Pyrrolo[1,2-d][1,2,4]triazines and pyrazolo[1,5-d][1,2,4]triazines as NLRP3 inhibitors

Abstract

Compounds for use as inhibitors of the NLRP3 inflammasome pathway are provided, such compounds being as defined by the compounds of formula (I) and containing a group R 1 , R 2 , R 3 , R 4 , and R 5 is defined herein, and the compounds may be useful, for example, as pharmaceutical agents for use in the treatment of diseases or disorders associated with NLRP3 inflammasome activity. [Formula 1] TIFF2026504422000218.tif35128

Description

[Technical Field]

[0001] Described herein are pyrrolo[1,2-d][1,2,4]triazines and pyrazolo[1,5-d][1,2,4]triazines useful as inhibitors of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway. Also described herein are processes for the preparation of 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 / 278438.

[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 is a compound of formula (I):

[0010] [ka] or a pharmaceutically acceptable salt thereof, A is N or CH; R 1 teeth,

[0011] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0012] [ka] R 7 and R 8 are each independently H, CH3, or F.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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. DETAILED DESCRIPTION OF THE INVENTION

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

[0018] [ka] or a pharmaceutically acceptable salt thereof, A is N or CH; R 1 teeth,

[0019] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0020] [ka] R 7 and R 8 are each independently H, CH3, or F.

[0021] In one embodiment, provided herein is a compound of formula (I):

[0022] [ka] or a pharmaceutically acceptable salt thereof, A is N or CH; R 1 teeth,

[0023] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0024] [ka] R 7 and R 8 are each independently H, CH3, or F; However, the compound

[0025] [ka] isn't it.

[0026] In some particular embodiments, provided herein are compounds of Formula (I) having the formula (IA):

[0027] [ka] During the ceremony, R 1 teeth,

[0028] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0029] [ka] R7 and R 8 are each independently H, CH3, or F.

[0030] In some particular embodiments, provided herein are compounds of Formula (I) having the formula (IA):

[0031] [ka] During the ceremony, R 1 teeth,

[0032] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0033] [ka] R 7 and R 8 are each independently H, CH3, or F; However, the compound

[0034] [ka] isn't it.

[0035] In some embodiments, provided herein are compounds of Formula (I) having the formula (IB):

[0036] [ka] During the ceremony, R 1 teeth,

[0037] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0038] [ka] R 7 and R 8 are each independently H, CH3, or F; however, a)R 6 is selected from CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3, in which case R 7 and R 8 are each independently H, CH3, or F; b)R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, and CD3, in which case R 7 is CH3 or F, R 8 is H, CH3, or F.

[0039] In some embodiments, provided herein are compounds of Formula (I) having the formula (IB):

[0040] [ka] During the ceremony, R 1 teeth,

[0041] [ka] and R 2 is H or CH3, R 3 is CF3, OCF3, OCH3, CH3, Cl, or OCHF2, R 4 is H or CH3, R 5 is H or CH3, R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3,

[0042] [ka] R 7 and R 8 are each independently H, CH3, or F; however, a)R 6 is selected from CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3, in which case R 7 and R 8 are each independently H, CH3, or F; b)R 6 are CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, and CD3, in which case R 7 is CH3 or F, R 8 is H, CH3, or F, provided that the compound

[0043] [ka] isn't it.

[0044] In one embodiment, R 1 teeth,

[0045] [ka] is.

[0046] In one embodiment, R 1 teeth

[0047] [ka] and R 6 is CH3 or CH2CH3, and R 7 is H, CH3, or F, and R 8 is H or R 7 and R 8 Both are F.

[0048] In a further embodiment, R 1 teeth

[0049] [ka] and R 6 is CH3 or CH2CH3, and R 7 is CH3 or F, and R 8 is H or R 7 and R 8 Both are F.

[0050] In a further embodiment, R 1 teeth

[0051] [ka] and R 6is CH2CH3 and R 7 is H, CH3, or F, and R 8 is H or R 7 and R 8 Both are F.

[0052] In one embodiment, R 1 teeth,

[0053] [ka] is.

[0054] In another embodiment, R 1 teeth,

[0055] [ka] is.

[0056] In one embodiment, R 1 teeth,

[0057] [ka] and During the ceremony, a)R 6 is CH3 or CH2CH3, and R 7 is H, CH3, or F, and R 8 is H or R 7 and R 8 are both F, or b)R 6 is CH3 or CH2CH3, and R 7 is CH3 or F, and R 8 is H or R 7 and R 8 are both F, or c)R 6 is CH2CH3 and R 7 is H, CH3, or F, and R 8 is H or R 7 and R8 are both F, or d)R 6 is CH3 or CH2CH3, and R 7 and R 8 are both F, or R 1 teeth,

[0058] [ka] is.

[0059] In one embodiment, R 2 , R 4 , and R 5 is hydrogen.

[0060] In one embodiment, R 2 is hydrogen and R 3 is CF3.

[0061] In one embodiment, R 3 is CF3.

[0062] In one embodiment, R 6 is CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, or CD3.

[0063] In one embodiment, R 6 is CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, or CD3.

[0064] In one embodiment, provided herein are compounds of Formula (I) as depicted in the Examples as final compounds, particularly final compounds 2-8, 10-17, 19-61.

[0065] 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.

[0066] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids.

[0067] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

[0068] 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.

[0069] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0070] 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.

[0071] 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.

[0072] The compounds may contain double bonds and thus may exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The compounds may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] The term "halo", as used herein, preferably includes fluoro, chloro, bromo and iodo.

[0082] 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.

[0083] 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.

[0084] Compound names were generated according to the nomenclature rules agreed upon by the Chemical Abstracts Service (CAS) using software from Advanced Chemical Development, Inc. (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 software from Advanced Chemical Development, Inc. (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.

[0085] 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.

[0086] 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.

[0087] 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 enantiomers obtained by asymmetric synthesis, followed by vibrational circular dichroism (VCD) analysis of the specific enantiomers.

[0088] Preparation of compounds Preparation of Final Compounds of the Invention Final compounds described by formula (II) can be prepared according to, but not limited to, general scheme 1. The intermediate compound of formula (IIa) can be synthesized from the intermediate of formula (Ia) or (Ib) by nucleophilic substitution with hydrazine in a suitable solvent, such as methanol, under thermal conditions by heating the mixture at a suitable temperature, such as the boiling point of the solvent. The intermediate compound of formula (IIIa) can be synthesized from (IIa) by condensation reaction in the presence of triethyl orthoformate under thermal conditions (e.g., 165°C) in a suitable solvent such as dimethylacetamide. The intermediate compound of formula (IVa) can be obtained by reacting (IIIa) with a suitable brominating agent, such as benzyltrimethylammonium tribromide, in a suitable solvent, such as DMF. The intermediate compound of formula (Va) is R 4 When R = H, it can be synthesized from (IVa) by reaction with a primary amine derivative via a Buchwald-Hartwig cross-coupling reaction using a palladium-based precatalyst such as G3t-BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base such as t-BuONa, and a solvent such as 2-methyl-2-butanol. 4 When =CH3, (Va) can be obtained by nucleophilic substitution of (IVa) under thermal conditions using DIPEA as base, CsF and tetrabutylammonium chloride as additives in a solvent such as acetonitrile. Intermediate compounds of formula (VIa) can be obtained from (Va) by reaction with TfO in a solvent such as dichloromethane in the presence of a base such as pyridine. Intermediate compounds of formula (VIb) can be obtained from (Va) by reaction with a chlorinating agent such as POCl in a suitable solvent such as acetonitrile. Intermediates of formula (VIIa), (VIIb), and (VIIc) can be obtained from (VIa) or (VIb) by Suzuki cross-coupling with a suitable boronic acid or ester derivative using a palladium precatalyst such as G4 cataCxium Chemistry - A European Journal, 2008, 14, 4267-4279) and a base such as CsCO or KPO in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). Alternatively, the reaction can be carried out using Pd(dppf)Cl as a source of palladium catalyst, dichloromethane, a base such as KCO in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). The final compounds of formula (II) can be obtained from intermediates (VIIa), (VIIb), and (VIIc) by removing the protecting group PG in an acidic medium such as HCl in 1,4-dioxane (intermediates (VIIb) and (VIIc)), or, in the case of intermediate (VIIa), by catalytic hydrogenolysis using palladium on carbon and H2 as catalyst in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar). Alternatively, the benzyl group in (VIIa) can be removed using BBr3. The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0089] [ka]

[0090] Alternatively, the final compounds described by formula (II) can be prepared according to, but not limited to, general scheme 2. The final compound of formula (II) can be obtained from (VIa) or (VIb) by Suzuki cross-coupling reaction. Typical non-limiting conditions may include the use of Pd(dppf)Cl, dichloromethane or Pd(PPh) as a catalyst, NaCO or KPO as a base, in 1,4-dioxane or 1,4-dioxane / water as a solvent at an appropriate temperature (e.g., 100°C). The final compound may be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0091] [ka]

[0092] Final compounds described by formula (III) can be prepared according to, but not limited to, general scheme 3. The intermediate compound of formula (IXa) can be synthesized from (VIIIa) by reaction with a primary amine derivative via a Buchwald-Hartwig cross-coupling reaction using a palladium-based precatalyst such as G3t-BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base such as t-BuONa, and a solvent such as 2-methyl-2-butanol. The intermediate compound of formula (Xa) can be obtained from (IXa) by reaction with Tf2O in the presence of a base such as pyridine in a solvent such as dichloromethane. Intermediate compounds of formula (XIa) can be obtained from (Xa) by Suzuki cross-coupling reaction with an appropriate boronic acid or ester derivative using Pd(dppf)Cl2 as a source of palladium catalyst and a base such as dichloromethane, K2CO3 or K3PO4 in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). Intermediate compounds of formula (XIIa) can be obtained from (XIa) by removal of the Boc protecting group under acidic conditions such as HCl or TFA in 1,4-dioxane. The final compounds of general formula (III) can be obtained from (XIIa) by reductive alkylation (e.g., with formaldehyde in the presence of related agents such as sodium triacetoxyborohydride or sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0093] [ka]

[0094] Alternatively, the final compounds described by formula (III) can be prepared according to, but not limited to, general scheme 4. Intermediate compounds of formula (XIIIa) can be obtained from (IXa) by removal of the Boc protecting group under acidic conditions such as HCl or TFA in 1,4-dioxane. Intermediate compounds of formula (XIVa) can be obtained from (XIIIa) by reductive alkylation (for example with formaldehyde in the presence of sodium triacetoxyborohydride or another reducing agent such as NaBH3CN). - Intermediate compounds of formula (XVa) can be obtained from (XIVa) by reaction with a chlorinating agent such as POCl3 in a suitable solvent such as acetonitrile. The final compound of formula (III) can be obtained from (XVa) by Suzuki cross-coupling reaction. Typical non-limiting conditions can include the use of Pd(dppf)Cl, dichloromethane or Pd(PPh), as a catalyst, NaCO or KPO as a base, in 1,4-dioxane or 1,4-dioxane / water as a solvent at an appropriate temperature (e.g., 100°C). The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0095] [ka]

[0096] Alternatively, intermediate compounds of general formula (XIa) can be synthesized using the non-limiting synthetic route shown in general Scheme 5. Intermediate compounds of formula (XVIa) can be obtained from (Xa) by Suzuki cross-coupling reaction with an appropriate boronic acid or ester derivative using Pd(dppf)Cl2 as a source of palladium catalyst and a base such as dichloromethane, K2CO3, Cs2CO3, or K3PO4 in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). The intermediate compound of formula (XIa) can be obtained from intermediate (XVIa) by removing the benzyl protecting group by catalytic hydrogenolysis using catalyst palladium on carbon and H2 in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar). Alternatively, the benzyl group in (XVIa) can be removed using BBr3.

[0097] [ka]

[0098] Alternatively, intermediate compounds of general formula (XIIa) can be synthesized using the non-limiting synthetic route shown in general Scheme 6. Intermediate compounds of formula (XVIIa) can be obtained from (Xa) by Suzuki cross-coupling reaction with an appropriate boronic acid or ester derivative using Pd(dppf)Cl2 as a source of palladium catalyst and a base such as dichloromethane, K2CO3, Cs2CO3, or K3PO4 in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). Intermediate compounds of general formula (XIIa) can be obtained from (XVIIa) by global deprotection of both the Boc and methoxymethyl protecting groups by treatment with an acidic medium such as HCl in 1,4-dioxane.

[0099] [ka]

[0100] Final compounds described by formula (IV) can be prepared according to, but not limited to, general scheme 7. The intermediate compound of formula (XVIIIa) can be synthesized from (VIIIa) by reaction with a primary amine derivative via a Buchwald-Hartwig cross-coupling reaction using a palladium-based precatalyst such as G3t-BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base such as t-BuONa, and a solvent such as 2-methyl-2-butanol. - Intermediate compounds of formula (XIXa) can be obtained from (XVIIIa) by reaction with a chlorinating agent such as POCl3 in a suitable solvent such as acetonitrile. Intermediate compounds of formula (XXa) and (XXb) can be obtained from (XIXa) by Suzuki cross-coupling reaction with a suitable boronic acid or ester derivative using a palladium precatalyst such as G4 cataCxium Chemistry - A European Journal, 2008, 14, 4267-4279) and a base such as CsCO or KPO in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). Alternatively, the reaction can be carried out using Pd(dppf)Cl as a source of palladium catalyst, dichloromethane, a base such as KCO in a suitable solvent such as 1,4-dioxane / water at a suitable temperature (e.g., 100°C). The intermediate compounds of formula (XXIa) and (XXIb) can be obtained from intermediates (XXa) and (XXb) by removal of the benzyl protecting group by catalytic hydrogenolysis using catalyst palladium on carbon and H2 in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar). Intermediate compounds of formula (XXIIa) and (XXIIb) can be obtained from (XXIa) and (XXIb) by reductive alkylation (e.g., using formaldehyde in the presence of related agents such as sodium triacetoxyborohydride or sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds of formula (IV) can be obtained from (XXIIa) or (XXIIb) by removing the methoxymethyl (XXIIa) or ethoxymethyl (XXIIb) protecting group in an acidic medium such as HCl in 1,4-dioxane. The final compounds can be further purified by supercritical fluid chromatography or chiral reversed-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0101] [ka]

[0102] The final compounds described by formula (V) can be prepared according to, but not limited to, general scheme 8. The intermediate compounds of formula (XXIIIa) and (XXIIIb) can be obtained from (XXa) and (XXb) by a one-pot procedure involving the use of palladium on carbon, H2 as a catalyst, in the presence of a polyoxymethylene homopolymer in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar) at a suitable temperature (e.g., room temperature). The final compounds of formula (V) can be obtained from (XXIIIa) or (XXIIIb) by removing the methoxymethyl (XXIIIa) or ethoxymethyl (XXIIIb) protecting group in an acidic medium such as HCl in 1,4-dioxane. The final compounds can be further purified by supercritical fluid chromatography or chiral reversed-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0103] [ka]

[0104] Final compounds described by formula (VI) can be prepared according to, but not limited to, general scheme 9. The intermediate compound of formula (XXIVa) can be obtained by treatment with a suitable base, such as NaH, in a suitable solvent, such as DMF, at a suitable temperature, such as room temperature, followed by reaction with a methylating agent, such as methyl iodide, according to (XVIIa). Intermediate compounds of formula (XXVa) can be obtained from formula (XXIVa) by global deprotection of both the Boc and methoxymethyl protecting groups by treatment with an acidic medium such as HCl in 1,4-dioxane. The final compounds of general formula (VI) can be obtained from (XXVa) by reductive alkylation (e.g., with formaldehyde in the presence of related agents such as sodium triacetoxyborohydride or sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0105] [ka]

[0106] Final compounds described by formula (VII) can be prepared according to, but not limited to, general scheme 10. The intermediate compound of formula (Va) can be synthesized from (VIIIa) by reaction with a primary amine derivative via a Buchwald-Hartwig cross-coupling reaction using a palladium-based precatalyst such as G3t-BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base such as t-BuONa, and a solvent such as 2-methyl-2-butanol. Intermediate compounds of formula (XXVIIa) can be obtained from (XXVIa) by removal of the Boc protecting group under acidic conditions such as HCl or TFA in 1,4-dioxane. Intermediate compounds of formula (XXVIIa) can be obtained from (XXVIIa) by reductive alkylation (for example with formaldehyde in the presence of sodium triacetoxyborohydride or another reducing agent such as NaBH3CN). - Intermediate compounds of formula (XXIXa) can be obtained from (XXVIIIa) by reaction with a chlorinating agent such as POCl3 in a suitable solvent such as acetonitrile. The final compounds of formula (VII) can be obtained from (XXIXa) by Suzuki cross-coupling reaction. Typical non-limiting conditions can include the use of Pd(dppf)Cl, dichloromethane or Pd(PPh), as a catalyst, NaCO or KPO as a base, in 1,4-dioxane or 1,4-dioxane / water as a solvent at an appropriate temperature (e.g., 100°C). The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0107] [ka]

[0108] Final compounds described by formula (VIII) can be prepared according to, but not limited to, general scheme 11. Intermediate compounds of formula (XXXIa) can be synthesized from intermediates of formula (XXXa) or (XXXb) by nucleophilic substitution with hydrazine in a solvent such as ethanol under thermal conditions by heating the mixture at a suitable temperature such as 90°C. The intermediate compound of formula (XXXIIa) can be synthesized from the intermediate of formula (XXXIa) by condensation with an electrophilic carbonyl source such as methyl chloroformate. Non-limiting conditions can be as follows: An electrophilic carbonyl source such as methyl chloroformate is added to a mixture of the intermediate compound (XXXIa) in a suitable solvent such as dichloromethane at a suitable temperature such as room temperature in the presence of a suitable base such as DIPEA. The mixture is stirred for a suitable reaction time, for example, 16 hours, and then concentrated. The condensation can be completed, for example, by dissolving the residue in a suitable solvent such as ethanol and stirring the mixture in the presence of a suitable base such as KOH at a suitable temperature such as 100 ° C. The intermediate compound of formula (XXXIIIa) can be obtained from (XXXIIa) by reaction with a chlorinating agent such as POCl3 in a suitable solvent such as toluene by heating at a suitable temperature (e.g. 135°C). Intermediate compounds of formula (XXXIVa) can be synthesized from (XXXIIIa) by Suzuki cross-coupling reaction with an appropriate boronic acid or ester derivative using a palladium-based pre-catalyst such as XPhos Pd G3, an appropriate base such as K3PO4 in a suitable solvent such as 1,4-dioxane / water under suitable non-limiting conditions, for example under microwave irradiation at 150°C for a suitable time (for example 1 hour). Intermediate compounds of formula (XXXVa) can be obtained from (XXXIVa) in a non-limiting manner by treatment with a suitable thiolating agent, such as diphosphorus pentasulfide, in a suitable solvent, such as pyridine, under thermal conditions (e.g., 150°C). Intermediate compounds of formula (XXXVIa) can be obtained from (XXXVa) in a non-limiting manner by treatment with an alkylating agent such as bromoethane in the presence of a suitable base such as K2CO3 in a suitable solvent such as THF / water. Intermediate compounds of formula (XXXVIIa) can be obtained in a non-limiting manner from (XXXVIa) by nucleophilic substitution of (XXXVIa) with a suitable amine under thermal conditions (for example, at a temperature of 130° C.) using an excess of the suitable amine for a suitable reaction time (for example, 48 hours). The final compounds of formula (VIII) can be obtained from intermediate (XXXVIIa) by removal of the benzyl protecting group by catalytic hydrogenolysis using catalyst palladium on carbon and H2 in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar).

[0109] [ka]

[0110] Final compounds described by formula (IX) can be prepared according to, but not limited to, general scheme 12. Intermediate compounds of formula (XXXVIIIa) can be obtained in a non-limiting manner from (XXXVIa) by nucleophilic substitution of (XXXVIa) with a suitable amine under thermal conditions (for example, at a temperature of 130° C.) using an excess of the suitable amine for a suitable reaction time (for example, 48 hours). Intermediate compounds of formula (XXXIXa) can be obtained from intermediate (XXXVIIa) by removal of the benzyl protecting group by catalytic hydrogenolysis using catalyst palladium on carbon and H2 in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar). Intermediate compounds of formula (XLa) can be obtained from (XXXIXa) by deprotection of the Boc protecting group by treatment with an acidic medium such as HCl in 1,4-dioxane. The final compounds of general formula (IX) can be obtained from (XLa) by reductive alkylation (e.g., with formaldehyde in the presence of related agents such as sodium triacetoxyborohydride or sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0111] [ka]

[0112] Alternatively, the final compounds described by formula (IX) can be prepared according to, but not limited to, general scheme 13. Intermediate compounds of formula (XLIa) can be obtained from (XXXVIIa) by deprotection of the Boc protecting group by treatment with an acidic medium such as HCl in 1,4-dioxane. Intermediate compounds of formula (XLIIa) can be obtained from (XLIa) by reductive alkylation (e.g., using formaldehyde in the presence of related agents such as sodium triacetoxyborohydride or sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds of formula (IX) can be obtained from intermediate (XLIIa) by catalytic hydrogenolysis using catalyst palladium on carbon and H2 in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar). Alternatively, the benzyl group in (VIIa) can be removed using BBr3. The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0113] [ka]

[0114] Final compounds described by formula (X) can be prepared according to, but not limited to, general scheme 14. Intermediate compounds of formula (XLIIIa) can be obtained from (XLIa) by nucleophilic substitution of an appropriate alkyl halide in the presence of a suitable base, such as DIPEA, in a suitable solvent, such as acetonitrile, at a suitable temperature, such as 85° C. The final compounds of formula (X) can be obtained from intermediate (XLIIIa) by global deprotection using BBr3 in a suitable solvent such as dichloromethane with a suitable additive such as aniline at a suitable temperature such as 0° C. The final compounds can be further purified by supercritical fluid chromatography or chiral reversed-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0115] [ka]

[0116] Final compounds described by formula (XI) can be prepared according to, but not limited to, general scheme 15. Intermediate compounds of formula (XLIVa) can be obtained in a non-limiting manner from (XXXVIa) by nucleophilic substitution with a suitable amine (XXXVIa) under thermal conditions (for example at a temperature of 120° C.) using a suitable amine used in excess in the presence of a suitable base such as DIPEA, in a suitable solvent such as DMSO, for a suitable reaction time (for example 96 hours). The intermediate compound of formula (XLVa) can be obtained from intermediate (XLIVa) by removal of the benzyl protecting group by catalytic hydrogenolysis using catalyst palladium on carbon and H2 in a suitable solvent (e.g., methanol, ethanol, or ethyl acetate) at a suitable pressure (e.g., atmospheric pressure or 10 bar). Intermediate compounds of formula (XLVIa) can be obtained from (XLVa) by deprotection of the Boc protecting group by treatment with an acidic medium such as HCl in 1,4-dioxane. The final compounds of general formula (XI) can be obtained from (XLVIa) by reductive alkylation (e.g., with formaldehyde in the presence of related agents such as sodium triacetoxyborohydride or sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds can be further purified by supercritical fluid chromatography or chiral reverse-phase HPLC to separate their diastereomerically or enantiomerically pure components.

[0117] [ka]

[0118] Pharmacology The compounds are potently brain penetrant 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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).

[0125] 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.

[0126] 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 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] In one embodiment, there is provided a compound according to any one of the embodiments described herein for use as a medicament.

[0133] In one embodiment, there is provided use of a compound 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.

[0134] 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.

[0135] 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 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).

[0136] 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 according to any one of the embodiments described herein).

[0137] 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.

[0138] 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.

[0139] Experimental Department General preparation and analytical methods Compounds according to the invention may generally be prepared by a series of steps, each of which is well known to those skilled in the art or may be described herein.

[0140] It is clear that in the reactions described above and below, the reaction products may be isolated from the reaction medium and, if necessary, further purified according to methods generally known in the art, such as extraction, crystallization, and chromatography. It is further clear that reaction products present in one or more enantiomeric forms may be isolated from their mixtures by well-known techniques, in particular preparative chromatography, such as preparative HPLC, chiral chromatography, etc. Individual diastereomers or individual enantiomers may also be obtained by supercritical fluid chromatography (SFC).

[0141] The starting materials and intermediates are compounds that are either commercially available or can be prepared according to conventional reaction procedures generally known in the art.

[0142] analysis part LC-MS (liquid chromatography / mass spectrometry)

[0143] General Procedure 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 if necessary (see methods table below).

[0144] 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.

[0145] 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] - , etc.). 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.

[0146] 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.

[0147] [Table 1]

[0148] NMR For some compounds, 1 H NMR spectra were recorded on a Bruker Avance III spectrometer operating at 300 or 400 MHz, a Bruker Avance III-HD spectrometer operating at 400 MHz, a Bruker Avance NEO spectrometer operating at 400 MHz, a Bruker Avance NEO spectrometer operating at 500 MHz, or a Bruker Avance 600 spectrometer operating at 600 MHz using chloroform-d (deuterated chloroform, CDCl), DMSO-d (deuterated DMSO, dimethyl-d sulfoxide), methanol-d (deuterated methanol), benzene-d (deuterated benzene, CD), or acetone-d (deuterated acetone, (CD)CO) as solvents. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as the internal standard.

[0149] Melting point Values ​​are either peak values ​​or melting ranges and are obtained with experimental uncertainties commonly associated with this analytical method.

[0150] Method A: For some compounds, melting points were determined using a DSC823e (Mettler Toledo) apparatus. Melting points were measured using a temperature gradient of 10°C / min. The standard maximum temperature was 300°C.

[0151] Method B: For some compounds, melting points were determined in open capillary tubes on a Mettler Toledo MP50. Melting points were measured with a temperature gradient of 10°C / min. The maximum temperature was 300°C. Melting point data were read from a digital display and checked from a video recording system.

[0152] Note the stereochemistry. Whenever the designation "RS" appears herein, it indicates that the compound is a racemic mixture at the indicated center, unless otherwise specified. The stereochemical configuration of some compound centers can be designated "(R)" or "(S)" when the mixture is isolated or derived from enantiomerically pure starting materials, and for some compounds, the stereochemical configuration at the designated center may be designated "(R)" or "(S)" when the compound itself has been isolated as a single stereoisomer and is enantiomerically / diastereomerically pure, but the absolute stereochemistry has not been determined. * (R)" or " * In compounds reported herein, the enantiomeric excess was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC), followed by SFC comparison of the separated enantiomers. In (RS) intermediates / compounds where the bond is shown as either a thick wedge or a parallel-lined wedge and a stereocenter is designated, the designation indicates that the sample is a mixture of stereoisomers, with one stereoisomer having the indicated substituent or group projected above or below the plane of the depicted drawing and one stereoisomer having the substituent or group in the opposite projection below or above the plane of the drawing, e.g.,

[0153] [ka] teeth,

[0154] [ka] represents a mixture of

[0155] The absolute configuration of the chiral centers (designated as R and / or S) can be rationalized. The synthesis of all final compounds started from intermediates of known absolute configuration consistent with literature precedent or was obtained from appropriate synthetic procedures. The assignment of the absolute configuration of additional stereocenters could then be assigned by standard NMR methods. [Example]

[0156] Several methods for preparing the compounds of this invention are illustrated in the following examples. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification, or could be synthesized by one skilled in the art using published methods.

[0157] [Table 2-1]

[0158] [Table 2-2]

[0159] Preparation of intermediates For intermediates that were used in the next reaction step as crude or partially purified intermediates, in some cases no molar amount is stated for such intermediate in the next reaction step or an estimated molar amount or theoretical molar amount for such intermediate in the next reaction step is given in the reaction protocols set out below.

[0160] Synthesis of Intermediate 1 (I-1), 2-iodo-5-(trifluoromethyl)phenol

[0161] [ka]

[0162] Sodium hydride [7646-69-7] (4.9 g, 124 mmol, 2 equiv.) was added to anhydrous toluene [108-88-3] (182 mL) under a nitrogen atmosphere at 0 °C. 3-Trifluoromethylphenol [98-17-9] (10 g, 61.7 mmol, 1 equiv.) was then added portionwise. The mixture was stirred at 0 °C for 30 min. Iodine [7553-56-2] (15.7 g, 61.7 mmol, 1 equiv.) was 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 EtOAc and washed twice with brine. The organic layer was separated, dried over MgSO4, filtered and concentrated in vacuo to give 2-iodo-5-(trifluoromethyl)phenol, I-1, as a pale red oil (18.7 g, quantitative).

[0163] Synthesis of Intermediate 2 (I-2), 2-(benzyloxy)-1-iodo-4-(trifluoromethyl)benzene

[0164] [ka]

[0165] 2-Iodo-5-(trifluoromethyl)phenol I-1 (18.7 g, 64.93 mmol, 1 equiv.) and K2CO3 [584-08-7] (13.6 g, 97.39 mmol, 1.5 equiv.) were dissolved in acetone [67-64-1] (324 mL). The reaction mixture was stirred at room temperature for 15 minutes. Benzyl bromide [100-39-0] (8.7 mL, 71.42 mmol, 1.1 equiv.) was then added dropwise, and the reaction mixture was stirred at reflux (56 °C) for 4 hours. The solvent was removed in vacuo. Water was added to the resulting crude product, and the suspension was extracted with EtOAc. The organic layers were combined, dried over MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (silica 330 g, 100% heptane). The desired fractions were collected and concentrated in vacuo to afford 2-(benzyloxy)-1-iodo-4-(trifluoromethyl)benzene I-2 as a white solid (19.6 g, 79%).

[0166] Synthesis of Intermediate 3 (I-3), 2-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0167] [ka]

[0168] Isopropylmagnesium chloride (2 M in THF) [1068-55-9] (15.6 mL, 31.1 mmol, 1.2 equiv.) was added dropwise to a stirred solution of 2-(benzyloxy)-1-iodo-4-(trifluoromethyl)benzene I-2 (9.8 g, 25.92 mmol, 1 equiv.) in anhydrous THF [109-99-9] (207 mL) at 0 °C under a N atmosphere. The reaction mixture was stirred at 0 °C for 2 h. Then, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane [61676-62-8] (7.9 mL, 38.88 mmol, 1.5 equiv.) was added dropwise. The reaction mixture was allowed to warm slowly to room temperature and stirred for 18 h. Excess THF was removed, and the reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was separated, dried over MgSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (silica 330 g, heptane, 0 / 100 to 10 / 90). The desired fractions were collected and concentrated under reduced pressure to give 2-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-3 as a white solid (6.69 g, 68%).

[0169] Synthesis of Intermediate 4 (I-4), 2-Bromo-5-(difluoromethoxy)phenol

[0170] [ka]

[0171] N-Bromosuccinimide [128-08-5] (5.84 g, 32.79 mmol) was added to a solution of 3-(difluoromethoxy)phenol [88798-13-4] (5 g, 31.23 mmol) in DCM (30 mL). The reaction mixture was stirred at room temperature for 2 h. The crude reaction mixture was washed twice with water (30 mL). The organics were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give a brown oil. The crude product was purified by flash column chromatography on silica gel (heptane / EtOAc: 100 / 0 to 80 / 20). The desired fractions were collected and concentrated in vacuo to give 2-bromo-5-(difluoromethoxy)phenol I-4 (4.6 g, 52% yield) as a colorless oil.

[0172] Synthesis of Intermediate 5 (I-5), 5-(difluoromethoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol

[0173] [ka]

[0174] A mixture of 2-bromo-5-(difluoromethoxy)phenol I-4 (1 g, 4.18 mmol), pinacolborane (2.14 g, 16.74 mmol), TEA (2.32 mL, 16.74 mmol), and toluene (20 mL) in a round-bottom flask (100 mL) was purged with nitrogen for 10 min. XPhos Pd G4 (108 mg, 0.13 mmol) was added, and the reaction mixture was heated at 80 °C for 1 h. The mixture was filtered through decalite and washed with toluene (2 × 20 mL). The filtrate was concentrated to dryness and purified using silica gel column chromatography (0–100% ethyl acetate in heptane). The desired fractions were combined and concentrated in vacuo to give 5-(difluoromethoxy)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol I-5 (420 mg, 35% yield).

[0175] Synthesis of Intermediate 6 (I-6), 1-iodo-2-(methoxymethoxy)-4-(trifluoromethyl)benzene

[0176] [ka]

[0177] N,N-Diisopropylethylamine [7087-68-5] (21.5 mL, 102 mmol) was added to a cooled solution of 2-iodo-5-(trifluoromethyl)phenol I-5 (34 g, 102 mmol) in DCM (1.2 L) at 0 °C, followed by the dropwise addition of chloromethyl methyl ether [107-30-2] (9.4 mL, 121.8 mmol). The reaction mixture was gradually warmed to room temperature and reacted for 18 h. The mixture was concentrated, and the residue was purified by flash column chromatography (silica 330 g, 0 / 100 to 10 / 90 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to give 1-iodo-2(methoxymethoxy)-4-(trifluoromethyl)benzene I-6 (25.5 g, 70% yield) as a colorless oil.

[0178] Synthesis of Intermediate 7 (I-7), 2-(2-(ethoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0179] [ka]

[0180] Isopropylmagnesium chloride (2 M in THF) [1068-55-9] (14.5 mL, 28.9 mmol) was added dropwise to a stirred solution of 1-iodo-2-(methoxymethoxy)-4-(trifluoromethyl)benzene I-6 (8 g, 24.09 mmol) in anhydrous THF (190 mL) at 0 °C under a N atmosphere. The reaction mixture was stirred at 0 °C for 2 h. Then, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (CAS [61676-62-8]) (7.4 mL, 36.1 mmol) was added dropwise to the mixture. The reaction mixture was allowed to warm slowly to room temperature and stirred for 16 h. The reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic phase was separated, dried (MgSO4), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (SiO2 120 g, EtOAc in heptane, 0 / 100 to 20 / 80). The desired fractions were collected and concentrated under reduced pressure to give 2-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-7 (6.4 g, yield: 76%) as a yellowish oil.

[0181] Synthesis of Intermediate 8 (I-8), 3-methyl-5-(trifluoromethyl)phenol

[0182] [ka]

[0183] A mixture of 1-bromo-3-methyl-5-(trifluoromethyl)benzene [86845-28-5] (120 g, 502.01 mmol, 1.00 equiv.) and lithium hydroxide [1310-65-2] (36.07 g, 1506.05 mmol, 3.00 equiv.), Pd2(dba)3 [51364-51-3] (22.99 g, 25.10 mmol, 0.05 equiv.), and BippyPhos [894086-00-1] (382 g, 753.02 mmol, 1.50 equiv.) in 1,4-dioxane (1.2 L) and water (240 mL) was stirred overnight at 100 °C under a N2 atmosphere. The mixture was cooled to room temperature. The resulting mixture was diluted with ice water (3 L). The resulting mixture was extracted with EtOAc (3×3 L). The combined organic layers were washed with brine (2×2 L) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography eluting with PE to give 3-methyl-5-(trifluoromethyl)phenol I-8 (66 g, 75%) as a yellow oil.

[0184] Synthesis of Intermediate 9 (I-9), 2-iodo-3-methyl-5-(trifluoromethyl)phenol

[0185] [ka]

[0186] NaH (30 g, mmol, 749 mmol, 2.00 equiv, 60%) was added portionwise to a solution of 3-methyl-5-(trifluoromethyl)phenol I-8 (66 g, 375 mmol, 1.00 equiv) in toluene (0.99 L) at 0 °C under a nitrogen atmosphere. Iodine [7553-56-2] (71.33 g, 281.02 mmol, 0.75 equiv) was added to the mixture at 0 °C. The solution was stirred at room temperature for 3 h. The resulting mixture was diluted with ice water (2 L). The resulting mixture was extracted with EtOAc (2 × 3 L). The combined organic layers were washed with brine (2 × 3 L) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with petroleum ether to afford 2-iodo-3-methyl-5-(trifluoromethyl)phenol I-9 (71 g, 63%) as a yellow oil.

[0187] Synthesis of Intermediate 10 (I-10), 1-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene

[0188] [ka]

[0189] Chloromethyl ethyl ether [3188-13-4] (44.5 g, 470 mmol, 2.00 equiv.) was added dropwise to a mixture of 2-iodo-3-methyl-5-(trifluoromethyl)phenol I-9 (71 g, 235 mmol, 1.00 equiv.) and CsCO (153 g, 470 mmol, 2.00 equiv.) in DMF (0.71 L) at 0 °C under a nitrogen atmosphere. The solution was stirred overnight at room temperature. The reaction mixture was diluted with ice / water (2 L). The resulting mixture was extracted with EtOAc (2 × 3 L). The combined organic layers were washed with brine (2 × 3 L) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with petroleum ether to afford 1-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene I-10 (42 g, 50%) as a yellow oil.

[0190] Synthesis of Intermediate 11 (I-11), 2-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0191] [ka]

[0192] 4,4,5,5-Tetramethyl-1,3,2-dioxaborolane [25015-63-8] (59.70 g, 466.51 mmol, 4.00 equiv.) was added to a mixture of 1-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene I-10 (42 g, 117 mmol, 1.00 equiv.), [1,1'-biphenyl]-2-yldicyclohexylphosphane [247940-06-3] (4.0 g, 0.10 equiv.), TEA (70.8 g, 700 mmol, 6.00 equiv.), and Pd(AcO) [3375-31-3] (1.31 g, 5.83 mmol, 0.05 equiv.) in 1,4-dioxane (0.42 L) under a nitrogen atmosphere. The reaction mixture was stirred at 100°C for 2 hours. The mixture was cooled to room temperature. The reaction mixture was diluted with ice / water (2 L). The resulting mixture was extracted with EtOAc (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 afforded 31.0106 g (73.82%) of 2-[2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-11 as a white solid.

[0193] Synthesis of Intermediate 12 (I-12), Methyl (2S,4S)-4-hydroxy-1-tritylpyrrolidine-2-carboxylate

[0194] [ka]

[0195] Triethylamine [121-44-8] (64 mL, 459 mmol, 6 equiv.) and trityl chloride [76-83-5] (21.3 g, 76.41 mmol, 1 equiv.) were added to a solution of methyl (2S,4S)-4-hydroxypyrrolidine-2-carboxylate hydrochloride [227935-34-4] (13.85 g, 76.26 mmol, 1 equiv.) in chloroform (191 mL) under a nitrogen atmosphere at 0 °C. The reaction mixture was stirred at room temperature for 16 h. A solution of saturated NH4Cl and aqueous NH3 (2:1) was added to the crude mixture. After phase separation, the aqueous phase was extracted with DCM. The organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2 330 g, 0 / 100 to 20 / 80 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to afford (2S,4S)-4-hydroxy-1-tritylpyrrolidine-2-carboxylate I-12 as a white solid (25.3 g, 81%).

[0196] Synthesis of Intermediate 13 (I-13), (2S,4S)-4-((methylsulfonyl)oxy)-1-tritylpyrrolidine-2-carboxylate

[0197] [ka]

[0198] Triethylamine [121-44-8] (40 mL, 287 mmol, 4.4 equiv) and methanesulfonyl chloride [124-63-0] (17 mL, 150.9 mmol, 2.3 equiv) were added to a stirred solution of (2S,4S)-4-hydroxy-1-tritylpyrrolidine-2-carboxylate I-12 (25.3 g, 65.30 mmol, 1 equiv) in dichloromethane (255 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 16 h. The reaction mixture was diluted with DCM and washed with saturated aqueous NaCO, water, and brine. The organic solution was dried over MgSO, filtered, and concentrated under reduced pressure to give methyl (2S,4S)-4-((methylsulfonyl)oxy)-1-tritylpyrrolidine-2-carboxylate I-13 as an orange solid (30.4 g, estimated quantitative yield), which was used in the next step without further purification.

[0199] Synthesis of Intermediate 14 (I-14), Methyl (2S,4R)-4-Azido-1-tritylpyrrolidine-2-carboxylate

[0200] [ka]

[0201] Sodium azide [26628-22-8] (5.3 g, 81.62 mmol, 2.5 equiv.) was added to a stirred solution of methyl (2S,4S)-4-((methylsulfonyl)oxy)-1-tritylpyrrolidine-2-carboxylate I-13 (15.2 g, 32.65 mmol, 1 equiv.) in DMF (180 mL) under a nitrogen atmosphere. The mixture was stirred at 90 °C for 16 h. The reaction mixture was poured into a saturated solution of NaHCO and extracted with EtOAc. The organic layer was washed several times with brine, dried over MgSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (120 g of SiO, 0 / 100 to 10 / 90 EtOAc in heptane). The desired fractions were collected to give methyl (2S,4R)-4-azido-1-tritylpyrrolidine-2-carboxylate I-14 as a white solid (10.7 g, 79%).

[0202] Synthesis of Intermediate 15 (I-15), ((2S,4R)-4-amino-1-tritylpyrrolidin-2-yl)methanol

[0203] [ka]

[0204] Methyl (2S,4R)-4-azido-1-tritylpyrrolidine-2-carboxylate I-14 (10.3 g, 25.0 mmol, 1 equiv.) in anhydrous THF (67 mL) was added dropwise to a stirred solution of lithium aluminum hydride [16853-85-3] (3.7 g, 96.9 mmol, 3.9 equiv.) in anhydrous THF (90 mL) under a nitrogen atmosphere at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. The reaction mixture was then stirred at room temperature for 3 h. The reaction mixture was cooled to 0 °C, and water (3.7 mL) was added dropwise. After stirring for 5 min, 3.75 M aqueous NaOH (3.8 mL) was added. After stirring for 5 min, water (9.2 mL) was added, and the mixture was stirred at room temperature for 1 h. The resulting precipitate was dried over MgSO4, collected on a Celite pad, and washed with DCM. The organic solvent was removed in vacuo to give ((2S,4R)-4-amino-1-tritylpyrrolidin-2-yl)methanol I-15 as a white solid (8.64 g, 96%).

[0205] Synthesis of Intermediate 16 (I-16), tert-butyl ((3R,5S)-5-(hydroxymethyl)-1-tritylpyrrolidin-3-yl)carbamate

[0206] [ka]

[0207] To a stirred solution of ((2S,4R)-4-amino-1-tritylpyrrolidin-2-yl)methanol I-15 (220 g, 614 mmol, 1 equiv.) in DCM (2200 mL) under a N atmosphere, di-tert-butyl dicarbonate [24424-99-5] (161 g, 736 mmol, 1.2 equiv.) was added portionwise. The mixture was stirred at room temperature for 18 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EtOAc (100 / 0 to 60 / 40) to give tert-butyl ((3R,5S)-5-(hydroxymethyl)-1-tritylpyrrolidin-3-yl)carbamate I-16 as a white foam (180 g, 64%).

[0208] Synthesis of Intermediate 17 (I-17), tert-butyl ((3R,5R)-5-fluoro-1-tritylpiperidin-3-yl)carbamate

[0209] [ka]

[0210] To a stirred solution of tert-butyl ((3R,5S)-5-(hydroxymethyl)-1-tritylpyrrolidin-3-yl)carbamate I-16 (180 g, 392 mmol, 1 equiv.) in THF (3.6 L) was added diethyl(trifluoro-lambda 4-sulfanyl)amine [38078-09-0] (88.6 g, 549.5 mmol, 1.4 equiv.) dropwise at 0 °C under a N atmosphere. The reaction was stirred at 0 °C for 1 h. The mixture was then stirred at room temperature for 1 h. The mixture was cooled to 0 °C and adjusted to pH = 12 with saturated aqueous NaCO solution. The aqueous phase was extracted with EtOAc (3 × 500 mL). The organics were dried over NaSO. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / EtOAc (100 / 0 to 75 / 25) to afford tert-butyl ((3R,5R)-5-fluoro-1-tritylpiperidin-3-yl)carbamate I-17 as a white solid (135 g, 74%).

[0211] Synthesis of Intermediate 18 (I-18), tert-butyl ((3R,5R)-5-fluoropiperidin-3-yl)carbamate

[0212] [ka]

[0213] To a stirred solution of tert-butyl ((3R,5R)-5-fluoro-1-tritylpiperidin-3-yl)carbamate I-17 (135 g, 293 mmol, 1 equiv.) in EtOH (2700 mL) was added dropwise hydrogen chloride (190 mL, 2 M) at room temperature. The reaction mixture was stirred at room temperature for 1.5 h. Then, NaHCO3 (32.2 g) was added to the solution, and the reaction mixture was stirred at room temperature for 1.5 h. The solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with EtOAc / MeOH (100 / 0 to 85 / 15) to give tert-butyl ((3R,5R)-5-fluoropiperidin-3-yl)carbamate I-18 as a white solid (30.1 g, 47%).

[0214] Synthesis of Intermediate 19 (I-19), tert-butyl ((3R,5R)-5-fluoropiperidin-3-yl)carbamate

[0215] [ka]

[0216] Formaldehyde solution [50-00-0] (1.4 mL, 37% in water, 1.09 g / mL, 18.78 mmol, 2 equiv.) followed by formic acid [64-18-6] (700 μL, 1.22 g / mL, 18.55 mmol, 2 equiv.) was added to a solution of tert-butyl ((3R,5R)-5-fluoropiperidin-3-yl)carbamate I-18 (2 g, 9.16 mmol, 1 equiv.) in dry 2-methyl-THF (45.0 mL). The resulting colorless solution was stirred at room temperature for 1.5 hours and then heated at 80 °C for 2.5 hours. After cooling at room temperature, the solution was concentrated in vacuo to give tert-butyl (3R,5R)-5-fluoro-1-methylpiperidin-3-yl)carbamate I-19 as an off-white residue (2.85 g), which was used in the next step without further purification.

[0217] Synthesis of Intermediate 20 (I-20), tert-butyl ((3R,5R)-1-ethyl-5-fluoropiperidin-3-yl)carbamate

[0218] [ka]

[0219] Iodoethane [75-03-6] (0.5 mL, 6.22 mmol, 1.4 equiv.) was added to a stirred solution of tert-butyl ((3R,5R)-5-fluoropiperidin-3-yl)carbamate I-18 (1 g, 4.58 mmol, 1 equiv.) and DIPEA (1.5 mL, 8.7 mmol, 1.9 equiv.) in CH3CN (25 mL). The resulting reaction mixture was stirred overnight at room temperature (monitored by TLC stained with Dragendorf). The reaction mixture was concentrated in vacuo. The residue was taken up in a water / EtOAc mixture, the organic layer was separated, and the aqueous layer was extracted twice more with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered off and concentrated in vacuo to afford tert-butyl ((3R,5R)-1-ethyl-5-fluoropiperidin-3-yl)carbamate I-20 as a white solid (950 mg, 84%).

[0220] Synthesis of Intermediate 21 (I-21), tert-butyl (R)-methyl(1-methylpiperidin-3-yl)carbamate

[0221] [ka]

[0222] (R)-tert-Butyl methyl(piperidin-3-yl)carbamate [309962-67-2] (2.5 g, 11.7 mmol) was dissolved in MeOH (200.5 mL). Then, Pd / C (10%) (1.24 g, 1.17 mmol) and polyoxymethylene homopolymer (0.5 g) were added. The solution was stirred at room temperature under 1 atmosphere of H2 for 3 hours. The solution was filtered through dicalite, washed with EtOH, and concentrated in vacuo. The resulting residue was dissolved in diluted ammonia and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated in vacuo to give tert-butyl (R)-methyl(1-methylpiperidin-3-yl)carbamate I-21 as an oil (2.5 g, 94% yield).

[0223] Synthesis of Intermediate 22 (I-22), tert-butyl (R)-(1-ethylpiperidin-3-yl)(methyl)carbamate

[0224] [ka]

[0225] A solution of iodoethane [75-03-6] (2.37 g, 1.94 g / mL, 15.17 mmol) in DMF (2.99 mL) was added to a mixture of (R)-tert-butyl methyl(piperidin-3-yl)carbamate [309962-67-2] (2.5 g, 11.67 mmol) and DIPEA (4.02 mL, 23.33 mmol) in DMF (93.6 mL). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into water plus 0.5 g of NaHCO and extracted three times with EtOAc. The combined organic phases were washed with brine, dried over MgSO, filtered, and evaporated to give tert-butyl (R)-(1-ethylpiperidin-3-yl)(methyl)carbamate I-22 (2.5 g, 88% yield) as a yellow oil.

[0226] Synthesis of Intermediate 23 (I-23), (3R,5R)-1-ethyl-5-fluoropiperidin-3-amine dihydrochloride

[0227] [ka]

[0228] tert-Butyl ((3R,5R)-1-ethyl-5-fluoropiperidin-3-yl)carbamate I-20 (950 mg, 3.86 mmol, 1 equiv.) was dissolved in 5N-6N HCl [7647-01-0] in IPA (11.8 mL, 59 mmol, excess). The reaction mixture was stirred at room temperature for 4 hours (monitored by TLC stained with Dragendorff). The solid was filtered and washed with iPrOH and DIPE to give (3R,5R)-1-ethyl-5-fluoropiperidin-3-amine dihydrochloride S-23 as a white solid (780 mg, 92%).

[0229] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0230] [Table 3]

[0231] Synthesis of Intermediate 27 (I-27), Synthesis of 1H-Pyrrole-2-carbohydrazide

[0232] [ka]

[0233] Hydrazine hydrate [10217-52-4] (175 mL, 1796 mmol) was added to a solution of ethyl pyrrole-2-carbonate [2199-43-1] (25 g, 179.7 mmol) in EtOH (383 mL) at room temperature in a sealed iron reactor under nitrogen. The reaction mixture was stirred at 90° C. for 24 hours. The reaction mixture was cooled to room temperature. The mixture was filtered, and the solid was washed with cold EtOH to give 1H-pyrrole-2-carbohydrazide I-27 (14.5 g, 64% yield) as a white solid.

[0234] Synthesis of Intermediate 28 (I-28), Pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0235] [ka]

[0236] Methyl chloroformate [79-22-1] (13.38 mL, 173 mmol) was added to a stirred suspension of 1H-pyrrole-2-carbohydrazide I-27 (14.5 g, 115.5 mmol) and DIPEA [7087-68-5] (60.5 mL, 346.4 mmol) in dichloromethane (600 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo. The residue was dissolved in EtOH (800 mL) and KOH [1310-58-3] (22.7 g, 404.2 mmol) was added. The reaction was stirred at reflux for 2 h. The formed precipitate was filtered off and dissolved in water. The resulting solution was acidified to pH 4 with 1 N HCl and stirred at room temperature for 30 min. The resulting precipitate was collected and dried in vacuo to afford pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-28 (16.3 g, 92% yield) as a white solid.

[0237] Synthesis of Intermediate 29 (I-29), 1-chloropyrrolo[1,2-d][1,2,4]triazin-4(3H)-one

[0238] [ka]

[0239] Phosphorus oxychloride [10025-87-3] (5.6 mL, 59.55 mmol) was added dropwise over 15 min to a stirred suspension of pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-28 (1.8 g, 11.91 mmol), DIPEA [7087-68-5] (4.2 mL, 23.82 mmol), and distilled water [7732-18-5] (279 μL, 15.48 mmol) in toluene (41 mL). The suspension was stirred at 135 °C for 8 h in a sealed glass reactor. The reaction mixture was cooled to room temperature and added dropwise to EtOH (90 mL) with stirring over 15 min, followed by stirring at room temperature for 15 min. The solvent was evaporated in vacuo. The crude product was absorbed onto Celite and purified by flash column chromatography (silica 120 g, 0 / 100 to 30 / 70 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DCM, filtered, and dried in vacuo to afford 1-chloropyrrolo[1,2-d][1,2,4]triazin-4(3H)-one I-29 (1.4 g, 69% yield) as an off-white solid.

[0240] Synthesis of Intermediate 30 (I-30), 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4(3H)-one

[0241] [ka]

[0242] Potassium phosphate tripotassium phosphate [7778-53-2] (6.4 g, 29.5 mmol) and Pd(PPh) [14221-01-3] (510 mg, 0.6 mmol) were added sequentially to a stirred solution of 1-chloropyrrolo[1,2-d][1,2,4]triazin-4(3H)-one I-29 (2 g, 11.8 mmol) and 2-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-3 (5.4 g, 14.2 mmol) in 1,4-dioxane (91 mL) and water (9 mL) (previously purged with nitrogen for 5 min) at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at 105 °C overnight. The mixture was concentrated, and the residue was purified by flash column chromatography (80 g silica, dry-loaded onto Celite, 0 / 100 to 30 / 70 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo. The product was triturated with heptane to give 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4(3H)-one I-30 (3.9 g, 65% yield) as a white foamy solid.

[0243] Synthesis of Intermediate 31 (I-31), 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazine-4(3H)-thione

[0244] [ka]

[0245] Diphosphorus pentasulfide (8.7 g, 38.73 mmol, 2.5 equiv.) was added to a stirred solution of 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4(3H)-one I-30 (5.97 g, 15.49 mmol) in pyridine (77 mL). The mixture was stirred at 150° C. for 8 h. The reaction was recharged with diphosphorus pentasulfide (4.35 g, 19.37 mmol, 1.25 equiv.). The mixture was stirred at 150° C. for 8 h. It was then cooled to room temperature, diluted with water, and extracted with EtOAc (3 x 600 mL). The extracts were combined, dried over anhydrous MgSO, filtered, and concentrated in vacuo to give a brown solid. The brown solid was subjected to Celite 545 and subsequently purified by chromatography (silica 120 g, dry-loaded onto Celite, 0-89% EtOAc / heptane). The desired fractions were collected and concentrated in vacuo to give 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazine-4(3H)-thione I-31 (5.7 g, 72%) as a yellowish solid.

[0246] Synthesis of Intermediate 32 (I-32), 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[1,2-d][1,2,4]triazine

[0247] [ka]

[0248] Potassium carbonate [584-08-7] (3.2 g, 22.87 mmol, 6 equiv.) diluted with water (19.3 mL) was added dropwise to a stirred solution of 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazine-4(3H)-thione I-31 (1.53 g, 3.81 mmol) in THF (38.5 mL) at room temperature. Bromoethane [74-96-4] (0.6 mL, 7.62 mmol, 2 equiv.) was then added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water and extracted with EtOAc (×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 80 g, 0 / 100 to 9 / 91 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to afford 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[1,2-d][1,2,4]triazine I-32 (577 mg, yield: 35%) as a white solid.

[0249] Synthesis of Intermediate 33 (I-33), tert-butyl (R)-3-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate

[0250] [ka]

[0251] 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[1,2-d][1,2,4]triazine I-32 (526 mg, 1.23 mmol) and (R)-1-boc-3-aminopiperidine [188111-79-7] (3.16 g, 15.31 mmol) were added to a sealed glass reactor. The mixture was then stirred at 130 °C for 48 h. The mixture was cooled to room temperature, diluted with saturated aqueous NaHCO3, and extracted with EtOAc (x3). The combined organic layers were dried (MgSO4), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g, 0 / 100 to 60 / 40 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to afford tert-butyl (R)-3-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-33 (568 mg, yield: 81%) as a beige foamy solid.

[0252] Synthesis of Intermediate 34 (I-34), (1R,2R)-2-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)cyclohexan-1-ol

[0253] [ka]

[0254] 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[1,2-d][1,2,4]triazine I-32 (60 mg, 0.14 mmol) and (1R,2R)-2-aminocyclohexanol [931-16-8] (500 mg, 4.25 mmol) were added to a sealed glass tube. The mixture was then stirred at 130 °C for 48 h. The mixture was cooled to room temperature and extracted with aqueous NaHCO and EtOAc (3 times). The combined organic layers were dried (MgSO), filtered, and the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (40 g silica, 0 / 100 to 75 / 25 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to afford (1R,2R)-2-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)cyclohexan-1-ol I-34 (40 mg, yield: 45%) as a dark yellow sticky solid.

[0255] Synthesis of Intermediate 35 (I-35), tert-butyl 6-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)-1,4-oxazepane-4-carboxylate

[0256] [ka]

[0257] 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[1,2-d][1,2,4]triazine I-32 (306 mg, 0.71 mmol), tert-butyl 6-amino-1,4-oxazepane-4-carboxylate [1170390-54-1] (649 mg, 2.85 mmol), DIPEA (248 μL, 1.43 mmol), and DMSO (1 mL) were added to a sealed glass tube. The mixture was stirred at 120 °C for 4 days. The solvent was concentrated in vacuo. The crude was purified by flash column chromatography (silica 12 g, 0 / 100 to 50 / 50 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to afford tert-butyl 6-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)-1,4-oxazepane-4-carboxylate I-35 (239 mg, yield: 57%) as a yellow oil.

[0258] Additional analogs were synthesized in an analogous manner starting from I-32 and using the appropriate reagents.

[0259] [Table 4-1]

[0260] [Table 4-2]

[0261] Synthesis of Intermediate 41 (I-41), tert-butyl (R)-3-((1-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate

[0262] [ka]

[0263] 10% Palladium on carbon [7440-05-3] (199 mg, 0.19 mmol) was added to a stirred solution of tert-butyl (R)-3-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-33 (568 mg, 1.00 mmol) in methanol (28 mL) at 0 °C under a nitrogen atmosphere. The nitrogen atmosphere was then replaced with hydrogen (1 atm, balloon), and the reaction mixture was stirred at room temperature for 4 h. The mixture was filtered through a pad of Celite and washed with DCM / MeOH (9:1). The solvent was evaporated in vacuo to give tert-butyl (R)-3-((1-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylic acid I-41 (478 mg, yield: 99%) as a dark yellow foamy solid.

[0264] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0265] [Table 5]

[0266] Synthesis of Intermediate 44a (I-44a), (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol trifluoroacetate

[0267] [ka]

[0268] Trifluoroacetic acid [76-05-1] (5.6 mL, 74.32 mmol) was added dropwise to a stirred solution of tert-butyl (R)-3-((1-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-41 (478 mg, 1.00 mmol) in anhydrous DCM (5.6 mL) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated in vacuo to give (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol trifluoroacetate I-44a (495 mg, 99% yield) as a brown foamy solid. The product was used in the next step without further purification.

[0269] Synthesis of Intermediate 44b (I-44b), (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol dihydrochloride

[0270] [ka]

[0271] Hydrochloric acid (4 M) in dioxane [7647-01-0] (1 mL, 4.23 mmol) was added to a solution of tert-butyl (R)-3-((1-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-41 (105 mg, 0.220 mmol) in 1,4-dioxane (0.175 mL) at room temperature. The mixture was stirred at room temperature for 1 hour. The solvent was evaporated in vacuo to give (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol dihydrochloride I-44b (95 mg, 100% yield) as an orange solid.

[0272] Synthesis of Intermediate 44c (I-44c), (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol hydrochloride

[0273] [ka]

[0274] HCl (4N in dioxane) [7647-01-0] (1.9 mL, 7.62 mmol) was added to tert-butyl (R)-3-((1-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-41 (130 mg, 0.27 mmol), and the mixture was stirred at room temperature for 30 minutes. The solvent was concentrated in vacuo to give (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol hydrochloride I-44c (124 mg, 99% yield) as a brown solid. The crude product was used directly in the next step.

[0275] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0276] [Table 6]

[0277] Synthesis of Intermediate 47a (I-47a), (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine trifluoroacetate

[0278] [ka]

[0279] Trifluoroacetic acid [76-05-1] (1.43 mL, 19 mmol) was added dropwise to a stirred solution of tert-butyl (R)-3-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-33 (150 mg, 0.26 mmol) in anhydrous DCM (1.5 mL) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated in vacuo to give (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-47a (153 mg, 99% yield) as a yellowish sticky solid. The product was used in the next step without further purification.

[0280] Synthesis of Intermediate 47a (I-47b), (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine hydrochloride

[0281] [ka]

[0282] HCl (4N in 1,4-dioxane) [7647-01-0] (2.0 mL, 7.9 mmol) was added to tert-butyl (R)-3-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)piperidine-1-carboxylate I-33 (150.0 mg, 0.3 mmol), and the mixture was stirred at room temperature for 30 minutes. The solvent was concentrated in vacuo to give (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine hydrochloride I-47b (142.0 mg, 99% yield) as an orange solid.

[0283] Synthesis of Intermediate 47c (I-47c), (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine

[0284] [ka]

[0285] (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine hydrochloride I-47b was dissolved in DCM / MeOH (4:1), the solution was basified with saturated NaHCO solution, and extracted three times with DCM / MeOH (4:1). The combined organics were dried over MgSO and concentrated in vacuo to give (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-47c as a brown solid.

[0286] Synthesis of Intermediate 48 (I-48), (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine

[0287] [ka]

[0288] 2-(2-Bromoethoxy)tetrahydro-2h-pyran [17739-45-6] (50 μL, 0.31 mmol) and DIPEA [7087-68-5] (0.28 mL, 1.57 mmol) were added to a stirred solution of (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine trifluoroacetate I-47a (152 mg, 0.26 mmol) in acetonitrile (3 mL). The mixture was stirred at 85° C. for 16 hours. The reaction was recharged with 2-(2-bromoethoxy)tetrahydro-2h-pyran [17739-45-6] (1.2 equiv., 50 μL, 0.31 mmol) and DIPEA [7087-68-5] (6 equiv., 0.28 mL, 1.57 mmol). The reaction mixture was cooled to room temperature, diluted with saturated aqueous NaHCO3, and extracted with EtOAc (x3). The combined organic layers were dried (MgSO4), filtered, and the solvent evaporated in vacuo. The crude was purified by flash column chromatography (silica 20 g, DCM / MeOH (9:1), 0 / 100 to 35 / 65) in DCM. The desired fractions were collected and concentrated in vacuo to afford 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-((3R)-1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-48 (150 mg, yield: 92%) as a beige foamy solid.

[0289] Synthesis of Intermediate 49 (I-49), (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(1-(2-fluoroethyl)piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine

[0290] [ka]

[0291] N,N-Diisopropylethylamine [7087-68-5] (112.9 μL, 0.6 mmol) followed by 1-fluoro-2-iodoethane [762-51-6] (23.5 μL, 0.3 mmol) were added sequentially to a stirred mixture of (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine hydrochloride I-47b (142.0 mg, 0.3 mmol) in N,N-dimethylformamide [68-12-2] (9 mL), and the resulting solution was stirred at room temperature for 16 h. N,N-Diisopropylethylamine [7087-68-5] (112.9 μL, 0.6 mmol) and 1-fluoro-2-iodoethane [762-51-6] (23.5 μL, 0.3 mmol) were added to the mixture, which was then stirred at room temperature for 72 hours. The mixture was concentrated, and the residue was purified by flash column chromatography (silica 20 g, AcOEt 100%). The desired fractions were collected and concentrated in vacuo to give (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(1-(2-fluoroethyl)piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-49 (160 mg, yield: 95%) as an orange sticky solid.

[0292] Synthesis of Intermediate 50 (I-50), (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(1-cyclopropylpiperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine

[0293] [ka]

[0294] Molecular sieves 4A (94 mg) were added to a 30 mL sealed glass reactor containing a stirred solution of (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-47c (220 mg, 0.47 mmol) and dry THF / MeOH (9:1) (3.3 mL) under N at room temperature. Then, (1-ethoxycyclopropoxy)trimethylsilane [27374-25-0] (284 μL, 1.41 mmol), acetic acid (323 μL, 5.65 mmol), and sodium cyanoborohydride [25895-60-7] (93 mg, 1.41 mmol) were added to this mixture under N at room temperature to give a brown solution. The resulting mixture was stirred at 65 °C for 16 h, then cooled to room temperature and extracted with DCM (3 × 60 mL) and NaCO (aq) (10 mL). The extracts were combined, dried over anhydrous MgSO, filtered, and concentrated to dryness in vacuo to give a yellow oil. The yellow oil was subjected to silica gel chromatography (12 g, irregular 40–60 μm, 0–3% MeOH / DCM) to give (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(1-cyclopropylpiperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-50 (203 mg, 84%) as a brown foamy solid.

[0295] Synthesis of Intermediate 51 (I-51), 1H-Pyrazole-5-carbohydrazide

[0296] [ka]

[0297] A mixture of methyl 1H-pyrazole-3-carboxylate [15366-34-4] (20 g, 0.16 mol) and hydrazine hydrate (10 mL, 1.03 g / mL, 0.22 mol) in 100 mL of MeOH was heated to reflux for 6 h. The reaction mixture was cooled to 0 °C, and the solid was filtered, washed with water and EtOH, and then dried under vacuum at 50 °C overnight to give 1H-pyrazole-5-carbohydrazide I-51 (19.2 g, 96% yield) as a bright white solid.

[0298] Synthesis of Intermediate 52 (I-52), Pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0299] [ka]

[0300] A mixture of 1H-pyrazole-5-carbohydrazide I-51 (18.2 g, 144.31 mmol) and triethyl orthoformate (26 mL, 158 mmol) in DMA (145 mL) in a sealed pressure tube was heated at 165° C. for 16 h. The reaction mixture was cooled to 0° C., and the solid was filtered and washed with EtOH to give pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-52 (14.2 g, 72% yield) as a white solid.

[0301] Synthesis of Intermediate 53 (I-53), 7-Bromopyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0302] [ka]

[0303] A suspension of pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-52 (10 g, 73.5 mmol) and K2CO3 (13.2 g, 95.5 mmol) in DMF (200 mL) was stirred at room temperature for 10 min. Benzyltrimethylammonium tribromide (37.2 g, 95.51 mmol) was then added, and the reaction mixture was heated at 40 °C for 2 h. The reaction mixture was cooled to 0 °C and quenched by the dropwise addition of aqueous Na2SO5 (28 g in 100 mL of water). The resulting mixture was stirred at 0 °C for an additional 30 min, then warmed to room temperature and further diluted with 700 mL of water. The suspension was allowed to stand overnight (without stirring) and filtered. The precipitate was washed with water and isopropyl ether and dried in vacuo at 50 °C overnight to give 7-bromopyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-53 (14.3 g, 91% yield) as a white solid.

[0304] Synthesis of Intermediate 54 (I-54), 7-Bromopyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0305] [ka]

[0306] (R)-1-Boc-3-aminopiperidine [188111-79-7] (1.12 g, 5.58 mmol), tBuXPhos-Pd G3 [1447963-75-8] (369.5 mg, 0.47 mmol), and tBuONa [865-48-5] (9.3 mL, 2 M in THF, 18.6 mmol) were added sequentially to a solution of 7-bromopyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-53 (1 g, 4.65 mmol) in 2-methyl-2-butanol (40 mL) at room temperature in a sealed tube under a nitrogen atmosphere. The reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was concentrated in vacuo, dissolved in saturated aqueous NaHCO3, and extracted with EtOAc (x3). The combined organic layers were washed with brine, dried over MgSO, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography (silica 80 g, eluent: EtOAc in heptane, 0–100%). The desired fractions were collected and concentrated in vacuo to afford (R)-3-((4-oxo-4,5-dihydropyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-54 (980 mg, 63% yield) as an orange foam.

[0307] Additional analogs were synthesized in a similar manner from I-53 using appropriate reagents.

[0308] [Table 7-1]

[0309] [Table 7-2]

[0310] [Table 7-3]

[0311] Synthesis of Intermediate 67 (I-67), 7-Bromopyrazolo[1,5-d][1,2,4]triazin-4(5H)-one (R)-7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one Hydrochloride

[0312] [ka]

[0313] To a solution of tert-butyl (R)-3-((4-oxo-4,5-dihydropyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-54 (2 g, 5.74 mmol) in DCM (3 mL) was added HCl (6 M in iPrOH) (10 mL, 6 M, 60 mmol). The reaction mixture was stirred at room temperature for 4 h. The solid was filtered off, washed with DCM (5 mL), and dried in a vacuum oven at 45 °C for 18 h to give (R-7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one hydrochloride salt I-67 (1.45 g, 93% yield) as a white solid.

[0314] Synthesis of Intermediate 68 (I-68), 7-((1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0315] [ka]

[0316] HCl (4 M in dioxane) (2.36 mL, 4 M, 9.42 mmol) was added to a solution of tert-butyl 6-((4-oxo-4,5-dihydropyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)-1,4-oxazepane-4-carboxylate I-57 (330 mg, 0.942 mmol) in methanol (4 mL). The reaction mixture was stirred at room temperature until complete conversion. The reaction mixture was concentrated under reduced pressure to give the crude product (HCl salt). The free amine was obtained by solid-phase extraction using Si-propylsulfonic acid SCX-2 resin (SiliCycle). The crude product was dissolved in MeOH and transferred to a column packed with Si-propylsulfonic acid SCX-2 resin (SiliCycle). The free amine was released by eluting the column first with MeOH and then with ammoniated methanol (7 N). The tube containing the product was concentrated under reduced pressure to give 7-((1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-68 (250 mg, 106% yield).

[0317] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0318] [Table 8]

[0319] Synthesis of Intermediate 70 (I-70), (R)-7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0320] [ka]

[0321] Formaldehyde solution [50-00-0] (1.28 mL, 12.93 mmol, 5 equiv.) was added to a solution of (R)-7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one hydrochloride I-67 (700 mg, 2.59 mmol) in MeOH (16 mL). The reaction mixture was stirred at room temperature for 10 minutes, after which sodium cyanoborohydride [25895-60-7] (490 mg, 7.76 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with HCl (1 M, 1 mL) and stirred at room temperature for 1 hour. The solution was concentrated to retain approximately 3 mL of solvent, and saturated aqueous NaHCO3 (3 mL) was added. The mixture was extracted with EtOAc (5 × 30 mL) and 2-MeTHF (5 × 30 mL). The combined organics were dried (Na2SO4) and concentrated in vacuo to afford (R)-7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-70 (0.79 g, 66% yield) as a colorless sticky oil.

[0322] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0323] [Table 9]

[0324] Synthesis of Intermediate 73 (I-73), (R)-4-chloro-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0325] [ka]

[0326] POCl3[10025-87-3] (1200 μL, 1.65 g / mL, 12.91 mmol, 4 equiv.) was added to a suspension of (R)-7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-70 (790 mg, 3.18 mmol) in acetonitrile (20 mL). The resulting reaction mixture was stirred at 80 °C for 10 h, and additional POCl3 (1200 μL, 12.91 mmol) was added. The reaction mixture was then heated at 80 °C for 42 h. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with dichloromethane (5x). The combined organic phase was washed with brine and concentrated under reduced pressure to give (R)-4-chloro-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-73 (570 mg, 60.5% yield, 90% purity). The crude product was used directly in the next step.

[0327] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0328] [Table 10-1]

[0329] [Table 10-2]

[0330] Synthesis of Intermediate 81 (I-81), t-butyl (R)-3-((4-(((trifluoromethyl)sulfonyl)oxy)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate

[0331] [ka]

[0332] To a mixture of (R)-3-((4-oxo-4,5-dihydropyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-54 (500 mg, 1.50 mmol) in DCM (8 mL) was added dry pyridine [110-86-1] (611 μL, 7.57 mmol) and TfO [358-23-6] (763.55 μL, 4.54 mmol) in DCM (2 mL) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with ice / water and NaHCO. The combined organic layers were dried over anhydrous MgSO, filtered, and concentrated in vacuo. The crude product was further purified by column chromatography (heptane / EtOAc: 100 / 0 to 50 / 50) to afford t-butyl (R)-3-((4-(((trifluoromethyl)sulfonyl)oxy)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-81 (540 mg, 77% yield) as a yellow solid.

[0333] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0334] [Table 11-1]

[0335] [Table 11-2]

[0336] Suzuki cross-coupling: Synthesis of Intermediate 87 (I-87), t-butyl(1R,2R)-2-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)cyclohexan-1-ol

[0337] [ka]

[0338] Method 1: A solution of potassium carbonate [584-08-7] (294 mg, 2.129 mmol) in water (0.9 mL) was added to a stirred solution of 7-(((1R,2R)-2-hydroxycyclohexyl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl trifluoromethanesulfonate I-86 (203 mg, 0.53 mmol) and intermediate I-3 (301 mg, 0.8 mmol) in 1,4-dioxane (3.6 mL). Nitrogen was bubbled through the mixture for 10 minutes, and Pd(dppf)Cl2DCM [95464-05-4] (87 mg, 0.1 mmol) was added at room temperature. The reaction mixture was heated at 90 °C and stirred under a nitrogen atmosphere for 16 hours. The reaction was cooled to room temperature, and water was added. The mixture was extracted with EtOAc. The organic layer was dried over MgSO, filtered, and concentrated in vacuo. The crude was purified by flash column chromatography (40 g silica, 0 / 100 to 50 / 50 EtOAc in heptane). The desired fractions were collected and concentrated in vacuo to afford (1R,2R)-2-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)cyclohexan-1-ol I-87 (112 mg, 43% yield) as a yellow sticky solid.

[0339] Synthesis of Intermediate 88 (I-88), tert-butyl (R)-3-((4-(2-hydroxy-4-(trifluoromethoxy)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate

[0340] [ka]

[0341] Method 2: A tube was charged with tert-butyl (R)-3-((4-(((trifluoromethyl)sulfonyl)oxy)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-81 (170 mg, 0.36 mmol), 2-hydroxy-4-(trifluoromethoxy)phenylboronic acid [1309768-22-6] (111 mg, 0.5 mmol), and potassium carbonate [584-08-7] (151.11 mg, 1.09 mmol). 1,4-Dioxane (2.43 mL, 1.03 g / mL, 28.43 mmol) and water (0.61 mL) were added, and the mixture was degassed with nitrogen. Pd(dppf)Cl₂·DCM [95464-05-4] (36 mg, 0.044 mmol) was added, and the mixture was stirred and heated at 90 °C for 3 h. The mixture was poured into saturated NaHCO₃ solution and extracted twice with EtOAc. The combined organic layers were washed with brine and dried over Na₂SO₄. After filtration, the solvent was evaporated. The residue was purified on a silica gel column eluted with 0–50% EtOAc in heptane. The pure fractions were combined and concentrated to give tert-butyl (R)-3-((4-(2-hydroxy-4-(trifluoromethoxy)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-88 (67 mg, 36% yield) as a pale yellow solid.

[0342] Synthesis of Intermediate 89 (I-89), (R)-N-(1-benzylpiperidin-3-yl)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0343] [ka]

[0344] Method 3: A reaction flask was charged with (R)—N-(1-benzylpiperidin-3-yl)-4-chloropyrazolo[1,5-d][1,2,4]triazin-7-amine I-75 (2.0 g, 5.83 mmol), 2-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-7 (2.13 g, 6.42 mmol), tribasic potassium phosphate [7778-53-2] (3.72 g, 17.50 mmol), Pd(dppf)Cl DCM [95464-05-4] (357 mg, 0.44 mmol), 1,4-dioxane (40 mL), and water (4 mL). Nitrogen was bubbled through the reaction mixture, which was then stirred at 90° C. for 3 h. The reaction mixture was cooled to room temperature, diluted with brine, and extracted with DCM (2x). The combined organic layers were concentrated in vacuo. The residue was purified by silica gel column chromatography (gradient elution: 30-60% EtOAc in heptane) to give (R)-N-(1-benzylpiperidin-3-yl)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-89 (2.13 g, 71% yield).

[0345] Synthesis of Intermediate 90 (I-90), (R)-N-(1-benzylpiperidin-3-yl)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0346] [ka]

[0347] Method 4: A reaction flask was charged with (R)—N-(1-benzylpiperidin-3-yl)-4-chloropyrazolo[1,5-d][1,2,4]triazin-7-amine I-75 (500 mg, 1.46 mmol), 2-[2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-11 (578 mg, 1.6 mmol), CsCO[534-17-8] (1426 mg, 4.38 mmol), cataCXium Pd G[2230788-67-5] (108.26 mg, 0.15 mmol), 1,4-dioxane (10 mL), and water (1 mL). The reaction mixture was bubbled with nitrogen and stirred at 90° C. for 4 h. The mixture was poured into water, and the resulting mixture was extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO, filtered, and concentrated. The residue was purified on a silica gel column eluted with 0% to 100% EtOAc in heptane. The pure fractions were concentrated to give (R)-N-(1-benzylpiperidin-3-yl)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-90 (625 mg, 79% yield) as a sticky oil.

[0348] Additional analogs were synthesized in a similar manner, using appropriate reagents following the cross-coupling method used to synthesize the compounds.

[0349] [Table 12-1]

[0350] [Table 12-2]

[0351] [Table 12-3]

[0352] Synthesis of Intermediate 100 (I-100), tert-butyl (R)-3-((4-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate

[0353] [ka]

[0354] Pd / C (10%) [7440-05-3] (230.0 mg, 0.22 mmol) was added to a solution of tert-butyl (R)-3-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-92 (613 mg, 1.1 mmol) in ethanol (7 mL) at room temperature under nitrogen. The nitrogen atmosphere was replaced with a hydrogen balloon, and the reaction mixture was stirred at room temperature for 2.5 h. The mixture was filtered through Celite and washed with EtOH. The solvent was removed in vacuo to give tert-butyl (R)-3-((4-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-100 (380 mg, 73% yield).

[0355] Synthesis of Intermediate 101 (I-101), (R)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0356] [ka]

[0357] (R)-N-(1-benzylpiperidin-3-yl)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-90 (312 mg, 0.58 mmol) was dissolved in MeOH (3.6 mL) and Pd / C (10%) (22.29 mg, 0.021 mmol) was added. The solution was stirred at room temperature under 1 atmosphere of H for 3 hours. The mixture was filtered through dicalite, washed with EtOH, and concentrated in vacuo to give (R)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine Int-101 (260 mg, 100% yield) as a sticky solid.

[0358] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0359] [Table 13]

[0360] Synthesis of Intermediate 103 (I-103), tert-butyl (R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)(methyl)amino)piperidine-1-carboxylate

[0361] [ka]

[0362] NaH (60% dispersion in mineral oil) [7646-69-7] (94.89 mg, 2.37 mmol, 5.3 equiv) was added portionwise to a mixture of tert-butyl (R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-93 (230 mg, 0.44 mmol, 1 equiv) in dry DMF (10 mL) at 0 °C and flushed with nitrogen. The reaction was stirred at room temperature for 30 min. After cooling again to 0 °C, iodomethane [74-88-4] (100 μL, 2.28 g / mL, 1.6 mmol, 3.6 equiv) was added, and the reaction mixture was then stirred at room temperature for 2.5 h. The reaction mixture was quenched with MeOH and concentrated in vacuo. The crude was purified by silica gel column chromatography (gradient elution: 0–2% MeOH in DCM) to give tert-butyl (R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)(methyl)amino)piperidine-1-carboxylate I-103 (64 mg, 24% yield).

[0363] Synthesis of Intermediate 104 (I-104), (R)-2-(7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol bistrifluoroacetate

[0364] [ka]

[0365] Trifluoroacetic acid [76-05-1] (11.5 mL, 153.5 mmol) was added dropwise to a stirred solution of tert-butyl (R)-3-((4-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-100 (380 mg, 0.80 mmol) in anhydrous DCM (11.5 mL) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 1.5 h. The solvent was evaporated in vacuo to give (R)-2-(7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol bistrifluoroacetate I-104 (475 mg, 98% yield) as a yellow oil. The product was used in the next step without further purification.

[0366] Synthesis of Intermediate 105 (I-105), (R)-5-methyl-2-(7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)phenol

[0367] [ka]

[0368] HCl (4 M in 1,4-dioxane) [7647-01-0] (1.12 mL, 4 M, 4.47 mmol) was added to a solution of tert-butyl (R)-3-((4-(2-hydroxy-4-methylphenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate I-91 (100 mg, 0.24 mmol) in 1,4-dioxane (1.13 mL), and the mixture was stirred at room temperature for 2 h. The mixture was poured into saturated NaHCO3 solution and extracted three times with EtOAc. The combined organics were washed with brine, dried over MgSO4, filtered, and evaporated to give (R)-5-methyl-2-(7-(piperidin-3-ylamino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)phenol I-105 (61 mg, 80% yield) as a yellow sticky solid.

[0369] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0370] [Table 14]

[0371] Synthesis of Intermediate 108 (I-108), 2-(7-(((3R,5R)-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry)

[0372] [ka]

[0373] Hydrochloric acid (4 M) in dioxane [7647-01-0] (0.84 mL, 3.4 mmol) was added to a solution of tert-butyl (3R,5R)-3-fluoro-5-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidine-1-carboxylate (relative stereochemistry) I-95 (90 mg, 0.17 mmol) in dioxane (0.12 mL) at room temperature. The mixture was stirred at room temperature for 1 hour. TLC showed the formation of the desired product. The solvent was evaporated in vacuo to give 2-(7-(((3R,5R)-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry) I-108 (79 mg, yield: 99%) as a white solid.

[0374] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0375] [Table 15]

[0376] Synthesis of Intermediate 110 (I-110), 2-(7-(((3R,5R)-5-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry)

[0377] [ka]

[0378] HCl (4 M in dioxane) [7647-01-0] (3 mL, 12 mmol, excess) was added to a solution of tert-butyl (3R,5R)-3-((4-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)-5-methylpiperidine-1-carboxylate (relative stereochemistry) I-99 (251 mg, 0.51 mmol, 1 equiv.) in 1,4-dioxane [123-91-1] (10 mL) at room temperature. The solution was stirred at room temperature overnight. LCMS showed complete conversion and a clean reaction profile. The reaction mixture was concentrated in vacuo to give 2-(7-(((3R,5R)-5-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry) as a brown residue (0.51 mmol, quantitative yield).

[0379] Synthesis of Intermediate 111 (I-111), (R)-N-(1-ethylpiperidin-3-yl)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0380] [ka]

[0381] DIPEA (122 μL, 0.71 mmol) and iodoethane [75-03-6] (43 μL, 0.53 mmol) were added to a stirred solution of (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-102 (150 mg, 0.355 mmol) in anhydrous DMF (2.8 mL). The resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc, washed with brine, and concentrated under reduced pressure (182 mg, quantitative yield). The crude product was used directly in the next step.

[0382] Additional analogs were synthesized in a similar manner using appropriate reagents.

[0383] [Table 16]

[0384] Synthesis of Intermediate 116 (I-116), (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(1-(oxetan-3-yl)piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0385] [ka]

[0386] 3-Oxetanone [6704-31-0] (102 mg, 1.42 mmol) was added to a solution of (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-102 (150 mg, 0.36 mmol) in MeOH (1.9 mL). The reaction mixture was stirred at room temperature for 30 minutes, after which sodium cyanoborohydride [25895-60-7] (45 mg, 0.71 mmol) was added. The reaction mixture was stirred at room temperature overnight. The crude reaction mixture was transferred to a column packed with Si-propylsulfonic acid SCX-2 resin (SiliCycle). The desired product was released by eluting the column first with MeOH and then with ammoniated methanol (7N). The tube containing the desired product was concentrated under reduced pressure to give crude (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(1-(oxetan-3-yl)piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-116 (132 mg, 75% purity, 58% yield). The crude product was used directly in the next step.

[0387] Synthesis of Intermediate 117 (I-117), (R)-N-(1-cyclopropylpiperidin-3-yl)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0388] [ka]

[0389] A solution of (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-102 (50 mg, 0.118 mmol), (1-ethoxycyclopropoxy)trimethylsilane [27374-25-0] (103 mg, 0.59 mmol), and acetic acid (14 μL, 0.24 mmol) in MeOH (1 mL) was treated with sodium cyanoborohydride [25895-60-7] (19 mg, 0.30 mmol) and stirred at 60° C. for 3 h. The reaction was repeated using the same amounts. The two reaction mixtures were combined, diluted with EtOAc, washed with water, and concentrated under reduced pressure to give (R)—N-(1-cyclopropylpiperidin-3-yl)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-117 (110 mg, quantitative yield). The crude product was used directly in the next step without further purification.

[0390] Synthesis of intermediate 118 (I-118), (1R,2R-relative stereochemistry)-2-((R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidin-1-yl)cyclobutan-1-ol

[0391] [ka]

[0392] A solution of (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazole[1,5-d][1,2,4]triazin-7-amine I-102 (210 mg, 0.50 mmol), 1,2-bis(trimethylsilyloxy)cyclobutene (229 mg, 0.99 mmol), and acetic acid (57 μL, 0.99 mmol) in MeOH (4 mL) was treated with sodium cyanoborohydride (78 mg, 1.24 mmol) and stirred overnight at room temperature. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic phases were concentrated in vacuo, and the resulting residue was purified by silica gel column chromatography (gradient elution: 0–5% MeOH in DCM) to afford (1R,2R-relative stereochemistry)-2-((R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidin-1-yl)cyclobutan-1-ol I-118 (85 mg, 12% yield).

[0393] Synthesis of Intermediate 119 (I-119), (R)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0394] [ka]

[0395] (R)—N-(1-benzylpiperidin-3-yl)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-90 (312 mg, 0.58 mmol) was dissolved in MeOH (3.6 mL) and Pd / C (10%) (22.29 mg, 0.021 mmol) and polyoxymethylene homopolymer [30525-89-4] (100 mg) were added. The solution was stirred at room temperature under 1 atmosphere of H2 for 3 hours. The solution was filtered through dicalite, washed with EtOH, and further concentrated in vacuo. The residue was taken up in diluted ammonia and extracted three times with EtOAc, and the combined organics were washed with brine, dried over MgSO, filtered, and evaporated to give (R)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-119 (250 mg, 93% yield) as a sticky solid.

[0396] Synthesis of Intermediate 120 (I-120), 3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0397] [ka]

[0398] Hydrazine hydrate [7803-57-8] (12.1 mL, 162.2 mmol) was added via syringe to a 750 mL sealed stainless steel reactor containing a solution of ethyl 4-methyl-1H-pyrazole-3-carboxylate [6076-12-6] (5 g, 32.4 mmol) in ethanol (80 mL) at room temperature under a nitrogen atmosphere to give a colorless solution. After sealing the reactor, the mixture was stirred at 90 °C for 18 h and then allowed to reach room temperature. The mixture was concentrated to dryness in vacuo and purified by flash column chromatography (80 g silica, dry-packed on silica, MeOH-DCM (10:1) in DCM from 0 / 100 to 70 / 30) to give 4-methyl-1H-pyrazole-5-carbohydrazide I-120 (4.94 g, 96% yield) as a white solid.

[0399] Synthesis of Intermediate 121 (I-121), 3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0400] [ka]

[0401] Triethyl orthoformate [122-51-0] (6.6 mL, 39.5 mmol) was added via syringe to a 100 mL stainless steel reactor containing a stirred solution of 4-methyl-1H-pyrazole-5-carbohydrazide I-120 (4.94 g, 35.25 mmol) and DMF (35 mL) at room temperature to give a colorless solution. After sealing the reactor, the mixture was stirred at 165 °C for 16 h. The mixture was cooled to give a brown solution. The mixture was co-distilled with toluene five times to give a light brown slurry. DCM (700 mL), pre-cooled to 0 °C, was added to the solid. The mixture was filtered through a glass Hirsch funnel and dried in vacuo to give 3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-121 (3.85 g, 72% yield) as a white solid.

[0402] Synthesis of Intermediate 122 (I-122), 7-Bromo-3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0403] [ka]

[0404] K2CO3 (4.6 g, 33.1 mmol) was added to a 10 mL round-bottom flask containing a solution of 3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-121 (3.8 g, 25.5 mmol) and DMF (69 mL) at room temperature. The suspension was stirred at room temperature for 10 min. Benzyltrimethylammonium tribromide [111865-47-5] (12.9 g, 33.1 mmol) was then added. The reaction mixture was stirred at 40 °C for 16 h. Saturated aqueous Na2SO3 was added, and the mixture was diluted with EtOAc (x8). The solid precipitated, which was then filtered and dried under high vacuum to give 7-bromo-3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-122 (4.1 g, 67% yield) as a white solid.

[0405] Synthesis of Intermediate 123 (I-123), tert-butyl (R)-(1-methylpiperidin-3-yl)carbamate

[0406] [ka]

[0407] Formaldehyde solution [50-00-0] (4 mL, 53.73 mmol) followed by formic acid [64-18-6] (2 mL, 53.03 mmol) was added to a solution of tert-butyl (R)-piperidin-3-ylcarbamate [309956-78-3] (5 g, 24.97 mmol) in 2-methyltetrahydrofuran (50 mL). The resulting solution was stirred at room temperature for 1 h and then at 80 °C for 1 h. After cooling to room temperature, the mixture was concentrated, and the residue was directly subjected to column chromatography (silica 80 g; DCM / MeOH (1:0 to 9:1)) to afford tert-butyl (R)-(1-methylpiperidin-3-yl)carbamate I-123 (5.63 g, 100% yield) as a clear oil that crystallized upon standing.

[0408] Synthesis of Intermediate 124 (I-124), (R)-1-methylpiperidin-3-amine dihydrochloride

[0409] [ka]

[0410] (R)-(1-Methylpiperidin-3-yl)carbamate I-123 (14.2 g, 66.26 mmol) was dissolved in 1,4-dioxane (53 mL). The reaction was cooled to 0 °C, and HCl 4N in 1,4-dioxane (332 mL, 1327.8 mmol) was added. The mixture was stirred at room temperature for 1 hour and 30 minutes. The solvent was evaporated in vacuo to give (R)-1-methylpiperidin-3-amine dihydrochloride I-124 (12.9 g, 100% yield) as a white solid.

[0411] Synthesis of Intermediate 125 (I-125), (R)-3-methyl-7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one

[0412] [ka]

[0413] 7-Bromo-3-methylpyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-122 (1.5 g, 6.5 mmol), tBuONa [865-48-5] (2.5 g, 25.9 mmol), and tBuXPhos-Pd G3 [1447963-75-8] (1 g, 1.3 mmol) were added to a solution of (R)-1-methylpiperidin-3-amine dihydrochloride (S_122) (1.1 g, 7.1 mmol) and 2-methyl-2-butanol [75-85-4] (51 mL) in a 150 mL screw-cap vial under N atmosphere. The mixture was sparged with N for 10 min and then stirred at 60 °C for 7 h. The vial was cooled to room temperature, and the mixture therein was concentrated to dryness in vacuo, diluted with water (150 mL), and extracted with DCM (150 mL). The organic layer was separated, dried (MgSO), filtered, and the solvent was concentrated in vacuo to give a yellow sticky solid. The yellow sticky solid was subjected to silica gel chromatography (80 g of yellow sticky solid, 0–8% MeOH / DCM) to give (R)-3-methyl-7-((1-methylpiperidin-3-yl)amino)pyrazole[1,5-d][1,2,4]triazin-4(5H)-one I-125 (420 mg, 24% yield) as a yellow oil.

[0414] Synthesis of Intermediate 126 (I-126), (R)-4-chloro-3-methyl-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0415] [ka]

[0416] Phosphorus oxychloride [10025-87-3] (1.2 mL, 12.84 mmol) was added to a solution of (R)-3-methyl-7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-125 (420 mg, 1.6 mmol) and anhydrous acetonitrile (12 mL) in a 35 mL screw-cap vial at room temperature under a N atmosphere. The mixture was sparged with N for 10 minutes and then stirred at 90 °C for 18 hours. The vial was cooled to room temperature, and the mixture therein was concentrated to dryness in vacuo, diluted with NaHCO (aq) (50 mL), and extracted with EtOAc (50 mL x 3). The organic layer was separated, dried (MgSO), filtered, and the solvent was evaporated in vacuo to give (R)-4-chloro-3-methyl-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-126 (260 mg, yield: 51%) as a yellow sticky solid. The crude product was used directly in the next step.

[0417] Synthesis of Intermediate 127 (I-127), (R)-4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-3-methyl-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine

[0418] [ka]

[0419] To a 35 mL screw-cap vial sparged with nitrogen for 5 minutes was added (R)-4-chloro-3-methyl-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-126 (260 mg, 0.93 mmol), 2-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane I-3 (385 mg, 1.02 mmol), potassium carbonate [584-08-7] (384 mg, 2.78 mmol), 1,4-dioxane (6.2 mL), and water (1.5 mL) to give a heterogeneous mixture. Pd(dppf)Cl·DCM [95464-05-4] (91 mg, 0.11 mmol) was then added to the vial, which was then capped and heated at 90 °C for 18 h. The vial was cooled to room temperature, then diluted with water (50 mL) and extracted with DCM / MeOH (50 mL × 2). The combined extracts were dried over anhydrous MgSO, filtered, and concentrated to dryness in vacuo to give a brown sticky solid. The solid was subjected to silica gel chromatography (25 g, brown sticky solid; 0–10% MeOH / DCM) to give (R)-4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-3-methyl-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-127 (230 mg, 35% yield) as a yellow oil.

[0420] Synthesis of Intermediate 128 (I-128), 4-chloro-N-((8S,8aR)-octahydroindolizin-8-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine (Relative Stereochemistry)

[0421] [ka]

[0422] POCl3[10025-87-3] (700 μL, 7.53 mmol) was added to a suspension of 7-(((8S,8aR)-octahydroindolizin-8-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one I-61 (520 mg, 1.87 mmol) in acetonitrile (20 mL) (relative stereochemistry). The resulting reaction mixture was stirred at 80 °C until complete conversion (2 h). The reaction mixture was concentrated to dryness, triturated in saturated aqueous NaHCO3 at 0 °C, and saturated with solid NaCl. The resulting mixture was extracted with 2-Me-THF (8 × 20 mL). The combined organic layers were washed with brine, dried (NaSO), and concentrated to give 4-chloro-N-((8S,8aR)-octahydroindolizin-8-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine (relative stereochemistry) I-128 (350 mg, 64% yield).

[0423] Preparation of final compounds Synthesis of Final Compound 1 (FC-1): (R)-2-(4-((1-methylpiperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0424] [ka]

[0425] Sodium triacetoxyborohydride [56553-60-7] (330 mg, 1.51 mmol) was added to a stirred solution of (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol trifluoroacetate I-44a (495 mg, 1.01 mmol), triethylamine [121-44-8] (988 μL, 7.05 mmol), and formaldehyde (37% aqueous solution) [50-00-0] (150 μL, 2.02 mmol) in methanol (12 mL) under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 16 h. It was then diluted with saturated aqueous NaHCO and extracted with EtOAc (x3). The combined organic layers were dried (MgSO), filtered, and concentrated. The crude product was purified by flash column chromatography (methanol / dichloromethane, 0 / 100 to 6 / 94). Fractions containing the desired product were collected and concentrated. The residue was further purified by preparative HPLC (column: Phenomenex Gemini C18 30 × 100 mm, 5 μm; gradient water 25 mM NH₄HCO₃ / MeCN, 70 / 30 to 27 / 73). The desired fractions were collected and lyophilized to give compound X, (R)-2-(4-((1-methylpiperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-1 (265 mg, yield: 50%) as a yellow solid.

[0426] Additional final compounds were synthesized in a similar manner using appropriate reagents.

[0427] [Table 17-1]

[0428] [Table 17-2]

[0429] Synthesis of final compound 8, (FC-8): 2-(4-(((1R,2R)-2-hydroxycyclohexyl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0430] [ka]

[0431] 10% Palladium on carbon [7440-05-3] (20 mg, 0.02 mmol) was added to a stirred solution of (1R,2R)-2-((1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[1,2-d][1,2,4]triazin-4-yl)amino)cyclohexan-1-ol I-34 (40 mg, 0.06 mmol) in methanol (3 mL) under a nitrogen atmosphere at 0 °C. The nitrogen atmosphere was replaced with hydrogen (1 atm), and the reaction mixture was stirred at room temperature for 4 h. The mixture was filtered through a pad of Celite. The pad was washed with dichloromethane / methanol (9:1). The organic mixture was concentrated, and the crude was purified by preparative HPLC (column: Phenomenex Gemini C18 30 × 100 mm 5 μm; gradient water 25 mM NH₄HCO₃ / acetonitrile, 70 / 30 to 27 / 73). The fractions containing the desired product were collected and lyophilized to give 2-(4-(((1R,2R)-2-hydroxycyclohexyl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-8 (10 mg, yield: 41%) as a yellow solid.

[0432] Additional final compounds were synthesized in a similar manner using appropriate reagents.

[0433] [Table 18-1]

[0434] [Table 18-2]

[0435] Synthesis of final compound 14 (FC-14): 2-(7-(((3R,5R)-1-ethyl-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0436] [ka]

[0437] A reaction vessel was charged with (2-hydroxy-4-(trifluoromethyl)phenyl)boronic acid [1072951-50-8] (297.4 mg, 1.44 mmol, 1.5 equiv.), Pd(dppf)Cl dichloromethane [95464-05-4] (94.4 mg, 0.12 mmol, 12 mol%), and KCO [584-08-7] (399 mg, 2.89 mmol, 3 equiv.) and flushed with nitrogen (three vacuum / nitrogen cycles). 7-(((3R,5R)-1-ethyl-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl trifluoromethanesulfonate I-84 (397 mg, 0.96 mmol, 1 equiv.) in 1,4-dioxane (6.7 mL) and water (1.5 mL) was added, and the resulting reaction mixture was stirred at 90 °C for 1.5 h. The reaction mixture was quenched with saturated aqueous NaHCO and extracted three times with EtOAc. The combined organic layers were dried over MgSO, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography (0-2% methanol:dichloromethane). The residue thus obtained was recrystallized in methanol to give 2-(7-(((3R,5R)-1-ethyl-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC-14 (16.8 mg, 4% yield) as a yellow solid.

[0438] Additional final compounds were synthesized in a similar manner using appropriate reagents.

[0439] [Table 19]

[0440] Synthesis of final compound 16, (FC-16): 2-(7-((4-methyl-1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0441] [ka]

[0442] A reaction flask was charged with N-(4-chloropyrazolo[1,5-d][1,2,4]triazin-7-yl)-4-methyl-1,4-oxazepan-6-amine I-74 (178 mg, 0.632 mmol), (2-hydroxy-4-(trifluoromethyl)phenyl)boronic acid [1072951-50-8] (142 mg, 0.69 mmol), potassium phosphate tribasic [7778-53-2] (401 mg, 1.89 mmol), Pd(dppf)Cl₂·dichloromethane [95464-05-4] (51 mg, 0.06 mmol), 1,4-dioxane (4.3 mL), and water (0.4 mL). The reaction mixture was purged with nitrogen and stirred at 90 °C for 3 h. The mixture was purified by solid-phase extraction using Si-propylsulfonic acid SCX-2 resin (SiliCycle). The crude reaction mixture was transferred to a column packed with Si-propylsulfonic acid SCX-2 resin (SiliCycle). The desired product was released by eluting the column first with MeOH and then with ammoniated methanol (7N). The tube containing the product was concentrated under reduced pressure. The crude product was purified using silica gel column chromatography (gradient elution: 0 to 4% methanol in dichloromethane) to give 2-(7-((4-methyl-1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC-16 (155 mg, 60% yield).

[0443] Additional final compounds were synthesized in a similar manner using appropriate reagents.

[0444] [Table 20-1]

[0445] [Table 20-2]

[0446] Synthesis of final compound 21 (FC-21): (R)-2-(7-((1-(2-fluoroethyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0447] [ka]

[0448] 4M HCl in 1,4-dioxane [7647-01-0] (0.89 mL, 4 M, 3.54 mmol) was added to a solution of (R)—N-(1-(2-fluoroethyl)piperidin-3-yl)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-115 (171 mg, 0.35 mmol) in methanol (3 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was transferred to a column packed with Si-propylsulfonic acid SCX-2 resin (SiliCycle). The column was first eluted with methanol. The desired product was released by eluting with 7N NH3 / methanol. The tube containing the desired product was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0–3% methanol in dichloromethane) to give (R)-2-(7-((1-(2-fluoroethyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC-21 (85 mg, 56% yield).

[0449] Additional final compounds were synthesized in a similar manner using appropriate reagents.

[0450] [Table 21-1]

[0451] [Table 21-2]

[0452] Synthesis of final compound 27 (FC-27): (R)-3-methyl-2-(7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0453] [ka]

[0454] 4 M HCl [7647-01-0] in 1,4-dioxane (2.4 mL, 9.6 mmol) was added to a solution of (R)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-N-(1-methylpiperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-119 (235 mg, 0.51 mmol) in 1,4-dioxane (2.42 mL), and the mixture was stirred at room temperature for 2 h. The mixture was poured into saturated NaHCO solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over MgSO, filtered, and evaporated. Purification was carried out by preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 30 × 150 mm, mobile phase: 0.25% aqueous NHHCO, CHCN). The pure fractions were concentrated and co-evaporated twice with MeOH. The residue was lyophilized to give (R)-3-methyl-2-(7-((1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (130 mg, 63.22% yield) FC-27 as a white foam.

[0455] Additional final compounds were synthesized in a similar manner using appropriate reagents.

[0456] [Table 22]

[0457] Synthesis of final compound 31 (FC-31): (R)-2-(4-((1-(oxetan-3-yl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0458] [ka]

[0459] 3-Oxetanone [6704-31-0] (0.020 mL, 0.31 mmol) was added to a mixture of (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol dihydrochloride I-44b (95 mg, 0.21 mmol) and triethylamine [121-44-8] (0.116 mL, 0.835 mmol) in dichloroethane (3 mL). The mixture was stirred for 15 minutes. Sodium triacetoxyborohydride [56553-60-7] (88 mg, 0.418 mmol) was added. The mixture was stirred at room temperature for 16 hours. The mixture was diluted with saturated NaHCO3 and then extracted with dichloromethane (x3). The combined organic layers were separated, dried over MgSO, filtered, and concentrated. The crude product was purified by flash column chromatography (dichloromethane / methanol (9:1), 0 / 100 to 100 / 0 in dichloromethane). The resulting material was further purified by reverse-phase chromatography (Phenomenex Gemini C18 30 × 100 mm, 5 μm column, 47% [25 mM NH4HCO3]-53% acetonitrile:methanol (1:1) to 18% [25 mM NH4HCO3]-82% acetonitrile:methanol (1:1)) to afford (R)-2-(4-((1-(oxetan-3-yl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-31 (38 mg, 41% yield) as a yellow foam.

[0460] Synthesis of Final Compound 32, (FC-32): 2-(4-((4-methyl-1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0461] [ka]

[0462] Sodium triacetoxyborohydride [56553-60-7] (112 mg, 0.51 mmol, 1.5 equiv.) was added to a stirred solution of 2-(4-((1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol hydrochloride I-46 (146.93 mg, 0.34 mmol), triethylamine [121-44-8] (335 μL, 2.39 mmol, 7 equiv.), and formaldehyde (37% aqueous solution) [50-00-0] (51 μL, 0.68 mmol, 2 equiv.) in methanol (3.4 mL) under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 16 h. The reaction was recharged with formaldehyde (37% aqueous solution) [50-00-0] (51 μL, 0.68 mmol, 2 equiv.) and sodium triacetoxyborohydride [56553-60-7] (112 mg, 0.51 mmol, 1.5 equiv.). The mixture was stirred at room temperature for an additional 16 h. The mixture was concentrated, and the crude was purified by flash column chromatography (dichloromethane:methanol (9:1) / dichloromethane 0 / 100 to 100 / 0) to give 2-(4-((4-methyl-1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (130 mg) as a yellow solid. Thirty mg of the product was repurified by reverse-phase chromatography (Phenomenex Gemini C18 30 × 100 mm 5 μm column, 59% [25 mM NH4HCO3]-41% [I:MeOH (1:1)] to 17% [25 mM NH4HCO3]-83% [I:MeOH (1:1)]) to give 2-(4-((4-methyl-1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-32 (17.2 mg, 12% yield) as a yellow solid.

[0463] Final Compound 33 and Final Compound 34: Synthesis of (R)-2-(4-((4-methyl-1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol and (S)-2-(4-((4-methyl-1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0464] [ka]

[0465] 2-(4-((4-Methyl-1,4-oxazepan-6-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (racemic form) FC-32 (100 mg, 0.25 mmol) was purified by SFC to isolate both chiral products. SFC purification was performed using an i-amylose-1 column with ISOC 30% ethanol + 0.1% diethylamine, followed by collection and concentration to give FC-33 (25.9 mg, yield: 25%) as a yellow foamy solid and FC-34 (21.4 mg, yield: 21%) as a yellow foamy solid. The absolute configurations were not determined.

[0466] Synthesis of final compound 35 (FC-35): (R)-2-(4-((1-(methyl-d3)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0467] [ka]

[0468] Sodium cyanoborodeuteride [25895-62-9] (30 mg, 0.45 mmol) was added to a stirred solution of (R)-2-(4-(piperidin-3-ylamino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol hydrochloride I-44c (124 mg, 0.3 mmol), triethylamine [121-44-8] (294 μL, 2.1 mmol), and formaldehyde-d2 solution (approximately 20 wt % in DO, 98 atom % D) [1664-98-8] (86 μL, 0.6 mmol) in methanol-d1 (99.5 atom % D) [1455-13-6] (4.2 mL) under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated. The residue was purified by reverse-phase chromatography (Phenomenex Gemini C18 30 × 100 mm, 5 μm column, 70% [25 mM NH4HCO3]-30% acetonitrile to 27% [25 mM NH4HCO3]-73% acetonitrile) to give (R)-2-(4-((1-(methyl-d3)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-35 (42 mg, 35% yield) as a yellow solid.

[0469] Synthesis of final compound 36 and final compound 37: 2-(4-(((8R,8aS)-octahydroindolizin-8-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol and 2-(4-(((8S,8aR)-octahydroindolizin-8-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0470] [ka]

[0471] FC-11 (92 mg, 0.22 mmol) was purified by SFC to isolate both chiral products. SFC purification was performed using an i-amylose-1 column with ISOC 25% 2-propanol + 0.1% diethylamine to give FC-36 (first eluting enantiomer) (27.4 mg, yield: 28%) as a yellow solid and FC-37 (second eluting enantiomer) (29.6 mg, yield: 31%) as a yellow solid. The absolute configurations were not determined.

[0472] Synthesis of Final Compound 38 (FC-38) and Final Compound 39 (FC-39): 2-(4-(((1R,8aS)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) and 2-(4-(((1S,8aS)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (relative stereochemistry)

[0473] [ka]

[0474] Process 1 10% Palladium on carbon [7440-05-3] (180 mg, 0.17 mmol) was added to a stirred solution of 1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(octahydroindolizin-1-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-38 (343 mg, 0.68 mmol) in methanol (10 mL) under a nitrogen atmosphere at 0 °C. The nitrogen atmosphere was then replaced with hydrogen [1333-74-0] (1 atm), and the reaction mixture was stirred at room temperature for 16 h. The mixture was filtered through a pad of Celite and washed with DCM / MeOH (4:1). The solvent was evaporated in vacuo to give 2-(4-((octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (271 mg, yield: 93%) as a yellow foamy solid.

[0475] Process 2 2-(4-((octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (270 mg, 0.63 mmol) was separated into its diastereomeric components by preparative HPLC (column: Phenomenex Gemini C18 30 × 100 mm 5 μm; gradient water 25 mM NH4HCO3 / acetonitrile 75 / 25 to 0 / 100). The fractions were collected and lyophilized to give 2-(4-(((1R,8aS)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) FC-38 (141 mg, 0.33 mmol) as a yellow solid and 2-(4-(((1S,8aS)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) FC-39 (38 mg, 0.09 mmol) as a yellow solid.

[0476] Synthesis of final compound 40 (FC-40): (R)-2-(4-((1-(2-hydroxyethyl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0477] [ka]

[0478] Boron tribromide [10294-33-4] (245 μL, 2.5 mmol) was added dropwise to a stirred solution of 2-(4-(((3R)-1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol I-48 (150 mg, 0.25 mmol) in anhydrous dichloromethane (3.7 mL) at −20° C. under a nitrogen atmosphere. The mixture was stirred at 0° C. for 30 min. Aniline [62-53-3] (45 μL, 0.5 mmol) was carefully added dropwise. The mixture was poured into an ice / saturated aqueous NaHCO3 mixture and extracted with a dichloromethane / methanol (9:1) mixture (×3). The combined organic layers were dried (MgSO), filtered, and concentrated. The crude product was purified by reverse phase (Phenomenex Gemini C18 30 × 100 mm 5 μm column; 50% [25 mM NH4HCO3]-50% [acetonitrile:methanol (1:1)] to 0% [25 mM NH4HCO3]-100% [acetonitrile:methanol (1:1)]) to give (R)-2-(4-((1-(2-hydroxyethyl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-40 (15.5 mg, 14% yield) as a yellow solid.

[0479] Synthesis of final compound 41 (FC-41) and final compound 42 (FC-42): 2-(4-(((1R,8aR)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol and 2-(4-(((1S,8aS)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0480] [ka]

[0481] FC-39 (38 mg, 0.09 mmol) was purified by SFC to isolate both chiral products. Stationary phase: Amylose-1 250 × 30 mm 5 μm. Mobile phase: 70% CO2-30% EtOH + 0.1% diethylamine. Fractions were collected and concentrated in vacuo to give FC-41, the first eluting enantiomer (11 mg, 0.03 mmol) as a dark yellow solid, and FC-42, the second eluting enantiomer (12 mg, 0.03 mmol) as a dark yellow solid. The absolute configuration was not determined.

[0482] Synthesis of final compound 43 (FC-43) and final compound 44 (FC-44): 2-(4-(((1R,8aS)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol and 2-(4-(((1S,8aR)-octahydroindolizin-1-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0483] [ka]

[0484] FC-38 (141 mg, 0.33 mmol) was purified by SFC to isolate both chiral components. Stationary phase: Amylose-1 250 × 30 mm 5 μm, Mobile phase: 60% CO2-40% MeOH + 0.1% diethylamine. Fractions were collected, concentrated in vacuo, and triturated with diisopropyl ether to give FC-43, the first eluting enantiomer (57 mg, 0.13 mmol) as a yellow solid, and FC-44, the second eluting enantiomer (57 mg, 0.13 mmol) as a yellow solid. The absolute configuration was not determined.

[0485] Synthesis of final compound 45 (FC-45): (R)-2-(4-((1-(2-fluoroethyl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0486] [ka]

[0487] Boron tribromide [10294-33-4] (114 μL, 1.2 mmol) was added dropwise to a stirred solution of (R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(1-(2-fluoroethyl)piperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine I-49 (120 mg, 0.23 mmol) in anhydrous dichloromethane (300 μL) at −20° C. under a nitrogen atmosphere. The reaction mixture was stirred at 0° C. for 30 minutes. Aniline [62-53-3] (45 μL, 0.5 mmol) was carefully added dropwise. The mixture was poured into an ice / saturated aqueous NaHCO mixture and extracted with a dichloromethane / methanol (9:1) mixture (×3). The combined organic layers were dried (MgSO), filtered, and concentrated. The residue was purified by reverse phase (Phenomenex Gemini C18 30 × 100 mm, 5 μm column, 59% [25 mM NH4HCO3]-41% [100% acetonitrile] to 17% [25 mM NH4HCO3]-83% [100% acetonitrile]) to give (R)-2-(4-((1-(2-fluoroethyl)piperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-45 (14.7 mg, 15% yield) as a yellowish solid.

[0488] Synthesis of Final Compound 46, (FC-46): 2-(7-(((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry)

[0489] [ka]

[0490] Sodium triacetoxyborohydride [56553-60-7] (76 mg, 0.35 mmol) was added to a stirred solution of 2-(7-(((3R,5R)-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry) I-108 (100 mg, 0.803 mmol), triethylamine [121-44-8] (228 μL, 1.62 mmol), and formaldehyde (37% aqueous solution) [50-00-0] (34 μL, 0.46 mmol) in methanol (3 mL) under a nitrogen atmosphere at 0° C. The mixture was stirred at room temperature for 16 hours. The reaction was recharged with triethylamine [121-44-8] (3 equiv., 114 μL, 0.81 mmol), formaldehyde (37% aqueous solution) [50-00-0] (2 equiv., 34 μL, 0.46 mmol), and sodium triacetoxyborohydride [56553-60-7] (1.5 equiv., 76 mg, 0.35 mmol), and stirred at room temperature for an additional 32 h. The mixture was diluted with NaHCO (saturated aqueous solution) and extracted with DCM. The organic layer was extracted, dried over MgSO, and concentrated in vacuo to recover the starting material as the free base (71 mg, 77%) as a pale yellow solid. To a stirred solution of the recovered 2-(7-(((3R,5R)-5-fluoropiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) (71 mg, 0.18 mmol) was added sodium cyanoborohydride [25895-60-7] (18 mg, 0.27 mmol). Triethylamine [121-44-8] (101 μL, 0.72 mmol) and formaldehyde (37% aqueous solution) [50-00-0] (27 μL, 0.36 mmol) in methanol (2.5 mL) were added under a nitrogen atmosphere at 0° C. The mixture was stirred at room temperature for 16 hours. The mixture was concentrated. The residue was purified by reverse-phase chromatography (Phenomenex Gemini C18 30 × 100 mm 5 μm column, 59% [25 mM NH4HCO3]-41% [acetonitrile:methanol (1:1)] to 17% [25 mM NH4HCO3]-83% [acetonitrile:methanol = (1:1)]).The relevant fractions were collected and the solvent was lyophilized to give 2-(7-(((3R,5R)-5-fluoro-1-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) FC-46 (18.7 mg, yield: 25%) as a white solid.

[0491] Synthesis of Final Compound 47 (FC-47) and Final Compound 48 (FC-48): (R)-2-(7-((4-methyl-1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and (S)-2-(7-((4-methyl-1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0492] [ka]

[0493] 2-(7-((4-Methyl-1,4-oxazepan-6-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC-16 (150 mg, 0.37 mmol) was purified by SFC to isolate both chiral products. SFC conditions: Stationary phase: Chiralpak Diacel AD 20 × 250 mm, Mobile phase: CO, EtOH + 0.4iPrNH. FC-47, the first eluting enantiomer (58 mg, yield: 77%) and FC-48, the second eluting enantiomer (57 mg, yield: 76%) were obtained. The absolute configurations were not determined.

[0494] Final compound 49 (FC-49), final compound 50 (FC-50), final compound 51 (FC-51) and final compound 52 (FC-52): 2-(7-(((1S,8aR)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol, 2-(7-(((1R,8aS)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol Synthesis of azin-4-yl)-5-(trifluoromethyl)phenol, 2-(7-(((1R,8aR)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol, and 2-(7-(((1S,8aS)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0495] [ka]

[0496] Process 1 A solution of 4-chloro-N-(octahydroindolizin-1-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine (200 mg, 0.68 mmol) I-76 and (2-hydroxy-4-(trifluoromethyl)phenyl)boronic acid [1072951-50-8] (85.95 mg, 0.42 mmol) in 1,4-dioxane (10 mL) containing water (1 mL) was purged with nitrogen for 5 minutes. To the solution was added tribasic potassium phosphate [7778-53-2] (204 mg, 0.96 mmol) and Pd(dppf)Cl2 dichloromethane [95464-05-4] (26.22 mg, 0.032 mmol). The reaction mixture was heated at 110 °C under microwave irradiation for 3 minutes. The reaction mixture was poured into saturated aqueous ammonium chloride solution. The organics were extracted with ethyl acetate (5 × 10 mL). The combined organic layers were dried (NaSO) and concentrated to dryness to give 2-(7-((octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (300 mg, 85% yield) as a brown solid as a mixture of two racemic diastereomers.

[0497] Process 2 The residue was purified by preparative HPLC (stationary phase: RP XBridge Prep C18 OBD-10 μm, 50×150 mm, mobile phase: 0.25% aqueous NH 4 HCO 3 , CH 3 CN) to give two diastereomers. The first eluting diastereomer was further purified by preparative chiral SFC (stationary phase: Chiralcel Diacel OD 20 × 250 mm, mobile phase: CO, EtOH + 0.4 iPrNH) to give the optically pure trans enantiomers 2-(7-(((1S,8aR)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((1R,8aS)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-49 and FC-50). The absolute configurations were not determined. 7.3 mg (10% yield) was obtained for the first eluting enantiomer, and 7.9 mg (11% yield) was obtained for the second eluting enantiomer. The second eluting diastereomer was further purified by preparative chiral SFC (stationary phase: Chiralcel Diacel OD 20 × 250 mm, mobile phase: CO , EtOH + 0.4 iPrNH ) to give the optically pure cis enantiomers 2-(7-(((1R,8aR)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((1S,8aS)-octahydroindolizin-1-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-51 and FC-52). The absolute configuration was not determined. 4.2 mg (6% yield) was obtained for the first eluting enantiomer and 4.3 mg (6% yield) for the second eluting enantiomer.

[0498] Synthesis of Final Compound 53 (FC-53) and Final Compound 54 (FC-54): 2-(7-(((8S,8aR)-octahydroindolizin-8-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((8R,8aS)-octahydroindolizin-8-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0499] [ka]

[0500] A reaction flask was charged with 4-chloro-N-((8S,8aR)-octahydroindolizin-8-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine (relative stereochemistry) I-128 (200 mg, 0.68 mmol), (2-hydroxy-4-(trifluoromethyl)phenyl)boronic acid [1072951-50-8] (155 mg, 0.75 mmol), 1,4-dioxane (5 mL), and water (0.5 mL). The resulting mixture was purged with nitrogen. Pd(dppf)Cl2 dichloromethane [95464-05-4] (56 mg, 0.068 mmol) and tribasic potassium phosphate [7778-53-2] (435 mg, 2.05 mmol) were added, and the mixture was heated at 90 °C for 18 h. The crude reaction mixture was diluted with dichloromethane. The aqueous layer was separated, and the organic layer was filtered through decalite, dried (NaSO), and concentrated. The crude product was purified using flash column chromatography (gradient elution: 0–10% MeOH in dichloromethane) to give the racemic mixture 2-(7-(((8S,8aR)-octahydroindolizin-8-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) (128 mg, 45% yield). The compounds were purified by preparative chiral SFC (stationary phase: Chiralcel Diacel OD 20 × 250 mm, mobile phase: CO, EtOH + 0.4 iPrNH) to give the optically pure enantiomers 2-(7-(((8S,8aR)-octahydroindolizin-8-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((8R,8aS)-octahydroindolizin-8-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-53 and FC-54). The absolute configurations were not determined. 51 mg (80% yield, relative to the separation) was obtained for the first eluting enantiomer and 51 mg (80% yield, relative to the separation) was obtained for the second eluting enantiomer.

[0501] Synthesis of Final Compound 55 (FC-55) and Final Compound 56 (FC-56): 2-(7-(((R)-1-((1S,2S)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((R)-1-((1R,2R)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0502] [ka]

[0503] Step 1: Lithium bromide [7550-35-8] (6 mg, 0.071 mmol) and cyclopentene oxide [285-67-6] (239 mg, 2.84 mmol) were added to a solution of (R)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrazolo[1,5-d][1,2,4]triazin-7-amine I-102 (300 mg, 0.71 mmol) in isopropanol (4 mL). The reaction mixture was stirred at 95° C. for 16 hours in a sealed reaction vessel. The reaction was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (gradient elution: 0 to 10% MeOH in dichloromethane) to give (1S,2S-relative stereochemistry)-2-((R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidin-1-yl)cyclopentan-1-ol (198 mg, 54% yield). 41 mg of 2-(7-(((R)-1-((1S,2S-relative stereochemistry)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (loss of the methoxymethyl protecting group) was obtained as a by-product.

[0504] Step 2: 4M HCl in 1,4-dioxane [7647-01-0] (0.70 mL, 4 M, 2.78 mmol) was added to a solution of (1S,2S-relative stereochemistry)-2-((R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidin-1-yl)cyclopentan-1-ol (198 mg, 0.39 mmol) in MeOH (3 mL). The reaction mixture was stirred at room temperature overnight. The crude reaction mixture was transferred to a column packed with Si-propylsulfonic acid SCX-2 resin (SiliCycle) and eluted with MeOH. The product compound was released by eluting with 7 N NH3 / MeOH. The tubes containing the product compound were combined and concentrated under reduced pressure. The crude product was combined with 41 mg of 2-(7-(((R)-1-((1S,2S-relative stereochemistry)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol obtained as a by-product from step 1 and purified by silica gel column chromatography (gradient elution: 0-8% MeOH in DCM) to give 191 mg of the product compound (58% over two steps).

[0505] Step 3: 2-(7-(((R)-1-((1S,2S-relative stereochemistry)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (191 mg, 0.41 mmol) was purified by preparative SFC (stationary phase: Chiralpak Diacel AD 20 × 250 mm, mobile phase: CO₂, iPrOH + 0.4 Separation with iPrNH gave 2-(7-(((R)-1-((1S,2S)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((R)-1-((1R,2R)-2-hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-55 and FC-56). The absolute configurations were not determined. 78 mg (24% yield over three steps) was obtained for the first eluting diastereomer and 72 mg (24% yield over two steps) was obtained for the second eluting diastereomer.

[0506] Synthesis of Final Compound 57 (FC-57) and Final Compound 58 (FC-58): 2-(7-(((R)-1-((1R,2R)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((R)-1-((1S,2S)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0507] [ka]

[0508] Process 1 4M HCl in 1,4-dioxane [7647-01-0] (0.6 mL, 4 M, 2.38 mmol) was added to a solution of (1R,2R-relative stereochemistry)-2-((R)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[1,5-d][1,2,4]triazin-7-yl)amino)piperidin-1-yl)cyclobutan-1-ol I-118 (0.17 mmol) in methanol (3 mL). The reaction mixture was stirred overnight at room temperature. The crude reaction mixture was transferred to a column packed with Si-propylsulfonic acid SCX-2 resin (SiliCycle) and eluted with MeOH. The reaction product was then released by elution with 7N NH3 / MeOH. The tube containing the reaction product was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0-5% MeOH in dichloromethane) to give 2-(7-(((R)-1-((1R,2R-relative stereochemistry)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (48 mg, 61% yield).

[0509] Process 2 2-(7-(((R)-1-((1R,2R-relative stereochemistry)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (48 mg, 0.11 mmol) was purified by preparative SFC (stationary phase: Chiralpak Diacel AD 20 × 250 mm, mobile phase: CO2, EtOH + 0.4 Separation with iPrNH gave 2-(7-(((R)-1-((1R,2R)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((R)-1-((1S,2S)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-57 and FC-58). The absolute configurations were not determined. 8.3 mg (11% yield over three steps) was obtained for the first eluting diastereomer, and 9.7 mg (13% yield over two steps) was obtained for the second eluting diastereomer.

[0510] Synthesis of Final Compound 59 (FC-59) and Final Compound 60 (FC-60): 2-(7-(((3R,5R)-1,5-dimethylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((3S,5S)-1,5-dimethylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol

[0511] [ka]

[0512] Process 1 37% formaldehyde [50-00-0] in HO (133 μL, 0.82 g / mL, 1.33 mmol, 3.1 equiv.) was added to a stirred solution of 2-(7-(((3R,5R)-5-methylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry) I-110 (198 mg, 0.43 mmol, 1 equiv.) in MeOH [67-56-1] (5.9 mL). The resulting mixture was stirred at room temperature for 1 h. After the mixture was cooled to 0 °C, sodium triacetoxyborohydride [56553-60-7] (225.5 mg, 1.06 mmol, 2.5 equiv.) was added in portions. The reaction mixture was allowed to warm to room temperature and further stirred for 1 h. The mixture was diluted with saturated aqueous NaHCO3 and extracted three times with EtOAc. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by SiO2 flash column chromatography (0–6% MeOH:dichloromethane) to give 2-(7-(((3R,5R)-1,5-dimethylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) (200 mg).

[0513] Process 2 2-(7-(((3R,5R)-1,5-dimethylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) was analyzed by preparative SFC (stationary phase: Chiralcel Diacel OJ 20 × 250 mm, mobile phase: CO2, EtOH + 0.4 Purification by iPrNH afforded 2-(7-(((3R,5R)-1,5-dimethylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((3S,5S)-1,5-dimethylpiperidin-3-yl)amino)pyrazolo[1,5-d][1,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-59 and FC-60). The absolute configurations were not determined. 30 mg (35% yield over two steps) was obtained for the first eluting enantiomer (gray solid), and 29 mg (34% yield over two steps) was obtained for the second eluting enantiomer (yellow solid).

[0514] Synthesis of final compound 61 (FC-61): (R)-2-(4-((1-cyclopropylpiperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol

[0515] [ka]

[0516] Boron tribromide [10294-33-4] (0.14 mL, 1.40 mmol) was added dropwise to a mixture of ((R)-1-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(1-cyclopropylpiperidin-3-yl)pyrrolo[1,2-d][1,2,4]triazin-4-amine) I-50 (203 mg, 0.40 mmol) in dichloromethane (4 mL) at −20° C. The resulting mixture was stirred at 0° C. for 2 hours. Aniline [62-53-3] (0.15 mL, 1.60 mmol) was added, and the reaction mixture was stirred for 10 minutes. The mixture was concentrated to dryness. The residue was purified by preparative HPLC (Phenomenex, Gemini 5 μm C18, 30 × 100 mm column, gradient 51–94% (v / v) MeCN / MeOH (1:1) / water (25 mM NH4HCO3)). Product-containing fractions were concentrated to dryness in vacuo to give (R)-2-(4-((1-cyclopropylpiperidin-3-yl)amino)pyrrolo[1,2-d][1,2,4]triazin-1-yl)-5-(trifluoromethyl)phenol FC-61 (77.8 mg, 44%) as a yellow foamy solid.

[0517] Analytical data - LC-MS LCMS: [M+H] + denotes the protonated mass of the free base of the compound, and R t means retention time (in minutes) and method refers to the method used for LCMS.

[0518] [Table 23-1]

[0519] [Table 23-2]

[0520] NMR 1 H NMR spectra were recorded on Bruker Avance III and Avance NEO spectrometers. Chemical shifts are expressed in ppm relative to tetramethylsilane.

[0521] [Table 24-1]

[0522] [Table 24-2]

[0523] [Table 24-3]

[0524] [Table 24-4]

[0525] [Table 24-5]

[0526] [Table 24-6]

[0527] [Table 24-7]

[0528] Example B - Pharmaceutical Composition A compound of the present invention (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 of the present invention (e.g., a compound of the Examples) is intimately mixed with a pharmaceutically acceptable carrier in a process for preparing a pharmaceutical composition.

[0529] Example C - Biological Examples The activity of the compounds according to the present invention can be evaluated by in vitro methods.The compounds according to the present invention exhibit beneficial pharmacological properties, for example, as shown in the following test, they are prone to inhibit NLRP3 activity, and are therefore suitable for the treatment of NLRP3 inflammasome activity-related diseases.

[0530] 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).

[0531] I C 50 The EC50 values ​​(for IL-1β) and EC50 values ​​(IL6 and TNFα) were obtained for the compounds of the invention / examples and are shown in the table below.

[0532] [Table 25-1]

[0533] [Table 25-2]

[0534] 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.

[0535] [Table 26]

[0536] Example E - Further Testing One or more compounds may be tested in several other ways to assess permeability, stability (including metabolic and blood stability), and solubility, among other properties.

[0537] 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 Proteome Research 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).

[0538] Different screening approaches are applied, for example, generating two or up to four concentration relationships per compound. 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 pIC upon compound addition are calculated. 50 are reported below.

[0539] [Table 27]

[0540] 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.

[0541] 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

[0542] In vitro intrinsic clearance (Cl int ) (ml / min / mg microsomal protein) is calculated using the following formula:

[0543]

number

[0544] [Table 28-1]

[0545] [Table 28-2]

[0546] In hepatocytes The metabolic stability of test compounds is tested using hepatocytes (1 milj cells) from human and preclinical species incubated with 1 μM of test compound at 37° C. for up to 120 minutes.

[0547] 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

[0548] In vitro intrinsic clearance (Cl int ) (μl / min / million cells) is calculated using the following formula:

[0549]

number

[0550] [Table 29]

[0551] 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.

[0552] 2.Purpose Determining the extent of plasma protein binding of the test compound.

[0553] 3.Customer provided · Compound identifier, molecular formula. 25 μL of 10 mM or 50 μL of 5 mM test compound in DMSO per species.

[0554] 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

[0555] 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.

[0556] 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.

[0557] 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.

[0558] 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 The calculated fu in 25% plasma (fu25%) is converted to fu in 100% plasma (fu100%) using the following formula: fu100%=fu25% / (4-(3fu25%)) 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

[0559] 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.

[0560] Brain tissue connection 1. Purpose The goal of this study was to determine the brain tissue binding of test compounds in rat and mouse brain tissue using equilibrium dialysis. The peak area ratios of test compounds in brain tissue homogenates and buffer were assessed by LC-MS / MS.

[0561] 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.

[0562] Na2HPO4, NaH2PO4, and NaCl are purchased from local suppliers.

[0563] Acetonitrile and methanol are purchased from Merck (Darmstadt, Germany). Other reagents are purchased from local suppliers.

[0564] Disposable RED plates with inserts (90006BLCS) are purchased from Thermo.

[0565] 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.

[0566] [Table 30]

[0567] 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.

[0568] Thaw frozen brain tissue homogenate (stored at -80 °C). Immediately thaw the frozen brain tissue homogenate in a 37 °C water bath.

[0569] Preparation of stock and working solutions Prepare stock solutions of the test compounds 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.

[0570] 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.

[0571] 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.

[0572] 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.

[0573] 4. Data Analysis All calculations were performed using Microsoft Excel.

[0574] From the peak area ratio, the peak area ratio of the test compound and the control compound in the buffer compartment and the brain tissue homogenate compartment is determined. The percentage of bound test compound and control compound is calculated as follows:

[0575]

number

[0576] [Table 31] data

[0577] 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: PO Oral by 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.

[0578] 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. 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 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. 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. The sample volume does not exceed the maximum recommended blood sample volume from the animal. 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. Anesthesia Isoflurane: Induction: 4% (O2 and room air) Maintenance: 2% (O2 and room air)

[0579] 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.

[0580] 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.

[0581] Sample transport Freezing to bioanalysis department The animals are observed throughout the experiment.

[0582] During the acclimation period (after transfer), animals are monitored daily by LAM personnel.

[0583] 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.

[0584] 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.

[0585] Determination of partition coefficients kpuu,brain Kpuu,brain was calculated as follows: 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

[0586] [Table 32]

[0587] 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.

[0588] [Table 33]

[0589] 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 for 30 minutes at 37°C, 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 was measured using 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.

[0590] [Table 34]

[0591] 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).

[0592] [Table 35]

[0593] 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.

[0594] [Table 36]

[0595] 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.

[0596] [Table 37]

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 During the ceremony, A is N or CH; R １ teeth, 【Chemistry 2】 and R ２ is H or CH ３ and R ３ is CF ３ , OCF ３ , OCH ３ , C.H. ３ , Cl, or OCHF ２ and R ４ is H or CH ３ and R ５ is H or CH ３ and R ６ は、CH ３ 、CH ２ CH ３ 、CH(CH ３ ) ２ 、CH ２ CH ２ OH、CH ２ CH ２ F、CD ３ 、 【Transformation 3】 and R ７ and R ８ are each independently H, CH ３ or F.

2. R １ teeth, 【Chemistry 4】 2. The compound of claim 1, wherein:

3. R １ teeth 【Transformation 5】 and R ６ is CH ３ or CH ２ CH ３ and R ７ is H, CH ３ , or F, and R ８ is H or R ７ and R ８ and both are F.

4. R １ teeth, 【Transformation 6】 2. The compound of claim 1, wherein:

5. R ２ , R ４ , and R ５ The compound of claim 1 , wherein is hydrogen.

6. R ３ is CF ３ 2. The compound of claim 1, wherein:

7. R ６ is CH ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , C.H. ２ CH ２ OH, CH ２ , C.H. ２ F or CD ３ 2. The compound of claim 1, wherein:

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. A process for preparing a pharmaceutical composition according to claim 8, comprising intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound according to any one of claims 1 to 7.

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