2-(pyridazin-3-yl)-5-(trifluoromethyl)phenol as an NLRP3 inhibitor

Abstract

The present invention relates to compounds for use as inhibitors of the NLRP3 inflammasome pathway, such compounds being as defined by the compounds of formula (I): [Formula 1] TIFF2026504419000104.tif23128In formula, group R 1 , R 2 , R 3 wherein Z is as defined herein, and the compounds may be useful, for example, as pharmaceutical agents for use in treating diseases or disorders associated with NLRP3 inflammasome activity.

Description

[Technical Field]

[0001] Described herein are 2-(pyridazin-3-yl)-5-(trifluoromethyl)phenols useful as inhibitors of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway. Also described herein are processes for preparing the compounds, pharmaceutical compositions containing the compounds, and methods of using the compounds in the treatment of various diseases and disorders mediated by the NLRP3 inflammasome pathway. [Background technology]

[0002] Inflammasomes, considered central signaling hubs of the innate immune system, are multiprotein complexes that assemble upon activation of a specific set of intracellular pattern recognition receptors (PRRs) by a wide variety of pathogen-associated or risk-associated molecular patterns (PAMPs or DAMPs). To date, it has been shown that inflammasomes can be formed by nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and pyrin and HIN200 domain-containing proteins (Van Opdenbosch N. and Lamkanfi M. Immunity, 2019 Jun 18;50(6):1352-1364). NLRP3 inflammasomes assemble upon detection of environmental crystals, pollutants, host-derived DAMPs, and protein aggregates (Tartey S and Kanneganti TD. Immunology, 2019 Apr;156(4):329-338). Clinically relevant DAMPs that engage NLRP3 include uric acid and cholesterol crystals that cause gout and atherosclerosis, amyloid-β fibrils that are neurotoxic in Alzheimer's disease, and asbestos particles that cause mesothelioma (Kelley et al., Int J Mol Sci, 2019 Jul 6;20(13)). Furthermore, NLRP3 is activated by infectious agents such as fungal pathogens such as Vibrio cholerae, Aspergillus fumigatus, and Candida albicans, adenovirus, influenza A virus, and SARS-CoV-2 (Tartey and Kanneganti, 2019; Fung et al. Emerg Microbes Infect, 2020 Mar 14;9(1):558-570).

[0003] Although the exact mechanism of NLRP3 activation remains unclear, it has been suggested that a single-step activation is sufficient in human monocytes, whereas a two-step mechanism is in place in mice. Given the multiple triggers, the NLRP3 inflammasome requires add-on regulation at both the transcriptional and post-transcriptional levels (Yang Y et al., Cell Death Dis, 2019 Feb 12;10(2):128).

[0004] The NLRP3 protein consists of an N-terminal pyrin domain followed by a nucleotide-binding site domain (NBD) and a leucine-rich repeat (LRR) motif on the C-terminus (Sharif et al., Nature, 2019 Jun;570(7761):338-343). Upon recognition of PAMPs or DAMPs, NLRP3 aggregates with adaptor proteins, apoptosis-associated speck-like protein (ASC), and the protease caspase-1 to form a functional inflammasome. Upon activation, procaspase-1 undergoes autoproteolysis, resulting in the cleavage of gasdermin D (Gsdmd) to generate an N-terminal Gsdmd molecule, which ultimately leads to pore formation in the plasma membrane and a lytic form of cell death called pyroptosis. Alternatively, caspase-1 cleaves the pro-inflammatory cytokines pro-IL-1β and pro-IL-18, allowing the release of their biologically active forms by pyroptosis ( Kelley et al., 2019 ).

[0005] Dysregulation of the NLRP3 inflammasome or its downstream mediators has been implicated in many pathologies, ranging from immune / inflammatory diseases, autoimmune / autoinflammatory diseases (cryopyrin-associated periodic syndrome (Miyamae T. Paediatr Drugs, 2012 Apr 1;14(2):109-17), sickle cell disease, and systemic lupus erythematosus (SLE)), to liver disorders (e.g., nonalcoholic steatohepatitis (NASH), chronic liver disease, viral hepatitis, alcoholic steatohepatitis, nonalcoholic fatty acid liver disease, and alcoholic liver disease) (Szabo G and Petrasek J. Nat Rev Gastroenterol Hepatol, 2015 Jul;12(7):387-400), and inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis) (Zhen Y and Zhang H. Front Immunol, 2019 Feb 28;10:276). Inflammatory joint disorders (e.g., gout, pseudogout (chondrocalcinosis), arthropathy, osteoarthritis, and rheumatoid arthritis) have also been associated with NLRP3 activation (Vande Walle L et al., Nature, 2014 Aug 7;512(7512):69-73). Furthermore, kidney-related diseases (hyperoxaluria (Knauf et al., Kidney Int, 2013 Nov;84(5):895-901), lupus nephritis, hypertensive nephropathy (Krishnan et al., Br J Pharmacol, 2016 Feb;173(4):752-65), hemodialysis-associated inflammation and diabetic nephropathy, a kidney-related complication of diabetes (type 1, type 2, and diabetes mellitus), also known as diabetic kidney disease (Shahzad et al., Kidney Int, 2015 Jan;87(1):74-84)) have been associated with NLRP3 inflammasome activation.There are reports linking the onset and progression of neuroinflammatory disorders (e.g., brain infection, acute injury, multiple sclerosis, Alzheimer's disease) and neurodegenerative diseases (e.g., Parkinson's disease) with NLRP3 inflammasome activation (Sarkar et al., NPJ Parkinson's Dis, 2017 Oct 17;3:30). Furthermore, cardiovascular or metabolic disorders (e.g., cardiovascular risk reduction (CvRR), atherosclerosis, type I and type II diabetes and related complications (e.g., nephropathy, retinopathy), peripheral arterial disease (PAD), acute heart failure, and hypertension (Ridker et al., CANTOS Trial Group. N Engl J Med, 2017 Sep 21;377(12):1119-1131, and Toldo S and Abbate A. Nat Rev Cardiol, 2018) Apr;15(4):203-214) has recently been associated with NLRP3. Skin-related diseases have also been described (e.g., wound healing and scar formation, inflammatory skin diseases such as acne, hidradenitis suppurativa (Kelly et al., Br J Dermatol, 2015 Dec;173(6)). In addition, respiratory conditions have been associated with NLRP3 inflammasome activity (e.g., asthma, sarcoidosis, severe acute respiratory syndrome (SARS) (Nieto-Torres et al., Virology, 2015 Nov;485:330-9)), silicosis, pneumonia, as well as age-related macular degeneration (Doyle et al., Nat Med, 2012 May;18(5):791-8). Several cancer-related diseases / disorders have been described in association with NLRP3 (e.g., myeloproliferative neoplasms, leukemia, myelodysplastic syndromes (MOS), myelofibrosis, lung cancer, and colon cancer (Ridker et al., Lancet, 2017 Oct 21;390(10105):1833-1842; Derangere et al., Cell Death Differ. 2014 Dec;21(12):1914-24; Basiorka et al., Lancet Haematol, 2018 Sep;5(9):e393-e402, Zhang et al., Hum Immunol, 2018 Jan;79(1):57-62).

[0006] Several patent applications have described NLRP3 inhibitors, recent examples of which include WO 2020 / 234715, WO 2021 / 193897, WO 2022 / 135567, U.S. Pat. No. 11,319,319, and WO 2023 / 003002.

[0007] For example, inhibitors of the NLRP3 inflammasome pathway are needed to study neurodegenerative disorders such as Alzheimer's disease. Summary of the Invention [Means for solving the problem]

[0008] Described herein are compounds that inhibit the NLRP3 inflammasome pathway.

[0009] In some embodiments, provided herein are compounds of formula (I):

[0010] [ka] The compound or a pharmaceutically acceptable salt thereof; R 1 is hydrogen, methyl, or chloro; R 2 teeth,

[0011] [ka] and In the formula, Y is CH, NR 21 , or O, R 20 is oxetan-3-yl; CD3; C optionally substituted with halo, hydroxy, or cyano 1~4 is alkyl, R 21 is hydrogen or C 1~2 is alkyl, R 3is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; R 4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; R 3 and R 4 are taken together to form a divalent group -R selected from the list below 3 -R 4 - and a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) —CH—CH—CH—NH—, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-, f) —O—CH—CH—O—, g) -CH=CH-CH=N-, and h) -N=CH-CH=CH-, Each Z is independently hydrogen or fluoro.

[0012] In another aspect, there are provided compounds for use as pharmaceuticals. In another aspect, there are provided pharmaceutical compositions comprising a therapeutically effective amount of a compound provided herein.

[0013] In a further aspect, there are provided compounds and pharmaceutical compositions comprising such compounds for use in the treatment of diseases or disorders mediated by the NLRP3 inflammasome pathway, for example neurodegenerative disorders such as Alzheimer's disease.

[0014] In another aspect, there is provided a use of the compound in the manufacture of a medicament for the treatment of a disease or disorder mediated by the NLRP3 inflammasome pathway, for example a neurodegenerative disorder such as Alzheimer's disease.

[0015] In another aspect, provided is a method of treating a disease or disorder mediated by the NLRP3 inflammasome pathway, e.g., a neurodegenerative disorder such as Alzheimer's disease. In a further aspect, provided is a method of inhibiting NLRP3 inflammasome activity in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound provided herein. [Brief explanation of the drawings]

[0016] [Figure 1] In vivo long-term potentiation (LTP) experiments: Measurement of the effect of the NLRP3 inhibitor compound 9 on LPS-induced pro-inflammatory cytokine IL1β. Figure 1(A) Measurement of IL1β, Figure 1(B) Measurement of IL6, Figure 1(C) Measurement of TNFα. [Figure 2] In vivo long-term potentiation (LTP) experiments: Measurement of the effect of the NLRP3 inhibitor compound 51 on LPS-induced pro-inflammatory cytokine IL1β. Figure 2(A) Measurement of IL1β, Figure 2(B) Measurement of IL6, Figure 2(C) Measurement of TNFα. DETAILED DESCRIPTION OF THE INVENTION

[0017] Provided herein are compounds of formula (I):

[0018] [ka] The compound and pharmaceutically acceptable salts thereof, R 1 is hydrogen, methyl, or chloro; R 2 teeth,

[0019] [ka] and In the formula, Y is CH, NR 21 , or O, R 20is oxetan-3-yl; C optionally substituted with halo, hydroxy, or cyano 1~4 is alkyl, R 21 is hydrogen or C 1~2 is alkyl, R 3 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; R 4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; R 3 and R 4 are taken together to form a divalent group -R selected from the list below 3 -R 4 - and a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) —CH—CH—CH—NH—, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-, f) —O—CH—CH—O—, g) -CH=CH-CH=N-, and h) -N=CH-CH=CH-, Each Z is independently hydrogen or fluoro.

[0020] In one embodiment, R 1 is hydrogen or methyl.

[0021] In one embodiment, R 2 teeth,

[0022] [ka] and In the formula, R 20 is C 1~2 alkyl, CD3, 2-hydroxyethyl, 2-fluoroethyl, 3-fluoropropyl, or cyanomethyl.

[0023] In one embodiment, R 2 teeth,

[0024] [ka] and In the formula, R 20 is methyl.

[0025] In one embodiment, R 21 is hydrogen or methyl.

[0026] In one embodiment, R 3 is hydrogen or methyl.

[0027] In one embodiment, R 4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl.

[0028] In one embodiment, R 3 and R 4 are taken together to form a divalent group -R selected from the list below 3 -R 4 -It is. a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) —CH—CH—CH—NH—, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-, f) —O—CH—CH—O—, g) -CH=CH-CH=N-, and h) -N=CH-CH=CH-.

[0029] In one embodiment, R 1 is hydrogen or methyl, R 2 teeth,

[0030] [ka] and wherein Y is CH or O; R 20 is methyl, R 3 is hydrogen or R 4 is hydrogen, or R 3 and R 4 are taken together to form a divalent group -R selected from the list below 3 -R 4 - and a) -CH2-CH2-CH2-, e) -CH2-CH2-O-CH2-, Each Z is hydrogen.

[0031] Compounds of particular interest are (S)-2-(4-((1-methylpiperidin-3-yl)methyl)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)-5-(trifluoromethyl)phenol (S)-3-methyl-2-(6-((1-methylpiperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol, and (S)-2-(4-((4-methylmorpholin-2-yl)methyl)-7,8-dihydro-5H-pyrano[3,4-d]pyridazin-1-yl)-5-(trifluoromethyl)phenol.

[0032] In another aspect, provided herein is a compound

[0033] [ka] or a pharmaceutically acceptable salt thereof.

[0034] Pharmaceutically acceptable salts include acid addition salts and base addition salts.Such salts can be formed by conventional means, for example, by reacting the free acid form or free base form of the compound provided herein with one or more equivalents of a suitable acid or base, optionally in a solvent or in a medium in which the salt is insoluble, and then removing the solvent or medium using standard techniques (for example, in vacuo, by lyophilization, or by filtration).Salts can also be prepared by exchanging the counterion of the compound provided herein in the form of a salt with another counterion, for example, using a suitable ion exchange resin.

[0035] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids.

[0036] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0037] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.

[0038] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0039] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper. Particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0040] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Particular organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0041] The compounds may contain double bonds and thus may exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond.

[0042] The compounds provided herein may also contain one or more asymmetric carbon atoms and thus may exhibit enantiomerism and / or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, such as chromatography or fractional crystallization. The various stereoisomers may be isolated by separating a racemic mixture or other mixture of compounds using conventional techniques, such as fractional crystallization or HPLC (High Performance Liquid Chromatography) techniques. Alternatively, the desired isomer may be prepared by reaction of the appropriate optically active starting material under conditions that will not cause racemization or epimerization, or by reaction of the appropriate starting material with a "chiral auxiliary" that can be subsequently removed at a suitable stage, for example, by resolution, including dynamic resolution such as salt formation with a homochiral acid, followed by separation of the diastereomeric salts by conventional means, such as chromatography, or by reaction with a suitable chiral reagent or chiral catalyst.

[0043] In the structures shown herein, where the stereochemistry of any particular chiral atom is not specified, all stereoisomers are contemplated. Where stereochemistry is specified by a solid wedge or dashed line representing a particular configuration, that stereoisomer is so specified and defined.

[0044] Absolute configuration is specified according to the Cahn-Ingold-Prelog system. The configuration at the asymmetric atom is specified by either R or S. Resolved compounds whose absolute configuration is not known can be designated as (+) or (-) depending on the direction they rotate polarized light.

[0045] When a particular stereoisomer is specified, this means that the stereoisomer is substantially free, i.e., associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, particularly less than 2%, and most preferably less than 1% of other isomers. Thus, when a compound of formula (I) is, for example, specified as (R), this means that the compound is substantially free of the (S) isomer.

[0046] The compounds may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.

[0047] Also provided herein are isotopically labeled compounds in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature (or the most abundant atom found in nature). Exemplary isotopes include isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 O. 17 O. 18 O, and 18 F is an example of tritium ( 3 H) and carbon-l4( 14 C) Isotopes are useful due to their ease of preparation and detectability. Furthermore, substitution with heavier isotopes, such as deuterium, may offer therapeutic advantages resulting from greater metabolic stability. For example, 15 O. 13 N, 11 C, and 18Isotopes such as F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds can generally be prepared by following procedures similar to those disclosed in the Examples below.

[0048] Unless otherwise specified, C as defined herein 1~q The alkyl group (q is the upper limit of the range) can be straight or branched chain.

[0049] C 3~q Cycloalkyl (q is the upper limit of the range) refers to an alkyl group that is cyclic, for example, a cycloalkyl group can be monocyclic or, if there are enough atoms, bicyclic. In one embodiment, such a cycloalkyl group is monocyclic. Substituents may be attached at any point on the cycloalkyl group.

[0050] The term "halo", as used herein, preferably includes fluoro, chloro, bromo and iodo.

[0051] C 1~q The alkoxy group (q is the upper limit of the range) has the formula -OR a where R a is C as defined herein 1~q It is an alkyl group.

[0052] Haro C 1~q Alkyl (q is the upper limit of the range) groups are C 1~q refers to an alkyl group, such group being substituted by one or more halo. HydroxyC 1~q Alkyl (q is the upper limit of the range) is C as defined herein. 1~q refers to an alkyl group, such a group being substituted by one or more (e.g., one) hydroxy (—OH) groups (or one or more, e.g., one, hydrogen atoms replaced by —OH). 1~q Alkoxy and Hydroxy C1~q The alkoxy groups may each be substituted by one or more halo groups or by one or more (e.g., one) hydroxy groups, such as the corresponding -OC. 1~q represents an alkyl group.

[0053] Compound names were generated according to the nomenclature rules agreed upon by the Chemical Abstracts Service (CAS) using Advanced Chemical Development, Inc. software (ACD / Name product version 10.01; Build 15494, December 1, 2006) or according to the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC) using Advanced Chemical Development, Inc. software (ACD / Name product version 10.01.0.14105, October 2006). In the case of tautomeric forms, names were generated for the tautomeric form shown in the structure.

[0054] The compounds of the present invention can generally be prepared by a series of steps, each of which is known to those skilled in the art. In particular, the compounds can be prepared according to the following synthetic methods.

[0055] The compounds of formula (I) may be synthesized in the form of a racemic mixture of enantiomers, which can be separated from one another according to art-known resolution procedures. The racemic compounds of formula (I) may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. The diastereomeric salt forms are then separated, for example, by selective or fractional crystallization, and the enantiomers are liberated therefrom by alkalinization. Alternative ways of separating the enantiomeric forms of the compounds of formula (I) include liquid chromatography using chiral stationary phases or chiral supercritical fluid chromatography (SCF). The pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically.

[0056] The absolute configurations of the compounds reported herein were determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC), followed by SFC comparison of the separate enantiomer(s) obtained by asymmetric synthesis, followed by vibrational circular dichroism (VCD) analysis of the specific enantiomer(s).

[0057] Preparation of compounds The final compound of formula (Ia) (wherein each Z represents H) is

[0058] [ka] - the intermediate of formula (II) can be prepared by deprotecting the intermediate of formula (II) in a suitable acidic medium, such as, for example, a solution of hydrochloric acid in 1,4-dioxane, at a suitable temperature, such as, for example, room temperature, or by deprotecting the intermediate of formula (II) under suitable hydrogenation conditions, such as, for example, Pd / C in a hydrogen atmosphere, in a suitable solvent, such as, for example, ethyl acetate or ethanol, at a suitable temperature, such as, for example, room temperature. intermediates of formula (II) by reacting intermediates of formula (III) with a suitable organozinc reagent in the presence of a suitable nickel catalyst, such as, for example, nickel (1,2-dimethoxyethane) dibromide, in the presence of a suitable ligand, such as, for example, 4,4'-di-tert-butyl-2,2'-bipyridine, with a suitable base, such as, for example, pyridine, in a suitable solvent, such as, for example, DMA, at a suitable temperature, such as, for example, 100°C, or by reacting the intermediate of formula (III) with a suitable organozinc reagent via Negishi coupling in the presence of a suitable palladium catalyst, such as, for example, cataCXium Pd G4, in a suitable solvent, such as, for example, THF, at a suitable temperature, such as, for example, 50° C., Alternatively, by reacting the intermediate of formula (III) with a suitable alcohol derivative in the presence of a suitable photocatalyst, such as, for example, Ir[ppy]2(dtbbpy)PF6, using a suitable catalyst, such as, for example, NiBr2(dtbbpy), and a suitable photoactivator, such as, for example, 5,7-di-tert-butyl-3-phenyl-1,3-benzoxazol-3-ium, in a suitable base or mixture of bases, such as, for example, pyridine and quinuclidine, in a suitable solvent or mixture of solvents, such as, for example, MTBE and DMA, under LED irradiation at 450 nm with 100% light intensity, Alternatively, intermediates of formula (III) can be prepared by reacting them with a suitable vinylboronic acid or vinylboronic ester via Suzuki coupling in the presence of a suitable palladium catalyst, such as, for example, dichlorobis(triphenylphosphine)palladium(II), in the presence of a suitable base, such as, for example, potassium phosphate tribasic, in a suitable solvent, such as, for example, a mixture of 1,4-dioxane and water, at a suitable temperature, such as, for example, 100° C., followed by reduction of the double bond by hydrogenation with Pd / C under a hydrogen atmosphere in a suitable solvent, such as, for example, ethanol or ethyl acetate, at a suitable temperature, such as, for example, room temperature, or by mild reduction using sodium borohydride, sodium triacetoxyborohydride, or related agents, in a suitable solvent, such as, for example, methanol, at a suitable temperature, such as, for example, 0° C. or room temperature. The intermediate of formula (III) can be prepared by reacting the intermediate of formula (IV) with a suitable boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst, such as, for example, tetrakistriphenylphosphine palladium, in the presence of a suitable base, such as, for example, sodium carbonate, in a suitable solvent, such as, for example, a mixture of 1,4-dioxane and water, at a suitable temperature, such as, for example, 100° C.

[0059] Alternatively, the intermediate according to formula (II) is

[0060] [ka] The intermediate of formula (V) can be prepared by reacting it with a suitable boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst, such as, for example, tetrakistriphenylphosphine palladium, in the presence of a suitable base, such as, for example, sodium carbonate, in a suitable solvent, such as, for example, a mixture of 1,4-dioxane and water, at a suitable temperature, such as, for example, 100° C. intermediates of formula (V) can be obtained by reacting intermediates of formula (IV) with a suitable organozinc reagent in the presence of a suitable nickel catalyst, such as, for example, nickel (1,2-dimethoxyethane) dibromide, in the presence of a suitable ligand, such as, for example, 4,4'-di-tert-butyl-2,2'-bipyridine, with a suitable base, such as, for example, pyridine, in a suitable solvent, such as, for example, DMA, at a suitable temperature, such as, for example, 100°C, or by reacting the intermediate of formula (IV) with a suitable organozinc reagent via Negishi coupling in the presence of a suitable palladium catalyst, such as, for example, cataCXium Pd G4, in a suitable solvent, such as, for example, THF, at a suitable temperature, such as, for example, 50° C., Alternatively, by reacting the intermediate of formula (IV) with a suitable alcohol derivative in the presence of a suitable photocatalyst, such as, for example, Ir[ppy]2(dtbbpy)PF6, using a suitable catalyst, such as, for example, NiBr2(dtbbpy), and a suitable photoactivator, such as, for example, 5,7-di-tert-butyl-3-phenyl-1,3-benzoxazol-3-ium, with a suitable base or mixture of bases, such as, for example, pyridine and quinuclidine, in a suitable solvent or mixture of solvents, such as, for example, MTBE and DMA, under LED irradiation at 450 nm at 100% light intensity, Alternatively, intermediates of formula (IV) can be prepared by reacting them with a suitable vinylboronic acid or vinylboronic ester via Suzuki coupling in the presence of a suitable palladium catalyst, such as, for example, dichlorobis(triphenylphosphine)palladium(II), in the presence of a suitable base, such as, for example, potassium phosphate tribasic, in a suitable solvent, such as, for example, a mixture of 1,4-dioxane and water, at a suitable temperature, such as, for example, 100° C., followed by reduction of the double bond by hydrogenation with Pd / C under a hydrogen atmosphere in a suitable solvent, such as, for example, ethanol or ethyl acetate, at a suitable temperature, such as, for example, room temperature, or by mild reduction using sodium borohydride, sodium triacetoxyborohydride, or related agents in a suitable solvent, such as, for example, methanol, at a suitable temperature, such as, for example, 0° C. or room temperature.

[0061] Those skilled in the art will recognize that R 2 However, when a heteroatom is beta to the reactive site, as in the case of 2-(bromomethyl)morpholino derivatives, one will find that neither the Negishi nor Suzuki coupling can be performed, and that MacMillan chemistry (Dong, Z., MacMillan, D.W.C. Metalla photoredox-enabled deoxygenative arylation of alcohols. Nature 598: 451-456 (2021) (https: / / doi.org / 10.1038 / s41586-021-03920-6) is the best option.

[0062] The final compound of formula (Ib) (wherein each Z represents F) is

[0063] [ka] The intermediate of formula (VI) can be prepared by reacting it with a suitable boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst, such as, for example, tetrakistriphenylphosphine palladium, in the presence of a suitable base, such as, for example, sodium carbonate, in a suitable solvent, such as, for example, a mixture of 1,4-dioxane and water, at a suitable temperature, such as, for example, 100° C. The intermediate of formula (VI) can be prepared by reacting the intermediate of formula (VII) with a suitable fluorinating agent, such as, for example, DAST, in the presence of a suitable auxiliary, such as, for example, triethylamine trihydrofluoride, in a suitable solvent, such as, for example, anhydrous DCM, at a suitable temperature, such as, for example, 0° C. The intermediate of formula (VII) can be prepared by reacting the intermediate of formula (VIII) with an appropriate zincate reagent in the presence of a suitable catalyst, such as, for example, copper(I) cyanide, in the presence of a suitable auxiliary, such as, for example, anhydrous lithium chloride, in a suitable solvent, such as, for example, THF, at a suitable temperature, such as, for example, -20°C.

[0064] Alternatively, the final compound according to formula (Ib) (wherein each Z represents F) is

[0065] [ka] - can be prepared by deprotecting an intermediate of formula (IX) in a suitable acidic medium, such as, for example, a solution of hydrochloric acid in 1,4-dioxane, at a suitable temperature, such as, for example, room temperature, or by deprotecting an intermediate of formula (XI) under suitable hydrogenation conditions, such as, for example, Pd / C in a hydrogen atmosphere, in a suitable solvent, such as, for example, ethyl acetate or ethanol, at a suitable temperature, such as, for example, room temperature. The intermediate of formula (IX) can be prepared by reacting the intermediate of formula (X) with a suitable fluorinating agent, such as, for example, DAST, in the presence of a suitable auxiliary, such as, for example, triethylamine trihydrofluoride, in a suitable solvent, such as, for example, anhydrous DCM, at a suitable temperature, such as, for example, 0° C. The intermediate of formula (X) can be prepared by reacting the intermediate of formula (VII) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst, such as, for example, tetrakistriphenylphosphine palladium, in the presence of a suitable base, such as, for example, sodium carbonate, in a suitable solvent, such as, for example, a mixture of 1,4-dioxane and water, at a suitable temperature, such as, for example, 100° C.

[0066] The intermediate of formula (VIII) is

[0067] [ka] For example, it can be prepared by reacting an intermediate of formula (XI) with a suitable activating agent, such as, for example, oxalyl chloride, in the presence of a suitable catalyst, such as, for example, DMF, in a suitable solvent, such as, for example, DCM, at a suitable temperature, such as, for example, 0° C. Intermediates of formula (XI) can be prepared by saponification of an intermediate of formula (XII), which is first protected by a suitable protecting group PG, such as, for example, methyl or ethyl, using a suitable base, such as, for example, lithium hydroxide, in a suitable solvent, such as, for example, a mixture of THF, water, and methanol, at a suitable temperature, such as, for example, room temperature or 50° C. The intermediate of formula (XII) can be prepared by reacting the intermediate of formula (IV) in the presence of a suitable catalyst, such as, for example, Pd(dppf)Cl2, in the presence of a suitable base, such as, for example, sodium acetate or triethylamine, in a suitable solvent, such as, for example, methanol or ethanol, at a suitable temperature, such as, for example, 100°C, and at a suitable pressure, such as, for example, 10 bar, in a pressure vessel under a carbon monoxide atmosphere.

[0068] Those skilled in the art will recognize that R 2 , R 3 , and / or R 4It will be appreciated that if nor-compounds contain a protecting group such as, for example, Boc, deprotection of intermediates of formula (Ia) or (Ib) will provide deprotected compounds of formula (Ia) or (Ib). Further functionalization of these nor-compounds is possible using an aldehyde coupling partner in the presence of a reducing agent such as, for example, sodium triacetoxyborohydride, in a suitable solvent such as, for example, methanol or dichloromethane, at a suitable temperature such as, for example, 0° C.

[0069] Pharmacology The compounds are potently brain penetrant, have low cardiovascular burden, and may be useful in central nervous system diseases such as Parkinson's disease, Alzheimer's disease, dementia, motor neuron disease, Huntington's disease, traumatic brain injury, multiple sclerosis, and amyotrophic lateral sclerosis.

[0070] Pharmaceutical Compositions and Combinations In one embodiment, further described herein is a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound provided herein as an active ingredient. The compound can be formulated into various pharmaceutical forms for administration purposes. Suitable compositions may include any composition typically used for systemic drug administration. To prepare a pharmaceutical composition, an effective amount of the compound, optionally in salt form, as the active ingredient is combined and thoroughly mixed with a pharmaceutically acceptable carrier, which may take various forms depending on the preparation desired for administration. These pharmaceutical compositions are preferably in a unit dosage form suitable for administration, particularly oral administration or parenteral injection. For example, when preparing a composition in oral dosage form, any of the usual pharmaceutical media may be used, such as water, glycols, oils, alcohols, etc. for oral liquid preparations such as suspensions, syrups, elixirs, emulsions, and solutions, or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrants, etc. for powders, pills, capsules, and tablets. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, although other ingredients (e.g., to aid solubility) may be included. For example, injectable solutions may be prepared in which the carrier comprises saline, glucose solution, or a mixture of saline and glucose solution. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be used. Also included are solid form preparations which are intended to be converted to liquid form preparations shortly before use.

[0071] The pharmaceutical compositions may further contain various other ingredients well known in the art, such as lubricants, stabilizers, buffers, emulsifiers, viscosity adjusting agents, surfactants, preservatives, flavoring agents, or coloring agents.

[0072] It is particularly advantageous to formulate the above-mentioned pharmaceutical composition into unit dosage form in order to facilitate administration and ensure uniformity of dosage.As used herein, unit dosage refers to a physically separate unit suitable for single administration, and each unit contains a predetermined amount of active ingredient calculated to produce desired therapeutic effect together with necessary pharmacological carrier.The example of such unit dosage form is tablet (including scored tablet or coated tablet), capsule, pill, powder packet, wafer, suppository, injection solution or suspension etc., and their multiple portions.

[0073] The daily dosage of the compound will, of course, vary depending on the compound used, the mode of administration, the desired treatment and the mycobacterial disease being treated, but generally satisfactory results are obtained when the compound is administered in a daily dose not exceeding 1 gram, e.g., in the range of 10-50 mg / kg body weight.

[0074] As used herein, the term "pharmaceutical composition" refers to a compound provided herein, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier in a form suitable for oral or parenteral administration.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance useful in the preparation or use of a pharmaceutical composition, and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonicity agents, buffers, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegrants, lubricants, wetting agents, sweeteners, flavoring agents, dyes, and combinations thereof, as known to those skilled in the art (see, e.g., Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070).

[0076] The term "subject", as used herein, refers to an animal, preferably a mammal, most preferably a human, who is or has been the object of treatment, observation or experiment, for example.

[0077] The term "therapeutically effective amount," as used herein, refers to an amount of a compound that elicits a biological or medical response in a subject, such as, for example, reducing or inhibiting enzyme or protein activity, or improving symptoms, alleviating a condition, delaying disease progression, or preventing disease. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent, and / or ameliorate a condition, disorder, or disease (i) mediated by NLRP3, or (ii) associated with NLRP3 activity, or (iii) characterized by NLRP3 activity (normal or abnormal); or (2) reduce or inhibit NLRP3 activity; or (3) reduce or inhibit NLRP3 expression. In another non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound that, when administered to a cell, tissue, noncellular biological material, or culture medium, is effective to at least partially reduce or inhibit NLRP3 activity; or at least partially reduce or inhibit NLRP3 expression.

[0078] As used herein, the terms "inhibit," "inhibition," or "inhibiting" refer to the reduction or suppression of a given condition, symptom, or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process. Specifically, inhibition of NLRP3 or inhibition of the NLRP3 inflammasome pathway includes reducing the ability of NLRP3 or the NLRP3 inflammasome pathway to induce the production of IL-1 and / or IL-18. This can be achieved by mechanisms including, but not limited to, inactivation, destabilization, and / or alteration of the distribution of NLRP3.

[0079] As used herein, the term "NLRP3" is meant to include, but is not limited to, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide strands, complementary sequences, peptides, polypeptides, proteins, homologous and / or orthologous NLRP molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, different species, and active fragments thereof.

[0080] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or halting the onset of the disease or at least one of its clinical symptoms); or reducing or improving at least one physical parameter or biomarker associated with the disease or disorder, including those that may not be discernible to the patient.

[0081] As used herein, the terms "prevent," "preventing," or "prevention" of any disease or disorder refers to prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.

[0082] As used herein, a subject is "in need of" a treatment if such subject would benefit biologically, medically, or in quality of life from such treatment.

[0083] In one embodiment, there is provided a compound according to any one of the embodiments described herein for use as a medicament.

[0084] In one embodiment, there is provided use of a compound provided herein according to any one of the embodiments described herein (and / or a pharmaceutical composition comprising such a compound according to any one of the embodiments described herein) in the treatment of a disease or disorder associated with NLRP3 activity (including inflammasome activity); in the treatment of a disease or disorder in which NLRP3 signaling contributes to the pathology, and / or symptoms, and / or progression of the disease / disorder; in inhibiting NLRP3 inflammasome activity (including in a subject in need thereof), and / or as an NLRP3 inhibitor.

[0085] In one embodiment, there is provided use of a compound provided herein according to any one of the embodiments described herein (and / or a pharmaceutical composition comprising such a compound according to any one of the embodiments described herein) in the treatment of a disease or disorder associated with NLRP3 activity (including inflammasome activity); in the treatment of a disease or disorder in which NLRP3 signaling contributes to the pathology, and / or symptoms, and / or progression of the disease / disorder; in inhibiting NLRP3 inflammasome activity (including in a subject in need thereof), and / or as an NLRP3 inhibitor.

[0086] In one embodiment, there is provided use of a compound provided herein (and / or a pharmaceutical composition comprising such a compound according to any one of the embodiments described herein) in the manufacture of a medicament for treating a disease or disorder associated with NLRP3 activity (including inflammasome activity); for treating a disease or disorder in which NLRP3 signaling contributes to the pathology, and / or symptoms, and / or progression of the disease / disorder; and / or for inhibiting NLRP3 inflammasome activity (including in a subject in need thereof).

[0087] In one embodiment, there is provided a method of treating a disease or disorder in which NLRP3 signaling contributes to the pathology, and / or symptoms, and / or progression of the disease / disorder, comprising administering, e.g., to a subject (in need thereof), a therapeutically effective amount of a compound provided herein according to any one of the embodiments described herein (and / or a pharmaceutical composition comprising such a compound according to any one of the embodiments described herein). In a further embodiment, there is provided a method of inhibiting NLRP3 inflammasome activity in a subject (in need thereof), comprising administering to a subject in need thereof a therapeutically effective amount of a compound provided herein according to any one of the embodiments described herein (and / or a pharmaceutical composition comprising such a compound of the invention according to any one of the embodiments described herein).

[0088] The compounds of the present invention, whether for use in the above indications or not, may have the advantage that they may be more effective, less toxic, longer acting, more potent, have fewer side effects, be more easily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than compounds known in the prior art, and / or possess other useful pharmacological, physical, or chemical properties.

[0089] For example, the compounds may have the advantage of having good or improved thermodynamic solubility (e.g., compared to compounds known in the prior art; e.g., determined by known methods and / or methods described herein). The compounds may have the advantage of blocking pyroptosis and the release of pro-inflammatory cytokines (e.g., IL-1β) from cells. The compounds may also have the advantage of avoiding side effects that may result from the selectivity of NLRP3 inhibition, for example, compared to compounds of the prior art. The compounds provided herein may also have the advantage of having good or improved in vivo pharmacokinetics and oral bioavailability. The compounds of the present invention may also have the advantage of having good or improved in vivo efficacy. In particular, the compounds may also have advantages over prior art compounds when compared in the tests outlined below.

[0090] Experimental Department Several methods for preparing the compounds of the present disclosure are illustrated in the following examples. Unless otherwise specified, all starting materials were obtained from commercial suppliers and used without further purification, or could be synthesized by one of ordinary skill in the art using published methods.

[0091] Abbreviation

[0092] [Table 1]

[0093] Preparation of intermediates For intermediates that are used in a subsequent reaction step as a crude intermediate or as a partially purified intermediate, either no molar amount is stated for such intermediate in the subsequent reaction step, or an estimated molar amount or theoretical molar amount is stated for such intermediate in the subsequent reaction step.

[0094] 2-Iodo-5-(trifluoromethyl)phenol [CAS102771-00-6] I1

[0095] [ka]

[0096] Sodium hydride [CAS 7646-69-7] (2.47 g, 61.69 mmol) was added to anhydrous toluene (92 mL) under a nitrogen atmosphere at 0 °C. 3-Trifluoromethylphenol [CAS 98-17-9] (3.8 mL, 30.84 mmol) was then added dropwise. The mixture was stirred at 0 °C for 30 min. Iodine [7553-56-2] (7.83 g, 30.84 mmol) was then added portionwise, and the mixture was stirred from 0 °C to room temperature for 3 h. The mixture was acidified to pH 4-5 with 37% HCl in water at 0 °C, then extracted with EtOAc and washed twice with brine. The organic layer was separated, dried (MgSO ), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g, EtOAc in heptane, 0 / 100 to 10 / 90). The desired fractions were collected and concentrated in vacuo to give Intermediate 1 (6.5 g, yield: 72%) as a colorless oil.

[0097] 1-Iodo-2-(methoxymethoxy)-4-(trifluoromethyl)benzene I2

[0098] [ka]

[0099] Intermediate 1 (6.5 g, 22.6 mmol) and K2CO3 [CAS584-08-7] (5.3 g, 38.4 mmol) were dissolved in anhydrous DMF (95 mL). The reaction mixture was stirred at room temperature for 30 minutes. Then, chloromethyl methyl ether [CAS107-30-2] (2.4 mL, 29.3 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with EtOAc. The organic layers were combined, dried (MgSO4), filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (silica 80 g, heptane 100%). The desired fractions were collected and concentrated in vacuo to give Intermediate 2 (5.5 g, 72% yield) as a colorless oil.

[0100] 2-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I3

[0101] [ka]

[0102] Isopropylmagnesium chloride (2M in THF) [CAS 1068-55-9] (7.2 mL, 14.46 mmol) was added dropwise to a stirred solution of Intermediate 2 (4 g, 12.05 mmol) in anhydrous THF (96 mL) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 2 hours. 2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (CAS 61676-62-8]) (3.7 mL, 18.07 mmol) was then added dropwise to the mixture. The reaction mixture was allowed to warm slowly to room temperature and stirred for 18 hours. The reaction was quenched with saturated NH4Cl (saturated in water) and extracted with EtOAc. The organic layer was separated, dried (MgSO4), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g, EtOAc in heptane, 0 / 100 to 20 / 80). The desired fractions were collected and concentrated in vacuo to give intermediate 3 (2.6 g, yield: 63%) as a colorless oil.

[0103] 3-Methyl-5-(trifluoromethyl)phenol [CAS934180-46-8] I4

[0104] [ka]

[0105] Lithium hydroxide hydrate (2.8 g, 65.24 mmol) was added to a stirred solution of tris(dibenzylideneacetone)dipalladium(0) [CAS 51364-51-3] (616 mg, 0.652 mmol) and BippyPhos [CAS 894086-00-1] (681 mg, 1.31 mmol) in 50 mL of 1,4-dioxane and 5 mL of distilled water (previously bubbled with nitrogen for 5 min). The mixture was stirred at room temperature for 5 min. Then, 3-bromo-5-methylbenzotrifluoride [CAS 86845-28-5] (5.25 g, 21.74 mmol) was added. The reaction mixture was stirred at 100 °C for 16 h. The mixture was filtered through a pad of Celite and washed with AcOEt. The filtrate was washed with HCl (2 M in water). The organic layer was separated, dried (MgSO), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120 g, AcOEt in heptane, 0 / 100 to 2 / 98). The desired fractions were collected and concentrated in vacuo to give Intermediate 4 (3.4 g, yield: 80%) as a yellow oil.

[0106] 2-Iodo-3-methyl-5-(trifluoromethyl)phenol I5

[0107] [ka]

[0108] Sodium hydride (60% dispersion in mineral oil, 1.59 mg, 39.7 mmol) was added to a stirred solution of intermediate 4 (3.4 mg, 19.9 mmol) in 60 mL of anhydrous toluene at 0 °C under a nitrogen atmosphere. The mixture was stirred at 0 °C for 30 min. Iodine (5.05 g, 19.87 mmol) was then added portionwise, and the mixture was stirred from 0 °C to room temperature for 3 h. The mixture was acidified to pH 4-5 with concentrated HCl at 0 °C, then extracted with AcOEt and washed twice with brine. The organic layer was separated, dried (MgSO), filtered, and the solvent was evaporated in vacuo to give intermediate 5 (6.5 g, 74% yield) as a brown oil, which was used directly without further purification.

[0109] 2-Iodo-1-(methoxymethoxy)-3-methyl-5-(trifluoromethyl)benzene I6

[0110] [ka]

[0111] Intermediate 5 (9.05 g, 29.96 mmol) was dissolved in DCM (300 mL) and cooled to 0 °C. To this solution, N,N-diisopropylethylamine [CAS 7087-68-5] (6.34 mL, 35.96 mmol) was added, followed by dropwise addition of chloromethyl methyl ether [CAS 107-30-2] (2.8 mL, 35.96 mmol). The reaction mixture was gradually warmed to room temperature and stirred for 16 h. The mixture was concentrated, and the residue was purified by flash column chromatography (120 g dry-loaded silica, AcOEt in heptane, 0 / 100 to 3 / 97). The desired fractions were collected and concentrated in vacuo to give Intermediate 6 (7.39 g, yield: 71%) as a yellowish oil.

[0112] 2-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I7

[0113] [ka]

[0114] Intermediate 6 (7.4 g, 21.34 mmol) was added dropwise to a stirred solution of palladium(II) acetate [CAS 3375-31-3] (489 mg, 2.13 mmol), CyJohnPhos [CAS 247940-06-3] (787 mg, 0.57 mmol), triethylamine (15 mL, 106.72 mmol), and anhydrous 1,4-dioxane (92 mL) (previously bubbled with nitrogen for 5 minutes) in a sealed tube. The mixture was stirred at room temperature for 5 minutes, and then 4,4,5,5-tetramethyl[1,3,2]dioxaborolane [CAS 25015-63-8] (16 mL, 106.72 mmol) was added. The reaction mixture was stirred at 100 °C for 16 hours. The mixture was filtered through a pad of Celite and washed with EtOAc. The filtrate was washed with saturated aqueous NH4Cl. The organic layer was separated, dried (MgSO), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120 g, EtOAc in heptane, 0 / 100 to 1 / 99). The desired fractions were collected and concentrated in vacuo to give Intermediate 7 (6.87 g, yield: 90%) as an orange solid.

[0115] 2-(2-chloro-6-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I8

[0116] [ka]

[0117] Intermediate 8 was prepared by a similar sequence to Intermediate 7 using 3-bromo-5-chlorobenzotrifluoride as the starting material.

[0118] 1-(Benzyloxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene I9

[0119] [ka]

[0120] Intermediate 5 (9.457 g, 24.11 mmol) and K2CO3 [584-08-7] (5.049 g, 36.16 mmol) were dissolved in acetone (120 mL). The reaction mixture was stirred at room temperature for 15 minutes. Then, benzyl bromide [100-39-0] (3.3 mL, 26.52 mmol) was added dropwise, and the reaction mixture was stirred at reflux for 16 hours. The reaction mixture was diluted with water and extracted with EtOAc. The organic layers were combined, dried over MgSO4, filtered off, and concentrated in vacuo. The crude product was purified by flash column chromatography (silica, 120 g, heptane 100%). The desired fractions were collected and concentrated in vacuo to give a pinkish solid (6.44 g, purity: 66%). The impure product was dissolved in EtOAc and washed with NH3 (5% in water). The organic layer was separated, dried over MgSO4, filtered, and concentrated in vacuo. The solid was purified by flash column chromatography (silica, 120 g, heptane 100%). The desired fractions were collected and concentrated in vacuo to give Intermediate 9 (4.61 g, yield: 46%) as a white solid.

[0121] 2-(2-(benzyloxy)-6-methyl-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I10

[0122] [ka]

[0123] Intermediate 9 (4.575 g, 11.08 mmol) was added portionwise to a stirred solution of palladium(II) acetate [3375-31-3] (254 mg, 1.11 mmol) and CyJohnPhos [247940-06-3] (409 mg, 1.11 mmol) in triethylamine [121-44-8] (7.8 mL, 55.42 mmol) and anhydrous 1,4-dioxane (48 mL) (previously purged with nitrogen for 5 min). The mixture was stirred at room temperature for 5 min, and then 4,4,5,5-tetramethyl[1,3,2]dioxaborolane [25015-63-8] (8.5 mL, 55.42 mmol) was added. The reaction mixture was stirred in a sealed tube at 100 °C for 16 h. The mixture was filtered through a pad of Celite and washed with EtOAc. The filtrate was washed with NH4Cl (saturated in water). The organic layer was separated, dried (MgSO4), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120 g, EtOAc in heptane, 0 / 100 to 3 / 97). The desired fractions were collected and concentrated in vacuo to give Intermediate 10 (3345 mg, yield: 73%) as a light brown solid.

[0124] 1,4-Dichloro-6,7-dihydro-5H-cyclopenta[d]pyridazine [CAS5466-43-3]I11

[0125] [ka]

[0126] 1-(Trimethylsilyloxy)cyclopentene [CAS 19980-43-9] (17 mL, 0.878 g / mL, 95.496 mmol) was added to a stirred solution of 3,6-dichloro-1,2,4,5-tetrazine [CAS 106131-61-7] (10 g, 66.245 mmol) in 250 mL of toluene. The resulting mixture was heated at reflux for 5 hours. Upon completion, as observed by the disappearance of the red color, the mixture was cooled to room temperature, evaporated under reduced pressure, and dried in vacuo at room temperature to give Intermediate 11 (12.67 g, estimated quantitative yield) as a brown solid.

[0127] 5,8-Dichloro-2,3-dihydro-[1,4]dioxino[2,3-d]pyridazine [CAS1313733-23-1] I12

[0128] [ka]

[0129] Ethylene glycol [CAS107-21-1] (1.49 mL, 26.72 mmol) and sodium hydride (1.1 g, 27.5 mmol) were added to a solution of perchloropyridazine [CAS20074-67-3] (5 g, 22.95 mmol) in 130 mL of anhydrous DMF at 0 °C. The reaction mixture was stirred at room temperature for 18 h. Then, additional sodium hydride (1.1 g, 27.5 mmol) was added, and the mixture was stirred at 60 °C for 3 h. The solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120 g, EtOAc in heptane, 0 / 100 to 50 / 50). The desired fractions were collected and concentrated under reduced pressure to give intermediate 12 (315 mg, yield: 7%) as a white solid.

[0130] 6-Chloropyridazine-3-carbonyl chloride [CAS6531-04-0] I13

[0131] [ka]

[0132] Oxalyl chloride [CAS79-37-8] (1.0 mL, 12.05 mmol) was added dropwise to a stirred solution of 6-chloropyridazine-3-carboxylic acid [CAS5096-73-1] (1.5 g, 9.27 mmol) and anhydrous DMF (75 μL, 0.97 mmol) in 32 mL of anhydrous DCM at 0 °C under a nitrogen atmosphere. The suspension was stirred at 0 °C for 10 min and then at room temperature for 2 h until a clear brownish solution was obtained. The solvent was evaporated in vacuo to give intermediate 13 (1.65 g, estimated yield) as a light brown solid, which was used in the next step without further purification.

[0133] tert-Butyl 4-((6-chloropyridazine-3-carbonyl)piperidine-1-carboxylate I14

[0134] [ka]

[0135] A freshly prepared solution of (1-(tert-butoxycarbonyl)piperidin-4-yl)zinc(II) iodide (0.42 M in THF) (23.3 mL, 9.79 mmol, prepared from tert-butyl 4-iodopiperidine-1-carboxylate) was added dropwise to a stirred solution of copper(I) cyanide [CAS 544-92-3] (970 mg, 10.72 mmol) and anhydrous lithium chloride [CAS 7447-41-8] (910 mg, 21.44 mmol) in 40 mL of anhydrous THF at −20° C. under a nitrogen atmosphere. The mixture was stirred at −20° C. for 30 minutes. Then, intermediate 13 (1.65 g, 9.32 mmol) diluted in 10 mL of anhydrous THF was added dropwise. The reaction mixture was stirred at -20 °C for 30 min and at room temperature for 16 h, then diluted with saturated aqueous NH4Cl (50 mL) and extracted with EtOAc (x3). The combined organic layers were dried (MgSO4), filtered through a pad of Celite, and the solvent from the filtrate was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g, dry-loaded silica, EtOAc in heptane, 0 / 100 to 28 / 72). The desired fractions were collected and concentrated in vacuo to give Intermediate 14 (1.52 g, yield: 50%) as a beige solid.

[0136] tert-Butyl 4-((6-chloropyridazin-3-yl)difluoromethyl)piperidine-1-carboxylate I15

[0137] [ka]

[0138] Triethylammonium fluoride [CAS73602-61-6] (77 μL, 0.46 mmol) and DAST [CAS38078-09-0] (171 μL, 1.23 mmol) were added dropwise sequentially to a stirred solution of Intermediate 14 (100 mg, 0.31 mmol) in 2.5 mL of anhydrous DCM in a PTFE flask at 0° C. under a nitrogen atmosphere. The reaction was stirred at room temperature for 10 minutes and then at 40° C. for 16 hours. It was then cooled to room temperature, poured into a vigorously stirred ice / (saturated aqueous NaHCO) mixture, and extracted with DCM (×3). The combined organic layers were dried (MgSO), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica, EtOAc in heptane, 0 / 100 to 25 / 75) to give intermediate 15 (92 mg, yield: 71%) as a colorless sticky solid (18% impure with starting material).

[0139] tert-Butyl 3-((6-chloropyridazine-3-carbonyl)piperidine-1-carboxylate I16

[0140] [ka]

[0141] Intermediate 16 was made in a similar manner to intermediate 14 using tert-butyl 3-iodopiperidine-1-carboxylate [CAS850761-36-3] as the coupling partner in the previous step.

[0142] tert-Butyl 3-((6-chloropyridazin-3-yl)difluoromethyl)piperidine-1-carboxylate I17

[0143] [ka]

[0144] Intermediate 17 was made similarly to Intermediate 15 using Intermediate 16 as the starting material.

[0145] 1-chloro-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-6,7-dihydro-5H-cyclopenta[d]pyridazine I18-1

[0146] [ka]

[0147] Two batches: A 20 mL microwave vial was charged with Intermediate 11 (680 mg, 3.597 mmol), Intermediate 3 (1.428 g, 4.085 mmol), and tribasic potassium phosphate [CAS 7778-53-2] (2.312 g, 10.892 mmol). 15 mL of 1,4-dioxane and 5 mL of water were added. The mixture was degassed with nitrogen for 5 minutes, followed by the addition of dichlorobis(triphenylphosphine)palladium(II) [CAS 13965-03-2] (122.4 mg, 0.174 mmol). The vial was sealed and heated under microwave irradiation at 100 °C for 15 minutes. Upon completion, the vials were combined, and the reaction mixture was diluted with water and extracted with EtOAc (×2). The combined organic layers were dried (Na SO ), filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography with heptane / EtOAc (1:0 to 4:1) to give intermediate 18-1 (745 mg, 58% yield) as a light brownish powder.

[0148] Alternatively, Intermediate 11 (1.5 g, 10.58 mmol), Intermediate 3 (1.8 g, 8.741 mmol), and sodium carbonate (1.6 g, 15.096 mmol) were dissolved in 45 mL of 1,4-dioxane and 15 mL of water. The mixture was degassed with nitrogen for 5 minutes, after which Pd(PPh3)4 [CAS14221-01-3] (500 mg, 0.433 mmol) was added. The mixture was stirred at 110 °C for 2 hours. Upon completion, the reaction mixture was diluted with water and extracted with EtOAc (×2). The combined organic layers were dried over (Na2SO4), filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50 × 150 mm, mobile phase: 0.25% aqueous NH4HCO3, CH3CN). The purest fractions were collected, evaporated under reduced pressure and co-evaporated with MeOH (x3) to give intermediate 18-1 (1.3 g, 46% yield) as a light brown oil that crystallized on standing.

[0149] The following intermediates were typically obtained using Suzuki cross-couplings using PdCl2(PPh3)2 [CAS 13965-03-2], Pd(PPh3)4 [CAS 14221-01-3], or cataCXium Pd G4 [CAS 2230788-67-5] as catalyst.

[0150] [Table 2-1]

[0151] [Table 2-2]

[0152] [Table 2-3]

[0153] [Table 2-4]

[0154] tert-Butyl S)-3-((6-chloropyridazin-3-yl)methyl)piperidine-1-carboxylate I19-1

[0155] [ka]

[0156] General Procedure A for Negishi Coupling: Activated zinc (233.4 mg, 3.57 mmol), magnesium chloride (25.49 mg, 0.27 mmol), (1,2-dimethoxyethane)nickel dibromide (110.16 mg, 0.36 mmol), 3,6-dichloropyridazine [CAS 141-30-0] (0.7 g, 1.96 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (95.8 mg, 0.36 mmol), and tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate [CAS 116574-71-1] (500 mg, 1.78 mmol) were mixed as solids. DMA (6.26 mL) and pyridine (244.39 μL, 3.03 mmol) were then added. The vial was flushed with nitrogen, sealed, and stirred at 100 °C for 2 h. Aqueous LiCl (5%) was then added, and the product was extracted into EtOAc (x3). The combined organic layers were washed with brine, dried over MgSO4, filtered, and the solvent was evaporated. The residue was purified on a silica gel column using 0-5% EtOAc in DCM as the eluent. The product-containing fractions were evaporated. Purification was carried out by preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 30 x 150 mm, mobile phase: 0.25% aqueous NH4HCO3, CH3CN). The pure fractions were evaporated and co-evaporated twice with MeOH to give intermediate 19-1 (130 mg, 14% yield) as a sticky oil.

[0157] General Procedure B via MacMillan Coupling: In a 20 mL vial (oven-dried overnight), 8 mL of anhydrous MTBE was added to a mixture of tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate [CAS116574-71-1] (380.9 mg, 1.769 mmol) and 5,7-di-tert-butyl-3-phenyl-1,3-benzoxazol-3-ium (498.3 mg, 1.616 mmol) previously flushed with nitrogen / vacuum three times. The suspension (white) was stirred at room temperature for 5 minutes. Then, a solution of pyridine [CAS110-86-1] (0.13 mL, 1.611 mmol) in 1 mL of anhydrous MTBE was added to the previous solution. The reaction mixture was stirred at room temperature for 30 minutes. In a 40 mL vial (oven-dried overnight), 10 mL of anhydrous DMA was added to a mixture of 3,6-dichloropyridazine [CAS 141-30-0] (151.4 mg, 1.016 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine nickel(II) dibromide (25 mg, 0.0513 mmol), (4,4'-di-tert-butyl-2,2'-bipyridine)bis[(2-pyridinyl)phenyl]iridium(III) hexafluorophosphate [CAS 676525-77-2] (15 mg, 0.0164 mmol), and quinuclidine [CAS 100-76-5] (196.4 mg, 1.766 mmol) that had been previously flushed with nitrogen / vacuum three times. The methyl tert-butyl ether suspension was transferred to a 6 mL syringe under air. A syringe filter and a new needle were then attached to the syringe, and the methyl tert-butyl ether solution was then injected into the dimethylacetamide solution through the syringe filter. The reaction mixture was sparged with nitrogen and then sealed with parafilm. The vial was stirred at 1500 rpm and irradiated for 4 hours under a 450 nm LED module at 100% light intensity in a PennOC Integrated Photoreactor (Brookfield Integrated Photoreactor) with the fan speed at 1500 rpm and maximum stirring speed. The reaction mixture was filtered through a pad of dicalite, and the cake was washed with EtOAc (50 mL). The filtrate was quenched with water (50 mL).The product was extracted with DCM (approximately 50 mL) and again with EtOAc (50 mL). The different organic layers were combined, dried over MgSO, filtered, and concentrated under reduced pressure at 40°C. The crude product was purified by column chromatography on 25 g of Sfar silica HC in a Biotage system using a gradient of 0 to 60% EtOAc in heptane (25 CV). The different product fractions were combined and concentrated under reduced pressure at 40°C to give intermediate 19-1 (135 mg, 14% yield) as an orange oil.

[0158] The following intermediates were obtained using general procedures A and / or B exactly as indicated in the table.

[0159] [Table 3-1]

[0160] [Table 3-2]

[0161] [Table 3-3]

[0162] [Table 3-4]

[0163] 2-(6-(piperidin-3-ylmethyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol hydrochloride I20-1

[0164] [ka]

[0165] HCl (4 M in 1,4-dioxane) [7647-01-0] (2 mL, 8 mmol) was added to a solution of intermediate 19-7 (172.1 mg, 0.33 mmol) in 4 mL of DCM at room temperature. The colorless solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give intermediate 20-1 (124 mg, estimated quantitative) as a yellow residue, which was used in the next step without further purification.

[0166] Alternatively, intermediate 19-7 (43.7 mg, 0.1 mmol) was dissolved in DCM (0.5 mL), and then TFA [76-05-1] (0.084 mL, 1.49 g / mL, 1.1 mmol) was added, and the mixture was stirred at room temperature for 1 h. The solvent was evaporated, and the residue was redissolved in MeOH and purified on an SCX2 column eluted with MeOH / NH. Evaporation of the fractions gave intermediate 20-1 (32 mg, estimated quantitative yield) as a yellow residue, which was used in the next step without further purification.

[0167] The following intermediates were obtained accordingly:

[0168] [Table 4-1]

[0169] [Table 4-2]

[0170] [Table 4-3]

[0171] [Table 4-4]

[0172] (S)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-((4-methylmorpholin-2-yl)methyl)-7,8-dihydro-5H-pyrano[3,4-d]pyridazine I21

[0173] [ka]

[0174] Sodium triacetoxyborohydride (25 mg, 0.4 mmol) was added to a 10 mL round-bottom flask containing a stirred solution of I20-21 (140 mg, 0.3 mmol), triethylamine (0.26 mL, 1.8 mmol), and formaldehyde solution (40 μL, 0.5 mmol) in methanol (2.7 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred at room temperature for 16 h. The mixture was concentrated, and the residue was subjected to silica gel chromatography (12 g, irregular 40-60 μm; dry-packed silica, 0-8% MeOH / DCM) to afford intermediate 21 as a sticky solid (33 mg, 24%).

[0175] Benzyl S-2-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrophthalazin-1-yl)methyl)-4-methylpiperazine-1-carboxylate I22

[0176] [ka]

[0177] Intermediate 22 was synthesized in the same manner as Intermediate 21, using Intermediates 20-22 as starting materials.

[0178] 1-(Ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene I23

[0179] [ka]

[0180] A solution of 2-iodo-3-methyl-5-(trifluoromethyl)phenol (71 g, 235.07 mmol, 1.00 equiv.) and Cs2CO3 (153.18 g, 470.14 mmol, 2.00 equiv.) in DMF (0.71 L) was added under a nitrogen atmosphere, followed by the dropwise addition of chloromethyl ethyl ether (44.45 g, 470.14 mmol, 2.00 equiv.) at 0 °C. The solution was stirred overnight at room temperature. The resulting mixture was diluted with ice water (2 L). The resulting mixture was extracted with EA (2 × 3 L). The combined organic layers were washed with brine (2 × 3 L) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with PE to give 1-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene (42 g, 49.61%) as a yellow oil.

[0181] 2-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I24

[0182] [ka]

[0183] To a solution of 1-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene (42 g, 116.62 mmol, 1.00 equiv.), [1,1'-biphenyl]-2-yldicyclohexylphosphane (4.0 g, 0.10 equiv.), TEA (70.81 g, 699.7 mmol, 6.00 equiv.), and Pd(AcO) (1.31 g, 5.83 mmol, 0.05 equiv.) in dioxane (0.42 L) was added 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (59.70 g, 466.51 mmol, 4.00 equiv.) under a nitrogen atmosphere. The solution was stirred at 100 °C for 2 h. The mixture was cooled to room temperature. The resulting mixture was diluted with ice water (2 L). The resulting mixture was extracted with EA (2 x 2 L). The combined organic layers were washed with brine (2 x 2 L) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied onto a silica gel column using petroleum ether. The residue was purified by trituration with n-hexane (300 mL) at -30 °C. The precipitated solid was collected by filtration. This gave 31.0106 g (73.82%) of 2-[2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a white solid.

[0184] 1 H NMR: (300MHz, CDCl3, ppm) δ7.13-7.03(m,2H),5.23(s,2H),3.74(q,J=7.1Hz,2H),2.41(s,3H),1.41(s,12H),1.29-1.18(m,3H).

[0185] tert-Butyl (R)-3-((6-chloro-1,2,4,5-tetrazin-3-yl)amino)piperidine-1-carboxylate I25

[0186] [ka]

[0187] A solution of (R)-tert-butyl 3-aminopiperidine-1-carboxylate [CAS 188111-79-7] (10.61 g, 53 mmol) and TEA (8.82 mL, 63.6 mmol) in 80 mL of DCM was added via syringe pump to a stirred solution of 3,6-dichloro-1,2,4,5-tetrazine [CAS 106131-61-7] (8 g, 53 mmol) in dry DCM (400 mL) over 15 minutes at temperatures between -5 °C and 0 °C in an Optimax reactor. The reaction mixture was stirred at 0 °C for 30 minutes. LC / MS indicated the formation of the desired product. The solution was washed with a solution of 10 g of K2CO3 in 300 mL of water. The organic layer was separated, dried over MgSO4, and evaporated (25 °C) to give the desired product as a red solid (16.7 g, 100% yield). This was used in the next step without further purification.

[0188] tert-Butyl (R)-3-((4-chloro-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)amino)piperidine-1-carboxylate I26

[0189] [ka]

[0190] 1-Pyrrolidino-1-cyclopentene [CAS 7148-07-4] (8 g, 58.3 mmol) was added dropwise to a mixture of tert-butyl (R)-3-((6-chloro-1,2,4,5-tetrazin-3-yl)amino)piperidine-1-carboxylate I25 (16.68 g, 53 mmol) in dry toluene (390.98 mL) in a 1 L four-neck reactor. The mixture was stirred at room temperature for 30 min (Caution: Some nitrogen was released). The mixture was then heated at 50 °C for 30 min, and then the mixture was heated at reflux for 6 h. The first 10 mL of reflux effluent was removed. After 6 h, LC / MS showed no more SM or intermediates. The reaction mixture was cooled and washed with water and 8 g of K2CO3. The organic layer was separated, dried over MgSO4, filtered, and concentrated. The residue was purified on a silica gel column, eluent: EtOAc in heptane, 0-100%. Evaporation of the pure fractions afforded the desired product (9.09 g, 48.6% yield) as a pink foam.

[0191] tert-Butyl (3R)-3-((4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)amino)piperidine-1-carboxylate I27

[0192] [ka]

[0193] In a pressure tube, 1,4-dioxane (9.06 mL) and deionized water (2.56 mL) were degassed with nitrogen for 5 minutes. Then, tert-butyl (3R)-3-((4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)amino)piperidine-1-carboxylate I27 (500 mg, 1.42 mmol), 2-[2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I24 (612.44 mg, 1.7 mmol), and tribasic potassium phosphate [CAS 7778-53-2] (932.41 mg, 4.39 mmol) were added. Methanesulfonato(2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-1,1'biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) [CAS 1445085-55-1] (239.88 mg, 0.28 mmol) was added, and the tube was flushed with nitrogen and then closed. The reaction mixture was heated at 105 °C for 2 h. LC / MS showed complete conversion. The reaction mixture was cooled, poured into saturated NaHCO3 solution, and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified on a silica gel column, eluting with 0-80% EtOAc in heptane. Pure fractions were combined and concentrated to give the desired product (703 mg, 90.1% yield) as a white foam.

[0194] 3-Methyl-2-(4-(((R)-piperidin-3-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)-5-(trifluoromethyl)phenol I28

[0195] [ka]

[0196] HCl (4 M in 1,4-dioxane) (6.04 mL, 4 M, 24.17 mmol) was added to a solution of dihydro-5H-cyclopenta[d]pyridazin-1-yl)amino)piperidine-1-carboxylate I27 (701 mg, 1.27 mmol) in 1,4-dioxane (6.1 mL), and the mixture was stirred at room temperature for 2 h. LC / MS showed complete conversion. The mixture was poured into saturated NaHCO3 solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to give the desired product (495 mg, 99.08% yield) as a white solid.

[0197] Preparation of final compounds In general, the preparation of final compounds was carried out using racemic mixtures separated by preparative SFC, and the absolute configuration was assigned by synthesizing enantiopure compounds starting from chiral intermediates.

[0198] (R)-2-(6-((1-methylpiperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 1 and (S)-2-(6-((1-methylpiperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 2

[0199] [ka]

[0200] A 37% aqueous solution of formalin [CAS 50-00-0] (600 μL, 0.815 g / mL, 6.026 mmol, 4.2 equiv.) was added to a stirred solution of intermediate 20-1 (577 mg, 1.406 mmol, 1 equiv.) and EtN (500 μL, 0.728 g / mL, 3.597 mmol, 2.5 equiv.) in 20 mL of MeOH, and the resulting mixture was stirred at room temperature for 3 h. Sodium triacetoxyborohydride [CAS 56553-60-7] (600 mg, 2.831 mmol, 2 equiv.) was then added portionwise, and the mixture was further stirred overnight. The mixture was diluted with saturated aqueous NaHCO and extracted with EtOAc. The organic layer was separated, washed with brine, dried (NaSO), filtered, and evaporated under reduced pressure. The crude product was purified by short column chromatography on silica gel using DCM / 7N NH in MeOH (1:0 to 9:1) followed by preparative SFC (stationary phase: Chiralpak Daicel IC 20 × 250 mm, mobile phase: CO, EtOH + 0.4 iPrNH) to give compounds 1 (57 mg, 23% yield) and 2 (56 mg, 23% yield) as pale yellow solids.

[0201] The following final compounds were synthesized accordingly:

[0202] [Table 5-1]

[0203] [Table 5-2]

[0204] [Table 5-3]

[0205] [Table 5-4]

[0206] [Table 5-5] 1 Compound 29 was synthesized as a comparison with the corresponding 3-piperidinyl (compound 30)

[0207] (S)-2-(4-((4-methylmorpholin-2-yl)methyl)-7,8-dihydro-5H-pyrano[3,4-d]pyridazin-1-yl)-5-(trifluoromethyl)phenol 24 and (S)-2-(1-((4-methylmorpholin-2-yl)methyl)-7,8-dihydro-5H-pyrano[3,4-d]pyridazin-4-yl)-5-(trifluoromethyl)phenol 25

[0208] [ka]

[0209] 10% Palladium on carbon (0.028 g, 0.026 mmol) was added to a 10 mL round-bottom flask containing a stirred solution of intermediate 21 (0.066 g, 0.132 mmol) and MeOH (2.6 mL) under a nitrogen atmosphere to give a black heterogeneous mixture (s / l). The nitrogen atmosphere was then replaced with hydrogen (1 atm, balloon), and the resulting mixture was stirred at room temperature for 16 h. The heterogeneous mixture was filtered through a pad of Celite and rinsed with MeOH (3 × 5 mL). The solvent was concentrated to dryness under vacuum. The crude product was subjected to SFC (Phenomenex Lux Amylose-1 250 × 30 mm, 5 μm; ISOC 20% ethanol + 0.1% DEA) and concentrated to give compounds 24 (14.4 mg, 26%) and 25 (8.3 mg, 15%) as off-white solids.

[0210] (R)-2-(4-((4-methylpiperazin-2-yl)methyl)-5,6,7,8-tetrahydrophthalazin-1-yl)-5-(trifluoromethyl)phenol 26 and (S)-2-(4-((4-methylpiperazin-2-yl)methyl)-5,6,7,8-tetrahydrophthalazin-1-yl)-5-(trifluoromethyl)phenol 27

[0211] [ka]

[0212] Compounds 26 (respectively 27) were synthesized analogously to compounds 24 (respectively 25) using intermediate 22 as starting material.

[0213] (S)-2-(8-((1-methylpiperidin-3-yl)methyl)-1,2,3,4-tetrahydropyrido[2,3-d]pyridazin-5-yl)-5-(trifluoromethyl)phenol 31 and (R)-2-(8-((1-methylpiperidin-3-yl)methyl)-1,2,3,4-tetrahydropyrido[2,3-d]pyridazin-5-yl)-5-(trifluoromethyl)phenol 32

[0214] [ka]

[0215] A racemic mixture of compounds 5 and 6 (60 mg, 0.15 mmol) and Pd / C (10%) (10 mg, 0.0095 mmol) in 6 mL of EtOH was stirred at 50 °C for 16 h under 15 bar of H. The mixture was filtered through a syringe with a 45 μm filter, washed with EtOH, and the solvent was evaporated under vacuum. Purification was carried out via preparative SFC (stationary phase: Chiralcel Daicel IC 20 × 250 mm, mobile phase: CO, EtOH + 0.4 iPrNH) to give compound 31 (18 mg, 30% yield) and compound 32 (15 mg, 25% yield) as white solids.

[0216] (S)-3-Methyl-2-(6-((1-(oxetan-3-yl)piperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 33

[0217] [ka]

[0218] MeOH (1.5 mL) was added to a mixture of (S)-intermediate 20-6 (106.8 mg, 0.275 mmol) and 3-oxetanone [6704-31-0] (83.4 mg, 1.157 mmol). The mixture was stirred at room temperature for 15 minutes. Sodium cyanoborohydride [25895-60-7] (58.7 mg, 0.934 mmol) was added to the previous solution. The reaction was stirred at room temperature for 6 hours. An additional amount of 3-oxetanone [6704-31-0] (58.3 mg, 0.809 mmol) was added, and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with saturated NaHCO3 in water and diluted with EtOAc. The product was extracted with EtOAc (x3) and DCM. The combined organic layers were dried (MgSO4), filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on a 10 g Sfar silica HC column in a Biotage system using a gradient of 0-10% NH in MeOH (3.5 M) in DCM. The purest fractions were combined and concentrated under reduced pressure. The resulting product was precipitated with DIPE and evaporated in vacuo to give compound 33 (42 mg, 37% yield) as a white powder.

[0219] (R)-3-Methyl-2-(6-((1-(oxetan-3-yl)piperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 34

[0220] [ka]

[0221] Compound 34 was prepared similarly to compound 33 using (R)-intermediate 18-6 as the starting material.

[0222] (S)-3-Methyl-2-(6-((1-(methyl-d3)piperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 35 and (R)-3-Methyl-2-(6-((1-(methyl-d3)piperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 36

[0223] [ka]

[0224] Formaldehyde-d2 [1664-98-8] (38 μL, 0.261 mmol) was added to a solution of intermediate 20-6 (49.8 mg, 0.128 mmol) in dry DCM (1.4 mL) and dry DMF (0.5 mL). The mixture was stirred at room temperature for 30 minutes. An additional amount of sodium cyanoborohydride [25895-62-9] (20 mg, 0.304 mmol) was added at room temperature, and the reaction mixture was stirred at room temperature for 2 hours and 30 minutes. The reaction mixture was quenched with saturated NaHCO3 solution and DCM and extracted with DCM (×3). The combined organic layers were dried (MgSO4), filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on a 10 g Sfar silica HC column in a Biotage system using a gradient of 0–10% NH3 (3.5 M) in MeOH in DCM (15 CV) and 10% NH3 (3.5 M) in MeOH in DCM (15 CV). The enantiomers were then separated by preparative SFC (stationary phase: Chiralpak Daicel IG 20 × 250 mm, mobile phase: CO2, EtOH + 0.4% iPrNH2). The purest fractions were combined, concentrated under reduced pressure, and dried in vacuo to give compound 35 (9 mg, 18% yield) and compound 36 (15 mg, 32% yield) as white solids.

[0225] (R)-2-(6-((1-ethylpiperidin-3-yl)methyl)pyridazin-3-yl)-3-methyl-5-(trifluoromethyl)phenol 37

[0226] [ka]

[0227] A solution of iodoethane [75-03-6] (35 μL, 0.435 mmol) and DIPEA [7087-68-5] (0.25 mL, 1.451 mmol) in 2 mL of anhydrous DMF was added to (R)-intermediate 20-6 (107.5 mg, 0.277 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with EtOAc and washed with saturated NaHCO solution. The aqueous layer was back-extracted with EtOAc (x2) and DCM. The combined organic layers were dried (MgSO), filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on a 10 g SfAr silica HC column in a Biotage system using a gradient of 0–10% DCM / MeOH (3.5 M) / DCM (15 CV) and 10% DCM / MeOH (3.5 M) / DCM (10 CV). The purest fractions were collected and evaporated under reduced pressure. The product was precipitated with DIPE and dried in vacuo to give compound 37 (56 mg, 53% yield) as a beige solid.

[0228] The following final compounds were synthesized accordingly:

[0229] [Table 6]

[0230] 3-Methyl-2-(4-(R)-1-methylpiperidin-3-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)-5-(trifluoromethyl)phenol 51

[0231] [ka]

[0232] Formalin (0.39 mL, 0.82 g / mL, 3.96 mmol) was added to a stirred solution of 3-methyl-2-(4-((R)-piperidin-3-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)-5-(trifluoromethyl)phenol I28 (495 mg, 1.26 mmol) in MeOH (19.7 mL), and the resulting mixture was stirred at room temperature for 1 h. After the mixture was cooled to 0 °C, sodium triacetoxyborohydride [CAS 56553-60-7] (0.67 g, 3.15 mmol) was added portionwise. The mixture was then warmed to room temperature and stirred for an additional 1 h. LC / MS showed complete conversion. The reaction mixture was poured into saturated NaHCO3, extracted with EtOAc, washed with brine, dried over MgSO4, and concentrated. The residue was purified on a silica gel column using 0-10% MeOH / NH3 7N in DCM as an eluent. Pure fractions were collected and concentrated. The residue was crystallized from ACN to give 3-methyl-2-(4-(((R)-1-methylpiperidin-3-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyridazin-1-yl)-5-(trifluoromethyl)phenol 51 (437 mg, 99.28% yield) as a white solid.

[0233] The following final compounds were synthesized according to the procedures described for compounds 1-50.

[0234] [Table 7]

[0235] Analytical and Biological Data Example A - Analytical Data LCMS High-performance liquid chromatography (HPLC) measurements were performed using the LC pump, diode-array (DAD) or UV detector, and column specified in each method. Additional detectors were included as needed (see methods table below).

[0236] The flow from the column was delivered to a mass spectrometer (MS) configured with an atmospheric pressure ion source. It is within the knowledge of one skilled in the art to set tuning parameters (e.g., scan range, dwell time, etc.) to obtain ions that allow identification of the nominal monoisotopic molecular weight (MW) of the compound. Data collection was performed with appropriate software.

[0237] Compounds were analyzed by their experimental retention times (R t ) and ions. Unless otherwise specified in the tables of data, the reported molecular ions are [M+H] + (protonated molecule) and / or [MH] - (deprotonated molecule). If the compound is not directly ionizable, the type of adduct is specified (i.e., [M+NH4] + , [M+HCOO] - For molecules with multiple isotopic patterns (Br, Cl), the reported values ​​are those obtained for the lowest isotopic mass. All results are obtained with experimental uncertainties typically associated with the methods used.

[0238] Hereinafter, "SQD" means single quadrupole detector, "MSD" means mass selected detector, "RT" means room temperature, "BEH" means crosslinked ethylsiloxane / silica hybrid, "DAD" means diode array detector, and "HSS" means high strength silica.

[0239] [Table 8]

[0240] LCMS data for all final compounds are shown below.

[0241] [Table 9-1]

[0242] [Table 9-2]

[0243] NMR 1 H NMR spectra were recorded on Bruker Avance III and Avance NEO spectrometers. CDCl3 was used as the solvent unless otherwise stated. Chemical shifts are expressed in ppm relative to tetramethylsilane. OH signals may be lost in CDCl3.

[0244] [Table 10-1]

[0245] [Table 10-2]

[0246] [Table 10-3]

[0247] [Table 10-4]

[0248] Example B - Pharmaceutical Composition A compound (e.g., a compound of the Examples) is associated with a pharmaceutically acceptable carrier, thereby providing a pharmaceutical composition comprising such an active compound. A therapeutically effective amount of a compound provided herein (e.g., a compound of the Examples) is intimately mixed with a pharmaceutically acceptable carrier in a method for preparing a pharmaceutical composition.

[0249] Example C - Biological Examples The activity of the compound can be evaluated by in vitro methods.The compound may exhibit beneficial pharmacological properties, such as the ability to inhibit NLRP3 activity, as shown in the following test, and thus be suitable for the treatment of NLRP3 inflammasome activity.

[0250] PBMC assay Peripheral venous blood was collected from healthy individuals, and human peripheral blood mononuclear cells (PBMCs) were isolated from the blood by Ficoll-Histopaque (Sigma-Aldrich, A0561) density gradient centrifugation. After isolation, PBMCs were stored in liquid nitrogen for later use. Once thawed, PBMC cell viability was determined in growth medium (RPMI medium supplemented with 10% fetal bovine serum, 1% Pen-Strep, and 1% L-glutamine). Compounds were spotted in 1:3 serial dilutions in DMSO and diluted to final concentrations in 30 μl of medium in a 96-well plate (Falcon, 353072). PBMCs were added at a density of 7.5 × 10 cells per well and incubated at 37 °C in a 5% CO incubator for 30 minutes. LPS stimulation was performed by adding 100 ng / mL LPS (final concentration, Invivogen, tlrl-smlps) for 6 h, after which cell supernatants were collected and analyzed for IL-1β (μM), IL6, and TNF cytokine levels (μM) by MSD technology according to the manufacturer's guidelines (MSD, K151A0H).

[0251] I C 50 Values ​​(for IL-1β) and EC 50 Values ​​(IL6 and TNFα) were obtained for compounds of the invention / examples and are shown in the table below.

[0252] [Table 11]

[0253] Example D - Outflow Ratio The purpose of this assay is to measure the permeability and efflux of test compounds using MDCK cells transfected with P-glycoprotein (MDR1). Two control compounds, propranolol (highly permeable) and prazosin (a P-glycoprotein substrate), are screened alongside the test compounds. MDCK cells are an epithelial cell line derived from canine kidneys. These cells can be stably transfected to express active P-glycoprotein (MDR1-MDCK), making them ideal for studying drug efflux via P-gp. Test compounds are added to either the apical or basolateral side of confluent monolayers of MDR1-MDCK cells, and permeability in the apical-to-basolateral (AB) and basolateral-to-apical (BA) directions is measured by monitoring the appearance of the test compound on opposite sides of the membrane using LCMS / MS. The efflux ratio (BA permeability relative to AB permeability) is calculated to determine whether the test compound is subject to P-gp efflux. We provide the apparent permeability (Papp) coefficient and efflux ratio. Experimental recovery values ​​are also provided. Further details can be found on the Cyprotex website at https: / / www.cyprotex.com / admepk / in-vitro-permeability-and-drug-transporters / mdr1-mdck-permeability.

[0254] [Table 12]

[0255] Example E-hERG Inhibition The whole-cell patch clamp technique on transfected cells allows the study of ion channels with limited or no interference from other ion channels. The effects of compounds on hERG currents are tested using an automated planar patch clamp system, SyncroPatch 384 PE (Obergrussberger, A., Bruggemann, A., Goetze, T.A., Rapedius, M., Haarmann, C., Rinke, I., Becker, N., Oka, T., Ohtsuki, A., Stengel, T., Vogel, M., Steindl, J., Muller, M., Stiehler, J., George, M. & Fertig, N. (2016). Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module. Journal of Laboratory Automation 21(6):779-793. All cells were patch-clamped in whole-cell mode. The SyncroPatch 384 PE is an automated patch-clamp system that allows for parallel recording from 384 wells. The module is integrated into a liquid-handling pipetting robotic system, Biomek FXP, for cell and compound application. Voltage protocols are built on the SyncroPatch 384 PE, and data are acquired using PatchControl 384 and analyzed using DataControl 384 (both from Nanion Technologies).

[0256] Different screening approaches are applied, for example, two or up to four concentration relationships per compound are generated. Different concentrations are applied either as a single dose or at two cumulatively increasing concentrations. hERG currents are determined as the maximum tail current at -30 mV, and the percentage inhibition and pIC50 upon compound addition are reported below.

[0257] [Table 13]

[0258] Example F - Further Testing One or more compound(s) may be tested in several other ways to assess permeability, stability (including metabolic and blood stability), and solubility, among other properties.

[0259] Metabolic Stability Test In liver microsomes The metabolic stability of test compounds is tested using liver microsomes (0.5 mg / mL protein) from human and preclinical species incubated with 1 μM test compound at 37° C. for up to 60 minutes.

[0260] In vitro metabolic half-life (t 1 / 2 ) is calculated using the slope of the log-linear regression from the parent compound survival versus time relationship (κ). t 1 / 2 =-ln(2) / κ

[0261] In vitro intrinsic clearance (Cl int ) (ml / min / mg microsomal protein) is calculated using the following formula:

[0262]

number

[0263] [Table 14]

[0264] In hepatocytes The metabolic stability of the test compounds was evaluated by measuring the metabolic stability of the test compounds in 37 oTests are performed using hepatocytes (1 milj cells) from human and preclinical species incubated at C for up to 120 min.

[0265] In vitro metabolic half-life (t 1 / 2 ) is calculated using the slope of the log-linear regression from the parent compound survival versus time relationship (κ). t 1 / 2 =-ln(2) / κ

[0266] In vitro intrinsic clearance (Cl int ) (μl / min / million cells) is calculated using the following formula:

[0267]

number

[0268] [Table 15]

[0269] Plasma and brain tissue binding Plasma protein binding 1. Protocol Summary Test compounds are prepared in species-specific plasma (diluted to 25% plasma in buffer). The plasma solution is added to one side of the membrane of an equilibrium dialysis system, and buffer (pH 7.4) is added to the other side. The system is allowed to reach equilibrium at 37°C. Compounds on both sides of the membrane are measured by LC-MS / MS, and the fraction of unbound compound is calculated. We provide the unbound fraction (fu) in plasma for each test compound, along with the recovery rate.

[0270] 2.Purpose Determining the extent of plasma protein binding of the test compound.

[0271] 3.Customer provided · Compound identifier, molecular formula. 25 μL of 10 mM or 50 μL of 5 mM test compound in DMSO per species.

[0272] 4.Material Plasma from the following strain and species combinations is used: Humans from ethnically diverse donors (mixed male and female - collected in tubes (not bags)) Male SD rat Mouse, male CD Dog, male beagle Monkey, male cynomolgus monkey Guinea pig, male, Dunkin Hartley

[0273] 5. Experimental Procedure A solution of test compound (1 μM test compound concentration; 0.5% final DMSO concentration) is prepared in species-specific plasma diluted with buffer to 25% plasma. Experiments are performed using equilibrium dialysis with two compartments separated by a semipermeable membrane. 500 μL of buffer (pH 7.4) is added to one side of the membrane, and 300 μL of plasma solution containing the test compound is added to the other side. After equilibration for 6 hours at 37°C in an incubator containing 5% CO2 and agitation at 250 rpm on an orbital shaker, samples are taken from both sides of the membrane.

[0274] Samples are matrix-matched by adding either buffer or diluted plasma to the relevant sample (i.e., 45 μL of buffer is added to 45 μL of plasma sample, and 45 μL of diluted plasma (25%) is added to 45 μL of buffer sample). Proteins are then precipitated from the matrix-matched sample by adding 180 μL of methanol containing the internal standard, followed by centrifugation at 2500 rpm for 30 minutes at 4°C. The supernatant (20 μL per compound x 4 compounds) is then diluted with water (100 μL) before analysis. Test compound incubations are performed in triplicate. Two control compounds are included in each experiment, as specified in the vendor documentation guidance.

[0275] 6.Quantitative analysis Solutions of each batch of compound are combined into cassettes of up to four compounds prior to cassette analysis by LC-MS / MS using Cyprotex's generic LC-MS / MS conditions.

[0276] 7. Data Analysis The unbound fraction in 25% plasma (fu25%) is calculated using the following formula: fu25% = peak area ratio buffer / peak area ratio plasma

[0277] The calculated fu in 25% plasma (fu25%) is converted to fu in 100% plasma (fu100%) using the following formula: fu100%=fu25% / (4-(3fu25%))

[0278] The % recovery is calculated using the following formula: Recovery % = 100 x ((Buffer F x VB) + (Plasma F x VP) / (Plasma I x VP)) During the ceremony, BufferF=final buffer compartment concentration (after dialysis) PlasmaF=Final plasma component concentration (after dialysis) PlasmaI = initial concentration in plasma VB = Volume within the buffer compartment VP = volume in the plasma compartment

[0279] 8. Deliverables The unbound fraction (fu) in plasma and recovery rate are returned in the form of an Excel spreadsheet. In addition, the sheet includes an indication of whether the data should be further reviewed by an internal Japanese reviewer (based on predefined rules provided in the Janssen guidance document) along with any relevant comments.

[0280] Brain tissue connection 1. Purpose The goal of this study is to determine the brain tissue binding of test compounds in rat and mouse brain tissue using equilibrium dialysis. The peak area ratio of the test compound(s) in brain tissue homogenate and buffer solution is assessed by LC-MS / MS.

[0281] 2. Materials and Reagents The sponsor will provide the test compounds. The control compounds verapamil and fluoxetine will be purchased from Sigma Chemical Co. The control compound venlafaxine will be purchased from MedChemExpress LLC.

[0282] Na2HPO4, NaH2PO4, and NaCl are purchased from local suppliers.

[0283] Acetonitrile and methanol are purchased from Merck (Darmstadt, Germany). Other reagents are purchased from local suppliers.

[0284] Disposable RED plates with inserts (90006 BLCS) are purchased from Thermo.

[0285] Brain tissue homogenate is prepared by diluting 1 volume of whole brain tissue with 9 volumes of buffer (PBS, pH 7.4), and the mixture is homogenized using a tissue homogenizer. Brain tissue homogenate is frozen at -80°C before use. Brain tissue from three or more individual animals is usually pooled.

[0286] [Table 16]

[0287] 3. Experimental Procedure Preparation of 100 mM sodium phosphate and 150 mM NaCl buffer (PBS) Prepare a basic solution by dissolving 14.2 g / L Na2HPO4 and 8.77 g / L NaCl in deionized water. Store at 4°C for up to 7 days. Prepare an acidic solution by dissolving 12 g / L NaH2PO4 and 8.77 g / L NaCl in deionized water. Store at 4°C for up to 7 days. Titrate the basic solution with the acidic solution to pH 7.4. Store at 4°C for up to 7 days. Check the pH on the day of the experiment and adjust if it is outside the specification of 7.4 ± 0.1.

[0288] Thaw frozen brain tissue homogenate (stored at -80 °C).

[0289] Immediately thaw the frozen brain tissue homogenate in a 37 °C water bath.

[0290] Preparation of stock and working solutions Prepare stock solutions of the test compound(s) and the control compounds verapamil, fluoxetine, and venlafaxine in DMSO at a concentration of 10 mM. Dilute 2 µL of the stock solution (10 mM) with 198 µL of DMSO to obtain a working solution (100 µM). Then, remove 12 µL of the working solution and mix it with 1,200 µL of brain tissue homogenate to achieve a final concentration of 1 µM (1% DMSO). Mix the spiked brain tissue homogenate by pipetting 5-6 times and vortex thoroughly.

[0291] Methods for Equilibrium Dialysis Assemble a 48-well RED device. Add 500 μL of PBS to the buffer side of the designated well. Immediately add 300 μL of spiked brain homogenate to the opposite side of the designated well. Assays were performed in triplicate. Seal the RED device and place it in an incubator at 37°C, 5% CO2, 150 RPM for 6 hours. At the end of the incubation, remove the seal and pipette 50 μL of sample from both the buffer and brain tissue homogenate chambers into separate wells of a new 96-well plate.

[0292] Preparation of equilibrium dialysis samples Add 50 μL of blank brain tissue homogenate to the buffer sample and an equal volume of PBS to the collected brain tissue homogenate sample. Add 400 μL of room temperature quench solution (acetonitrile containing internal standard (IS), 200 nM labetalol, 100 nM alprazolam, 200 nM imipramine, and 2 μM ketoprofen)) to precipitate the proteins. Vortex for 5 minutes. Centrifuge the samples in the plate at 3,220 g for 30 minutes at room temperature. Transfer 100 μL of the supernatant to a new plate. The supernatant can be diluted with 100 μL or 200 μL of water depending on the LC / MS signal response and peak shape. Mix well and analyze the samples using LC / MS / MS.

[0293] Preparation of stability samples For the time 0 sample, transfer 50 μL of spiked brain tissue homogenate sample to a new plate containing 50 μL of PBS, then add 400 μL of acetonitrile containing internal standards (IS, 200 nM labetalol, 100 nM alprazolam, 200 nM imipramine, and 2 μM ketoprofen) to precipitate proteins. Vortex for 5 minutes. Transfer 50 μL of spiked brain tissue homogenate sample to a new plate, and incubate the plate at 37°C, 5% CO2 for 6 hours. After incubation, add 50 μL of PBS and 400 μL of acetonitrile containing internal standards (IS, 200 nM labetalol, 100 nM alprazolam, 200 nM imipramine, and 2 μM ketoprofen) to precipitate proteins. Vortex for 5 minutes. All stability samples are centrifuged at 3,220 g for 30 minutes at room temperature. 100 μL of the supernatant is transferred to a new plate. The supernatant can be diluted with 100 μL or 200 μL of water according to the LC / MS signal response and peak shape. Mix well and analyze the samples using LC / MS / MS.

[0294] 4. Data Analysis All calculations were performed using Microsoft Excel.

[0295] From the peak area ratio, the peak area ratio of the test compound and the control compound in the buffer chamber and the brain tissue homogenate chamber is determined. The percentage of bound test compound(s) and control compound is calculated as follows:

[0296]

number

[0297] Recovery rate (%) = (peak area ratio of buffer chamber * Peak area ratio of V buffer chamber + tissue homogenate chamber * V tissue homogenate chamber) / peak area ratio T=0 sample * V Tissue Homogenate Chamber * 100% Fuapp = apparent unbound fraction measured in brain tissue homogenates D = dilution factor of brain tissue % Binding = Brain tissue binding

[0298] [Table 17]

[0299] Pharmacokinetics Administration Test System Mouse:Swiss Crl:CD1 Balb / cAnNCrl NOD Cg-Prkdcscidll2rgtmWjl / SzJ-NSG C57BL / 6JRj Rat: Sprague Dawley Wister Supplier:Charles River Germany Age: 6-8 weeks Acclimatization period: at least 3 days Diet and Feeding: SAFE A04 maintenance diet and water available ad libitum Administration: Oral administration by PO gavage IV intravenous tail vein SC rear Dosage volume: PO 10mL / kg IV 2 mL / kg SC 10mL / kg According to good practice guide for administration volume N=3 per time point.

[0300] Blood sampling Sampling site: At each time point, animals are sacrificed by decapitation, and blood is collected by exsanguination into capillary tubes for microsampling, or into BD Vacutainers for other samples. Blood samples are immediately placed on melting ice and centrifuged at approximately 1900 x g for 10 minutes at 4°C to obtain plasma.

[0301] Decapitation under isoflurane anesthesia Induction: 4% (O2 and room air) Volume: Microsampling: 32 μl onto EDTA Collection tube: EDTA coated 75 mm capillary tube, Vitrex® Capillary EDTA, Catalog No. 164113 BD Vacutainer 2mL K2E (EDTA) 3.6mg. BD (REF.368841) Sampling time: Flexible; dependent on route of administration and expected PK profile Suggestions for PO: 1 hour, 4 hours, 7 hours, 24 hours after administration

[0302] In rats, continuous blood sampling is performed via the tail vein. The choice of sampling method (Microvette tubes or capillary tubes) depends on the amount of plasma required for bioanalysis.

[0303] In mice, serial blood sampling is usually performed by puncturing the saphenous vein. Occasionally, blood sampling in mice can be performed via the tail vein.

[0304] The sample volume does not exceed the maximum recommended blood sample volume from the animal.

[0305] Following general guidelines for blood collection for laboratory animals, For tissue or terminal blood sampling: Animals are anesthetized with an isoflurane mixture. Blood sampling is performed by decapitation and the animal can be bled prior to collection of tissues.

[0306] Anesthesia Isoflurane: Induction: 4% (O2 and room air) Maintenance: 2% (O2 and room air)

[0307] Tissue sampling Sampling: After bleeding, individual samples of brain are dissected and weighed. Collection tube: Super polyethylene vial 20 mL Perkin Elmer (REF. 6008117) (Polytron) Dissolving Matrix D Tube 1mL MP Biomedicals (REF.6913-500) (Fast Prep) Equipment: Polytron PT3100 High-speed preparative sample preparation equipment Tissue homogenization: Tissue samples were homogenized in demineralized water (1 / 9 w / v or +3 mL if tissue weight <0.33 g). Homogenization was performed under reduced light conditions.

[0308] plasma preparation Centrifugation: Start within 1 hour of sampling Centrifugation conditions: 4°C, 1900 x g, ±10 minutes Collection method: 10 μL of plasma is collected into a 96-well format holder using a Vitrex® end-to-end pipette (catalog number 174313). If less than 10 μL of plasma can be collected, 4 μL of plasma is collected (Vitrex® end-to-end 4-tL, catalog number 174213). If less than 4 μL can be collected, no sample is transferred. Storage: All samples are protected from sunlight and stored at -18°C prior to analysis.

[0309] Sample transportation and freezing to the bioanalysis department The animals are observed throughout the experiment.

[0310] During the acclimation period (after transfer), animals are monitored daily by LAM personnel.

[0311] During the experiment, animals will be visually observed by the animal research staff after dosing and at each sample time point for appearance, behavior, and potential side effects. Any abnormalities will be registered on the remarks sheet of the study protocol.

[0312] If the animals are not in optimal condition, check for weight loss ≥ 20%, changes in body temperature, mobility, behavior, and pain manifestations. If the body temperature is ≤ 33°C, the animals are euthanized and removed from the experiment. In case of doubt, a veterinarian is consulted, who will decide the fate of these animals. Deviations are registered in the study file.

[0313] Determination of partition coefficients kpuu,brain Kpuu,brain was calculated as follows:

[0314] Kpuu,brain=(AUC,last,brain * BTB,r) / (AUC,last,plasma * PPB,m) AUC,last is defined as the area under the concentration-time curve from administration (time 0) to the time of the last measured concentration in brain and plasma, respectively. BTB,r is the brain tissue binding in rats as defined above PPB,m is the plasma protein binding in mice as defined above

[0315] [Table 18]

[0316] Solubility assay Aliquots of the DMSO solution containing the test compound are dispensed into 96-well plates, the DMSO evaporated, and the pellet redissolved by adding buffer. The solubility of the compound in pH 2.0 or 7.0 buffer is measured after stirring at 25 °C for 3 days. The samples are centrifuged and the supernatant is filtered. The filtrate is pooled and the concentration is measured by liquid chromatography / tandem mass spectrometry (LC-MS / MS). The solid state properties of the residue are evaluated by polarized light microscopy (PLM). data:

[0317] [Table 19]

[0318] Phospholipidosis assay The phospholipid-inducing potential of compounds was assessed according to a published procedure (Mesens, N.; Steemans, M.; Hansen, E.; Peters, A.; Verheyen, G.; Vanparys, PA. 96-well flow cytometric screening assay for detecting in vitro phospholipidosis-induction in the drug discovery phase, Toxicology in Vitro 23, (2009), 217-226). Data are reported as the concentration showing a two-fold increase in fluorescence. data:

[0319] [Table 20]

[0320] In vivo LPS experiments: Determination of the effects of NLRP3 inhibitors on LPS-induced pro-inflammatory cytokine IL1β. Animals were treated with an NLRP3 inhibitor before LPS administration, and the effect of the NLRP3 inhibitor on inflammasome activation was assessed by measuring IL1β. IL6 and TNFα were also measured to exclude effects on LPS-induced NF-kB signaling. Compounds were administered by oral gavage (PO) 30 min before intraperitoneal LPS injection (10 mg / kg) (Escherichia coli O111:B4; L4130, Sigma-Aldrich). Three doses of each compound were tested. Control, wild-type, and NLRP3 knockout mice were orally administered vehicle. NLRP3 knockout mice were included as a negative control, i.e., to define the endogenous levels of IL1β in these experiments. Each treatment group contained eight animals. However, in certain experiments, the NLRP3 knockout group contained fewer animals (usually n = 4). Four hours after LPS injection, animals were sacrificed by decapitation, and plasma samples were collected for bioanalysis and cytokine (IL1β, IL6, and TNFα) analysis using ELISA (IL1β, Quantikine MLB00C, R&D Systems Minneapolis, Canada) and MSD (IL6 and TNFα, V-Plex K15048D MSD, Meso Scale Diagnostics, Maryland, USA). Plasma samples were diluted 1 / 20 for IL1β and TNFα measurements and further diluted 1 / 60 for IL6 analysis. Plates were read using a SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA, USA) or an MSD reader (Meso Scale reader sector S600) for the Quantikine and MSD assays, respectively. Data were further analyzed in Excel and GraphPad Prism, including statistical analysis (one-way ANOVA). Plasma concentrations were determined according to the procedure described in the pharmacokinetics section. The free concentration was determined by multiplying the plasma concentration by the free fraction in plasma (free concentration = plasma concentration x fu,p). The free fraction in plasma is defined as follows: fu,p = PPB (% free) / 100.Measurement of PPB (% free) is described in the Plasma Protein Binding section. Working Example:

[0321] [Table 21]

[0322] [Table 22]

[0323] Human Whole Blood Assay For the human whole blood assay, 125 μl of undiluted blood was added to each well of a 96-well plate, followed by 25 μl of lipopolysaccharide (LPS E. coli, L4130, Sigma-Aldrich) at a concentration of 30 ng / mL. After priming the blood with LPS for 1 hour at 37°C, compound dilutions (dose-response, 25 μl / well) were added for 30 minutes at 37°C. The NLRP3 pathway was then activated by adding 25 μl of BzATP (A-385, Alomone Labs) to each well at a concentration of 1 mM. After 1.5 hours at 37°C, the plate was centrifuged (2000 rpm, 5 minutes), and the supernatant was collected and stored at -80°C before IL1β analysis using MSD (V-PLEX Human IL-1β Kit, K151QPD-2, Meso Scale) according to the manufacturer's instructions. Data were analyzed in GraphPad. Efficacy is IC 50 The potency is expressed as (the concentration required to inhibit 50% of the effect). When a compound is tested multiple times, the potency is reported as the geometric mean of the different replicates. Free potency was determined by multiplying the whole blood potency by the free fraction in plasma, assuming a blood / plasma ratio of 1 (free potency = whole blood potency x free fraction in plasma). The free fraction in plasma is defined as follows: fu, p = PPB (% free) / 100. Measurement of PPB (% free) is described in the Plasma Protein Binding section.

[0324] [Table 23]

[0325] Mouse whole blood assay For the mouse whole blood assay, blood from several mice was pooled (approximately 300 μl), and then 75 μl of undiluted mouse blood was added to each well of a 96-well plate. The NLRP3 pathway was primed by adding 25 μl of LPS (1 μg / mL) to each well for 3 hours at 37°C. Compounds were added at different concentrations (dose-response) and incubated at 37°C for 30 minutes, after which the pathway was activated with BzATP (5 mM, 25 μl / well) for 1 hour. At the end of the experiment, the plate was centrifuged at 2000 rpm for 5 minutes, and the supernatant was collected and stored at -80°C. IL1β analysis was performed using MSD (V-PLEX Mouse IL-1β Kit, K152QPD, Meso Scale) according to the manufacturer's instructions. Data were analyzed in GraphPad. Potency is expressed as IC50 (the concentration required to inhibit 50% of the effect). When a compound is tested multiple times, potency is reported as the geometric mean of the different replicates. Free potency was determined by multiplying the whole blood potency by the free fraction in plasma, assuming a blood / plasma ratio of 1 (free potency = whole blood potency x free fraction in plasma). The free fraction in plasma is defined as follows: fu, p = PPB (% free) / 100. Measurement of PPB (% free) is described in the Plasma Protein Binding section.

[0326] [Table 24]

[0327] Chromatographic Hydrophobicity Index (CHI) CHI LogD values, also referred to in the literature as ChromLogD, were determined for the compounds of the present invention. For a description of the assay, see, for example, Rombouts et al., J. Med. Chem. 2021, 64, 19, 14175-14191.

[0328] [Table 25]

[0329] Without wishing to be bound by theory, as already indicated above, the compounds of the invention exhibit advantageous properties such as brain penetration, phospholipidosis potential and / or polarity.

[0330] For example, the following compounds having a monocyclic core structure can be tested in the same assays described herein for brain penetration and phospholipidosis potential and compared to the final compound 9 according to the present invention.

[0331] [Table 26]

[0332] The following compounds with a bicyclic core structure can also be tested in the same assays described herein for CHI LogD and compared to the final compounds described herein.

[0333] [Table 27]

[0334] Substitution of -NH- for the -CH2- spacer in these compounds unexpectedly increased the polarity of the compounds.

Claims

1. Formula (I): 【Chemistry 1】 The compound or a pharmaceutically acceptable salt thereof, In the formula, R １ is hydrogen, methyl, or chloro; R ２ teeth, 【Chemistry 2】 and In the formula, Y is CH ２ , N.R. ２１ , or O, R ２０ is oxetan-3-yl; CD ３ C optionally substituted with halo, hydroxy, or cyano; １－４ is alkyl, R ２１ is hydrogen or C １－２ is alkyl, R ３ is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; R ４ is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; R ３ and R ４ together form a divalent group -R selected from the list ３ -R ４ - and a)-CH ２ -CH ２ -CH ２ - b)-CH ２ -CH ２ -CH ２ -CH ２ -、 c)-CH ２ -CH ２ -CH ２ -NHH-、 d)-CH ２ -O-CH ２ -CH ２ -、 e)-CH ２ -CH ２ -O-CH ２ -、 f)-O-CH ２ -CH ２ -O-、 g) -CH=CH-CH=N-, h) -N=CH-CH=CH-; A compound or a pharmaceutically acceptable salt thereof, wherein each Z is independently hydrogen or fluoro.

2. R １ is hydrogen or methyl.

3. R ２ teeth, 【Transformation 3】 and In the formula, R ２０ is C １－２ Alkyl, CD ３ , 2-hydroxyethyl, 2-fluoroethyl, 3-fluoropropyl, or cyanomethyl.

4. R ２ teeth, 【Chemistry 4】 and In the formula, R ２０ The compound of claim 1 , wherein represents methyl.

5. R ４ The compound of claim 1 , wherein is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl.

6. R ３ and R ４ together form a divalent group -R selected from the list ３ -R ４ -form, a)-CH ２ -CH ２ -CH ２ - b)-CH ２ -CH ２ -CH ２ -CH ２ - c)-CH ２ -CH ２ -CH ２ -NH- d)-CH ２ -O-CH ２ -CH ２ - e)-CH ２ -CH ２ -O-CH ２ - f)-O-CH ２ -CH ２ -O- g) -CH=CH-CH=N- h) -N=CH-CH=CH-, the compound of claim 1.

7. 2. The compound of claim 1, wherein each Y represents hydrogen.

8. R １ is hydrogen or methyl; R ２ teeth, 【Transformation 5】 and In the formula, Y is CH ２ or O, R ２０ is methyl, R ３ is hydrogen or R ４ is hydrogen, or R ３ and R ４ together form a divalent group -R selected from the list ３ -R ４ - and a)-CH ２ -CH ２ -CH ２ - e)-CH ２ -CH ２ -O-CH ２ -、 2. The compound of claim 1, wherein each Z is hydrogen.

9. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. A process for preparing a pharmaceutical composition according to claim 9, comprising intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound according to any one of claims 1 to 8.

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