3-b][1,4]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 h. After completion of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by reversed-phase flash chromatography (column: Xtimate C 18 , 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% TFA, v / v)-ACN]; B%: 5%-40%, 13 min) to give the title compound as a yellow solid (20.0 mg, 82% yield). LCMS: m / z 368.2 [M+H] + , ESIpos.

[0515] Step F: 4-[8-[(3R,5S)-1-Ethyl-5-hydroxy-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1, 4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile

[0516] 3-Hydroxy-4-[8-[(3R,5S)-5-hydroxy-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile; Iodoethane (5.8 mg, 0.04 mmol, 1.2 eq) was added to a solution of 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). The reaction mixture was then stirred at 20 °C for 1 h. After completion of the reaction, water (0.1 mL) was added and the mixture was purified by preparative HPLC (column: Phenomenex Gemini, 150 mm * 25 mm * 10 um; mobile phase: [water (0.1% NH4HCO3, V / V) - ACN]; B%: 22% - 52%, 12 min) to give the title compound as a white solid (5.47 mg, 45% yield). LCMS: m / z 396.2 [M+H] + , ESI pos.

[0517] Example 8:

[0518] 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0519]

[0520] Step A: 3,6-Dichloro-4-(2-iodoethoxy)pyridazine

[0521] 2-Iodoethanol (107.0 μL, 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 portionwise 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 was stirred at 40 °C overnight (18 h). The reaction was stirred for 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 (Na2SO4) and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel (40 g column, 0% - 40% EtOAc in heptane) eluting with 30% to give the title compound as a light yellow solid (88.0 mg, 20% yield). LCMS: m / z 318.9 2x 35 Cl [M+H] + , ESIpos.

[0522] Step B: (1R,2R)-2-[2-(3,6-Dichloropyridazin-4-yl)oxyethylamino]cyclohexanol

[0523] Dissolve 3,6-dichloro-4-(2-iodoethoxy)pyridazine (418.0 mg, 1.31 mmol, 1.0 eq), (1R,2R)-2-aminocyclohexanol (227.0 mg, 1.97 mmol, 1.5 eq) and N,N-diisopropylethylamine (0.9 mL, 5.17 mmol, 3.94 eq) in DMSO (8 mL) and stir at 50 °C for 2 h. Leave the reaction to react overnight (16 h). Then, dilute the reaction mixture with EtOAc (50 mL) and 50 w% brine (50 mL) and separate the layers. Extract the aqueous phase with EtOAc (2 x 50 mL), then dry the combined organic matters (Na2SO4), filter and concentrate in vacuo. Combine the crude product with another batch and purify by silica gel chromatography (40 g column, 0%-10% (MeOH:EtOAc) eluting with 10%) to obtain the title compound as a pale yellow solid (124.0 mg, 28% yield). LCMS: m / z 306.1 [M+H] + , ESIpos.

[0524] Step C: (1R,2R)-2-(3-Chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexanol

[0525] Add Pd-176 (360.0 mg, 0.45 mmol, 0.17 eq) and cesium carbonate (2.6 g, 7.98 mmol, 2.96 eq) to a flask containing (1R,2R)-2-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]-cyclohexanol (825.0 mg, 2.69 mmol, 1.0 eq) in tert-butanol (35 mL), which has been rinsed (bubbled with N2 for 5 minutes while ultrasonically treated). Heat the reaction mixture to 80 °C and stir for 2 h. Combine the mixture with other batches and filter through a Celite plug, washing the Celite with EtOAc (30 mL). Dry-load the concentrated filtrate (1.69 g) onto Celite and purify by reverse-phase flash chromatography (C 18 , 43 g column, 10%-100% MeCN[0.1% formic acid]:aqueous solution of 0.1% formic acid) eluting with 25%. After extracting the appropriate fractions with DCM (3 x 50 mL), drying (Na2SO4), filtering and concentrating, obtain the title compound as a pale yellow solid (76.0 mg, 10% yield). LCMS: m / z 270.1 / 272.1 [M+H] + , ESIpos.

[0526] Step D: (1R,2R)-2-[3-(4-Benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-6,7-dihydropyrido [4,3-b][1,4]oxazin-8-yl]cyclohexanol

[0527] 2-(4-Benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (CAS# 2923540-31-0, 82.0 mg, 0.22 mmol, 1.59 eq), (1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]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) were degassed with N2 (sonication, 5 min) in water (0.400 mL) and 1,4-dioxane (2 mL). 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 stirred over the weekend, during which no change was observed. Additionally, 2-(4-benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (27.0 mg, 0.07 mmol, 0.52 eq) and cesium carbonate (49.0 mg, 0.15 mmol, 1.07 eq) were added, the reaction mixture was sparged as described above, then XPhos Pd G3 (12.0 mg, 0.01 mmol, 0.1 eq) was added and the reaction mixture was heated for 2 h as described above. 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 with 5% to afford the title compound as a pale yellow solid (13.0 mg, 18% yield). LCMS: m / z 474.3 [M+H] + , ESI pos.

[0528] Step E: 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3- yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0529] Pd / C (type 87) (16.0 mg, 0.01 mmol, 0.1 eq) was added to a stirred solution of (1R,2R)-2-[3-(4-benzyloxy-6-methyl-2,3-dihydrobenzofuran-5-yl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl]cyclohexanol (36.0 mg, 0.08 mmol, 1.0 eq) in ethanol (3 mL). The hydrogenation vessel was placed under a hydrogen atmosphere (2 bar) at room temperature and stirred vigorously for 2 h. The reaction mixture was filtered through celite, rinsed with EtOH and concentrated under reduced pressure to give a crude residue (34 mg). The crude was dissolved in DMSO in 1.58 mL and purified by reverse-phase preparative HPLC (Waters 2767 Sample Manager, Waters 2545 Binary Gradient Module, Waters System Fluid Organizer, Waters 515 ACD Pump, Waters 515 Make-up Pump, Waters 2998 Photodiode Array Detector, Waters QDa) on a Waters X-Select CSH C 18 ODB preparative column( 5 μm, 30 mm X 100 mm) at a flow rate of 40 mL min-1 with a 0.1% formic acid in water-MeCN gradient over 8.5 min using UV across all wavelengths with PDA as well as QDA and ELS detectors. The at-column dilution pump generated 2 mL min-1 of methanol throughout the method which was included in the following MeCN percentages. Gradient information: 0.0 to 0.5 min, 12.5% MeCN; 0.5 to 5.5 min, ramping from 12.5% MeCN to 42.5% MeCN; 5.5 to 5.6 min, ramping from 42.5% MeCN to 100% MeCN; 5.6 to 8.5 min, held at 100% MeCN. The clean fractions were evaporated in a Genevac. The dried fractions were transferred to vials and lyophilized to afford the title compound as an off-white amorphous lyophilized solid (16.0 mg, 53% yield). LCMS: m / z 384.2 [M+H] + , ESI pos.

[0530] Example 9:

[0531] 2-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethyl)phenol

[0532]

[0533] 3-Methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (CAS# 2557358-38-8, 66.0 mg, 0.22 mmol, 1.55 eq), (1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexanol (38.0 mg, 0.14 mmol, 1.0 eq; Example 8, Step C), and cesium carbonate (131.0 mg, 0.4 mmol, 2.85 eq) were degassed with N2 in water (0.400 mL) and 1,4-dioxane (2 mL). 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 dried and 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 a yellow gummy product containing some minor impurities. The residue was triturated with TBME and PE 40:60. The resulting precipitate was filtered off and dried for 24 h to afford the title compound as a yellow solid (24.0 mg, 39% yield). LCMS: m / z 410.2 [M+H] + , ESI pos.

[0534] Example 10:

[0535] 4-[8-[(3R)-1-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; 2,2,2-Trifluoroacetic acid

[0536]

[0537] Step A: (3R)-3-[2-(3,6-Dichloropyridazin-4-yl)oxyethylamino]pyrrolidine-1-carboxylic acid tert-butyl ester

[0538] To a solution of 2-(3,6-dichloropyridazin-4-yl)oxyethyl ethanesulfonate (2600.0 mg, 8.63 mmol, 1.0 eq) and (R)-(+)-1-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). The reaction mixture was then stirred at 80 °C for 12 h. After completion of the reaction, the mixture was poured into water (30 mL) and extracted with EtOAc (200 mL × 2). The combined organic phases were washed with brine (100 mL × 2), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 0:1 to 1:1) to afford the title compound as a yellow solid (2.0 g, 61% yield). 1 1H NMR (400 MHz, CDCl3) δ [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).

[0539] Step B: (3R)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0540] At 25 °C under N2, to a solution of tert-butyl (3R)-3-[2-(3,6-dichloropyridazin-4-yl)oxyethylamino]pyrrolidine-1-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) solution was added Pd(OAc)2 (95.2 mg, 0.42 mmol, 0.08 eq). The reaction mixture was then 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 vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 5:1) to afford the title compound as a yellow solid (160.0 mg, 9% yield). LCMS: m / z 341.1 [M+H] + , ESIpos.

[0541] Step C: 3-chloro-8-[(3R)-pyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazine

[0542] A solution of tert-butyl (3R)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)pyrrolidine-1-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. After completion of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (column: Xtimate C 18 , 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% ammonia hydroxide v / v) - ACN]; B%: 5% - 35%, 10 min) and then lyophilized to afford the title compound as a yellow oil (30.0 mg, 27% yield). LCMS: m / z 241.0 [M+H] + , ESIpos.

[0543] Step D: 3-chloro-8-[(3R)-1-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazine

[0544] To a solution of 3-chloro-8-[(3R)-pyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]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). The mixture was then stirred at 20 °C for 1 h. After completion of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (column: Xtimate C 18 , 250 mm * 40 mm * 10 μm; mobile phase: [water (0.1% ammonia hydroxide v / v) - MeCN]; B%: 5% - 38%, 15 min) and then lyophilized to afford the title compound as a yellow solid (22.0 mg, 66% yield). LCMS: m / z 269.1 [M+H] + , ESIpos.

[0545] Step E: 4-[8-[(3R)-1-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3- yl]-3-methyl-5-(2-trimethylsilylethoxymethoxy)benzonitrile

[0546] At 25 °C under N2, XPhos Pd G3 (6.9 mg, 0.01 mmol, 0.1 eq) was added 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-[(3R)-1-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazine (22.0 mg, 0.08 mmol, 1.0 eq) in 1,4-dioxane (1 mL) and water (0.2 mL). The mixture was stirred at 95 °C for 2.5 h. After completion of the reaction, the reaction mixture was cooled to room temperature. EtOAc (30 mL) and brine (20 mL) were added to the mixture, and the layers were separated. The aqueous phase was extracted with EtOAc (30 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:1 to 0:1) to afford the title compound as a yellow solid (21.0 mg, 42% yield). 1 1H NMR (400 MHz, CD3OD) δ [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).

[0547] Step F: 4-[8-[(3R)-1-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3- yl]-3-hydroxy-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid

[0548] A solution of 4-[8-[(3R)-1-ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(2-trimethylsilyloxymethoxy)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. After completion of the reaction, the reaction mixture was concentrated in vacuo and dissolved in MeOH (2 mL), basified to pH = 7 with NH3·H2O. Then the reaction was filtered and the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC (column: Xtimate C18 , 250 mm * 50 mm * 10 μm; Mobile phase: [Water (0.1% TFA, v / v) - ACN]; B%: 5% - 35%, 10 minutes), and then lyophilized to give the title compound as a pale yellow solid (10.98 mg, 62% yield). LCMS: m / z 366.1 [M + H] + , ESIpos.

[0549] Example 11:

[0550] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-dimethyl-phenol; 2,2,2-Trifluoroacetic acid

[0551]

[0552] Step A: 1-benzyloxy-2-bromo-3,5-dimethyl-benzene

[0553] To a solution of 2-bromo-3,5-dimethyl-phenol (5.0 g, 24.9 mmol, 1.0 eq), benzyl bromide (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 a nitrogen atmosphere. The above reaction mixture was diluted with water (200 mL) and 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 vacuo. The residue was purified by silica gel chromatography (petroleum ether) to give the title compound as a colorless oil (7.0 g, 87% yield). 1 1H NMR (400 MHz, DMSO-d6) δ [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).

[0554] Step B: 2-(2-benzyloxy-4,6-dimethyl-phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane ane

[0555] To a solution of 1-benzyloxy-2-bromo-3,5-dimethyl-benzene (7.0 g, 24.04 mmol, 1.0 eq), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (12.21 g, 48.08 mmol, 2.0 eq), Cs2CO3 (15.67 g, 48.08 mmol, 2.0 eq), and tris(4-methoxy-3,5-dimethylphenyl)phosphine (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). The mixture was then stirred at 95 °C under N2 for 2 h. 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 x 3). The combined organic phases were washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:0 to 0:1) to afford the title compound as a colorless oil (3.5 g, 39% yield). 1 1H NMR (400 MHz, DMSO-d6) δ [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).

[0556] Step C: 3,5-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol

[0557] To a solution of 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-1,3,2-dioxaborolane (0.65 g, 1.92 mmol, 1.0 eq) in EtOAc (1 mL). The reaction mixture was then stirred at 25 °C under H2 (1100 mmHg) for 2 h. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to afford the title compound as an orange oil (420.0 mg, 79% yield). 1 1H NMR (400 MHz, CD3OD) δ [ppm]: 6.49 (s, 1H), 6.43 (s, 1H), 2.38 (s, 3H), 2.21 (s, 3H), 1.38 (s, 12H).

[0558] Step D: 2-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4, 3-b][1,4]oxazin-3-yl]-3,5-dimethylphenol

[0559] tert-Butyl-[(1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane (50.0 mg, 0.13 mmol, 1.0 eq), Na2CO3 (41.4 mg, 0.39 mmol, 3.0 eq), 3,5-dimethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (48.5 mg, 0.2 mmol, 1.5 eq) were added to a mixture of 1,4-dioxane (2.5 mL) and water (0.5 mL), followed by XphosPdG3 (22.1 mg, 0.03 mmol, 0.2 eq). The reaction mixture was then stirred at 95 °C under N2 for 1.5 h. The above reaction mixture was cooled to room temperature. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 3:1) to afford the title compound as an orange oil (40.0 mg, 31% yield). LCMS: m / z 470.4 [M+H] + , ESI pos.

[0560] Step E: 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-dimethyl-phenol; 2,2,2-Trifluoroacetic acid

[0561] A solution of 2-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-methyl-phenol (20.0 mg, 0.04 mmol, 1.0 eq) and HCl / dioxane (0.65 mL, 1.3 mmol, 2 M in dioxane) was stirred at 25 °C for 10 min. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150 mm * 30 mm * 15 um; mobile phase: [water (0.1% TFA, v / v)-ACN]; B%: 1%-55%, 12 min) to give the title compound as a white solid (19.4 mg, 49% yield). LCMS: m / z 356.1 [M+H] + , ESI pos.

[0562] Example 12:

[0563] 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol; 2,2,2-trifluoroacetic acid

[0564]

[0565] Step A: 3-methyl-5-(trifluoromethoxy)aniline

[0566] 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 three times. Then Pd(dppf)Cl2 (1.43 g, 1.95 mmol, 0.05 eq) was added to the above mixture. The mixture was stirred at 100 °C under N2 for 12 h. Then after the reaction was completed, the reaction mixture was cooled to room temperature. EtOAc (150 mL) and water (50 mL) were added to the above mixture, and the layers were separated. The aqueous phase was extracted with EtOAc (200 mL × 2). The combined extracts were washed with brine (100 mL x 2), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 5:1) to afford the title compound as a yellow oil (6.2 g, 56% yield). LCMS: m / z 192.2 [M+H] + , ESIpos

[0567] Step B: 3-methyl-5-(trifluoromethoxy)phenol

[0568] 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 NaNO2 (5.2 g, 75.33 mmol, 2.0 eq) and purged with N2 three times. The mixture was stirred at 80 °C for 3 h. After the reaction was completed, the mixture was poured into water (50 mL) at 0 °C. EtOAc (100 mL) was added to the above mixture, and the layers were separated. The aqueous phase was extracted with EtOAc (100 mL × 2). The combined phases were washed with brine (50 mL x 2), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 10 / 0 to 5 / 1) to afford the title compound as a brown oil (1.3 g, 16% yield). LCMS: m / z 191.2 [M-H] -, ESI neg.

[0569] Step C: 2-iodo-3-methyl-5-(trifluoromethoxy)phenol

[0570] Under N2, NaH (1.33 g, 33.3 mmol, 2.0 eq, 60%, in mineral oil) was added to a mixture of 3-methyl-5-(trifluoromethoxy)phenol (3.2 g, 16.65 mmol, 1.0 eq) in toluene (60 mL), and then the reaction mixture was stirred for 30 minutes. 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. After completion of the reaction, the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (20 mL). The aqueous phase was extracted with EtOAc (30 mL × 2). The combined phases were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:0 to 100:1) to afford the title compound as a yellow oil (2.3 g, 39% yield). LCMS: m / z 317.0 [M+H] + , ESI pos.

[0571] Step D: 1-benzyloxy-2-iodo-3-methyl-5-(trifluoromethoxy)benzene

[0572] To a mixture of 2-iodo-3-methyl-5-(trifluoromethoxy)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. After completion of the reaction, the reaction mixture was quenched with 1N HCl (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 × 2). The combined phases were washed with brine (60 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to give the title compound as a colorless oil (1.2 g, 78% yield). 1 H NMR (400 MHz, CD3OD) δ [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).

[0573] Step E: 2-[2-benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-di oxaborolane

[0574] At 25 °C under N2, Pd(OAc)2 (121.0 mg, 0.54 mmol, 0.2 eq) was added to a solution of 1-benzyloxy-2-iodo-3-methyl-5-(trifluoromethoxy)benzene (1.10 g, 2.7 mmol, 1.0 eq), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (1.37 g, 5.39 mmol, 2.0 eq), Cs2CO3 (1.76 g, 5.39 mmol, 2.0 eq), and tris(4-methoxy-3,5-dimethylphenyl)phosphine (235.3 mg, 0.54 mmol, 0.2 eq) in 1,4-dioxane (20 mL). The reaction mixture was then stirred at 95 °C under N2 for 4 h. After completion of the reaction, the reaction mixture was cooled to room 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 × 2). The combined phases were washed with brine (20 mL x 2), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to afford the title compound as a white solid (620.0 mg, 54% yield). 1 1H NMR (400 MHz, CD3OD) δ [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).

[0575] Step F: [(1R,2R)-2-[3-[2-benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-6,7-dihydropyridaz ino[4,3-b][1,4]oxazin-8-yl]cyclohexyloxy]-tert-butyl-dimethyl-silane

[0576] Under N2, XphosPdG3 (8.8 mg, 0.01 mmol, 0.2 eq) was added to a solution of 2-[2-benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (31.9 mg, 0.08 mmol, 1.5 eq), Na2CO3 (16.6 mg, 0.16 mmol, 3.0 eq), tert-butyl-[(1R,2R)-2-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane (20.0 mg, 0.05 mmol, 1.0 eq) in 1,4-dioxane (1 mL) / water (0.20 mL). The mixture was stirred at 95 °C for 16 h. After completion of the reaction, 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 × 2). The combined phases were washed with brine (10 mL x 2), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate 5:1) to afford the title compound as a yellow solid (12.0 mg, 35% yield). LCMS: m / z 630.4 [M+H] + , ESIpos.

[0577] Step G: 2-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4, 3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol

[0578] Under N2, [(1R,2R)-2-[3-[2-benzyloxy-6-methyl-4-(trifluoromethoxy)phenyl]-6,7-dihydropyridazino[4,3b][1,4]oxazin-8-yl]cyclohexyloxy]-tert-butyl-dimethyl-silane (70.0 mg, 0.11 mmol, 1.0 eq) was added to a solution of Pd / C (10.0 mg) in EtOAc (1 mL) and methanol (0.1 mL). The reaction mixture was stirred at 25 °C under H2 (1100 mmHg) for 1 h. After completion of the reaction, the suspension was filtered through a pad of diatomaceous earth, and the cake was washed with EtOAc (20 mL × 4). The combined filtrates were concentrated in vacuo to afford the title compound as a yellow oil (60.0 mg, 85% yield). LCMS: m / z 540.3 [M+H] + , ESIpos.

[0579] Step H: 2-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3- yl]-3-methyl-5-(trifluoromethoxy)phenol; 2,2,2-trifluoroacetic acid

[0580] A solution of 2-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]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, 2 M in dioxane) was stirred at 25 °C for 10 minutes. After completion of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150 mm * 30 mm * 15 μm; mobile phase: [water (0.1% TFA, v / v)-ACN]; B%: 3% - 53%, 12 minutes) to give the title compound as a white solid (33.1 mg, 65% yield). LCMS: m / z 426.2 [M+H] + , ESIpos.

[0581] Example 13:

[0582] 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-benzonitrile; 2,2,2-Trifluoroacetic acid

[0583]

[0584] Step A: 3-chloro-8-[(3R)-1-ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4] oxazine

[0585] Under N2, DIEA (162.1 mg, 1.26 mmol, 1.8 eq) was added to a solution of 2-(3,6-dichloro-5-methyl-pyridazin-4-yl)oxyethyl ethanesulfonate (220.0 mg, 0.7 mmol, 1.0 eq) and (3R)-1-ethylpiperidin-3-amine (116.3 mg, 0.91 mmol, 1.3 eq) in DMSO (1 mL). Then the reaction mixture was stirred at 80 °C for 16 h. After completion of the reaction, the above reaction mixture was cooled to 25 °C. The solution was passed through reverse-phase flash chromatography (column: Xtimate C18, 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% FA, v / v)-ACN]; B%: 5% - 35%, 10 minutes), and then lyophilized to give the title compound as a yellow solid (18.0 mg, 8.32% yield). LCMS: m / z 297.2 [M+H] + , ESIpos.

[0586] Step B: 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxa zine-3-yl]-3-(2-trimethylsilylethoxymethoxy)benzonitrile

[0587] To a solution of 3-chloro-8-[(3R)-1-ethylpiperidin-3-yl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazine (18.0 mg, 0.06 mmol, 1.0 eq), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(2-trimethylsilylethoxymethoxy)benzonitrile (34.1 mg, 0.09 mmol, 1.5 eq), and CsF (27.5 mg, 0.18 mmol, 3.0 eq) in dioxane (1.0 mL) and water (0.2 mL) was added XphosPdG3 (5.1 mg, 0.01 mmol, 0.1 eq). The reaction mixture was then stirred under nitrogen at 95 °C for 4 h. The above reaction mixture was cooled to room temperature. The mixture was concentrated in vacuo. The residue was purified by reverse-phase flash chromatography (column: Xtimate C 18 , 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% ammonium hydroxide v / v) - ACN]; B%: 5% - 35%, 10 min), and then lyophilized to give the title compound as a yellow solid (11.0 mg, 34% yield). LCMS: m / z 510.5 [M+H] + , ESI pos.

[0588] Step C: 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxa zine-3-yl]-3-hydroxy-benzonitrile; 2,2,2-Trifluoroacetic acid

[0589] A solution of 4-[8-[(3R)-1-ethylpiperidin-3-yl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-(2-trimethylsilylethoxymethoxy)benzonitrile (11.0 mg, 0.02 mmol, 1.0 eq) and TFA (104.7 mg, 1.08 mmol, 50.0 eq) in DCM (1 mL) was stirred at 25 °C under nitrogen for 1 h. After completion of the reaction, the reaction mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Xtimate C 18 , 250 mm * 50 mm * 10 μm; mobile phase: [water (0.1% TFA, v / v) - ACN]; B%: 10% - 100%, 15 min), and then lyophilized to give the title compound as a yellow solid (5.3 mg, 50% yield). LCMS: m / z 380.2 [M+H] + , ESIpos.

[0590] Example 14:

[0591] 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]indan-4-ol; 2,2,2-trifluoroacetic acid

[0592]

[0593] Step A: 5-[8-[(1R,2R)-2-[(tert-Butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7-dihydropyrida zino[4,3-b][1,4]oxazin-3-yl]indan-4-ol

[0594] To a solution of intermediate 6 tert-butyl-[(1R,2R)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane (10.0 mg, 0.03 mmol, 1.0 eq) in 1,4-dioxane (1 mL) / water (0.200 mL) was added Na2CO3 (7.99 mg, 0.08 mmol, 3.0 eq), 5-(4,4,5,5-tetramethyl-1,3,2-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 h. The reaction mixture was then cooled to room temperature, combined with another batch, and concentrated under reduced pressure. The crude product was purified by preparative TLC (petroleum ether:ethyl acetate = 5:1, R f = 0.5) to afford the title compound as an orange oil (10.0 mg, 71% yield). LCMS m / z: 496.2 [M+H] + , ESIpos.

[0595] Step B: 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxa zine-3-yl]indan-4-ol; 2,2,2-Trifluoroacetic acid

[0596] To a solution of 5-[8-[(1R,2R)-2-[(tert-butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]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 h. The mixture was then concentrated in vacuo, and the crude product was purified by preparative HPLC (column Phenomenex Luna C18 150*25 mm*10 um, conditions: water (TFA)-ACN, start: B 20, end: B 50, gradient time (min): 9, 100% B hold time (min): 2, flow rate (ml / min): 25) to afford 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] + , ESIpos.

[0597] Example 15:

[0598] 3-[8-[(1R,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol; 2,2,2-trifluoroacetic acid

[0599]

[0600] Step A: 3,6-Dichloro-4-(2-iodoethoxy)-5-methyl-pyridazine

[0601] 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 NaI (1.03 g, 6.9 mmol, 1.5 eq.), and the reaction mixture was stirred at 80 °C for 1 h. The above reaction mixture was cooled to room temperature, 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 vacuo. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 2:1) to afford the title compound as a yellow oil (1.0 g, 59% yield). 1 H NMR (400 MHz, CD3OD) δ [ppm]: 4.46 (t, 2H), 3.57 (t, 2H), 2.47 (s, 3H).

[0602] Step B: tert-Butyl-[(1R,2R)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin- 8-yl)cyclohexyloxy]-dimethyl-silane

[0603] 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-(1R,2R)-2-[(tert-butyl(dimethyl)silyl]oxycyclohexanamine (516.8 mg, 2.25 mmol, 1.5 eq.), and then the mixture was stirred at 50 °C for 12 h. The above reaction mixture was cooled to room temperature, diluted with water (50 mL), and 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 vacuo. The residue was purified by preparative TLC (PE:EA = 5:1) to give the title compound as a yellow oil (15.0 mg, 2% yield). LCMS: m / z 398.2 [M+H] + , ESIpos.

[0604] Step C: [(1R,2R)-2-[3-(2-Benzyloxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-6, 7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl]cyclohexyloxy]-tert-butyl-dimethyl-silane

[0605] To a solution of tert-butyl-[(1R,2R)-2-(3-chloro-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane (15.0 mg, 0.02 mmol, 1.0 eq) in 1,4-dioxane (2 mL) / water (0.4 mL) was added Na2CO3 (5.9 mg, 0.06 mmol, 3.0 eq.), 2-(2-benzyloxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (19.0 mg, 0.03 mmol, 1.5 eq.), and XphosPdG3 (6.4 mg, 0.01 mmol, 0.2 eq.), and then added for 1.5 h at 95 °C under N2 atmosphere. The above reaction mixture was cooled to room temperature, and diluted with water (20 mL), and 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 vacuo. The residue was purified by preparative TLC (petroleum ether:ethyl acetate = 3:1) to give the title compound as an orange oil (20.0 mg, 38% yield). LCMS: m / z 572.3 [M+H] + , ESI pos.

[0606] Step D: 3-[8-[(1R,2R)-2-[(tert-Butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7-dihydropyrida zino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol

[0607] [(1R,2R)-2-[3-(2-benzyloxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl]cyclohexyloxy]-tert-butyl-dimethyl-silane (20.0 mg, 0.03 mmol, 1.0 eq) was added to Pd / C (60.0 mg, purity: 10%) in EtOAc (2 mL) / methanol (0.20 mL), and the mixture was stirred at 25 °C for 1 h under a H2 atmosphere. The mixture was filtered through celite, and the filtrate was concentrated in vacuo to afford the title compound as an orange oil (15.0 mg, 26% yield). LCMS: m / z 482.2 [M+H] + , ESIpos.

[0608] Step E: 3-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxa zine-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol; 2,2,2-Trifluoroacetic acid

[0609] A solution of 3-[8-[(1R,2R)-2-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol (15.0 mg, 0.03 mmol, 1.0 eq) in HCl / dioxane (0.5 mL, 2 M in dioxane) was stirred at 25 °C for 0.08 h. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150 mm * 25 mm * 10 um; mobile phase: [water (0.1% TFA, v / v)-MeCN]; B%: 12% - 42%, 12 min) to give the title compound as a white solid (3.7 mg, 24% yield). LCMS: m / z 368.2 [M+H] + , ESIpos.

[0610] Example 16:

[0611] 5-[8-(4-hydroxycyclohexyl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0612]

[0613] Step A: trans-4-[(tert-Butyl(dimethyl)silyl]oxy-N-[2-(3,6-dichloropyridazin-4-yl)oxyethyl]cycl ohexanamine

[0614] A solution of 2-(3,6-dichloropyridazin-4-yl)oxyacetaldehyde (600.0 mg, 2.9 mmol, 1.0 eq) and trans-4-[tert-butyl(dimethyl)silyl]oxycyclohexanamine (665.01 mg, 2.9 mmol, 1.0 eq) in DCE (12 mL) was stirred at 25 °C for 10 minutes. Then NaBH(OAc)3 (1225.97 mg, 5.8 mmol, 2.0 eq) was added portionwise at 25 °C. The reaction mixture was stirred at 25 °C for 30 minutes. After completion of the reaction, the mixture was quenched with water (20 mL), then EtOAc (40 mL) was added and the layers were separated. The aqueous phase was extracted with EtOAc (30 mL × 3). The combined extracts were washed with brine (30 mL x 2), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (EA:MeOH = 1:0 to 5:1) to give the title compound as a white solid (490.0 mg, 40% yield). LCMS m / z: 420.2 [M+H] + , ESI pos.

[0615] Step B: tert-Butyl-[4-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]- dimethyl-silane

[0616] Under N2, Xphos Pd G4 (0.2 g, 0.23 mmol, 0.2 eq) was added to a mixture of trans-4-[tert-butyl(dimethyl)silyl]oxy-N-[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), and the mixture was stirred at 90 °C for 2 hours. After completion of the reaction, the mixture was cooled to room temperature. Then, EtOAc (20 mL) and water (10 mL) were added, and the layers were separated. The aqueous phase was extracted with EtOAc (20 mL × 3). The combined extracts were washed with brine (10 mL x 2), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (petroleum ether:ethyl acetate = 1:2 to 2:1) to give the title compound as a yellow solid (80.0 mg, 16% yield). LCMS m / z: 384.1 [M+H] + , ESI pos.

[0617] The isomer tert-butyl-[4-(3-chloro-6,7-dihydropyridazino[3,4-b][1,4]oxazin-5-yl)cyclohexyloxy]-dimethyl-silane was obtained as a yellow solid (60.0 mg, 9% yield).

[0618] Step C: 5-[8-[4-[(tert-Butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1, 4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0619] Under N2, to a solution of tert-butyl-[4-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)-cyclohexyloxy]-dimethyl-silane (70.0 mg, 0.18 mmol, 1.01 eq) and 6-methyl-5-(4,4,5,5-tetramethyl-1,3,2-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 Na2CO3 (57.57 mg, 0.54 mmol, 3.0 eq) and XPhos Pd g3 (30.69 mg, 0.04 mmol, 0.2 eq). The reaction mixture was heated to 95 °C under N2 under microwave irradiation for 2 h. After completion of the reaction, the reaction mixture was cooled to room temperature. Then EtOAc (10 mL) and water (5 mL) were added, and the layers were separated. The aqueous phase was extracted with EtOAc (10 mL × 3). The combined extracts were washed with brine (10 mL x 3), dried over Na2SO4, filtered, and concentrated in vacuo, and the crude product was combined with another batch. The residue was purified by preparative TLC (PE / EA 4:1, R f = 0.3) to give the title compound as a yellow solid (35.0 mg, 34% yield). LCMS m / z: 498.3 [M+H] + , ESIpos.

[0620] Step D: 5-[8-(4-Hydroxycyclohexyl)-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-meth yl-2,3-dihydrobenzofuran-4-ol

[0621] To a solution of 5-[8-[4-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]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 min. After completion of the reaction, the mixture was concentrated in vacuo. The residue was purified by C 18 column chromatography (0.1% aqueous TFA / MeCN, MeCN: 20%-30%, 4 min) to afford the title compound, by LCMS m / z: 384.1 [M+H] + , ESIpos.

[0622] Example 17:

[0623] 5-[8-[(1R,3S)-3-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0624]

[0625] Step A: (1R,3S)-3-[tert-Butyl(dimethyl)silyl]oxy-N-[2-(3,6-dichloropyridazin-4-yl)oxyethyl]cyclohexanamine Step B: tert-Butyl-[(1R,3S)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane

[0626] 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 (1R,3S)-3-[tert-butyl(dimethyl)silyl]oxycyclohexanamine (332.51 mg, 1.45 mmol, 1.0 eq), then NaBH(OAc)3 (614.46 mg, 2.90 mmol, 2.0 eq) was added, and the mixture was stirred at 25 °C for 0.5 h. The above reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography on silica gel (petroleum ether:ethyl acetate = 1:0 to 0:1) to give the title compound as a white solid (90.0 mg, 12% yield).

[0627] Step C: 5-[8-[(1R,3S)-3-[tert-Butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol Step D: 5-[8-[(1R,3S)-3-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0628] Under N2, to a solution of (1R,3S)-3-[tert-butyl(dimethyl)silyl]oxy-N-[2-(3,6-dichloropyridazin-4-yl)oxyethyl]cyclohexanamine (90.0 mg, 0.21 mmol, 1.0 eq) in 1,4-dioxane (3 mL) were added Cs2CO3 (208.7 mg, 0.64 mmol, 3.0 eq) and Xphos Pd G4 (36.84 mg, 0.04 mmol, 0.2 eq), and the reaction was stirred at 90 °C for 2 h under N2. The reaction mixture was cooled to room temperature and diluted with water (20 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was combined with another batch. The combined crude product was purified by column chromatography on silica gel (petroleum ether:ethyl acetate = 1:0 to 0:1) to give the title compound as a yellow oil (50.0 mg, 61% yield). LCMS m / z: 384.2 [M+H] +, ESI pos. Obtained the isomer tert-butyl-[(1S,3R)-3-(3-chloro-6,7-dihydropyridazino[3,4-b][1,4]oxazin-5-yl)cyclohexyloxy]-dimethyl-silane as a yellow oil (40.0 mg, 46% yield).

[0629] Example 18: Step A: (3S,4R)-3-[tert-Butyl(dimethyl)silyl]oxy-N-[2-(3,6-dichloropyridazin-4-yl)oxyethyl]tetrahydropyran-4-amine

[0630] To a solution of tert-butyl-[(1R,3S)-3-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)cyclohexyloxy]-dimethyl-silane (40.0 mg, 0.1 mmol, 1.0 eq) in 1,4-dioxane (2 mL) / water (0.200 mL) was added Na2CO3 (22.08 mg, 0.21 mmol, 2.0 eq), 6-methyl-5-(4,4,5,5-tetramethyl-1,3,2-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). Then the reaction mixture was stirred at 95 °C under N2 for 2 h. The above reaction mixture was combined with another batch and then diluted with water (20 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative TLC (petroleum ether:ethyl acetate = 4:1, R f = 0.4) to obtain the title compound as a yellow solid (30.0 mg, 34% yield). LCMS m / z: 498.3 [M+H] + , ESI pos.

[0631] Step B: tert-Butyl-[(3S,4R)-4-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]oxazin-8-yl)tetrahydropyran-3-yl]oxy-dimethyl-silane Step C: 5-[8-[(3S,4R)-3-[tert-Butyl(dimethyl)silyl]oxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0632] To a solution of 5-[8-[(1R,3S)-3-[tert-butyl(dimethyl)silyl]oxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]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 h. The mixture was then concentrated in vacuo and combined with another batch. The pH was adjusted to about 8 with aqueous NaHCO3 (0.4 mL), then filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The residue was purified by preparative HPLC (column: Phenomenex Gemini, 150 mm * 25 mm * 5 μm; mobile phase: [water (0.1% NH3·H2O, V / V)-ACN]; B%: 10% - 40%, 10 min) to afford the title compound as a white solid (10.3 mg, 66% yield). LCMS m / z: 384.2 [M+H] + , ESI pos.

[0633] Step D: 5-[8-[(3S,4R)-3-hydroxyoxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0634] 5-[8-[(3S,4R)-3-Hydroxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol

[0635]

[0636] Example A Example B

[0637] 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 (3S,4R)-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 minutes. Then NaBH(OAc)3 (655.24 mg, 3.09 mmol, 2.0 eq) was added portionwise to the above mixture. Then the reaction mixture was stirred at 20 °C for 5 minutes. After completion of the reaction, the reaction mixture was cooled to room temperature. EtOAc (40 mL) and water (40 mL) were added, and the layers were separated. The aqueous phase was extracted with EtOAc (30 mL × 2). The combined extracts were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated in vacuo 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 as a yellow oil (400.0 mg, 55% yield). LCMS m / z: 422.2 [M+H] + , ESI pos.

[0638] ​ ​

[0639] Under N2, to a mixture of the aforementioned (3S,4R)-3-[tert-butyl(dimethyl)silyl]oxy-N-[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), and then the mixture was stirred at 90 °C for 2 hours under N2. After completion of the reaction, the reaction mixture was cooled to room temperature. EtOAc (50 mL) and water (40 mL) were added, and the layers were separated. The aqueous phase was extracted with EtOAc (50 mL × 2). The combined extracts were washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated in vacuo 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 as a yellow oil (90.0 mg, 24% yield). LCMS m / z: 386.2 [M+H] + , ESI pos.

[0640] ​ ​

[0641] To a solution of tert-butyl-[(3S,4R)-4-(3-chloro-6,7-dihydropyridazino[4,3-b][1,4]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 Na2CO3 (28.3 mg, 0.27 mmol, 2.0 eq), 6-methyl-5-(4,4,5,5-tetramethyl-1,3,2-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), and then the mixture was stirred at 95 °C under N2 for 2 h. After completion of the reaction, the reaction mixture was cooled to room temperature. EtOAc (40 mL) and water (30 mL) were added, and the layers were separated. The aqueous phase was extracted with EtOAc (30 mL × 2). The combined extracts were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by preparative TLC (PE:EA = 2.5:1, R f = 0.2) to afford the title compound as a yellow oil (50.0 mg, 77% yield). LCMS m / z: 500.3 [M+H] + , ESIpos.

[0642] ​ ​

[0643] To a solution of 5-[8-[(3S,4R)-3-[tert-butyl(dimethyl)silyl]oxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][1,4]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 min. The reaction mixture was concentrated in vacuo at 30 °C. Then the residue was dissolved in MeOH (3 mL), and the pH was adjusted to about pH 7 with NaHCO3, and then purified by reverse phase CombiFlash chromatography (mobile phase: (water (0.1% ammonia v / v)-ACN); B%: 23%-25%) to afford the title compound as a white solid (20.3 mg, 64% yield). LCMS m / z: 386.1 [M+H] + , ESI pos.

[0644] ​

[0645] The compound of formula I can be used as an active ingredient in the production of tablets having the following composition in a manner known per se:

[0646]

[0647] ​

[0648] The compound of formula I can be used as an active ingredient in the production of capsules having the following composition in a manner known per se:

[0649]

Claims

1. A compound of formula I, wherein, R 1 is H or an alkyl group; R 2 Selected from: i. a 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted with 1 to 2 substituents selected from alkyl, OH, halo, haloalkyl, hydroxyalkyl or oxo; ii. a 5- to 6-membered heterocycle containing a single O heteroatom; iii. a 4- to 6-membered cycloalkyl optionally substituted with 1 to 2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle containing 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 R 3 is H, halogen, alkyl, alkoxy or alkoxyalkyl, where R 1 and R 3 one of them 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 form with the atoms to which they are attached i. a 4- to 5-membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle containing a single O heteroatom optionally substituted with 1 to 2 substituents independently selected from alkyl and halo; R 6 is halogen, haloalkyl or OH; R 7 is H or F; and its pharmaceutically acceptable salts.

2. The compound according to claim 1, 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.

3. The compound according to claim 1 or claim 2, wherein R 1 is H or alkyl and R 3 is H or alkyl, wherein R 1 or R 3 one of them is H and the other is alkyl.

4. The compound according to any one of claims 1 to 3, wherein R 2 is selected from i. a 6-membered heterocycle containing a single N heteroatom substituted with alkyl; ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom; iii. a 4-membered cycloalkyl substituted with alkyl and OH; and iv. a 9-membered bicyclic heterocycle containing a single N heteroatom optionally substituted with alkyl.

5. The compound according to any one of claims 1 to 4, wherein R 2 is selected from A 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted with alkyl or with both alkyl and OH; and a 4- to 6-membered cycloalkyl optionally substituted with 1 to 2 substituents independently selected from alkyl and OH.

6. The compound according to any one of claims 1 to 5, wherein X is O or –CH2-.

7. The compound according to any one of claims 1 to 6, wherein W is selected from ring systems A, C and D wherein Y is CH2 or –O-.

8. The compound according to any one of claims 1 to 7, wherein W is ring system A 9. A compound according to any one of claims 1 to 8, wherein R 4 is alkyl, cyano, haloalkyl or haloalkoxy.

10. The compound according to any one of claims 1 to 9, wherein R 4 is a cyano group.

11. The compound according to any one of claims 1 to 10, wherein R 5 is H, or R 4 and R 5 and form with the atoms to which they are attached i. a 4- to 5-membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle containing a single O heteroatom.

12. The compound according to any one of claims 1 to 10, wherein R 5 is H, or R 4 and R 5 and the atoms to which they are attached form a 5-membered cycloalkyl ring or a 5-membered heterocycle containing a single O heteroatom.

13. A compound according to any one of claims 1 to 11, wherein R 5 is H.

14. The compound according to any one of claims 1 to 12, wherein R 6 is OH.

15. The compound according to claim 1, wherein R 1 is H or an alkyl group; R 2 Selected from: i. a 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein the heterocycle is optionally substituted with alkyl, halo, haloalkyl, hydroxyalkyl or oxo; ii. -CH2-heterocycle, wherein the heterocycle is a 5- to 6-membered heterocycle containing a single O heteroatom; iii. a 4- to 6-membered cycloalkyl optionally substituted with 1 to 2 substituents independently selected from alkyl and OH; and iv. a 9-membered bicyclic heterocycle containing 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 R 3 is H, halogen, alkyl, alkoxy or alkoxyalkyl, where R 1 and R 3 one of them is H and the other is not H; R 4 is halogenated, alkyl, alkoxy, cyano, haloalkyl or haloalkoxy; R 5 is H; or R 4 and R 5 and form with the atoms to which they are attached i. a 4- to 5-membered cycloalkyl ring optionally substituted with oxo, or ii. a 5-membered heterocycle containing a single O heteroatom optionally substituted with 1 to 2 substituents independently selected from alkyl and halo; R 6 is halogen, haloalkyl or OH; R 7 is H or F; and its pharmaceutically acceptable salts.

16. The compound according to any one of claims 1 to 13, wherein R 1 is H or alkyl and R 3 is H or alkyl, where R 1 or R 3 one of them is H and the other is alkyl; R 2 Selected from: i. a 6-membered heterocycle containing 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 a cyano group; R 5 is H; R 6 is OH; and its pharmaceutically acceptable salts.

17. The compound according to claim 1, wherein R 1 is H or an alkyl group; R 2 Selected from: i. A 5- to 6-membered heterocycle containing a single O or single N heteroatom, wherein said heterocycle is optionally substituted by alkyl or by both alkyl and OH; and ii. A 4- to 6-membered cycloalkyl optionally substituted by 1 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 an alkyl group, where R 1 and R 3 one of them is H and the other is not H; R 4 is an alkyl group, a cyano group, a haloalkyl group or a haloalkoxy group; R 5 is H; R 6 is OH; Y is CH2 or O; and its pharmaceutically acceptable salts.

18. The compound according to claim 1, wherein R 1 is H or an alkyl group; R 2 is a 5- to 6-membered heterocycle containing a single O or a single N heteroatom, wherein said heterocycle is optionally substituted by an alkyl or by both an alkyl and OH; X is –O- or –CH2-; W is selected from ring systems C and D Among them R 3 is H or an alkyl group, where R 1 and R 3 one of them is H and the other is not H; R 4 is an alkyl group, a cyano group, a haloalkyl group or a haloalkoxy group; R 5 is H; R 6 is OH; Y is CH2 or O; and its pharmaceutically acceptable salts.

19. The compound according to any one of claims 1 to 14, wherein the compound is selected from 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; formic acid; 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-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][1,4]oxazin-3-yl]-5-methyl-benzonitrile; and its pharmaceutically acceptable salts.

20. The compound according to any one of claims 1 to 14, wherein the compound is selected from 3-Hydroxy-4-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid; 3-Hydroxy-4-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-5-methyl-benzonitrile; 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol; 2,2,2-trifluoroacetic acid; 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-indan-4-ol; 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol; 2,2,2-trifluoroacetic acid; 5-[8-[(1R,2R)-2-hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-2,3-dihydrobenzofuran-4-ol; 4-[8-[(3R,5S)-1-Ethyl-5-hydroxy-3-piperidinyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethyl)phenol; 4-[8-[(3R)-1-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; 2,2,2-trifluoroacetic acid; 4-[8-[(3R)-1-Ethylpyrrolidin-3-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-5-methyl-benzonitrile; 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-dimethyl-phenol; 2,2,2-trifluoroacetic acid 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3,5-dimethyl-phenol; 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol; 2,2,2-trifluoroacetic acid; 2-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-methyl-5-(trifluoromethoxy)phenol; 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-benzonitrile; 2,2,2-trifluoroacetic acid; 4-[8-[(3R)-1-Ethyl-3-piperidinyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-3-hydroxy-benzonitrile; 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]indan-4-ol; 2,2,2-trifluoroacetic acid; 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]indan-4-ol; 3-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol; 2,2,2-trifluoroacetic acid; 3-[8-[(1R,2R)-2-Hydroxycyclohexyl]-4-methyl-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]bicyclo[4.2.0]octa-1,3,5-trien-2-ol; 5-[8-[(1R,3S)-3-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; 5-[8-[(3S,4R)-3-Hydroxytetrahydropyran-4-yl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; and its pharmaceutically acceptable salts.

21. The 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][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; and its pharmaceutically acceptable salts.

22. The compound according to any one of claims 1 to 14, wherein the compound is selected from 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-2,3-dihydrobenzofuran-4-ol; 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-inden-4-ol; 2,2,2-trifluoroacetic acid; 5-[8-[(1R,2R)-2-Hydroxycyclohexyl]-6,7-dihydropyridazino[4,3-b][1,4]oxazin-3-yl]-6-methyl-inden-4-ol; and its pharmaceutically acceptable salts.

23. The compound according to any one of claims 1 to 22, which is used as a therapeutic active substance.

24. The compound according to any one of claims 1 to 22, which is used for treating or preventing a disease, disorder or condition, wherein the disease, disorder or condition responds to NLRP3 inhibition.

25. A pharmaceutical composition, which comprises the compound according to any one of claims 1 to 22 and a therapeutically inert carrier.

26. Use of the compound according to any one of claims 1 to 22 for treating or preventing a disease, disorder or condition, wherein the disease, disorder or condition responds to NLRP3 inhibition.

27. The compound according to any one of claims 1 to 22, which is used for treating or preventing a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

28. Use of the compound according to any one of claims 1 to 22 in treating or preventing a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease. Use of a compound according to any one of claims 1 to 22 for the manufacture of a medicament for the treatment or prophylaxis of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

30. A method of inhibiting NLRP3, the method comprising administering an effective amount of a compound as claimed in any one of claims 1 to 22 to inhibit NLRP3.

31. A method for the treatment or prophylaxis of a disease, disorder or condition, the method comprising 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.

32. The present invention as described in the specification.

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