EP4658654A1 - Pyrrolo[1,2-d][1,2,4]triazines and pyrazolo[1,5-d] [1,2,4]triazines as nlrp3 inhibitors - Google Patents

Pyrrolo[1,2-d][1,2,4]triazines and pyrazolo[1,5-d] [1,2,4]triazines as nlrp3 inhibitors

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Publication number
EP4658654A1
EP4658654A1 EP24702714.7A EP24702714A EP4658654A1 EP 4658654 A1 EP4658654 A1 EP 4658654A1 EP 24702714 A EP24702714 A EP 24702714A EP 4658654 A1 EP4658654 A1 EP 4658654A1
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EP
European Patent Office
Prior art keywords
mmol
triazin
trifluoromethyl
mixture
compounds
Prior art date
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EP24702714.7A
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German (de)
French (fr)
Inventor
Oscar MAMMOLITI
Soufyan JERHAOUI
Laura PEREZ BENITO
Edgar Jacoby
Santiago CAÑELLAS ROMAN
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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Publication of EP4658654A1 publication Critical patent/EP4658654A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • pyrrolo[l,2-d][l,2,4]triazines and pyrazolo[l,5- d][l,2,4]triazines that are useful as inhibitors of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway.
  • processes for the preparation of said compounds, pharmaceutical compositions comprising said compounds, methods of using said compounds in the treatment of various diseases and disorders mediated by the NLRP3 inflammasome pathway are also described herein.
  • Inflammasomes considered as central signalling hubs of the innate immune system, are multi-protein complexes that are assembled upon activation of a specific set of intracellular pattern recognition receptors (PRRs) by a wide variety of pathogen- or danger- associated molecular patterns (PAMPs or DAMPs).
  • PRRs pattern recognition receptors
  • PAMPs or DAMPs pathogen- or danger- associated molecular patterns
  • the NLRP3 inflammasome is assembled 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-P fibrils that are neurotoxic in Alzheimer’s disease and asbestos particles that cause mesothelioma (Kelley et al., IntJMol Sci, 2019 Jul 6;20(13)).
  • NLRP3 is activated by infectious agents such as Vibrio cholerae, fungal pathogens such as Aspergillus fumigatus and Candida albicans,' adenoviruses, influenza A virus and SARS-CoV-2 (Tartey and Kanneganti, 2019; Fung et al. Emerg Microbes Infect, 2020 Mar 14;9(l):558-570).
  • infectious agents such as Vibrio cholerae, fungal pathogens such as Aspergillus fumigatus and Candida albicans,' adenoviruses, influenza A virus and SARS-CoV-2 (Tartey and Kanneganti, 2019; Fung et al. Emerg Microbes Infect, 2020 Mar 14;9(l):558-570).
  • NLRP3 activation mechanism Although the precise NLRP3 activation mechanism remains unclear, for human monocytes, it has been suggested that a one-step activation is sufficient while in mice a two-step mechanism is in place. Given the multitude in triggers, the NLRP3 inflammasome requires add-on regulation at both transcriptional and post- transcriptional level (Yang Y et al., Cell Death Dis, 2019 Feb 12; 10(2): 128).
  • 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 C- terminal end (Sharif et al., Nature, 2019 Jun; 570(7761):338-343).
  • NBD nucleotide-binding site domain
  • LRR leucine-rich repeat
  • NLRP3 aggregates with the adaptor protein, apoptosis-associated speck-like protein (ASC), and with the protease caspase- 1 to form a functional inflammasome.
  • ASC apoptosis-associated speck-like protein
  • procaspase-1 Upon activation, procaspase-1 undergoes autoproteolysis and consequently cleaves gasdermin D (Gsdmd) to produce the N-terminal Gsdmd molecule that will ultimately lead to pore-formation in the plasma membrane and a lytic form of cell death called pyroptosis.
  • Gsdmd gasdermin D
  • caspase-1 cleaves the pro- inflammatory cytokines pro-IL-ip and pro-IL-18 to allow release of its biological active form by pyroptosis (Kelley et al., 2019).
  • Dysregulation of the NLRP3 inflammasome or its downstream mediators are associated with numerous pathologies ranging from immune/inflammatory diseases, auto-immune/auto-inflammatory diseases (Cryopyrin-associated Periodic Syndrome (Miyamae T. Paediatr Drugs, 2012 Apr 1; 14(2): 109-17); sickle cell disease; systemic lupus erythematosus (SLE)) to hepatic disorders (e.g. non-alcoholic steatohepatitis (NASH), chronic liver disease, viral hepatitis, alcoholic steatohepatitis, non-alcoholic fatty acid liver disease, and alcoholic liver disease) (Szabo G and Petrasek J.
  • NASH non-alcoholic steatohepatitis
  • NASH non-alcoholic steatohepatitis
  • chronic liver disease viral hepatitis
  • viral hepatitis alcoholic steatohepatitis
  • non-alcoholic fatty acid liver disease non-alcoholic fatty acid liver disease
  • kidney related diseases hypertensive nephropathy (Krishnan et al., Br J Pharmacol, 2016 Feb;173(4):752-65), hemodialysis related inflammation and diabetic nephropathy which is a kidney -related complication of diabetes (Type 1, Type 2 and mellitus diabetes), also called diabetic kidney disease (Shahzad et al., Kidney Int, 2015 Jan;87(l):74-84) are associated to NLRP3 inflammasome activation.
  • 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 artery disease (PAD), acute heart failure and hypertension.
  • myeloproliferative neoplasms myeloproliferative neoplasms, leukemias, myelodysplastic syndromes (MOS), myelofibrosis, lung cancer, colon cancer (Ridker et al., Lancet, 2017 Oct 21;390(10105): 1833-1842; Derangere et al., Cell Death Differ.
  • Described herein are compounds which inhibit the NLRP3 inflammasome pathway.
  • compositions comprising a therapeutically effective amount of a compound provided herein.
  • compositions comprising such compounds for use in the treatment of a disease or disorder mediated by the NLRP3 inflammasome pathway, for example neurodegenerative disorders such as Alzheimer’s Disease.
  • the instant compounds in the manufacture of a medicament for the treatment of a disease or disorder mediated by the NLRP3 inflammasome pathway, for example neurodegenerative disorders such as Alzheimer’s Disease.
  • a method of treating a disease or disorder mediated by the NLRP3 inflammasome pathway for example neurodegenerative disorders such as Alzheimer’s Disease.
  • R 7 and R 8 each independently are H, CH 3 or F.
  • R 7 and R 8 each independently are H, CH 3 or F, with the proviso that the compound is not
  • compounds of Formula (I) having Formula wherein with the proviso that a) R6 is selected from CH 2 CH 3 , CH(CH 3 ) 2 , CH 2 CH 2 OH, CH 2 CH 2 F, CD 3 , when R7 and R8 each independently are H, CH 3 or F; and b) R6 is CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , CH 2 CH 2 OH, CH 2 CH 2 F, CD 3 , when R7
  • R 7 and R 8 each independently are H, CH3 or F; with the proviso that a) R 6 is selected from CH2CH3, CH(CH 3 ) 2 , CH2CH2OH, CH2CH2F, CD 3 , when
  • R 7 and R 8 each independently are H, CH3 or F; and b) R 6 is CH 3 , CH2CH3, CH(CH 3 ) 2 , CH2CH2OH, CH2CH2F, CD 3 , when
  • R 7 is CH3 or F
  • R 8 is H, CH3 or F; with the proviso that the compound is not is H, CH3 or F, and R 8 is H, or both R 7 and R 8 are F.
  • R 6 is CH3 or CH2CH3; R 7 is CH3 or F, and R 8 is H, or both R 7 and R 8 are F.
  • R 6 is CH2CH3; R 7 is H, CH3 or F, and R 8 is H, or both R 7 and R 8 are F.
  • R 1 is wherein a) R 6 is CH3 or CH2CH3; R 7 is H, CH3 or F, and R 8 is H, or both R 7 and R 8 are F; or b) R 6 is CH3 or CH2CH3; R 7 is CH3 or F, and R 8 is H, or both R 7 and R 8 are F; or c) R 6 is CH2CH3; R 7 is H, CH3 or F, and R 8 is H, or both R 7 and R 8 are F; or d) R 6 is CH3 or CH2CH3; and both R 7 and R 8 are F; or
  • R 2 , R 4 and R 5 are hydrogen.
  • R 2 is hydrogen and R 3 is CF3.
  • R 3 is CF3.
  • R 6 is CH 3 , CH2CH3, CH(CH 3 ) 2 , CH2CH2OH, CH2CH2F or CD 3 .
  • R 6 is CH2CH3, CH(CH 3 ) 2 , CH2CH2OH, CH2CH2F or CD 3 .
  • salts include acid addition salts and base addition salts.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound as provided herein with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound provided herein in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • 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.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table.
  • the 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.
  • 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, and the like.
  • Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.
  • the instant compounds may contain double bonds and may thus exist as E (entgegeri) and Z (ziisammeri) geometric isomers about each individual double bond.
  • Compounds as provided herein may contain one or more asymmetric carbon atoms and may therefore exhibit enantiomerism or diastereoisomerism.
  • Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation.
  • the various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques.
  • the desired isomers may be made by reaction of an appropriate enantiomeric starting material under conditions which will not cause racemisation or epimerisation, or by reaction of an appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by resolution, including dynamic resolution, for example salt formation with a homochiral acid followed by separation of the diastereomeric salts by conventional means such as crystallization, or by reaction with an appropriate chiral reagent or chiral catalyst.
  • resolution including dynamic resolution, for example salt formation with a homochiral acid followed by separation of the diastereomeric salts by conventional means such as crystallization, or by reaction with an appropriate chiral reagent or chiral catalyst.
  • stereochemistry of any particular chiral atom is not specified, then all stereoisomers are contemplated. Where stereochemistry is specified by a solid or dashed wedge representing a particular configuration, then that stereoisomer is so specified and defined.
  • Absolute configurations are specified according to the Cahn-Ingold-Prelog system.
  • the configuration at an asymmetric atom is specified by either R or S.
  • Resolved compounds whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate polarized light.
  • a specific stereoisomer is identified, this means that said 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%, in particular less than 2% and most preferably less than 1%, of the other isomers.
  • a compound of formula (I) is for instance specified as (R)
  • the compounds may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • isotopically-labelled compounds wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (or the most abundant one found in nature).
  • Exemplary isotopes include isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, such as 2H, 3H, 11C, 13C, 14C , 13N, 15O, 17O, 18O, and 18F.
  • Tritiated (3H) and carbon-l4 (14C) isotopes are useful for their ease of preparation and detectability.
  • Isotopes such as 15O, 13N, 11C and 18F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy.
  • PET positron emission tomography
  • Isotopically labelled compounds can generally be prepared by following procedures analogous to those disclosed in the Examples hereinafter.. Unless otherwise specified, C1-q alkyl groups (where q is the upper limit of the range) defined herein may be straight-chain or be branched-chain.
  • C3-q cycloalkyl refers to an alkyl group that is cyclic, for instance cycloalkyl groups may be monocyclic or, if there are sufficient atoms, bicyclic. In an embodiment, such cycloalkyl groups are monocyclic. Substituents may be attached at any point on the cycloalkyl group..
  • halo when used herein, preferably includes fluoro, chloro, bromo and iodo.
  • C1-q alkoxy groups refers to the radical of formula -ORa, where Ra is a C1-q alkyl group as defined herein.
  • HaloC1-q alkyl (where q is the upper limit of the range) groups refer to C1-q alkyl groups, as defined herein, where such group is substituted by one or more halo.
  • HydroxyC1-q alkyl (where q is the upper limit of the range) refers to C1-q alkyl groups, as defined herein, where such group is substituted by one or more (e.g. one) hydroxy (-OH) groups (or one or more, e.g. one, of the hydrogen atoms is replaced with -OH).
  • haloCi- q alkoxy and hydroxyCi- q alkoxy represent corresponding -OCi- q alkyl groups that are substituted by one or more halo, or, substituted by one or more (e.g. one) hydroxy, respectively.
  • the instant compounds can generally be prepared by a succession of steps, each of which is known to the skilled person.
  • the compounds can be prepared according to the following synthesis methods.
  • the compounds of Formula (I) may be synthesized in the form of racemic mixtures of enantiomers which can be separated from one another following 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. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkalination.
  • An alternative manner of separating the enantiomeric forms of the compounds of Formula (I) involves liquid chromatography using a chiral stationary phase or a chiral supercritical fluid chromatography (SFC).
  • Said 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.
  • the absolute configuration of the compounds reported herein was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC) followed by SFC comparison of the separate enantiomer(s) which were obtained by asymmetric synthesis, followed by vibrational circular dichroism (VCD) analysis of the particular enantiomer(s).
  • Intermediate compounds of formula (Ila) can be synthesized from Intermediates of formulae (la) or (lb) by nucleophilic substitution with hydrazine under thermal conditions in an appropriate solvent, such as methanol, by heating the mixture at an appropriate temperature, such as at the boiling point temperature of the solvent.
  • an appropriate solvent such as methanol
  • Intermediate compounds of formula (Illa) can be synthesized from (Ila) by a condensation reaction in presence of triethyl orthoformate under thermal conditions (e.g. at 165 °C) in an appropriate solvent, such as dimethyl acetamide.
  • Intermediate compounds of formula (IVa) can be obtained by reacting (Illa) with an appropriate brominating agent, such as benzyl trimethyl ammonium tribromide, in an appropriate solvent, such as DMF.
  • a palladium-based pre-catalyst such as the G3 t- BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473)
  • a base such as Z-BuONa
  • solvent such as 2-methyl-2 -butanol.
  • (Va) can be obtained by a nucleophilic substitution of (IVa) under thermal conditions, using DIPEA as a base, CsF and tetrabutylammonium chloride as additives in a solvent such as acetonitrile.
  • Intermediates compounds of formula (Via) can be obtained from (Va) by reaction with Tf2O in a solvent such as dichloromethane in presence of a base, such as pyridine.
  • Intermediates compounds of formula (VIb) can be obtained from (Va) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent, such as acetonitrile.
  • final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
  • General Scheme 1 final compounds described by formula (II) can be prepared according to, but not limited to, the General Scheme 2.
  • - Final compounds of formula (II) can be obtained from from (VIa) or (VIb) by a Suzuki cross-coupling reaction.
  • Typical, non-limiting conditions may involve the use of Pd(dppf)Cl2.dichloromethane or Pd(PPh3)4 as catalyst, Na2CO3 or K3PO4 as base in 1,4-dioxane or 1,4-dioxane/water as a solvent at an appropriate temperature (e.g., at 100 °C).
  • the final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
  • General Scheme 2 Final compounds described by formula (III) can be prepared according to, but not limited to, the General Scheme 3.
  • Intermediate compounds of formula (IXa) can be synthesized from (Villa) by a reaction with a primary amine derivative by a Buchwald-Hartwig cross-coupling reaction using a palladium-based pre-catalyst, such as the G3 t-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.
  • a palladium-based pre-catalyst such as the G3 t-BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473)
  • a base such as t- BuONa
  • solvent such as 2-methyl-2 -butanol.
  • Intermediate compounds of formula (Xia) can be obtained from (Xa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water, at an appropriate temperature (e.g., at 100 °C).
  • Intermediate compounds of formula (Xlla) can be obtained from (Xia) by removal of the Boc protecting group under acidic conditions, such as HC1 in 1,4- dioxane or TFA.
  • Intermediate compounds of formula (XVa) can be obtained from (XlVa) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent such as acetonitrile.
  • a chlorinating agent such as POCI3
  • an appropriate solvent such as acetonitrile.
  • Intermediate compounds of formula (XVIa) can be obtained from (Xa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3, CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water, at an appropriate temperature (e.g., at 100 °C).
  • Intermediate compounds of formula (Xia) can be obtained from intermediates (XVIa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
  • an appropriate solvent e.g., methanol, ethanol, or ethyl acetate.
  • the benzylic group in (XVIa) can be removed using BBr3.
  • Intermediate compounds of formula (XVIIa) can be obtained from (Xa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3, CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water, at an appropriate temperature (e.g., at 100 °C).
  • Intermediate compounds of formula (XVIIIa) can be synthesized from (Villa) by a reaction with a primary amine derivative by a Buchwald-Hartwig cross- coupling reaction using a palladium-based pre-catalyst, such as the G3 t- BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base, such as Z-BuONa, and a solvent, such as 2-methyl-2-butanol.
  • a palladium-based pre-catalyst such as the G3 t- BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473)
  • a base such as Z-BuONa
  • a solvent such as 2-methyl-2-butanol.
  • Intermediate compounds of formula (XXa) and (XXb) can be obtained from (XIXa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using a palladium-based pre-catalyst such as the G4 cataCxium (Chemistry-A European Journal, 2008, 14, 4267 ⁇ 1279), a base such as CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water at an appropriate temperature (e.g., at 100 °C).
  • the reaction may be performed using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3 in an appropriate solvent, such as 1,4-dioxane/water at an appropriate temperature (e.g., at 100 °C).
  • Intermediate compounds of formulae (XXIa) and (XXIb) can be obtained from intermediates (XXa) and (XXb) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
  • a suitable pressure such as atmospheric pressure or 10 bars
  • an appropriate solvent e.g., methanol, ethanol, or ethyl acetate
  • Intermediate compounds of formulae (XXIIa) and (XXIIb) can be obtained from (XXIa) and (XXIb) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such a sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides.
  • reductive alkylation e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such a sodium cyanoborohydride
  • Final compounds of formula (IV) can be obtained from (XXIIa) or (XXIIb) by removal of the methoxymethyl (XXIIa) or ethoxymethyl (XXIIb) protecting groups in an acidic medium, such as HC1 in 1,4-di oxane.
  • the final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
  • Intermediate compounds of formulae (XXIIIa) and (XXIIIb) can be obtained from (XXa) and (XXb) by a one-pot procedure involving the use of palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate) in presence of polyoxymethylene - homopolymer at a suitable temperature (e.g., room temperature).
  • a suitable pressure such as atmospheric pressure or 10 bars
  • an appropriate solvent e.g., methanol, ethanol, or ethyl acetate
  • Final compounds of formula (V) can be obtained from (XXIIIa) or (XXIIIb) by removal of the methoxymethyl (XXIIIa) or ethoxymethyl (XXIIIb) protecting groups in an acidic medium, such as HC1 in 1,4-di oxane.
  • the final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
  • Intermediate compounds of formula (XXIVa) can be obtained by (XVIIa) by reaction with a methylating agent, such as methyl iodide, after treatment with an appropriate base, such as NaH, in an appropriate solvent, such as DMF at an appropriate temperature, such as room temperature.
  • a methylating agent such as methyl iodide
  • an appropriate base such as NaH
  • an appropriate solvent such as DMF
  • - Intermediate compounds of formula (XXVIIa) can be obtained from (XXVIa) by removal of the Boc protecting group under acidic conditions, such as HCl in 1,4- dioxane or TFA.
  • - Intermediate compounds of formula (XXVIIIa) can be obtained from (XXVIIa) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or another reducing agent, such as NaBH3CN).
  • - Intermediates compounds of formula (XXIXa) can be obtained from (XXVIIIa) by reaction with a chlorinating agent, such as POCl3 in an appropriate solvent such as acetonitrile.
  • Intermediate compound of formula (XXXIa) can be synthesized from Intermediates of formulae (XXXa) or (XXXb) by nucleophilic substitution with hydrazine under thermal conditions in solvents such as ethanol by heating the mixture at an appropriate temperature, such as 90 °C.
  • Intermediate compounds of formula (XXXIIa) can be synthesized from Intermediates of formula (XXXIa) by condensation with an electrophilic carbonyl source, such as methyl chloroformate.
  • an electrophilic carbonyl source such as methyl chloroformate
  • the electrophilic carbonyl source such as methyl chloroformate
  • an appropriate solvent such as dichloromethane
  • the mixture is stirred for an appropriate reaction time, such as 16 hours and then it is concentrated.
  • the condensation can be finalized, for example, by taking up the residue in an appropriate solvent, such as ethanol, and stirring the mixture in presence of an appropriate base, such as KOH, at an appropriate temperature, such as 100 °C.
  • Intermediate compounds of formula (XXXIIIa) can be obtained from (XXXIIa) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent, such as toluene by heating at an appropriate temperature (for example 135 °C).
  • a chlorinating agent such as POCI3
  • an appropriate solvent such as toluene
  • Intermediate compounds of formula (XXXIVa) can be synthesized from (XXXIIIa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using a palladium -based pre-catalyst such as the XPhos Pd G3, an appropriate base such as K3PO4, in an appropriate solvent, such as 1,4- dioxane/water under appropriate non-limiting conditions, such as at 150 °C under microwave irradiation for an appropriate time (e.g., 1 hour).
  • a palladium -based pre-catalyst such as the XPhos Pd G3, an appropriate base such as K3PO4
  • an appropriate solvent such as 1,4- dioxane/water under appropriate non-limiting conditions, such as at 150 °C under microwave irradiation for an appropriate time (e.g., 1 hour).
  • Intermediate compounds of formula (XXXVa) can be obtained from (XXXIVa) in a non-limiting manner by treatment with an appropriate thionation agent, such as phosphorous pentasulfide, in an appropriate solvent, such as pyridine under thermal conditions, (e.g., at 150 °C).
  • an appropriate thionation agent such as phosphorous pentasulfide
  • an appropriate solvent such as pyridine
  • 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 presence of an appropriate base, such as K2CO3, in an appropriate solvent, such as THF/water.
  • an alkylating agent such as bromoethane
  • an appropriate base such as K2CO3
  • an appropriate solvent such as THF/water.
  • Intermediate compounds of formula (XXXVIIa) can be obtained from (XXXVIa) in a non-limiting manner by a nucleophilic substitution of (XXXVIa) with an appropriate amine under thermal conditions (e.g., at a temperature of 130 °C) with the appropriate amine used in excess, for an appropriate reaction time (e.g., 48 hours).
  • Intermediate compounds of formula (XXXVIIIa) can be obtained from (XXXVIa) in a non-limiting manner by a nucleophilic substitution of (XXXVIa) with an appropriate amine under thermal conditions (e.g., at a temperature of 130 °C) with the appropriate amine used in excess, for an appropriate reaction time (e.g., 48 hours).
  • Intermediate compounds of formula (XXXIXa) can be obtained from intermediates (XXXVIIIa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
  • a suitable pressure such as atmospheric pressure or 10 bars
  • an appropriate solvent e.g., methanol, ethanol, or ethyl acetate
  • Intermediates compounds of formula (XLIIa) can be obtained from (XLIa) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides.
  • reductive alkylation e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride
  • nucleophilic substitution on alkyl halides or epoxides e.g., sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride
  • Intermediates compounds of formula (XLIIIa) can be obtained from (XLIa) by nucleophilic substitution of an appropriate alkyl halide in presence of an appropriate base, such as DIPEA, in an appropriate solvent, such as acetonitrile, at an appropriate temperature, such as 85 °C.
  • an appropriate base such as DIPEA
  • an appropriate solvent such as acetonitrile
  • Intermediates compounds of formula (XLIVa) can be obtained from (XXXVIa) in a non-limiting manner by a nucleophilic substitution of (XXXVIa) with an appropriate amine under thermal conditions (e.g., at a temperature of 120 °C) with the appropriate amine used in excess, for an appropriate reaction time (e.g., 96 hours), in an appropriate solvent, such as DMSO, in presence of an appropriate base, such as DIPEA.
  • Intermediate compounds of formula (XLVa) can be obtained from intermediates (XLIVa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
  • a suitable pressure such as atmospheric pressure or 10 bars
  • an appropriate solvent e.g., methanol, ethanol, or ethyl acetate.
  • the instant compounds are potent, 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.
  • 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.
  • compositions comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound as provided herein.
  • the compounds may be formulated into various pharmaceutical forms for administration purposes.
  • compositions there may be cited all compositions usually employed for systemically administering drugs.
  • an effective amount of the particular compound, optionally in salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • a pharmaceutically acceptable carrier which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
  • These pharmaceutical compositions are desirable in unitary dosage form suitable, in particular, for administration orally or by parenteral injection.
  • any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed.
  • the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
  • injectable solutions for example, may be prepared in which the carrier comprises saline solution, 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 employed.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations.
  • the pharmaceutical composition may additionally contain various other ingredients known in the art, for example, a lubricant, stabilising agent, buffering agent, emulsifying agent, viscosity-regulating agent, surfactant, preservative, flavouring or colorant.
  • a lubricant for example, a lubricant, stabilising agent, buffering agent, emulsifying agent, viscosity-regulating agent, surfactant, preservative, flavouring or colorant.
  • Unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.
  • the daily dosage of the compound will, of course, vary with the compound employed, the mode of administration, the treatment desired and the mycobacterial disease indicated. However, in general, satisfactory results will be obtained when the compound is administered at a daily dosage not exceeding 1 gram, e.g. in the range from 10 to 50 mg/kg body weight.
  • pharmaceutical composition refers to a compound as 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.
  • 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, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070) .
  • subject refers to an animal, preferably a mammal, most preferably a human, for example who is or has been the object of treatment, observation or experiment.
  • terapéuticaally effective amount means that amount of compound that elicits a biological or medicinal response in a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.
  • a therapeutically effective amount refers to the amount of the compound that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by NLRP3, or (ii) associated with NLRP3 activity, or (iii) characterised by activity (normal or abnormal) of NLRP3; or (2) reduce or inhibit the activity ofNLRP3; or (3) reduce or inhibit the expression of NLRP3.
  • a therapeutically effective amount refers to the amount of the compound that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reduce or inhibit the activity of NLRP3; or at least partially reduce or inhibit the expression of NLRP3.
  • inhibiting NLRP3 or inhibiting NLRP3 inflammasome pathway comprises reducing the ability ofNLRP3 or 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, inactivating, destabilizing, and/or altering distribution of NLRP3.
  • NLRP3 is meant to include, without limitation, nucleic acids, polynucleotides, oligonucleotides, sense and anti-sense 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.
  • treat refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient.
  • the term “prevent”, “preventing” or “prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.
  • a subject is "in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.
  • a compound for use as a medicament.
  • a compound for use 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 the NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder; in inhibiting NLRP3 inflammasome activity (including in a subject in need thereof); and/or as an NLRP3 inhibitor.
  • NLRP3 activity including inflammasome activity
  • NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder
  • inhibiting NLRP3 inflammasome activity including in a subject in need thereof.
  • a method of treating a disease or disorder in which the NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder comprising administering a therapeutically effective amount of a compound as provided herein, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compound, according to any one of the embodiment described herein), for instance to a subject (in need thereof).
  • a method of inhibiting the NLRP3 inflammasome activity in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a compound as provided herein, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compound, according to any one of the embodiment described herein).
  • the instant compounds may have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the above-stated indications or otherwise.
  • a better pharmacokinetic profile e.g. higher oral bioavailability and/or lower clearance
  • the instant compounds may have the advantage that they have a good or an improved thermodynamic solubility (e.g. compared to compounds known in the prior art; and for instance as determined by a known method and/or a method described herein).
  • the instant compounds may have the advantage that they will block pyroptosis, as well as the release of pro-inflammatory cytokines (e.g. IL-10) from the cell.
  • the instant compounds may also have the advantage that they avoid side-effects, for instance as compared to compounds of the prior art, which may be due to selectivity of NLRP3 inhibition.
  • Compounds as provided herein may also have the advantage that they have good or improved in vivo pharmacokinetics and oral bioavailability. They may also have the advantage that they have good or improved in vivo efficacy.
  • the instant compounds may also have advantages over prior art compounds when compared in the tests outlined hereinafter.
  • the compounds according to the invention can generally be prepared by a succession of steps, each of which may be known to the skilled person or described herein.
  • reaction products may be isolated from the reaction medium and, if necessary, further purified according to methodologies generally known in the art, such as extraction, crystallization, and chromatography. It is further evident that reaction products that exist in more than one enantiomeric form, may be isolated from their mixture by known techniques, in particular preparative chromatography, such as preparative HPLC, chiral chromatography. Individual diastereomers or individual enantiomers can also be obtained by Supercritical Fluid Chromatography (SFC).
  • SFC Supercritical Fluid Chromatography
  • the starting materials and the intermediates are compounds that are either commercially available or may be prepared according to conventional reaction procedures generally known in the art.
  • HPLC High-Performance Liquid Chromatography
  • MS Mass Spectrometer
  • tune parameters e.g. scanning range, dwell time. . .
  • ions nominal monoisotopic molecular weight (MW).
  • Data acquisition was performed with appropriate software.
  • Compounds are described by their experimental retention times (Rt) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H] + (protonated molecule) and/or [M-H]' (deprotonated molecule).
  • SQL Single Quadrupole Detector
  • MSD Mass Selective Detector
  • RT room temperature
  • BEH bridged ethylsiloxane/silica hybrid
  • DAD Diode Array Detector
  • HSS High Strength silica.
  • Chemical shifts (5) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as internal standard.
  • Values are either peak values or melt ranges and are obtained with experimental uncertainties that are commonly associated with this analytical method.
  • Method A For a number of compounds, melting points were determined with a DSC823e (Mettler Toledo) apparatus. Melting points were measured with a temperature gradient of 10 °C/minute. Standard maximum temperature was 300 °C.
  • Method B For a number of 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/minute. Maximum temperature was 300 °C. The melting point data was read from a digital display and checked from a video recording system
  • RS Whenever the notation “RS” is indicated herein, it denotes that the compound is a racemic mixture at the indicated center, unless otherwise indicated.
  • the stereochemical configuration for centers in some compounds has been designated “(R)” or “(5)” when the mixture(s) was separated or originated from enantiomerically pure starting materials; for some compounds, the stereochemical configuration at the indicated centers has been designated as “*(R/’ or when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure.
  • the enantiomeric excess of compounds reported herein was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC) followed by SFC comparison of the separated enantiomer(s).
  • the absolute configuration of chiral centers (indicated as R and/or S) can be rationalized.
  • the synthesis of all final compounds started from intermediates of known absolute configuration in agreement with literature precedent or obtained from appropriate synthetic procedures.
  • the assignment of the absolute configuration of additional stereocenters could then be assigned by standard NMR methods.
  • N,N-Diisopropylethylamine [7087-68-5] (21.5 mL, 102 mmol) was added to a coold, 0 °C, solution of 2-iodo-5-(trifluoromethyl)phenol 1-5 (34 g, 102 mmol) in DCM (1.2 L), followed by dropwise addition of chloromethyl methyl ether [107-30-2] (9.4 mL, 121.8 mmol).
  • the reaction mixture was allowed to gradually warm to rt and react for 18 h.
  • the mixture was concentrated, and the residue was purified by flash column chromatography (silica 330 g; EtOAc in heptane 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to yield l-iodo-2-(methoxymethoxy)-4- (trifluoromethyl)benzene 1-6 (25.5 g, yield: 70%) as a colorless oil.
  • the reaction mixture was stirred for 2 h at 100 °C. The mixture was allowed to cool down to rt. 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), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied onto a silica gel column with petroleum ether. The residue was purified by trituration with n-hexane (300 mL) at -30 °C. The precipitated solids were collected by filtration.
  • 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-l-tritylpyrrolidine-2-carboxylate 1-12 (25.3 g, 65.30 mmol, 1 equiv) in dichloromethane (255 mL) at 0 °C.
  • the reaction mixture was stirred at 0 °C to rt for 16 h.
  • Methyl (25,4A)-4-azido-l-tritylpyrrolidine-2-carboxylate 1-14 (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) at 0 °C under nitrogen atmosphere.
  • the reaction mixture was stirred at 0 °C for 30 min. Afterwards, the reaction mixture was stirred at rt for 3h.
  • the reaction mixture was cooled at 0 °C and water (3.7 mL) was added dropwise.
  • 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 ⁇ L, 1.22 g/mL, 18.55 mmol, 2 equiv) were added to a solution of tert-butyl ((3A,5A)-5-fluoropiperidin-3-yl)carbamate 1-18 (2 g, 9.16 mmol, 1 equiv) in dry 2-methyl-THF (45.0 mL). The resulting colourless solution was stirred at room temperature for 1.5 h and then, heated at 80 °C for 2.5 h.
  • Phosphorus oxychloride [10025-87-3] (5.6 mL, 59.55 mmol) was added dropwise over 15 min to a stirred suspension of pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-28 (1.8 g, 11.91 mmol), DIPEA [7087-68-5] (4.2 mL, 23.82 mmol) and distilled water [7732-18- 5] (279 ⁇ L, 15.48 mmol) in toluene (41 mL) at rt. The suspension was stirred at 135 °C for 8 h in a sealed glass reactor.
  • reaction mixture was stirred at 105 °C overnight.
  • the mixture was concentrated and the residue was purified by flash column chromatography (silica 80 g; dry load in celite; EtOAc in heptane 0/100 to 30/70).
  • the desired fractions were collected and concentrated in vacuo.
  • the product was triturated with heptane to yield to yield l-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)pyrrolo[l,2-d][l,2,4]triazin-4(3H)-one 1-30 (3.9 g, yield: 65%) as a white foamy solid.
  • Phosphorous pentasulfide (8.7 g, 38.73 mmol, 2.5 eq.) was added to a stirred solution of 1 -(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin-4(3H)-one 1-30 (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 phosphorous pentasulfide (4.35 g, 19.37 mmol, 1.25 eq.). The mixture was stirred at 150 °C for 8 h.
  • Trifluoroacetic acid [76-05-1] (1.43 mL, 19 mmol) was added dropwise to a stirred solution of tert-butyl (R)-3-((l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[l,2- d][l, 2, 4]triazin-4-yl)amino)piperi dine- 1 -carboxylate 1-33 (150 mg, 0.26 mmol) in DCM anhydrous (1.5 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at rt for 1 h.
  • the reaction was recharged with 2-(2- Bromoethoxy)tetrahydro-2h-pyran [17739-45-6] (1.2 eq, 50 ⁇ L, 0.31 mmol) and DIPEA [7087-68-5] (6 eq, 0.28 mL, 1.57 mmol)
  • the reaction mixture was cooled down to rt, diuted with sat. NaHCCf aqueous solution and extracted with EtOAc (x3).
  • the combined organic layers were dried (MgSCU), filtered and solvents evaporated in vacuo.
  • the crude was purified by flash column chromatography (silica 20 g, DCM/MeOH (9: 1) in DCM 0/100 to 35/65).
  • the yellow oil was subjected to silica gel chromatography (12 g irregular 40-60 um; 0-3% MeOH/DCM) to give (R)- l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(l-cy cl opropylpiperi din-3 - yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine 1-50 (203 mg, 84%) as a brown foam solid.
  • reaction mixture was stirred at 60 °C for 2h.
  • the reaction mixture was concentrated in vacuo, taken up in sat. aqueous NaHCO3 solution, extracted with EtOAC (x3).
  • the combined organic phases were washed with brine, dried on MgSCU, filtered, and evaporated under reduced pressure.
  • HC1 (4M in dioxane) (2.36 mL, 4 M, 9.42 mmol) was added to a solution of tert-butyl 6-((4-oxo-4,5-dihydropyrazolo[l,5-d][l,2,4]triazin-7-yl)amino)-l,4-oxazepane-4- carboxylate 1-57 (330 mg, 0.942 mmol) in methanol (4 mL). The reaction mixture was stirred at rt until complete conversion. The reaction mixture was concentrated under reduced pressure to give crude product (HC1 salt). The free amine was obtained by solid phase extraction using Si-Propylsulfonic acid SCX-2 resin (SiliCycle).
  • Method 3 A reaction flask was charged with (R)-N-(l-benzylpiperi din-3 -yl)-4- chloropyrazolo[l,5-d][l,2,4]triazin-7-amine 1-75 (2.0 g, 5.83 mmol), 2-(2- (methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-7 (2.13 g, 6.42 mmol), potassium phosphate tribasic [7778-53-2] (3.72 g, 17.50 mmol), Pd(dppf)C12 DCM [95464-05-4] (357 mg, 0.44 mmol), 1,4-dioxane (40 mL) and water (4 mL).
  • the reaction mixture was bubbled with nitrogen and stirred at 90°C for 4 hours.
  • 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 on MgSO4, filtered, and concentrated.
  • the residue was purified on a column with silica gel, eluent EtOAc in Heptane, from 0 to 100 %.
  • iodomethane [74-88-4] 100 ⁇ L, 2.28 g/mL, 1.6 mmol, 3.6 equiv was added and then the reaction mixture was allowed to stir at rt for 2.5 h. The reaction mixture was quenched with MeOH and concentrated in vacuo.
  • the crude was purified by silicagel column chromatography (gradient elution: 0 to 2% MeOH in DCM) to deliver tert- butyl (A > )-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[l,5- d][l,2,4]triazin-7-yl)(methyl)amino)piperidine-l-carboxylate 1-103 (64 mg, yield 24%).
  • Trifluoroacetic acid [76-05-1] (11.5 mL, 153.5 mmol) was added dropwise to a stirred solution of tert-butyl (A)-3-((4-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[l,5- d][l,2,4]triazin-7-yl)amino)piperidine-l-carboxylate 1-100 (380 mg, 0.80 mmol) in DCM anhydrous (11.5 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at rt for 1.5 h at rt.
  • HC1 (4M in 1,4-dioxane) [7647-01-0] (1.12 mL, 4M, 4.47 mmol) was added to a solution of tert-butyl (A)-3-((4-(2-hydroxy-4-methylphenyl)pyrazolo[l,5- d][l,2,4]triazin-7-yl)amino)piperidine-l-carboxylate 1-91 (100 mg, 0.24 mmol) in 1,4- dioxane (1.13 mL) and the mixture was stirred at rt for 2 h. The mixture was poured out in sat. NaHCCh solution and extracted three times with EtOAc.
  • 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, consisting of ethyl 4-methyl-lH- pyrazole-3 -carboxylate [6076-12-6] (5 g, 32.4 mmol) in ethanol (80 mL) at room temperature under nitrogen atmosphere to give colourless solution. After sealing the reactor, the mixture was stirred at 90 °C for 18 h and then, allowed to rt.
  • 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, consisting of 4-methyl-lH- pyrazole-5-carbohydrazide 1-120 (4.94 g, 35.25 mmol) and DMF (35 mL) at room temperature to give a colourless solution.
  • a stirred solution consisting of 4-methyl-lH- pyrazole-5-carbohydrazide 1-120 (4.94 g, 35.25 mmol) and DMF (35 mL) at room temperature to give a colourless solution.
  • the mixture was stirred at 165 °C for 16 h.
  • the mixture was allowed to cool to give a brown solution.
  • the mixture was co-distilled wit toluene, 5 times, to give a pale brown slurry.
  • Formaldehyde solution [50-00-0] (4 mL, 53.73 mmol) followed by formic acid [64-18- 6] (2 mL, 53.03 mmol) were added to a solution of tert-butyl (R)-piperi din-3 - ylcarbamate [309956-78-3] (5 g, 24.97 mmol) in 2-methyltetrahydrofuran (50 mL). The resulting solution was stirred 1 h at rt, then Ih at 80 °C.
  • the yellow sticky solid was subjected to silica gel chromatography (80 g of yellow sticky solid, 0-8% MeOH/DCM) to afford (7?)-3-methyl-7-((l-methylpiperidin-3-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4(5H)-one 1-125 (420 mg, yield: 24 %) as a yellow oil.
  • Phosphorus oxychloride [10025-87-3] (1.2 mL, 12.84 mmol) were added to a solution consisting of (R)-3-methyl-7-((l-methylpiperidin-3-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4(5H)-one 1-125 (420 mg, 1.6 mmol)and acetonitrile anhydrous (12 mL) at rt in a 35 mL screw-cap vial under N2 atmosphere. The mixture was sparged with N2 for 10 min and then stirred at 90 °C for 18 h.
  • 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)C12- dichloromethane [95464-05-4] (94.4 mg, 0.12 mmol, 12 mol%) and K2CO3 [584-08-7] (399 mg, 2.89 mmol, 3 equiv) and flushed with nitrogen (3 vacuum/nitrogen cycles).
  • the resulting mixture was purged with nitrogen and stirred at 90 °C for 3 hours.
  • the mixture was purified by solid phase extraction using Si-Propylsulfonic acid SCX-2 resin (SiliCycle).
  • the crude reaction mixture was transfered to a column loaded with Si-Propylsulfonic acid SCX-2 resin (SiliCycle).
  • the column was first eluted with MeOH after which the desired product was released by elution with ammoniated methanol (7 N). Tubes containing the product were concentrated under reduced pressure.
  • the column was first eluted with methanol.
  • the desired product was released by elution with 7 N NH3/methanol. Tubes containing the desired product were concentrated under reduced pressure.
  • the residue was purified using by silica gel column chromatography (gradient elution: 0 to 3% methanol in dichloromethane) to give (R)-2-(7-((l-(2-fluoroethyl)piperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC-21 (85 mg, yield 56%).
  • FC-32 (100 mg, 0.25 mmol) was purified by SFC to isolate both chiral products. SFC purification was made using a i-amylose-1 column with ISOC 30% Ethanol + 0.1% diethylamine to collect and concentrate to yield FC-33 (25.9 mg, yield: 25%) as a yellow foam solid and FC-34 (21.4 mg, yield: 21%) as a yellow foam solid. The absolute configuration was not determined.
  • FC-11 (92 mg, 0.22 mmol) was purified by SFC to isolate both chiral products. SFC purification was made using a i-amylose-1 column with ISOC 25% 2-propanol + 0.1% diethylamine to yield 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 configuration was not determined.
  • FC-39 (38 mg, 0.09 mmol) was purified by SFC to isolate both chiral products.
  • Stationary phase Amylose-1 250 x 30 mm 5 pm.
  • Mobile phase 70% CO2 - 30% EtOH + 0.1 % diethylamine. The fractions were collected and concentrated in vacuo to yield FC-41, first eluting enantiomer, (11 mg, 0.03 mmol) as a dark yellow solid and FC-42, second eluting enantiomer, (12 mg, 0.03 mmol) as a dark yellow solid.
  • the absolute configuration was not determined.
  • FC-38 (141 mg, 0.33 mmol) was purified by SFC to isolate both chiral components. Stationary phase: Amylose-1 250 x 30 mm 5 pm, Mobile phase: 60% CO2 - 40% MeOH + 0.1 % diethylamine. The fractions were collected, concentrated in vacuo, and triturated with diisopropyl ether to yield FC-43, first eluting enantiomer, (57 mg, 0.13 mmol) as a yellow solid and FC-44, second eluting enantiomer, (57 mg, 0.13 mmol) as a yellow solid. The absolute configuration was not determined.
  • the mixture was stirred at rt for 16 h.
  • the reaction was recharged with triethylamine [121-44-8] (3 eq, 114 ⁇ L, 0.81 mmol), Formaldehyde (37% aqueous solution) [50-00-0] (2 eq, 34 ⁇ L, 0.46 mmol) and sodium triacetoxyborohydride [56553-60-7] (1.5 eq, 76 mg, 0.35 mmol) and it was stirred at rt for 32 h more.
  • the mixture was diluted with NaHCCL (sat. aq) and extracted with DCM.
  • FC-16 (150 mg, 0.37 mmol) was purified by SFC to isolate both chiral products. SFC conditions: Stationary phase: Chiralpak Diacel AD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2. FC-47, first eluting enantiomer, (58 mg, yield: 77%) and FC-48, second eluting enantiomer, (57 mg, yield: 76%) were obtained.
  • Step 1 A solution of 4-chloro-N-(octahydroindolizin-l-yl)pyrazolo[l,5-d][l,2,4]triazin-7- amine, (200 mg, 0.68 mmol) 1-76 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.
  • Step 2 The residue was purified by preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-lOpm, 50x150mm, Mobile phase: 0.25% NH4HCO3 solution in water, CH3CN) to yield the two diastereomers.
  • the first eluting diastereomer was further purified by preparative chiral SFC (Stationary phase: Chiralcel Diacel OD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to yield the optically pure TRANS enantiomers 2-(7-(((lS,8aR)-octahydroindolizin-l-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((lR,8aS)- octahydroindolizin- 1 -yl)amino)pyrazolo[ 1 , 5-d] [ 1 ,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol (FC-49 and FC-50).
  • the compound was purified by preparative chiral SFC (Stationary phase: Chiralcel Diacel OD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to yield 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[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-53 and FC- 54).
  • the absolute configuration was not determined. 51 mg (80% yield, for the separation) were obtained for the first eluting enantiomer and 51 mg (80% yield, for the separation
  • the crude reaction mixture was transferred to a column loaded with Si-Propyl sulfonic acid SCX-2 resin (SiliCycle) and eluted with MeOH.
  • the product compound was released by elution with 7 N NH3/MeOH.
  • the tubes containing the product compound were combined and concentrated under reduced pressure.
  • a compound of the invention for instance, a compound of the examples
  • a pharmaceutically acceptable carrier for instance, a compound of the examples
  • a therapeutically effective amount of a compound of the invention is intimately mixed with a pharmaceutically acceptable carrier, in a process for preparing a pharmaceutical composition.
  • a compound according to the present invention exhibits valuable pharmacological properties, e.g. properties susceptible to inhibit NLRP3 activity, for instance as indicated the following test, and are therefore indicated for therapy related to NLRP3 inflammasome activity.
  • PBMCs peripheral blood mononuclear cells
  • Ficoll-Histopaque Sigma- Aldrich, A0561 density gradient centrifugation. After isolation, PBMCs were stored in liquid nitrogen for later use. Upon thawing, PBMC cell viability was determined in growth medium (RPMI media supplemented with 10% fetal bovine serum, 1% Pen- Strep and 1% L-glutamine). Compounds were spotted in a 1 :3 serial dilution in DMSO and diluted to the final concentration in 30 pl medium in 96 well plates (Falcon, 353072).
  • PBMCs peripheral blood mononuclear cells
  • LPS stimulation was performed by addition of 100 ng/ml LPS (final concentration, Invivogen, tlrl-smlps) for 6 hrs followed by collection of cellular supernatant and the analysis of IL-ip (pM), IL6 and TNFa cytokines levels (pM) via MSD technology according to manufacturers’ guidelines (MSD, K151A0H).
  • IC50 values for IL-ip
  • EC 50 values IL6 and TNFa
  • the objective of this assay is to measure the permeability and efflux of test compounds, using MDCK cells transfected with the P-glycoprotein (MDR1). Two control compounds are screened alongside the test compounds, propranolol (highly permeable) and prazosin (a substrate for P-glycoprotein).
  • MDCK cells are an epithelial cell line of canine kidney origin. These cells can be stably transfected to express active P-glycoprotein (MDR1- MDCK) and are ideal for studying drug efflux due to P-gp.
  • Test compound is added to either the apical or basolateral side of a confluent monolayer of MDR1-MDCK cells and permeability in the apical to basolateral (A-B) and basolateral to apical (B-A) direction is measured by monitoring the appearance of the test compound on the opposite side of the membrane using LCMS/MS.
  • Efflux ratios (B-A permeability over A-B permeability) are calculated to determine if the test compound is subject to P-gp efflux.
  • Papp apparent permeability
  • One or more compound(s) may be tested in a number of other assays to evaluate, amongst other properties, permeability, stability (including metabolic stability and blood stability) and solubility. hERG inhibition
  • the whole cell patch clamp technique on transfected cells allows the study of ion channels with no - or limited interference from other ion-channels.
  • the effect of compounds on the hERG current are studied with an automated planar patch clamp system, SyncroPatch 384PE (as described in Obergrussberger, A., Briiggemann, A., Goetze, T.A., Rapedius, M., Haarmann, C., Rinke, I., Becker, N., Oka, T., Ohtsuki, A., Stengel, T., Vogel, M., Steindl, J.,
  • the SyncroPatch 384PE is an automated patch clamp system which allows to conduct parallel recordings from 384 wells.
  • the module is incorporated in a liquid handling pipetting robot system, Biomek FXP, for application of cells and compounds.
  • voltage protocols are constructed, and data acquired using PatchControl384 and analyzed using DataControl384 (both Nanion Technologies).
  • hERG current is determined as the maximal tail current at -30 mV and percent inhibition upon compound addition as well as pICso are reported below.
  • liver microsomes 0.5 mg/ml protein
  • preclinical species incubated up to 60 minutes at 37°C with 1 pM test compound.
  • Fine incubation volume
  • liver hepatocytes (1 milj cells) from human and preclinical species incubated up to 120 minutes at 37°C with 1 pM test compound.
  • the in vitro intrinsic clearance (Clint) (pl/min/million cells) is calculated using the following formula:
  • # cellS/n C number of cells (xlO 6 ) in the incubation
  • Test compound is prepared in species specific plasma (diluted to 25% plasma in buffer). The plasma solution is added to one side of the membrane in an equilibrium dialysis system while buffer (pH 7.4) is added to the other side. The system is allowed to reach equilibrium at 37 °C. Compound on both sides of the membrane is measured by LC-MS/MS and the fraction of unbound compound is calculated. We deliver the fraction unbound in plasma (fu) for each test compound, along with percentage recovery.
  • Plasma from the following strains and species combinations will be used:
  • test compound (1 pM test compound concentration; 0.5 % final DMSO concentration) are prepared in species specific plasma diluted to 25% plasma with buffer. The experiment is performed using equilibrium dialysis with the two compartments separated by a semi-permeable membrane. 500 pL of buffer (pH 7.4) is added to one side of the membrane and 300 pL of the plasma solution containing the test compound is added to the other side. After equilibration for 6 hr at 37°C in an incubator with 5% CO2 and agitation at 250 rpm on an orbital shaker, samples are taken from both sides of the membrane.
  • buffer pH 7.4
  • Samples are matrix matched by addition of either buffer or diluted plasma to relevant samples (i.e. 45 pL of buffer is added to 45 pL of the plasma samples and 45 pL of diluted plasma (25%) is added to 45 pL of the buffer samples). Protein is then precipitated from the matrix-matched samples by addition of 180 pL of methanol containing internal standard followed by centrifugation at 4 °C at 2500 rpm for 30 min. Supernatant (20 pL per compound x 4 compounds) is then diluted with water (100 pL) prior to analysis. Test compound incubations are performed in triplicate. Two control compounds, as specified in the guidance to vendor document, are included in each experiment.
  • BufferF Final Buffer compartment concentration (after dialysis)
  • PlasmaF Final Plasma compartment concentration (after dialysis)
  • the fraction unbound in plasma (fu) and percent recovery is returned in the form of an Excel spreadsheet.
  • the sheet will contain an indication whether the data should be further scrutinized by an internal Janssen Reviewer (based on pre-defined rules supplied in the Janssen guidance document) along with any relevant comments.
  • the objective of this study is to determine brain tissue bindings of test compound(s) in rat and mouse brain tissue using Equilibrium Dialysis Method.
  • the peak area ratios of test compound(s) in brain tissue homogenate and buffer are evaluated by LC-MS/MS.
  • Control compounds verapamil and fluoxetine are purchased from Sigma Chemical Co.
  • Control compound venlafaxine is purchased from MedChemExpress LLC.
  • Na2HPO4, NaH2PO4 and NaCl are purchased from local supplier.
  • Acetonitrile and methanol are purchased from Merck (Darmstadt, Germany).
  • Other reagents are purchased from local supplier.
  • Brain tissue homogenate is prepared by diluting one volume of the whole brain tissue with nine volumes of buffer (PBS, pH 7.4), and the mixture is homogenized using a tissue homogenate machine. Brain tissue homogenate is frozen at -80 °C prior to use. Usually, the brain tissues from three or more individual animals are pooled.
  • Assemble the 48-well RED device apparatus Add 500 pL of PBS to the buffer side of the designated wells. Add 300 uL of spiked brain homogenate immediately to the opposite sides of the designated wells. The assay was performed triplicate. Seal the RED device and place the device in an incubator at 37°C with 5% CO2 at 150 RPM for 6 hours. At the end of incubation, remove the seal and pipette 50 pL of samples from both buffer and brain tissue homogenate chambers into separate wells of a new 96-well plate.
  • test compound(s) and control compound in the buffer and brain tissue homogenate chambers from peak area ratios. Calculate the percentages of test compound(s) and control compound bound as follows:
  • Fu ⁇ FUapp brain D+Fu app -(D*Fu app )
  • Rat Sprague Dawley Wistar Supplier: Charles River Germany
  • Sampling site At each time point, animals are sacrificed by decapitation and blood is collected by exsanguination into capillaries in case of micro sampling and otherwise in BD vacutainers. Blood samples are placed immediately on melting ice and plasma is obtained following centrifugation at 4° C for 10 minutes at approximately 1900 x g.
  • Amount Micro sampling: 32 pl on EDTA
  • rat serial blood sampling is performed through the tail vein.
  • Selection of sampling method depends of the amount of plasma needed for bioanalysis.
  • Collection tubes Super Polyethylene vial 20 ml Perkin Elmer (REF.
  • Homogenization tissue Tissue samples were homogenised in demineralised water (1/9 w/v or + 3 ml if tissue weight ⁇ 0.33 g). Homogenisation is carried out under dimmed light conditions.
  • Body weight loss > 20 % body temperature is checked when animals are in sub optimal condition, mobility, changed behavior, pain expression.
  • body temperature ⁇ 33 °C animals will be euthanized and excluded from the experiment.
  • the veterinarian physician will be consulted and he/she decide of the fate of these animals. Deviations will be registered in the amendments of the study file.
  • 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 dosing (time 0) to the time of the last measured concentration respectively in the brain and in the plasma
  • BTB,r is the brain tissue binding, as defined above, in rat
  • PPB,m is the plasma protein binding, as defined above, in mouse Human whole blood assay
  • 125 pl of undiluted blood was added to each well of a 96-well plate, followed by the administration of 25 pl of lipopolysaccharide (LPS E. Coli, L4130, Sigma-Aldrich) at a concentration of 30 ng/ml.
  • LPS E. Coli lipopolysaccharide
  • Blood was primed with LPS for one hour at 37°C before compound dilutions (dose-response, 25 pl/well) were added for 30 minutes at 37°C.
  • the NLRP3 pathway was activated by adding 25 pl of BzATP (A-385, Alomone Labs) to each well at a concentration of ImM.
  • mice blood of several mice was pooled (approximately 300 pl) before adding 75 pl of undiluted mouse blood to each well of a 96-well plate.
  • the NLRP3 pathway was primed by adding 25 pl of LPS (1 pg/ml) to each well for a duration of three hours at 37°C. Compounds were added at different concentrations (dose- response) and incubated for 30 minutes at 37°C before activation of the pathway with BzATP (5 mM, 25 pl/well) for 1 hour.
  • Solubility Assay An aliquot of a DMSO solution containing the test compound is dispensed in a 96-well plate, the DMSO is evaporated, and the pellet is re-dissolved by adding the buffer. The compound solubility in pH 2.0 or 7.0 buffer is measured after three days of agitation at 25 °C. The samples are centrifuged, and the super-natant is filtered. The filtrates are pooled, and the concentration is measured by liquid chromatography/tandem mass spectroscopy (LC-MS/MS). An assessment of the solid-state character of the residues is conducted by polarized light microscopy (PLM).
  • PLM polarized light microscopy
  • the phospholipidogenic potential of the compounds was assessed according to a reported procedure (Mesens, N.; Steemans, M.; Hansen, E.; Peters, A.; Verheyen, G.; Vanparys, P. A 96-well flow cytometric screening assay for detecting in vitro phospholipidosisinduction in the drug discovery phase, Toxicology in Vitro 23, (2009), 217-226. Data are reported as concentration showing a 2-fold increase in fluorescence.
  • CHI LogD Chromatography Hydrophobicity Index (CHI) CHI LogD, also referred as ChromLogD in the literature, values were determined for the compounds of the invention.
  • ChromLogD Chromatography Hydrophobicity Index

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Abstract

Provided are compounds for use as inhibitors of the NLRP3 inflammasone pathway, wherein such compounds are as defined by compounds of formula (I) and wherein the radicals R1, R2, R3, R4 and R5 are defined in the description, and where the compounds may be useful as medicaments, for instance for use in the treatment of a disease or disorder that is associated with NLRP3 inflammasome activity.

Description

PYRROLO[l,2-d][l,2,4]TRIAZINES AND PYRAZOLO[l,5-d]
[1,2,4] TRIAZINES AS NLRP3 INHIBITORS
TECHNICAL FIELD
Described herein are pyrrolo[l,2-d][l,2,4]triazines and pyrazolo[l,5- d][l,2,4]triazines that are useful as inhibitors of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway. Also described herein are processes for the preparation of said compounds, pharmaceutical compositions comprising said compounds, methods of using said compounds in the treatment of various diseases and disorders mediated by the NLRP3 inflammasome pathway.
BACKGROUND
Inflammasomes, considered as central signalling hubs of the innate immune system, are multi-protein complexes that are assembled upon activation of a specific set of intracellular pattern recognition receptors (PRRs) by a wide variety of pathogen- or danger- associated molecular patterns (PAMPs or DAMPs). To date, it was 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). The NLRP3 inflammasome is assembled 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-P fibrils that are neurotoxic in Alzheimer’s disease and asbestos particles that cause mesothelioma (Kelley et al., IntJMol Sci, 2019 Jul 6;20(13)). Additionally, NLRP3 is activated by infectious agents such as Vibrio cholerae, fungal pathogens such as Aspergillus fumigatus and Candida albicans,' adenoviruses, influenza A virus and SARS-CoV-2 (Tartey and Kanneganti, 2019; Fung et al. Emerg Microbes Infect, 2020 Mar 14;9(l):558-570).
Although the precise NLRP3 activation mechanism remains unclear, for human monocytes, it has been suggested that a one-step activation is sufficient while in mice a two-step mechanism is in place. Given the multitude in triggers, the NLRP3 inflammasome requires add-on regulation at both transcriptional and post- transcriptional level (Yang Y et al., Cell Death Dis, 2019 Feb 12; 10(2): 128).
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 C- terminal end (Sharif et al., Nature, 2019 Jun; 570(7761):338-343). Upon recognition of PAMP or DAMP, NLRP3 aggregates with the adaptor protein, apoptosis-associated speck-like protein (ASC), and with the protease caspase- 1 to form a functional inflammasome. Upon activation, procaspase-1 undergoes autoproteolysis and consequently cleaves gasdermin D (Gsdmd) to produce the N-terminal Gsdmd molecule that will ultimately lead 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-ip and pro-IL-18 to allow release of its biological active form by pyroptosis (Kelley et al., 2019).
Dysregulation of the NLRP3 inflammasome or its downstream mediators are associated with numerous pathologies ranging from immune/inflammatory diseases, auto-immune/auto-inflammatory diseases (Cryopyrin-associated Periodic Syndrome (Miyamae T. Paediatr Drugs, 2012 Apr 1; 14(2): 109-17); sickle cell disease; systemic lupus erythematosus (SLE)) to hepatic disorders (e.g. non-alcoholic steatohepatitis (NASH), chronic liver disease, viral hepatitis, alcoholic steatohepatitis, non-alcoholic 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 (eg. Crohn’s disease, ulcerative colitis) (Zhen Y and Zhang H. Front Immunol, 2019 Feb 28; 10:276). Also, inflammatory joint disorders (e.g. gout, pseudogout (chondrocalcinosis), arthropathy, osteoarthritis, and rheumatoid arthritis (Vande Walle L et al., Nature, 2014 Aug 7;512(7512):69-73) were linked to NLRP3. Additionally, 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 related inflammation and diabetic nephropathy which is a kidney -related complication of diabetes (Type 1, Type 2 and mellitus diabetes), also called diabetic kidney disease (Shahzad et al., Kidney Int, 2015 Jan;87(l):74-84) are associated to NLRP3 inflammasome activation. Reports link onset and progression of neuroinflammation-related disorders (e.g. brain infection, acute injury, multiple sclerosis, Alzheimer's disease) and neurodegenerative diseases (Parkinsons disease) to NLRP3 inflammasome activation (Sarkar et al., NPJ Parkinsons Dis, 2017 Oct 17;3:30). In addition, 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 artery 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) have recently been associated to NLRP3. Also, skin associated diseases were described (e.g. wound healing and scar formation; inflammatory skin diseases, eg. 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, but also age-related macular degeneration (Doyle et al., Nat Med, 2012 May;18(5):791-8). Several cancer related diseases/disorders were described linked to NLRP3 (e.g. myeloproliferative neoplasms, leukemias, myelodysplastic syndromes (MOS), myelofibrosis, lung cancer, 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(l):57-62).
Several patent applications describe NLRP3 inhibitors, with recent ones including for instance WO-2020/234715, WO-2021/193897, WO-2022/135567, US- 11,319,319, WO-2023/278438.
There is a need for inhibitors of the NLRP3 inflammasome pathway for example to study neurodegenerative disorders such as Alzheimer’s Disease.
SUMMARY
Described herein are compounds which inhibit the NLRP3 inflammasome pathway.
In some embodiments, provided herein are compounds of Formula (I), or pharmaceutically acceptable salts thereof, wherein
In another aspect, there are provided compounds for use as a medicament. In another aspect, there are provided pharmaceutical compositions comprising a therapeutically effective amount of a compound provided herein.
In a further aspect, there are provided compounds and pharmaceutical compositions comprising such compounds for use in the treatment of a disease or disorder mediated by the NLRP3 inflammasome pathway, for example neurodegenerative disorders such as Alzheimer’s Disease.
In another aspect, there is provided the use of the instant compounds in the manufacture of a medicament for the treatment of a disease or disorder mediated by the NLRP3 inflammasome pathway, for example neurodegenerative disorders such as Alzheimer’s Disease.
In another aspect, there is provided a method of treating a disease or disorder mediated by the NLRP3 inflammasome pathway, for example neurodegenerative disorders such as Alzheimer’s Disease. In a further aspect there is provided a method of inhibiting the NLRP3 inflammasome activity in a subject (in need thereof), the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound as provided herein.
DETAILED DESCRIPTION
Provided herein are compounds of formula (I) or pharmaceutically acceptable salts thereof, wherein A is N or CH;
R7 and R8 each independently are H, CH3 or F.
In an embodiment, provided herein are compounds of formula (I) or pharmaceutically acceptable salts thereof, wherein with the proviso that the compound is not
In some particular embodiments, provided herein are compounds of Formula (I), having Formula (I- A) , wherein
In some particular embodiments, provided herein are compounds of Formula (I), having F ormula (I- A)
R7 and R8 each independently are H, CH3 or F, with the proviso that the compound is not
In some embodiments, provided herein are compounds of Formula (I), having Formula wherein with the proviso that a) R6 is selected from CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3, when R7 and R8 each independently are H, CH3 or F; and b) R6 is CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3, when R7 is CH3 or F; and R8 is H, CH3 or F. In some embodiments, provided herein are compounds of Formula (I), having Formula
R7 and R8 each independently are H, CH3 or F; with the proviso that a) R6 is selected from CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3, when
R7 and R8 each independently are H, CH3 or F; and b) R6 is CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F, CD3, when
R7is CH3 or F; and
R8 is H, CH3 or F; with the proviso that the compound is not is H, CH3 or F, and R8 is H, or both R7 and R8 are F.
In a further embodiment wherein R6 is CH3 or CH2CH3; R7is CH3 or F, and R8 is H, or both R7 and R8 are F.
In a further embodiment wherein R6 is CH2CH3; R7 is H, CH3 or F, and R8 is H, or both R7 and R8 are F.
In an embodiment In another embodiment,
In an embodiment R1 is wherein a) R6 is CH3 or CH2CH3; R7 is H, CH3 or F, and R8 is H, or both R7 and R8 are F; or b) R6 is CH3 or CH2CH3; R7 is CH3 or F, and R8 is H, or both R7 and R8 are F; or c) R6 is CH2CH3; R7 is H, CH3 or F, and R8 is H, or both R7 and R8 are F; or d) R6 is CH3 or CH2CH3; and both R7 and R8 are F; or
In an embodiment R2, R4 and R5 are hydrogen.
In an embodiment, R2 is hydrogen and R3 is CF3.
In an embodiment R3 is CF3.
In an embodiment R6 is CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F or CD3.
In an embodiment R6 is CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F or CD3.
In an embodiment, provided herein are compounds of Formula (I), as described as final compounds in the examples, in particular, final compounds 2-8, 10-17, 19-61.
Pharmaceutically-acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound as provided herein with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound provided herein in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. 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.
Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
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, the 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.
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, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.
The instant compounds may contain double bonds and may thus exist as E (entgegeri) and Z (ziisammeri) geometric isomers about each individual double bond.
Compounds as provided herein may contain one or more asymmetric carbon atoms and may therefore exhibit enantiomerism or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired isomers may be made by reaction of an appropriate enantiomeric starting material under conditions which will not cause racemisation or epimerisation, or by reaction of an appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by resolution, including dynamic resolution, for example salt formation with a homochiral acid followed by separation of the diastereomeric salts by conventional means such as crystallization, or by reaction with an appropriate chiral reagent or chiral catalyst.
In the structures shown herein, where the stereochemistry of any particular chiral atom is not specified, then all stereoisomers are contemplated. Where stereochemistry is specified by a solid or dashed wedge representing a particular configuration, then that stereoisomer is so specified and defined.
Absolute configurations are specified according to the Cahn-Ingold-Prelog system. The configuration at an asymmetric atom is specified by either R or S. Resolved compounds whose absolute configuration is not known can be designated by (+) or (-) depending on the direction in which they rotate polarized light. When a specific stereoisomer is identified, this means that said 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%, in particular less than 2% and most preferably less than 1%, of the other isomers. Thus, when a compound of formula (I) is for instance specified as (R), this means that the compound is substantially free of the (S) isomer. The compounds may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. Also provided herein are isotopically-labelled compounds wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (or the most abundant one found in nature). Exemplary isotopes include isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, such as 2H, 3H, 11C, 13C, 14C , 13N, 15O, 17O, 18O, and 18F. Tritiated (3H) and carbon-l4 (14C) isotopes are useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium may afford therapeutic advantages resulting from greater metabolic stability. Isotopes such as 15O, 13N, 11C and 18F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labelled compounds can generally be prepared by following procedures analogous to those disclosed in the Examples hereinafter.. Unless otherwise specified, C1-q alkyl groups (where q is the upper limit of the range) defined herein may be straight-chain or be branched-chain. C3-q cycloalkyl (where q is the upper limit of the range) refers to an alkyl group that is cyclic, for instance cycloalkyl groups may be monocyclic or, if there are sufficient atoms, bicyclic. In an embodiment, such cycloalkyl groups are monocyclic. Substituents may be attached at any point on the cycloalkyl group.. The term “halo”, when used herein, preferably includes fluoro, chloro, bromo and iodo. C1-q alkoxy groups (where q is the upper limit of the range) refers to the radical of formula -ORa, where Ra is a C1-q alkyl group as defined herein. HaloC1-q alkyl (where q is the upper limit of the range) groups refer to C1-q alkyl groups, as defined herein, where such group is substituted by one or more halo. HydroxyC1-q alkyl (where q is the upper limit of the range) refers to C1-q alkyl groups, as defined herein, where such group is substituted by one or more (e.g. one) hydroxy (-OH) groups (or one or more, e.g. one, of the hydrogen atoms is replaced with -OH). Similarly, haloCi-q alkoxy and hydroxyCi-q alkoxy represent corresponding -OCi-q alkyl groups that are substituted by one or more halo, or, substituted by one or more (e.g. one) hydroxy, respectively.
The names of the compounds were generated according to the nomenclature rules agreed upon by the Chemical Abstracts Service (CAS) using Advanced Chemical Development, Inc., software (ACD/Name product version 10.01; Build 15494, 1 Dec 2006) or according to the nomenclature rules agreed upon by the International Union of Pure and Applied Chemistry (IUPAC) using Advanced Chemical Development, Inc., software (ACD/Name product version 10.01.0.14105, October 2006). In case of tautomeric forms, the name of the depicted tautomeric form of the structure was generated.
The instant compounds can generally be prepared by a succession of steps, each of which is known to the skilled person. In particular, the compounds can be prepared according to the following synthesis methods.
The compounds of Formula (I) may be synthesized in the form of racemic mixtures of enantiomers which can be separated from one another following 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. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkalination. An alternative manner of separating the enantiomeric forms of the compounds of Formula (I) involves liquid chromatography using a chiral stationary phase or a chiral supercritical fluid chromatography (SFC). Said 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. The absolute configuration of the compounds reported herein was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC) followed by SFC comparison of the separate enantiomer(s) which were obtained by asymmetric synthesis, followed by vibrational circular dichroism (VCD) analysis of the particular enantiomer(s).
PREPARATION OF THE COMPOUNDS
Preparation of the final compounds of the invention
Final compounds described by formula (II) can be prepared according to, but not limited to, the General Scheme 1.
Intermediate compounds of formula (Ila) can be synthesized from Intermediates of formulae (la) or (lb) by nucleophilic substitution with hydrazine under thermal conditions in an appropriate solvent, such as methanol, by heating the mixture at an appropriate temperature, such as at the boiling point temperature of the solvent.
Intermediate compounds of formula (Illa) can be synthesized from (Ila) by a condensation reaction in presence of triethyl orthoformate under thermal conditions (e.g. at 165 °C) in an appropriate solvent, such as dimethyl acetamide. Intermediate compounds of formula (IVa) can be obtained by reacting (Illa) with an appropriate brominating agent, such as benzyl trimethyl ammonium tribromide, in an appropriate solvent, such as DMF.
Intermediate compounds of formula (Va), when R4 = H, can be synthesized from (IVa) by a reaction with a primary amine derivative by a Buchwald-Hartwig cross-coupling reaction using a palladium-based pre-catalyst, such as the G3 t- BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base, such as Z-BuONa, and a solvent, such as 2-methyl-2 -butanol. When R4 = CH3, (Va) can be obtained by a nucleophilic substitution of (IVa) under thermal conditions, using DIPEA as a base, CsF and tetrabutylammonium chloride as additives in a solvent such as acetonitrile.
Intermediates compounds of formula (Via) can be obtained from (Va) by reaction with Tf2O in a solvent such as dichloromethane in presence of a base, such as pyridine. Intermediates compounds of formula (VIb) can be obtained from (Va) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent, such as acetonitrile.
Intermediates of formulae (Vila), (Vllb) and (Vile) can be obtained from (Via) or (VIb) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using a palladium-based pre-catalyst such as the G4 cataCxium (Chemistry-A European Journal, 2008, 14, 4267^1279), a base such as CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water at an appropriate temperature (e.g., at 100 °C). Alternatively, the reaction may be performed using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3 in an appropriate solvent, such as 1,4-dioxane/water at an appropriate temperature (e.g., at 100 °C).
- Final compounds of formula (II) can be obtained from intermediates (Vila), (Vllb) and (Vile) by removal of the protecting group PG in an acidic medium, such as HC1 in 1,4-di oxane (intermediates (Vllb) and (Vile)) or, in the case of intermediate (Vila), by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate). Alternatively, the benzylic group in (Vila) can be removed using BBr3. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components. General Scheme 1 Alternatively, final compounds described by formula (II) can be prepared according to, but not limited to, the General Scheme 2. - Final compounds of formula (II) can be obtained from from (VIa) or (VIb) by a Suzuki cross-coupling reaction. Typical, non-limiting conditions may involve the use of Pd(dppf)Cl2.dichloromethane or Pd(PPh3)4 as catalyst, Na2CO3 or K3PO4 as base in 1,4-dioxane or 1,4-dioxane/water as a solvent at an appropriate temperature (e.g., at 100 °C). The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components. General Scheme 2 Final compounds described by formula (III) can be prepared according to, but not limited to, the General Scheme 3.
Intermediate compounds of formula (IXa) can be synthesized from (Villa) by a reaction with a primary amine derivative by a Buchwald-Hartwig cross-coupling reaction using a palladium-based pre-catalyst, such as the G3 t-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.
Intermediates compounds of formula (Xa) can be obtained from (IXa) by reaction with Tf2O in a solvent such as dichloromethane in presence of a base, such as pyridine.
Intermediate compounds of formula (Xia) can be obtained from (Xa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water, at an appropriate temperature (e.g., at 100 °C).
Intermediate compounds of formula (Xlla) can be obtained from (Xia) by removal of the Boc protecting group under acidic conditions, such as HC1 in 1,4- dioxane or TFA.
- Final compounds of general formula (III) can be obtained from (Xlla) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 3
Alternatively, final compounds described by formula (III) can be prepared according to, but not limited to, the General Scheme 4.
Intermediate compounds of formula (Xllla) can be obtained from (IXa) by removal of the Boc protecting group under acidic conditions, such as HC1 in 1,4- dioxane or TFA.
Intermediate compounds of formula (XlVa) can be obtained from (Xllla) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or another reducing agent, such as NaBFFCN).
Intermediate compounds of formula (XVa) can be obtained from (XlVa) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent such as acetonitrile.
- Final compounds of formula (III) can be obtained from (XVa) by a Suzuki cross- coupling reaction. Typical, non-limiting conditions may involve the use of Pd(dppf)C12. dichloromethane or Pd(PPhs)4 as catalyst, Na2COs or K3PO4 as base in 1,4-di oxane or 1,4-dioxane/water as a solvent at an appropriate temperature (e.g., at 100 °C). The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 4
Alternatively, intermediate compounds of general formula (Xia) can be synthesized using the non-limiting synthetic route showed in General Scheme 5.
Intermediate compounds of formula (XVIa) can be obtained from (Xa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3, CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water, at an appropriate temperature (e.g., at 100 °C).
Intermediate compounds of formula (Xia) can be obtained from intermediates (XVIa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate). Alternatively, the benzylic group in (XVIa) can be removed using BBr3.
General Scheme 5 Alternatively, intermediate compounds of general formula (Xlla) can be synthesized using the non-limiting synthetic route showed in General Scheme 6.
Intermediate compounds of formula (XVIIa) can be obtained from (Xa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3, CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water, at an appropriate temperature (e.g., at 100 °C).
Intermediate compounds of general formula (Xlla) can be obtained from (XVIIa) by global deprotection of both the Boc and the methoxymethyl protecting groups by treatment with an acidic medium, such as HC1 in 1,4-di oxane.
General Scheme 6
Final compounds described by formula (IV) can be prepared according to, but not limited to, the General Scheme 7.
Intermediate compounds of formula (XVIIIa) can be synthesized from (Villa) by a reaction with a primary amine derivative by a Buchwald-Hartwig cross- coupling reaction using a palladium-based pre-catalyst, such as the G3 t- BuXPhos-Pd (Accounts of Chemical Research, 2008, 41, 1461-1473), a base, such as Z-BuONa, and a solvent, such as 2-methyl-2-butanol.
Intermediates compounds of formula (XIXa) can be obtained from (XVIIIa) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent such as acetonitrile.
Intermediate compounds of formula (XXa) and (XXb) can be obtained from (XIXa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using a palladium-based pre-catalyst such as the G4 cataCxium (Chemistry-A European Journal, 2008, 14, 4267^1279), a base such as CS2CO3 or K3PO4 in an appropriate solvent, such as 1,4-dioxane/water at an appropriate temperature (e.g., at 100 °C). Alternatively, the reaction may be performed using Pd(dppf)C12. dichloromethane as a source of palladium catalyst, a base such as K2CO3 in an appropriate solvent, such as 1,4-dioxane/water at an appropriate temperature (e.g., at 100 °C).
Intermediate compounds of formulae (XXIa) and (XXIb) can be obtained from intermediates (XXa) and (XXb) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
Intermediate compounds of formulae (XXIIa) and (XXIIb) can be obtained from (XXIa) and (XXIb) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such a sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides.
- Final compounds of formula (IV) can be obtained from (XXIIa) or (XXIIb) by removal of the methoxymethyl (XXIIa) or ethoxymethyl (XXIIb) protecting groups in an acidic medium, such as HC1 in 1,4-di oxane. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
(XXIa): Aik = CH3 (XXIIa): Aik = CH3
(XXIb): Aik = CH2CH3 (XXIIb): Aik = CH2CH3
General Scheme 7
Final compounds described by formula (V) can be prepared according to, but not limited to, the General Scheme 8.
Intermediate compounds of formulae (XXIIIa) and (XXIIIb) can be obtained from (XXa) and (XXb) by a one-pot procedure involving the use of palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate) in presence of polyoxymethylene - homopolymer at a suitable temperature (e.g., room temperature).
- Final compounds of formula (V) can be obtained from (XXIIIa) or (XXIIIb) by removal of the methoxymethyl (XXIIIa) or ethoxymethyl (XXIIIb) protecting groups in an acidic medium, such as HC1 in 1,4-di oxane. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 8
Final compounds described by formula (VI) can be prepared according to, but not limited to, the General Scheme 9.
Intermediate compounds of formula (XXIVa) can be obtained by (XVIIa) by reaction with a methylating agent, such as methyl iodide, after treatment with an appropriate base, such as NaH, in an appropriate solvent, such as DMF at an appropriate temperature, such as room temperature.
Intermediates compounds of formula (XXVa) can be obtained from (XXIVa) by global deprotection of both the Boc and the methoxymethyl protecting groups by treatment with an acidic medium, such as HC1 in 1,4-di oxane.
- Final compounds of general formula (VI) can be obtained from (XXVa) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 9 Final compounds described by formula (VII) can be prepared according to, but not limited to, the General Scheme 10. - Intermediate compounds of formula (XXVIa) can be synthesized from (VIIIa) by a reaction with a primary amine derivative by a Buchwald–Hartwig cross- coupling reaction using a palladium-based pre-catalyst, such as the G3 t- 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 in 1,4- dioxane or TFA. - Intermediate compounds of formula (XXVIIIa) can be obtained from (XXVIIa) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or another reducing agent, such as NaBH3CN). - Intermediates compounds of formula (XXIXa) can be obtained from (XXVIIIa) by reaction with a chlorinating agent, such as POCl3 in an appropriate solvent such as acetonitrile. - Final compounds of formula (VII) can be obtained from (XXIXa) by a Suzuki cross-coupling reaction. Typical, non-limiting conditions may involve the use of Pd(dppf)Cl2.dichloromethane or Pd(PPh3)4 as catalyst, Na2CO3 or K3PO4 as base in 1,4-dioxane or 1,4-dioxane/water as a solvent at an appropriate temperature (e.g., at 100 °C). The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 10
Final compounds described by formula (VIII) can be prepared according to, but not limited to, the General Scheme 11.
Intermediate compound of formula (XXXIa) can be synthesized from Intermediates of formulae (XXXa) or (XXXb) by nucleophilic substitution with hydrazine under thermal conditions in solvents such as ethanol by heating the mixture at an appropriate temperature, such as 90 °C.
Intermediate compounds of formula (XXXIIa) can be synthesized from Intermediates of formula (XXXIa) by condensation with an electrophilic carbonyl source, such as methyl chloroformate. Non-limiting conditions could be the following: the electrophilic carbonyl source, such as methyl chloroformate, is added to a mixture of Intermediate compound (XXXIa) in an appropriate solvent, such as dichloromethane, at the appropriate temperature, such as room temperature, in presence of an appropriate base, such as DIPEA. The mixture is stirred for an appropriate reaction time, such as 16 hours and then it is concentrated. The condensation can be finalized, for example, by taking up the residue in an appropriate solvent, such as ethanol, and stirring the mixture in presence of an appropriate base, such as KOH, at an appropriate temperature, such as 100 °C.
Intermediate compounds of formula (XXXIIIa) can be obtained from (XXXIIa) by reaction with a chlorinating agent, such as POCI3 in an appropriate solvent, such as toluene by heating at an appropriate temperature (for example 135 °C). Intermediate compounds of formula (XXXIVa) can be synthesized from (XXXIIIa) by a Suzuki cross-coupling reaction with appropriate boronic acid or ester derivatives using a palladium -based pre-catalyst such as the XPhos Pd G3, an appropriate base such as K3PO4, in an appropriate solvent, such as 1,4- dioxane/water under appropriate non-limiting conditions, such as at 150 °C under microwave irradiation for an appropriate time (e.g., 1 hour).
Intermediate compounds of formula (XXXVa) can be obtained from (XXXIVa) in a non-limiting manner by treatment with an appropriate thionation agent, such as phosphorous pentasulfide, in an appropriate solvent, such as pyridine under thermal conditions, (e.g., at 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 presence of an appropriate base, such as K2CO3, in an appropriate solvent, such as THF/water.
Intermediate compounds of formula (XXXVIIa) can be obtained from (XXXVIa) in a non-limiting manner by a nucleophilic substitution of (XXXVIa) with an appropriate amine under thermal conditions (e.g., at a temperature of 130 °C) with the appropriate amine used in excess, for an appropriate reaction time (e.g., 48 hours).
- Final compounds of formula (VIII) can be obtained from intermediates (XXXVIIa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
General Scheme 11
Final compounds described by formula (IX) can be prepared according to, but not limited to, the General Scheme 12.
Intermediate compounds of formula (XXXVIIIa) can be obtained from (XXXVIa) in a non-limiting manner by a nucleophilic substitution of (XXXVIa) with an appropriate amine under thermal conditions (e.g., at a temperature of 130 °C) with the appropriate amine used in excess, for an appropriate reaction time (e.g., 48 hours).
Intermediate compounds of formula (XXXIXa) can be obtained from intermediates (XXXVIIIa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
Intermediates compounds of formula (XLa) can be obtained from (XXXIXa) by deprotection of the Boc protecting group by treatment with an acidic medium, such as HC1 in 1,4-di oxane.
- Final compounds of general formula (IX) can be obtained from (XLa) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 12
Alternatively, final compounds described by formula (IX) can be prepared according to, but not limited to, the General Scheme 13. - Intermediates compounds of formula (XLIa) can be obtained from (XXXVIIa) by deprotection of the Boc protecting group by treatment with an acidic medium, such as HC1 in 1,4-di oxane.
Intermediates compounds of formula (XLIIa) can be obtained from (XLIa) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides.
- Final compounds of formula (IX) can be obtained from intermediates (XLIIa), by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate). Alternatively, the benzylic group in (Vila) can be removed using BBn. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
(XXXVIlla) (XLIa) (XLIIa) (lx)
General Scheme 13
Final compounds described by formula (X) can be prepared according to, but not limited to, the General Scheme 14.
Intermediates compounds of formula (XLIIIa) can be obtained from (XLIa) by nucleophilic substitution of an appropriate alkyl halide in presence of an appropriate base, such as DIPEA, in an appropriate solvent, such as acetonitrile, at an appropriate temperature, such as 85 °C.
- Final compounds of formula (X) can be obtained from intermediates (XLIIIa), by global deprotection, using BBn, in an appropriate solvent, such as dichloromethane, with an appropriate additive, such as aniline, at an appropriate temperature, such as 0 °C. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
(XLIa> (XLIIIa) (x)
General Scheme 14
Final compounds described by formula (XI) can be prepared according to, but not limited to, the General Scheme 15.
Intermediates compounds of formula (XLIVa) can be obtained from (XXXVIa) in a non-limiting manner by a nucleophilic substitution of (XXXVIa) with an appropriate amine under thermal conditions (e.g., at a temperature of 120 °C) with the appropriate amine used in excess, for an appropriate reaction time (e.g., 96 hours), in an appropriate solvent, such as DMSO, in presence of an appropriate base, such as DIPEA.
Intermediate compounds of formula (XLVa) can be obtained from intermediates (XLIVa) by removing the benzyl protecting group by catalytic hydrogenolysis, using palladium on charcoal as catalyst, H2 at a suitable pressure (such as atmospheric pressure or 10 bars) in an appropriate solvent (e.g., methanol, ethanol, or ethyl acetate).
Intermediates compounds of formula (XL Via) can be obtained from (XLVa) by deprotection of the Boc protecting group by treatment with an acidic medium, such as HC1 in 1,4-di oxane.
- Final compounds of general formula (XI) can be obtained from (XL Via) by reductive alkylation (e.g., with formaldehyde in presence of sodium triacetoxyborohydride or related agents, such as sodium cyanoborohydride) or nucleophilic substitution on alkyl halides or epoxides. The final compounds may be further purified by supercritical fluid chromatography or chiral reverse phase HPLC to separate their diastereomerically pure or enantiomerically pure components.
General Scheme 15
PHARMACOLOGY
The instant compounds are potent, 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.
PHARMACEUTICAL COMPOSITIONS AND COMBINATIONS
In an embodiment, further described herein are compositions comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound as provided herein. The compounds may be formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may be cited all compositions usually employed for systemically administering drugs. To prepare the pharmaceutical compositions, an effective amount of the particular compound, optionally in salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirable in unitary dosage form suitable, in particular, for administration orally or by parenteral injection. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in 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, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, 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 employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations.
The pharmaceutical composition may additionally contain various other ingredients known in the art, for example, a lubricant, stabilising agent, buffering agent, emulsifying agent, viscosity-regulating agent, surfactant, preservative, flavouring or colorant.
It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.
The daily dosage of the compound will, of course, vary with the compound employed, the mode of administration, the treatment desired and the mycobacterial disease indicated. However, in general, satisfactory results will be obtained when the compound is administered at a daily dosage not exceeding 1 gram, e.g. in the range from 10 to 50 mg/kg body weight.
As used herein, term "pharmaceutical composition" refers to a compound as 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.
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, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp. 1049-1070) .
The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human, for example who is or has been the object of treatment, observation or experiment.
The term "therapeutically effective amount" as used herein, means that amount of compound that elicits a biological or medicinal response in a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by NLRP3, or (ii) associated with NLRP3 activity, or (iii) characterised by activity (normal or abnormal) of NLRP3; or (2) reduce or inhibit the activity ofNLRP3; or (3) reduce or inhibit the expression of NLRP3. In another non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reduce or inhibit the activity of NLRP3; or at least partially reduce or inhibit the expression of NLRP3.
As used herein, the term "inhibit", "inhibition" or "inhibiting" refers 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, inhibiting NLRP3 or inhibiting NLRP3 inflammasome pathway comprises reducing the ability ofNLRP3 or 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, inactivating, destabilizing, and/or altering distribution of NLRP3.
As used herein, the term "NLRP3" is meant to include, without limitation, nucleic acids, polynucleotides, oligonucleotides, sense and anti-sense 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.
As used herein, the term "treat", "treating" or "treatment" of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient.
As used herein, the term "prevent", "preventing" or "prevention" of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.
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.
In an embodiment, there is provided a compound, according to any one of the embodiments described herein, for use as a medicament.
In an embodiment, there is provided a compound, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compound, according to any one of the embodiment described herein) for use 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 the NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder; in inhibiting NLRP3 inflammasome activity (including in a subject in need thereof); and/or as an NLRP3 inhibitor. In an embodiment, there is provided the use of compounds as provided herein, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compound, according to any one of the embodiment 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 the NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder; in inhibiting NLRP3 inflammasome activity (including in a subject in need thereof); and/or as an NLRP3 inhibitor.
In an embodiment, there is provided the use of compounds as provided herein, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compounds, according to any one of the embodiment described herein), in the manufacture of a medicament for: the treatment of a disease or disorder associated with NLRP3 activity (including inflammasome activity); the treatment of a disease or disorder in which the NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder; and/or inhibiting NLRP3 inflammasome activity (including in a subject in need thereof).
In an embodiment, there is provided a method of treating a disease or disorder in which the NLRP3 signalling contributes to the pathology, and/or symptoms, and/or progression, of said disease/disorder, comprising administering a therapeutically effective amount of a compound as provided herein, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compound, according to any one of the embodiment described herein), for instance to a subject (in need thereof). In a further embodiment, there is provided a method of inhibiting the NLRP3 inflammasome activity in a subject (in need thereof), the method comprising administering to the subject in need thereof a therapeutically effective amount of a compound as provided herein, according to any one of the embodiments described herein (and/or pharmaceutical compositions comprising such compound, according to any one of the embodiment described herein).
The instant compounds may have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the above-stated indications or otherwise.
For instance, the instant compounds may have the advantage that they have a good or an improved thermodynamic solubility (e.g. compared to compounds known in the prior art; and for instance as determined by a known method and/or a method described herein). The instant compounds may have the advantage that they will block pyroptosis, as well as the release of pro-inflammatory cytokines (e.g. IL-10) from the cell. The instant compounds may also have the advantage that they avoid side-effects, for instance as compared to compounds of the prior art, which may be due to selectivity of NLRP3 inhibition. Compounds as provided herein may also have the advantage that they have good or improved in vivo pharmacokinetics and oral bioavailability. They may also have the advantage that they have good or improved in vivo efficacy. Specifically, the instant compounds may also have advantages over prior art compounds when compared in the tests outlined hereinafter.
EXPERIMENTAL PART
GENERAL PREPARATION AND ANALYTICAL PROCESSES
The compounds according to the invention can generally be prepared by a succession of steps, each of which may be known to the skilled person or described herein.
It is evident that in the foregoing and in the following reactions, the reaction products may be isolated from the reaction medium and, if necessary, further purified according to methodologies generally known in the art, such as extraction, crystallization, and chromatography. It is further evident that reaction products that exist in more than one enantiomeric form, may be isolated from their mixture by known techniques, in particular preparative chromatography, such as preparative HPLC, chiral chromatography. Individual diastereomers or individual enantiomers can also be obtained by Supercritical Fluid Chromatography (SFC).
The starting materials and the intermediates are compounds that are either commercially available or may be prepared according to conventional reaction procedures generally known in the art.
Analytical Part
LC-MS (LIQUID CHROMATOGRAPH Y/MASS SPECTROMETRY)
General procedure
The High-Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below).
Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time. . .) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software. Compounds are described by their experimental retention times (Rt) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H]+ (protonated molecule) and/or [M-H]' (deprotonated molecule). In case the compound was 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 value is the one obtained for the lowest isotope mass. All results were obtained with experimental uncertainties that are commonly associated with the method used.
Hereinafter, “SQD” means Single Quadrupole Detector, “MSD” Mass Selective Detector, “RT” room temperature, “BEH” bridged ethylsiloxane/silica hybrid, “DAD” Diode Array Detector,” HSS” High Strength silica. Table: LCMS Method codes (Flow expressed in mL/min; column temperature (T) in
°C; Run time in minutes).
NMR
For a number of compounds, 1 H NMR spectra were recorded on a Bruker Avance III spectrometer operating at 300 or 400 MHz, on a Bruker Avance III-HD operating at 400 MHz, on a Bruker Avance NEO spectrometer operating at 400 MHz, on a Bruker
Avance Neo spectrometer operating at 500 MHz, or on a Bruker Avance 600 spectrometer operating at 600 MHz, using CHLOROFORM-t/ (deuterated chloroform, CDCh), DMSO4 (deuterated DMSO, dimethyl-d6 sulfoxide), METHANOL-tL (deuterated methanol), BENZENE-t/r, (deuterated benzene, CeDe) or ACETONE-t/r, (deuterated acetone, (CDs^CO) as solvents. Chemical shifts (5) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as internal standard.
Melting Points
Values are either peak values or melt ranges and are obtained with experimental uncertainties that are commonly associated with this analytical method.
Method A: For a number of compounds, melting points were determined with a DSC823e (Mettler Toledo) apparatus. Melting points were measured with a temperature gradient of 10 °C/minute. Standard maximum temperature was 300 °C.
Method B: For a number of 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/minute. Maximum temperature was 300 °C. The melting point data was read from a digital display and checked from a video recording system
Note on stereochemistry.
Whenever the notation “RS” is indicated herein, it denotes that the compound is a racemic mixture at the indicated center, unless otherwise indicated. The stereochemical configuration for centers in some compounds has been designated “(R)” or “(5)” when the mixture(s) was separated or originated from enantiomerically pure starting materials; for some compounds, the stereochemical configuration at the indicated centers has been designated as “*(R/’ or when the absolute stereochemistry is undetermined although the compound itself has been isolated as a single stereoisomer and is enantiomerically/diastereomerically pure. The enantiomeric excess of compounds reported herein was determined by analysis of the racemic mixture by supercritical fluid chromatography (SFC) followed by SFC comparison of the separated enantiomer(s). In intermediates/compounds wherein bonds are indicated either with a bold wedge or a wedge of parallel lines while the stereocenters are designated (RS), the representation indicates that the sample is a mixture of stereoisomers, one stereoisomer having the indicated substituents or groups projected above or below the plane of the drawing as represented, one stereoisomer having the substituents or groups in the opposite projection below or above the plane of the drawing, e.g.
The absolute configuration of chiral centers (indicated as R and/or S) can be rationalized. The synthesis of all final compounds started from intermediates of known absolute configuration in agreement with literature precedent or obtained from appropriate synthetic procedures. The assignment of the absolute configuration of additional stereocenters could then be assigned by standard NMR methods.
EXAMPLES
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 alternatively can be synthesized by a skilled person by using published methods.
Abbreviations Preparation of intermediates
For intermediates that were used in a next reaction step as a crude or as a partially purified intermediate, in some cases no mol amounts are mentioned for such intermediate in the next reaction step or alternatively estimated mol amounts or theoretical mol amounts for such intermediate in the next reaction step are indicated in the reaction protocols described below.
Sodium hydride [7646-69-7] (4.9 g, 124 mmol, 2 equiv) was added to anhydrous toluene [108-88-3] (182 mL) at 0 °C under nitrogen atmosphere. Then, 3- trifluoromethylphenol [98-17-9] (10 g, 61.7 mmol, 1 equiv) was 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 rt for 3 h. The mixture was acidified at 0 °C with cone. HC1 until pH 4-5, then was extracted with EtOAc and washed twice with brine. The organic layer was separated, dried over MgSO4, filtered off and concentrated in vacuo yielding 2-iodo-5- (trifluoromethyl)phenol, 1-1 as a pale red oil (18.7 g, quant). thyl)benzene
2-iodo-5-(trifluoromethyl)phenol 1-1 (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 rt for 15 minutes. Then benzyl bromide [100-39-0] (8.7 mL, 71.42 mmol, 1.1 equiv) was added dropwise and the reaction mixture was stirred at reflux (56 °C) for 4 h. 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 off 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 yielding 2- (benzyloxy)-l-iodo-4-(trifluoromethyl)benzene 1-2 as a white solid (19.6 g, 79%).
Isopropyl magnesium chloride (2M in THF) [1068-55-9] (15.6 mL, 31.1 mmol, 1.2 equiv) was added dropwise to a stirred solution of 2-(benzyloxy)-l-iodo-4- (trifluoromethyl)benzene 1-2 (9.8 g, 25.92 mmol, 1 equiv) in anhydrous THF [109-99- 9] (207 mL) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0 °C for 2 h. Afterwards, 2-isopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaborolane [61676-62-8] (7.9 mL, 38.88 mmol, 1.5 equiv) was added dropwise to the mixture. The reaction mixture was slowly warmed up to rt and stirred for 18 h. The excess of THF was removed and the reaction was quenched with a saturated aq. solution of NH4CI and extracted with EtOAc. The organic layer was separated, dried over MgSCU, filtered off and concentrated in vacuo. The crude product was purified by flash column chromatography (silica, 330 g, EtOAc: Heptane, 0/100 to 10/90). The desired fractions were collected and concentrated under reduced pressure yielding 2-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-3 as a white solid (6.69 g, 68%). n-bromosuccinimide [128-08-5] (5.84 g, 32.79 mmol) was added into the solution 3- (difluoromethoxy)phenol [88798-13-4 ] (5 g, 31.23 mmol) in DCM (30 mL). The reaction mixture was stirred at rt for 2 h. The reaction mixture was washed twice with water (30 mL). The organic was dried with anhydrous Na2SO4, filtered and filtrate was concentrated in vacuo to give a brown oil. This crude product was purified by flash column chromatography 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 1-4 (4.6 g, yield 52%) as colourless oil.
A mixture of 2-bromo-5 -(difluoromethoxy )phenol 1-4 (1 g, 4.18 mmol), pinacolborane (2.14 g, 16.74 mmol), TEA (2.32 mL, 16.74 mmol), toluene (20 mL) in a round bottomed flask (100 mL) was purged with nitrogen for 10 minutes. XPhos Pd G4 (108 mg, 0.13 mmol) was added, and the reaction mixture was heated at 80 °C for 1 hour. The mixture was filtered over decalite and washed with toluene (2 x 20 mL). The filtrate was concentrated to dryness, purified using silica gel column chromatography (ethyl acetate in heptane from 0 to 100%). The desired fractions were combined and concentrated in vacuo to afford 5-(difluoromethoxy)-2-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)phenol 1-5 (420 mg, yield 35%).
N,N-Diisopropylethylamine [7087-68-5] (21.5 mL, 102 mmol) was added to a coold, 0 °C, solution of 2-iodo-5-(trifluoromethyl)phenol 1-5 (34 g, 102 mmol) in DCM (1.2 L), followed by dropwise addition of chloromethyl methyl ether [107-30-2] (9.4 mL, 121.8 mmol). The reaction mixture was allowed to gradually warm to rt and react for 18 h. The mixture was concentrated, and the residue was purified by flash column chromatography (silica 330 g; EtOAc in heptane 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to yield l-iodo-2-(methoxymethoxy)-4- (trifluoromethyl)benzene 1-6 (25.5 g, yield: 70%) as a colorless oil.
Isopropyl magnesium chloride (2M in THF) [1068-55-9] (14.5 mL, 28.9 mmol) was added dropwise to a stirred solution of l-iodo-2-(methoxymethoxy)-4- (trifluoromethyl)benzene 1-6 (8g, 24.09 mmol) in anhydrous THF (190 mL) at 0 °C under N2 atmosphere. The reaction mixture was stirred at 0 °C for 2 h. Afterwards, 2- isopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaborolane [61676-62-8] (7.4 mL, 36.1 mmol) was added dropwise to the mixture. The reaction mixture was slowly warmed up to rt and stirred for 16 h. The reaction was quenched with a saturated solution of NH4CI aq. and extracted with EtOAc. The organic layer was separated, dried (MgSCU), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (SiCL 120 g, EtOAc in Heptane, 0/100 to 20/80). The desired fractions were collected and concentrated under reduced pressure to yield 2-(2- (methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-7 (6.4 g, yield: 76%) as a yellowish oil.
A mixture of l-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)s [51364-51-3] (22.99 g, 25.10 mmol, 0.05 equiv), 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 N2 atmosphere. The mixture was allowed to cool down to rt. The resulting mixture was diluted with ice water (3 L). The resulting mixture was extracted with EtOAc (3 x 3 L). The combined organic layers were washed with brine (2 x 2 L), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluted with PE to afford 3-methyl-5-(trifluoromethyl)phenol 1-8 (66 g, 75%) as a yellow oil.
NaH (30 g, mmol, 749 mmol, 2.00 equiv, 60%) was added in portions to a solution of 3-methyl-5-(trifluoromethyl)phenol 1-8 (66 g, 375 mmol, 1.00 equiv) in toluene (0.99 L) under nitrogen atmosphere at 0 °C. 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 for 3 h at rt. The resulting mixture was diluted with ice water (2 L). The resulting mixture was extracted with EtOAc (2 x 3 L). The combined organic layers were washed with brine (2 x 3 L), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluted with petroleum ether to afford 2-iodo-3-methyl-5-(trifluoromethyl)phenol 1-9 (71 g, 63%) as a yellow oil.
Chloromethyl ethyl ether [3188-13-4] (44.5 g, 470 mmol, 2.00 equiv) was added dropwise at 0 °C to a mixture of 2-iodo-3-methyl-5-(trifluoromethyl)phenol 1-9 (71 g, 235 mmol, 1.00 equiv) and CS2CO3 (153 g, 470 mmol, 2.00 equiv) in DMF (0.71 L) under nitrogen atmosphere. The solution was stirred overnight at rt. The reaction mixture was diluted with ice/water (2 L). The resulting mixture was extracted with EtOAc (2 x 3 L). The combined organic layers were washed with brine (2 x 3 L), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluted with petroleum ether to afford l-(ethoxymethoxy)-2-iodo-3-methyl-5- (trifluoromethyl)benzene 1-10 (42 g, 50%) as a yellow oil.
4,4,5,5-tetramethyl-l,3,2-dioxaborolane [25015-63-8] (59.70 g, 466.51 mmol, 4.00 equiv) was added to a mixture of l-(ethoxymethoxy)-2-iodo-3-methyl-5- (trifluoromethyl)benzene 1-10 (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)2 [3375-31-3] (1.31 g, 5.83 mmol, 0.05 equiv) in 1,4-dioxane (0.42 L) under nitrogen atmosphere. The reaction mixture was stirred for 2 h at 100 °C. The mixture was allowed to cool down to rt. 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), dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was applied onto a silica gel column with petroleum ether. The residue was purified by trituration with n-hexane (300 mL) at -30 °C. The precipitated solids were collected by filtration. This resulted in 31.0106 g (73.82%) of 2-[2-(ethoxymethoxy)-6-methyl-4- (trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-11 as a white solid.
Synthesis of Intermediate 12 (1-12), methyl (2S,4S)-4-hydroxy-l-tritylpyrrolidine-2- carboxylate
1-12
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) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. A solution of saturated aqueous of NH4CI and NH3 (2: 1) was added to the crude mixture. After separation of the phases, the aqueous phase was extracted with DCM. The organic phases were mixed, dried over MgSO4, filtered off and concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2 330g; EtOAc in heptane 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo to yield (25,45)- 4-hydroxy-l-tritylpyrrolidine-2-carboxylate 1-12 as a white solid (25.3 g, 81%).
1-13
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-l-tritylpyrrolidine-2-carboxylate 1-12 (25.3 g, 65.30 mmol, 1 equiv) in dichloromethane (255 mL) at 0 °C. The reaction mixture was stirred at 0 °C to rt for 16 h. The reaction mixture was diluted with DCM and washed with a saturated aqueous solution of Na2CO3, water, and brine. The organic solution was dried over MgSCL, filtered off and concentrated under reduced pressure to yield methyl (2S,4S)-4- ((methylsulfonyl)oxy)-l-tritylpyrrolidine-2-carboxylate 1-13 as an orange solid (30.4 g, assumed quant, yield) which was used without further purification in the next step. Synthesis of Intermediate 14 (1-14), methyl (2S,4R)-4-azido-l-tritylpyrrolidine-2- carboxylate
Sodium azide [26628-22-8] (5.3 g, 81.62 mmol, 2.5 equiv) was added to a stirred solution of methyl (25,45)-4-((methylsulfonyl)oxy)-l-tritylpyrrolidine-2-carboxylate I- 13 (15.2 g, 32.65 mmol, 1 equiv) in DMF (180 mL) under nitrogen atmosphere. The reaction mixture was stirred at 90 °C for 16 h. The reaction was diluted with a sat. solution of NaHCCh and extracted with EtOAc. The organic layer was washed with brine several times, dried over MgSCU, filtered off and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO2 120 g; EtOAc in Heptane 0/100 to 10/90). The desired fractions were collected to yield methyl (2S,4R)- 4-azido-l-tritylpyrrolidine-2-carboxylate 1-14 as a white solid (10.7 g, 79%).
Synthesis of Intermediate 15 (1-15), ((2S,4R)-4-amino-l-tritylpyrrolidin-2- yl) methanol
Methyl (25,4A)-4-azido-l-tritylpyrrolidine-2-carboxylate 1-14 (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) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 30 min. Afterwards, the reaction mixture was stirred at rt for 3h. The reaction mixture was cooled at 0 °C and water (3.7 mL) was added dropwise. After 5 min of stirring, an aqueous 3.75 M NaOH solution (3.8 mL) was added. And after 5 min of stirring, water (9.2 mL) was added, and the mixture was stirred at rt for 1 h. The obtained precipitate was dried over MgSCU and collected on a Celite pad and washed with DCM. The organic solvent was removed in vacuo to yield ((25,4A)-4-amino-l-tritylpyrrolidin-2- yl)methanol 1-15 as a white solid (8.64 g, 96%). Synthesis of Intermediate 16 (1-16), tert-butyl ((3R,5S)-5-(hydroxymethyl)-l- tritylpyrrolidin-3-yl) carbamate
To a stirred solution of ((25,4A)-4-amino-l-tritylpyrrolidin-2-yl)methanol 1-15 (220 g, 614 mmol, 1 equiv) in DCM (2200 mL) was added di-tert-butyl dicarbonate [24424- 99-5] (161 g, 736 mmol, 1.2 equiv) in portions under N2 atmosphere. The mixture was stirred for 18 h at room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EtOAc (100/0 to 60/40) to afford tert-butyl ((3R,5S)-5- (hydroxymethyl)-l-tritylpyrrolidin-3-yl)carbamate 1-16 as a white foam (180 g, 64%).
Synthesis of Intermediate 17 (1-17), tert-butyl ((3R,5R)-5-fluoro-l-tritylpiperidin-3- yl)carbamate
To a stirred solution of tert-butyl ((3A,55)-5-(hydroxymethyl)-l-tritylpyrrolidin-3- yl)carbamate 1-16 (180 g, 392 mmol, 1 equiv) in THF (3.6 L) was added diethyl(trifluoro-lambda4-sulfanyl)amine [38078-09-0] (88.6 g, 549.5 mmol, 1.4 equiv) dropwise at 0 °C under N2 atmosphere. The reaction was stirred for 1 h at 0 °C. Afterwards the mixture was stirred for 1 h at room temperature. The mixture was allowed to cool down to 0 °C and adjusted pH=12 with saturated aqueous solution of Na2COs. The aqueous phase was extracted with EtOAc (3 x5 00 mL). The organic was dried with Na2SO4. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether/EtOAc (100/0 to 75/25) to afford tert-butyl ((3/?,5/?)-5-fluoro- l -tritylpiperidin-3- yl)carbamate 1-17 as a white solid (135 g, 74%). Synthesis of Intermediate 18 (1-18), tert-butyl ((3R,5R)-5-fluoropiperidin-3- yl)carbamate
To a stirred solution of tert-butyl ((3R,5R)-5-fluoro-l-tritylpiperidin-3-yl)carbamate I- 17 (135 g, 293 mmol, 1 equiv) in EtOH (2700 mL) was added hydrogen chloride (190 mL, 2 M) dropwise at rt. The reaction mixture was stirred for 1.5 h at rt. Then NaHCCL (32.2 g) was added to the solution and the reaction mixture was stirred for 1.5 h at rt. The solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with EtOAc/MeOH (100/0 to 85/15) to afford tert-butyl ((3R,5R)-5-fluoropiperidin-3-yl)carbamate 1-18 as a white solid (30.1 g, 47%).
Synthesis of Intermediate 19 (1-19), tert-butyl ((3R,5R)-5-fluoropiperidin-3- yl)carbamate
CH2O
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) were added to a solution of tert-butyl ((3A,5A)-5-fluoropiperidin-3-yl)carbamate 1-18 (2 g, 9.16 mmol, 1 equiv) in dry 2-methyl-THF (45.0 mL). The resulting colourless solution was stirred at room temperature for 1.5 h and then, heated at 80 °C for 2.5 h. After cooling at room temperature, the solution was concentrated in vacuo yielding tert-butyl ((3A,5A)-5-fluoro-l-methylpiperidin-3-yl)carbamate 1-19 as an off-white residue (2.85 g) which was used in the next step without further purification.
Synthesis of Intermediate 20 (1-20), tert-butyl ((3R,5R)-l-ethyl-5-fluoropiperidin-3- yl)carbamate lodoethane [75-03-6] (0.5 mL, 6.22 mmol, 1.4 equiv) was added to a stirred solution of tert-butyl ((3A,5A)-5-fluoropiperidin-3-yl)carbamate 1-18 (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 at room temperature overnight (monitored by TLC stained with Dragendorff). The reaction mixture was concentrated in vacuo. The residue was taken in a mixture of water/EtOAc, the organic layer was separated, and the aqueous layer was further extracted with EtOAc twice. The combined organic layers were washed with brine, dried over MgSO4, filtered off and concentrated in vacuo yielding tert-butyl ((3A,5A)-l-ethyl-5-fluoropiperidin-3-yl)carbamate 1-20 as a white solid (950 mg, 84%).
Synthesis of Intermediate 21 (1-21), tert-butyl (R)-methyl(l-methylpiperidin-3- yl)carbamate
(A)-tert-butyl methyl(piperi din-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) was added. The solution was stirred at rt for 3 h at 1 atm H2. The solution was filtered over dicalite and washed with EtOH and concentrated in vacuo. The resulting residue was taken in diluted ammonia and extracted with EtOAc three times. The combined organic layers were washed with brine, dried over MgSO4, filtered off and concentrated in vacuo yielding tert-butyl (R)- methyl(l-methylpiperi din-3 -yl)carbamate 1-21 as an oil (2.5 g, yield 94%).
Synthesis of Intermediate 22 (1-22), tert-butyl (R)-(l-ethylpiperidin-3- ,
1-22
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 (A)-tert-butyl methyl(piperi din-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 18 h at rt. The reaction mixture was poured out in water + 0.5 g NaHCCL, extracted with EtOAc three times. The combined organic phases were washed with brine, dried on MgSCU, filtered and evaporated, yielding tert-butyl (A)-(l- ethylpiperidin-3-yl)(methyl)carbamate 1-22 (2.5 g, yield 88%) as a yellow oil.
Synthesis of Intermediate 23 (1-23), (3R,5R)-l-ethyl-5-fluoropiperidin-3-amine dihydrochloride tert-butyl ((3A,5A)-l-ethyl-5-fluoropiperidin-3-yl)carbamate 1-20 (950 mg, 3.86 mmol, 1 equiv) was dissolved in 5N to 6N HC1 in IPA [7647-01-0] (11.8 mL, 59 mmol, excess). The reaction mixture was stirred at room temperature for 4 h (monitoring by TLC stained with Dragendorff). The solids were filtered and washed with iPrOH and DIPE to yield (3A,5A)-l-ethyl-5-fluoropiperi din-3 -amine dihydrochloride S-23 as a white solid (780 mg, 92%).
Additional analogues were synthesized in an analogous manner using the reagents as appropriate.
1-27
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 yield lH-pyrrole-2- carbohydrazide 1-27 (14.5 g, yield: 64%) as a white solid.
Methyl chloroformate [79-22-1] (13.38 mL, 173 mmol) was added to a stirred suspension of lH-pyrrole-2-carbohydrazide 1-27 (14.5 g, 115.5 mmol) and DIPEA [7087-68-5] (60.5 mL, 346.4 mmol) in dichloromethane (600 mL) at rt. The reaction mixture was stirred at room temperature for 16 hours. 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 2h. The precipitate formed was filtered off, and it was taken up in water. The obtained solution was acidified with HC1 IN to pH 4, and stirred at rt for 30 min. The obtained precipitated was collected and dried in vacuo to afford pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-28 (16.3 g, yield: 92 %) as a white solid. Synthesis of Inter
1-29
Phosphorus oxychloride [10025-87-3] (5.6 mL, 59.55 mmol) was added dropwise over 15 min to a stirred suspension of pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-28 (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) at rt. The suspension was stirred at 135 °C for 8 h in a sealed glass reactor. The reaction mixture was cooled down to rt and was added dropwise to EtOH (90 mL) over 15 min under stirring, then was stirred at rt for 15 min. Solvent was evaporated in vacuo. The crude product was absorbed in celite and purified by flash column chromatography (silica 120 g; EtOAc in heptane 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DCM, filtered and dried in vacuo to yield l-chloropyrrolo[l,2- d][l,2,4]triazin-4(3H)-one 1-29 (1.4 g, yield: 69%) as a white off solid.
Potassium phosphate tribasic [7778-53-2] (6.4 g, 29.5 mmol) and Pd(PPhs)4 [14221-01- 3] (510 mg, 0.6 mmol) were added sequentially to a stirred solution of 1- chloropyrrolo[l,2-d][l,2,4]triazin-4(3H)-one 1-29 (2 g, 11.8 mmol) and 2-(2- (benzyloxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-3 (5.4 g, 14.2 mmol) in 1.4-dioxane (91 mL) and water (9 mL) (previously bubbled with nitrogen for 5 min) at rt under nitrogen atmosphere. The reaction mixture was stirred at 105 °C overnight. The mixture was concentrated and the residue was purified by flash column chromatography (silica 80 g; dry load in celite; EtOAc in heptane 0/100 to 30/70). The desired fractions were collected and concentrated in vacuo. The product was triturated with heptane to yield to yield l-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)pyrrolo[l,2-d][l,2,4]triazin-4(3H)-one 1-30 (3.9 g, yield: 65%) as a white foamy solid.
Phosphorous pentasulfide (8.7 g, 38.73 mmol, 2.5 eq.) was added to a stirred solution of 1 -(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin-4(3H)-one 1-30 (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 phosphorous pentasulfide (4.35 g, 19.37 mmol, 1.25 eq.). The mixture was stirred at 150 °C for 8 h. Then was cooled down to rt, diluted with water and extracted with EtOAc (3 x 600 mL). The extracts were combined, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to give brown solid. The brown solid was subjected to celite 545 followed by chromatography (silica 120 g; dry load in celite; 0-89% EtOAc/heptane). The desired fractions were collected and concentrated in vacuo to yield l-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)pyrrolo[l,2-d][l,2,4]triazine-4(3H)-thione 1-31 (5.7 g, 72%) as a yellowish solid.
Synthesis of Intermediate 32 (1-32), l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-
1-32
Potassium carbonate [584-08-7] (3.2 g, 22.87 mmol, 6 eq.) diluted in water (19.3 mL) was added dropwise to a stirred solution of l-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)pyrrolo[l,2-d][l,2,4]triazine-4(3H)-thione 1-31 (1.53 g, 3.81 mmol) in THF (38.5 mL) at rt. Then bromoethane [74-96-4] (0.6 mL, 7.62 mmol, 2 eq.) was added and the mixture was stirred at rt for 16 h. The reaction mixture was diluted with water and extracted with EtOAc (x3). The combined organic layers were dried (MgSCU), filtered and solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g; EtOAc in heptane 0/100 to 9/91). The desired fractions were collected and concentrated in vacuo to yield l-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[l,2-d][l,2,4]triazine 1-32 (577 mg, yield: 35%) as white solid. l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[l,2-d][l,2,4]triazine 1-32 (526 mg, 1.23 mmol) and (R)- l-boc-3 -aminopiperidine [188111-79-7] (3.16 g, 15.31 mmol) were added into a sealed glass reactor. The neat mixture was stirred at 130 °C for 48 h. The mixture was cooled down to rt, diluted with sat. NaHCOs aqueous solution and extracted with EtOAc (x3). The combined organic layers were dried (MgSO4), filtered and solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g; EtOAc in heptane 0/100 to 60/40). The desired fractions were collected and concentrated in vacuo to yield tert-butyl (A)-3-((l- (2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin-4- yl)amino)piperidine-l -carboxylate 1-33 (568 mg, yield: 81%) as a beige foamy solid.
1-34 l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-(ethylthio)pyrrolo[l,2-d][l,2,4]triazine 1-32 (60 mg, 0.14 mmol) and (lR,2R)-2-aminocyclohexanol [931-16-8] (500 mg, 4.25 mmol) were added into a sealed glass tube. The neat mixture was stirred at 130 °C for 48 h. The mixture was cooled down to rt, diluted with sat. NaHCO3 aqueous solution and extracted with EtOAc (x3). The combined organic layers were dried (MgSCU), filtered and solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 40 g; EtOAc in heptane 0/100 to 75/25). The desired fractions were collected and concentrated in vacuo to yield (lR,2R)-2-((l-(2- (benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin-4- yl)amino)cyclohexan-l-ol 1-34 (40 mg, yield: 45%) as a dark yellow sticky solid.
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 I-35 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 dry (1 mL) were added into a sealed glass tube. The mixture was stirred at 120ºC for 4 days. The solvent was concentrated in vacuo. The crude was purified by flash column chromatography (silica 12 g; EtOAc in Heptane 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo to yield 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. Additional analogues were synthesized in an analogous manner starting from I-32 using the reagents as appropriate.
10% Palladium on carbon [7440-05-3] (199 mg, 0.19 mmol) was added to a stirred solution of tert-butyl (R)-3-((l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[l,2- d][l, 2, 4]triazin-4-yl)amino)piperi dine- 1 -carboxylate 1-33 (568 mg, 1.00 mmol) in methanol (28 mL) at 0 °C under nitrogen atmosphere. Then, nitrogen atmosphere was replaced by hydrogen (1 atm, balloon) and the reaction mixture was stirred at rt for 4 h. The mixture was filtered off over a pad of celite and washed with DCM / MeOH (9: 1). Solvents were evaporated in vacuo to yield tert-butyl (R)-3-((l-(2-hydroxy-4- (trifluoromethyl)phenyl)pyrrolo[l,2-d] [1,2, 4]triazin-4-yl)amino)piperi dine- 1- carboxylate 1-41 (478 mg, yield: 99%) as a dark yellow foamy solid.
Additional analogues were synthesized in an analogous manner using the reagents as l-44a
Trifluoroacetic acid [76-05-1] (5.6 mL, 74.32 mmol) was added dropwise to a stirred solution of tert-butyl (R)-3-((l-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[l,2- d][l, 2, 4]triazin-4-yl)amino)piperi dine- 1 -carboxylate 1-41 (478 mg, 1.00 mmol) in DCM anhydrous (5.6 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at rt for 1 h. Solvent was evaporated in vacuo to yield (7?)-2-(4-(piperi din-3 - ylamino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifluoromethyl)phenol trifluoroacetic acid salt I-44a (495 mg, yield: 99%) as a brown foamy solid. Product was used in the next reaction step without further purification.
Hydrochloric acid (4M) in dioxane [7647-01-0] (1 mL, 4.23 mmol) was added to a solution of tert-butyl (R)-3-((l-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrrolo[l,2- d][l, 2, 4]triazin-4-yl)amino)piperi dine- 1 -carboxylate 1-41 (105 mg, 0.220 mmol) in 1,4- dioxane (0.175 mL) at rt. The mixture was stirred at rt for Ih. The solvent was evaporated in vacuo to yield (R)-2-(4-(piperi din-3 -ylamino)pyrrolo[l,2-d][ 1,2, 4]triazin- l-yl)-5-(trifluoromethyl)phenol dihydrochloride I-44b (95 mg, yield: 100 %) as an orange solid.
HC1 (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[l,2-d][ 1,2, 4]triazin-4- yl)amino)piperidine-l -carboxylate 1-41 (130 mg, 0.27 mmol) and the mixture was stirred at rt for 30 min. The solvent was concentrated in vacuo to yield (R)-2-(4- (piperidin-3-ylamino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifluoromethyl)phenol hydrochloride I-44c (124 mg, yield: 99%) as a brown solid. The crude product was used as such in the next step. Additional analogues were synthesized in an analogous manner using the reagents as appropriate:
Trifluoroacetic acid [76-05-1] (1.43 mL, 19 mmol) was added dropwise to a stirred solution of tert-butyl (R)-3-((l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[l,2- d][l, 2, 4]triazin-4-yl)amino)piperi dine- 1 -carboxylate 1-33 (150 mg, 0.26 mmol) in DCM anhydrous (1.5 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at rt for 1 h. Solvent was evaporated in vacuo to yield (R)-l-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine I-47a (153 mg, yield: 99%) as a yellowish sticky solid. Product was used in the next reaction step without further purification.
HC1 (4N in 1,4-dioxane) [7647-01-0] (2.0 mL, 7.9 mmol) was added to tert-butyl (R)-
3-((l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[l,2-d][l,2,4]triazin-4- yl)amino)piperidine-l -carboxylate 1-33 (150.0 mg, 0.3 mmol) and the mixture was stirred at rt for 30 min. The solvent was concentrated in vacuo to yield (R)-l-(2-
(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperi din-3 -yl)pyrrolo[l,2-d] [1,2, 4]triazin-
4-amine hydrochloride I-47b (142.0 mg, yield: 99%) as an orange solid.
(A)-l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[l,2- d][l,2,4]triazin-4-amine hydrochloride I-47b was dissolved in DCM/MeOH (4:1) and the solution was basified with saturated NaHCCL solution and extracted with DCM/MeOH (4: 1) three times. The combined organics were dried with MgSO4, filtered and concentrated in vacuo to give (/?)-! -(2-(benzyl oxy )-4- (trifluoromethyl)phenyl)-N-(piperidin-3-yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine I-47c as a brown solid.
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 (A)-l-(2- (benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperi din-3 -yl)pyrrolo[l,2-d][l, 2, 4]tri azin- 4-amine trifluoroacetic acid salt I-47a (152 mg, 0.26 mmol) in acetonitrile (3 mL). The mixture was stirred at 85 °C for 16 h. The reaction was recharged with 2-(2- Bromoethoxy)tetrahydro-2h-pyran [17739-45-6] (1.2 eq, 50 μL, 0.31 mmol) and DIPEA [7087-68-5] (6 eq, 0.28 mL, 1.57 mmol) The reaction mixture was cooled down to rt, diuted with sat. NaHCCf aqueous solution and extracted with EtOAc (x3). The combined organic layers were dried (MgSCU), filtered and solvents evaporated in vacuo. The crude was purified by flash column chromatography (silica 20 g, DCM/MeOH (9: 1) in DCM 0/100 to 35/65). The desired fractions were collected and concentrated in vacuo to yield l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-((3R)-l- (2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)piperi din-3 -yl)pyrrolo[l,2-d] [1,2, 4]triazin-4- amine 1-48 (150 mg, yield: 92%) as a beige foamy solid.
A,7V-Diisopropylethylamine [7087-68-5] (112.9 μL, 0.6 mmol) followed by l-fluoro-2- iodoethane [762-51-6] (23.5 μL, 0.3 mmol) were successively added to a stirred mixture of (A>)- l -(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(piperidin-3- yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine hydrochloride I-47b (142.0 mg, 0.3 mmol) in A A-Di methyl form ami de [68-12-2] (9 mL) and the resulting solution was stirred at rt for 16h. MA-Di isopropyl ethyl amine [7087-68-5] (112.9 μL, 0.6 mmol) and l-fluoro-2- iodoethane [762-51-6] (23.5 μL, 0.3 mmol) were added to the mixture and stirred at rt for 72h. 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 yield (R)-l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(l- (2-fluoroethyl)piperidin-3-yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine 1-49 (160 mg, yield: 95% ) as an orange sticky solid.
1-50
Molecular sieves 4 A (94 mg) was added to a 30 mL sealed glass reactor containing a stirring solution, consisting of (A)-l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N- (piperidin-3-yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine I-47c (220 mg, 0.47 mmol) and dry THF/MeOH (9: 1) (3.3 mL) at rt under N2. (l-ethoxycyclopropoxy)trimethylsilane [27374-25-0] (284 uL, 1.41 mmol), acetic acid (323 uL, 5.65 mmol) and sodium cyanoborohydride [25895-60-7] (93 mg, 1.41 mmol) were then added to the mixture at rt under N2 to give a brown solution. The resulting mixture was stirred at 65 °C for 16 h, and then was cooled to rt, extracted with DCM (3 x 60 mL) and ISfeCCL (aq. solution) (10 mL). The extracts were combined, dried over anhydrous MgSCU, filtered, and concentrated to dryness in vacuo to give yellow oil. The yellow oil was subjected to silica gel chromatography (12 g irregular 40-60 um; 0-3% MeOH/DCM) to give (R)- l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(l-cy cl opropylpiperi din-3 - yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine 1-50 (203 mg, 84%) as a brown foam solid.
A mixture of methyl lH-pyrazole-3 -carb oxy late [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 at reflux for 6 h. The reaction mixture was cooled at 0 °C and the solids were filtered, washed with water and EtOH and then dried in under vacuum at 50 °C overnight, yielding Synthesis of lH-pyrazole-5-carbohydrazide 1-51 (19.2 g, yield 96%) as a bright with solid. Synthesis of Intermediate 52 (1-52), pyrazolo[l,5-d][l,
O triethyl orthoformate - ► DMA
1-52
A mixture of lH-pyrazole-5-carbohydrazide 1-51 (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 solids were filtered and washed with EtOH to yield pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-52 (14.2 g, yield 72%) as a white solid.
Synthesis of Intermediate 53 (1-53), 7-bromopyrazolo[l,5-d][l,2,4]triazin-4(5H)-one
A suspension of pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-52 (10 g, 73.5 mmol) and K2CO3 (13.2 g, 95.5 mmol) in DMF (200 mL) was stirred at rt for 10 min. Then benzyltrimethylammonium tribromide (37.2 g, 95.51 mmol) was added and the reaction mixture was heated at 40 °C for 2 h. The reaction mixture was cooled 0 °C and quenched by the dropwise addition of aq. Na2S20s (28 g in 100 mL of water). The resulting mixture was stirred for an extra 30 min at 0 °C after which it was allowed to warm to rt and further diluted with 700 mL of water. The suspension was left overnight (without stirring) and filtered. The precipitate was washed with water and isopropyl ether, and dried in vacuo overnight at 50 °C to give 7-bromopyrazolo[l,5- d][l,2,4]triazin-4(5H)-one 1-53 (14.3 g, yield 91%) as a white solid.
(R)- l-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[l,5- d][l,2,4]triazin-4(5H)-one 1-53 (1 g, 4.65 mmol) in 2-methyl-2-butanol (40 mL) under nitrogen atmosphere at room temperature in a sealed tube. The reaction mixture was stirred at 60 °C for 2h. The reaction mixture was concentrated in vacuo, taken up in sat. aqueous NaHCO3 solution, extracted with EtOAC (x3). The combined organic phases were washed with brine, dried on MgSCU, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography (silica 80g, eluent: EtOAc in Heptane, from 0 to 100%) The desired fractions were collected and concentrated in vacuo to yield (A)-3-((4-oxo-4,5-dihydropyrazolo[l,5-d][l,2,4]triazin- 7-yl)amino)piperidine-l-carboxylate 1-54 (980 mg, yield 63%) as an orange foam.
Additional analogues were synthesized in an analogous manner from 1-53 using the
To a solution of tert-butyl (7?)-3-((4-oxo-4,5-dihydropyrazolo[l,5-d][l,2,4]triazin-7- yl)amino)piperidine-l -carboxylate 1-54 (2 g, 5.74 mmol) in DCM (3 mL) was added HC1 (6M in iPrOH) (10 mL, 6 M, 60 mmol). The reaction mixture was stirred at rt for 4 hours. The solids were filtered off and washed with DCM (5 mL) and dried in vacuum oven at 45 °C for 18 hours to afford (7?)-7-(piperidin-3-ylamino)pyrazolo[l,5- d][l,2,4]triazin-4(5H)-one hydrochloride 1-67 (1.45 g, yield 93%) as white solid.
HC1 (4M in dioxane) (2.36 mL, 4 M, 9.42 mmol) was added to a solution of tert-butyl 6-((4-oxo-4,5-dihydropyrazolo[l,5-d][l,2,4]triazin-7-yl)amino)-l,4-oxazepane-4- carboxylate 1-57 (330 mg, 0.942 mmol) in methanol (4 mL). The reaction mixture was stirred at rt until complete conversion. The reaction mixture was concentrated under reduced pressure to give crude product (HC1 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 transfered to a column loaded with Si- Propylsulfonic acid SCX-2 resin (SiliCycle). The column was first eluted with MeOH after which the free amine was released by elution with ammoniated methanol (7 N). Tubes containing the product were concentrated under reduced pressure to give 7-((l,4- oxazepan-6-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-68 (250 mg, yield 106%). An additional analogue was synthesized in an analogous manner using the reagents as appropriate: peridin-3-
Formaldehyde solution [50-00-0] (1.28 mL, 12.93 mmol, 5eq.) was added to a solution of (A)-7-(piperidin-3-ylamino)pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one hydrochloride I- 67 (700 mg, 2.59 mmol) in MeOH (16 mL). The reaction mixture was stirred for 10 min at room temperature after which sodium cyanoborohydride [25895-60-7] (490 mg, 7.76 mmol) was added. The reaction mixture was stirred at rt for 1 h. The mixture was diluted with HC1 (IM, 1 mL) and stirred at rt for Ih. The solution was concentrated to keep ~3 mL of solvent and sat. aq NaHCCh (3 mL) was added. The mixture was extracted with EtOAc (5 x 30 ml) and 2-MeTHF (5 x 30mL). The combined organics were dried (Na2SO4), and concentrated in vacuo to afford (A)-7-((l -m ethylpiperi din-3 - yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-70 (0.79 g, yield 66%) as colorless sticky oil.
Additional analogues were synthesized in an analogous manner using the reagents as appropriate:
1-73 POCI3 [10025-87-3] (1200 μL, 1.65 g/mL, 12.91 mmol, 4 eq.) was added to a suspension of (A)-7-((l-methylpiperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin- 4(5H)-one 1-70 (790 mg, 3.18 mmol) in acetonitrile (20 mL). The resulting reaction mixture was stirred at 80 °C for 10 hours and additional POCI3 (1200 μL, 12.91 mmol) was added. The reaction mixture was then heated at 80 °C for 42 hours. The reaction mixture was quenched by the addition sat. aq. NaHCCh and extracted with dichloromethane (5x). The combined organic phases were washed with brine and concentrated under reduced pressure to give (R)-4-chl oro-N-(l-m ethylpiperi din-3 - yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-73 (570 mg, yield 60.5%, 90% pure). The crude product was used as such in the next step. Additional analogues were synthesized in an analogous manner using the reagents as appropriate:
To a mixture of (A)-3-((4-oxo-4,5-dihydropyrazolo[l,5-d][l,2,4]triazin-7- yl)amino)piperidine-l -carboxylate 1-54 (500 mg, 1.50 mmol) in DCM (8 mL) was added dry pyridine [110-86-1] (611 μL, 7.57 mmol) and TfzO [358-23-6] (763.55 μL, 4.54 mmol) in DCM (2 mL) at 0 °C. Then the mixture was stirred at rt for 1 h. The reaction mixture was quenched with ice/water and NaHCO3. The combined organics were dried with MgSCU anhydrous, filtered, and concentrated in vacuo. The crude was further purified by column chromatography Heptane/EtOAc: from 100/0 to 50/50) to give Lbutyl (A)-3-((4-(((trifluoromethyl)sulfonyl)oxy)pyrazolo[l,5-d][l,2,4]triazin-7- yl)amino)piperidine-l -carboxylate 1-81 (540 mg, yield 77 %) as a yellow solid.
Additional analogues were synthesized in an analogous manner using the reagents as appropriate:
Suzuki cross-couplings:
1-87
Method r. 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-(((7A,2A)-2- hydroxycyclohexyl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl trifluoromethanesulfonate 1-86 (203 mg, 0.53 mmol) and intermediate 1-3 (301 mg, 0.8 mmol) in 1,4-dioxane (3.6 mL). The mixture was bubbled with nitrogen for 10 min, Pd(dppf)C12'DCM [95464-05-
4] (87 mg, 0.1 mmol) was added at rt. The reaction mixture was heated at 90 °C and stirred under nitrogen atmosphere for 16 h. The reaction was cooled down to rt and water was added. The mixture was extracted with EtOAc. The organic layers were combined, dried over MgSCU, filtered and concentrated in vacuo. The crude was purified by flash column chromatography (silica, 40 g; EtOAc in heptane from 0/100 to 50/50). The desired fractions were collected and concentrated in vacuo to yield (17?,27?)-2-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin- 7-yl)amino)cyclohexan-l-ol 1-87 (112 mg, yield 43%) as a yellow sticky solid. Synthesis of Intermediate 88 (1-88), tert-butyl (R)-3-((4-(2-hydroxy-4- (trifluoromethoxy)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-yl)amino)piperidine-l- carboxylate
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 1-81 (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)C12 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 out in sat NaHCCh solution and extracted twice with EtOAc. The combined organic layers were washed with brine and dried on Na2SO4. After filtration, the solvent was evaporated. The residue was purified on a column with silica gel, eluent EtOAc in Heptane, from 0 to 50 %. The pure fractions were combined and concentrated, yielding tert-butyl (R)-3-((4-(2-hydroxy-4-
(tri fluoromethoxy )phenyl)pyrazolo[l,5-d][l, 2, 4]tri azin-7-yl)amino)piperidine-l- carboxylate 1-88 (67 mg, yield 36%) as a light yellow solid.
Synthesis of Intermediate 89 (1-89), (R)-N-(l-benzylpiperidin-3-yl)-4-(2-
Method 3: A reaction flask was charged with (R)-N-(l-benzylpiperi din-3 -yl)-4- chloropyrazolo[l,5-d][l,2,4]triazin-7-amine 1-75 (2.0 g, 5.83 mmol), 2-(2- (methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-7 (2.13 g, 6.42 mmol), potassium phosphate tribasic [7778-53-2] (3.72 g, 17.50 mmol), Pd(dppf)C12 DCM [95464-05-4] (357 mg, 0.44 mmol), 1,4-dioxane (40 mL) and water (4 mL). The reaction mixture was bubbled with nitrogen and next stirred at 90 °C for 3 hours. 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 to 60% EtOAc in heptane) to give (R)-N-(l-benzylpiperi din-3 -yl)-4-(2- (methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-amine I- 89 (2.13 g, yield 71%)
Synthesis of Intermediate 90 (1-90), (R)-N-(l-benzylpiperidin-3-yl)-4-(2- (ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-
amine
1-90
Method 4\ A reaction flask was charged with (R)-N-(l-benzylpiperi din-3 -yl)-4- chloropyrazolo[l,5-d][l,2,4]triazin-7-amine 1-75 (500 mg, 1.46 mmol), 2-[2- (ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-l,3,2- dioxaborolane 1-11 (578 mg, 1.6 mmol), CS2CO3 [534-17-8] (1426 mg, 4.38 mmol), cataCXium Pd G4 [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 hours. 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 on MgSO4, filtered, and concentrated. The residue was purified on a column with silica gel, eluent EtOAc in Heptane, from 0 to 100 %. The pure fractions were concentrated, yielding (R)-N-(l-benzylpiperi din-3 -yl)-4-(2-(ethoxymethoxy)-6-m ethyl-4- (trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-90 (625 mg, yield 79%) as a sticky oil.
Additional analogues were synthesized in an analogous manner using the reagents as appropriate, according to the cross-coupling methods used to synthesize compounds:
1-100
Pd/C (10%) [7440-05-3] (230.0 mg, 0.22 mmol) was added to a solution of tert-butyl (7?)-3-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrazolo[l,5-d][l, 2, 4]tri azin-7- yl)amino)piperidine-l -carboxylate 1-92 (613 mg, 1.1 mmol) in ethanol (7 mL) at rt under nitrogen. Nitrogen atmosphere was changed by a hydrogen baloon and the reaction mixture was stirred for 2.5 h at rt. The mixture was filtered in celite and washed with EtOH. The solvent was removed in vacuo to yield tert-butyl (R)-3-((4-(2- hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7- yl)amino)piperidine-l -carboxylate 1-100 (380 mg, yield 73%). Synthesis of Intermediate 101 (1-101), (R)-4-(2-(ethoxymethoxy)-6-methyl-4-
(R)-N-(l-benzylpiperi din-3 -yl)-4-(2-(ethoxymethoxy)-6-m ethyl-4- (trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-90 (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 for 3 h at rt at 1 atm H2. The mixture was filtered over dicalite and washed with EtOH and further concentrated in vacuo yielding (K)-4- (2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-7V-(piperi din-3 - yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine Int-101 (260 mg, yield 100%) as a sticky solid.
An additional analogue was synthesized in an analogous manner using the reagents as appropriate: Synthesis of Intermediate 103 (1-103), tert-butyl (R)-3-((4-(2-(methoxymethoxy)-4-
(trifluoromethyl)phenyl)pyrazolo[l, 5-d][l,2,4]triazin- 7-yl) (methyl)amino)piperidine-
NaH (60% dispersion in mineral oil) [7646-69-7] (94.89 mg, 2.37 mmol, 5.3 equiv) was added portionwise into a mixture of tert-butyl (A’)-3-((4-(2-(methoxymethoxy)-4- (trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-yl)amino)piperidine-l- carboxylate 1-93 (230 mg, 0.44 mmol, 1 equiv) in dry DMF (10 mL) at 0 °C and flushed with nitrogen. The reaction was stirred 30 min at rt. 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 then the reaction mixture was allowed to stir at rt for 2.5 h. The reaction mixture was quenched with MeOH and concentrated in vacuo. The crude was purified by silicagel column chromatography (gradient elution: 0 to 2% MeOH in DCM) to deliver tert- butyl (A>)-3-((4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[l,5- d][l,2,4]triazin-7-yl)(methyl)amino)piperidine-l-carboxylate 1-103 (64 mg, yield 24%).
1-104
Trifluoroacetic acid [76-05-1] (11.5 mL, 153.5 mmol) was added dropwise to a stirred solution of tert-butyl (A)-3-((4-(2-hydroxy-4-(trifluoromethyl)phenyl)pyrazolo[l,5- d][l,2,4]triazin-7-yl)amino)piperidine-l-carboxylate 1-100 (380 mg, 0.80 mmol) in DCM anhydrous (11.5 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at rt for 1.5 h at rt. Solvent was evaporated in vacuo to yield (7?)-2-(7- (piperidin-3-ylamino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol bis trifluoroacetic acid salt 1-104 (475 mg, yield 98%) as a yellow oil. Product was used in the next reaction step without further purification.
HC1 (4M in 1,4-dioxane) [7647-01-0] (1.12 mL, 4M, 4.47 mmol) was added to a solution of tert-butyl (A)-3-((4-(2-hydroxy-4-methylphenyl)pyrazolo[l,5- d][l,2,4]triazin-7-yl)amino)piperidine-l-carboxylate 1-91 (100 mg, 0.24 mmol) in 1,4- dioxane (1.13 mL) and the mixture was stirred at rt for 2 h. The mixture was poured out in sat. NaHCCh solution and extracted three times with EtOAc. The combined organics were washed with brine, dried on MgSCU, filtered and evaporated, yielding (A)-5- methyl-2-(7-(piperi din-3 -ylamino)pyrazolo[ 1 , 5-d] [ 1 ,2,4]triazin-4-yl)phenol 1-105 (61 mg, yield 80%) as a yellow sticky solid.
Additional analogues were synthesized in an analogous manner using the reagents as appropriate:
Hydrochloric acid (4M) 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[l,5-d][l,2,4]triazin-7-yl)amino)piperidine-l- carboxylate (relative stereochemistry) 1-95 (90 mg, 0.17 mmol) in Dioxane (0.12 mL) at rt. The mixture was stirred at rt Ih. TLC showed the formation of the desired product. The solvent was evaporated in vacuo to yield 2-(7-(((3R,5R)-5- fluoropiperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol dihydrochloride (relative stereochemistry) 1-108 (79 mg, yield: 99%) as a white solid.
An additional analogue was synthesized in an analogous manner using the reagents as HC1 (4M 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[l,5- d][l,2,4]triazin-7-yl)amino)-5-methylpiperidine-l-carboxylate (relative stereochemistry) 1-99 (251 mg, 0.51 mmol, 1 equiv) in 1,4-dioxane [123-91-1] (10 mL) at rt. The solution was stirred at rt overnight. LCMS showed full conversion and clean reaction profile. The reaction mixture was concentrated in vacuo yielding 2-(7- (((3R,5R)-5-methylpiperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol dihydrochloride (relative stereochemistry) as a brown residue (0.51 mmol, quantitative yield).
1-111
DIPEA (122 μL, 0.71 mmol) and iodoethane [75-03-6] (43 μL, 0.53 mmol) were added to a stirred solution of (A>)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N- (piperidin-3-yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-102 (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 as such in the next step
Additional analogues were synthesized in an analogous manner using the reagents as appropriate:
Synthesis of Intermediate 116 (1-116), (R)-4-(2-(methoxymethoxy)-4- (trifluoromethyl)phenyl)-N-(l-(oxetan-3-yl)piperidin-3-yl)pyrazolo[l,5-
1-116
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[l,5- d][l,2,4]triazin-7-amine 1-102 (150 mg, 0.36 mmol) in MeOH (1.9 mL). The reaction mixture was stirred for 30 min at room temperature after which sodium cyanoborohydride [25895-60-7] (45 mg, 0.71 mmol) was added. The reaction mixture was stirred at rt overnight. The crude reaction mixture was transferred to a column loaded with Si-Propylsulfonic acid SCX-2 resin (SiliCycle). The column was first eluted with MeOH after which the desired product was released by elution with ammoniated methanol (7 N). Tubes containing the desired product were concentrated under reduced pressure to give crude (A)-4-(2-(methoxymethoxy)-4- (trifluoromethyl)phenyl)-N-( 1 -(oxetan-3 -yl)piperi din-3 -yl)pyrazolo [ 1,5- d][l,2,4]triazin-7-amine 1-116 (132 mg, purity 75%, yield 58%). The crude product was used as such in the next step.
1-117
A solution of (A)-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperi din-3 - yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-102 (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 quantities. The two reaction mixtures were combined and diluted with EtOAc, washed with water and concentrated under reduced pressure to obtain (R)-N-(\-cy cl opropylpiperi din-3 -yl)-4-(2-(m ethoxymethoxy)-4- (trifhioromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-117 (110 mg, quant yield). The crude product was used as such in the next step without further purification.
A solution of (A)-4-(2-(m ethoxymethoxy)-4-(trifluoromethyl)phenyl)-N-(piperi din-3 - yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-102 (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 at rt overnight. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic phases were concentrated in vacuo and the resulting residue purified by silica gel column chromatography (gradient elution: 0 to 5% MeOH in DCM) to give (1R,2R - relative stereochemistry)-2-((R)-3-((4-(2- (methoxymethoxy)-4-(trifluoromethyl)phenyl)pyrazolo[l,5-d][l, 2, 4]tri azin-7- yl)amino)piperidin-l-yl)cyclobutan-l-ol 1-118 (85 mg, yield 12%).
Synthesis of Intermediate 119 (1-119), (R)-4-(2-(ethoxymethoxy)-6-methyl-4- (trifluoromethyl)phenyl)-N-(l-methylpiperidin-3-yl)pyrazolo[l,5-d][l,2,4]triazin-7- amine
1-119
(A)-N-(l-benzylpiperi din-3 -yl)-4-(2-(ethoxymethoxy)-6-m ethyl-4- (trifhioromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-90 (312 mg, 0.58 mmol) was dissolved in MeOH (3.6 mL) and Pd/C (10%) (22.29 mg, 0.021 mmol) + Polyoxymethylene - Homopolymer [30525-89-4] (100 mg) was added. The solution was stirred for 3 h at rt at 1 atm H2. The solution was filtered over dicalite and washed with EtOH and further concentrated in vacuo. The residue was taken in diluted ammonia, extracted with EtOAc three times, the combined organics were washed with brine, dried on MgSCU, filtered and evaporated, yielding (A)-4-(2-(ethoxymethoxy)-6- methyl-4-(trifluoromethyl)phenyl)-N-(l-methylpiperidin-3-yl)pyrazolo[l,5- d][l,2,4]triazin-7-amine 1-119 (250 mg, yield 93%) as a sticky solid.
Synthesis of Intermediate 120 (1-120), 3-methylpyrazolo[l,5-d][l,2,4]triazin-4(5H)- one hydrazine monohydrate - ►
EtOH
1-120
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, consisting of ethyl 4-methyl-lH- pyrazole-3 -carboxylate [6076-12-6] (5 g, 32.4 mmol) in ethanol (80 mL) at room temperature under nitrogen atmosphere to give colourless solution. After sealing the reactor, the mixture was stirred at 90 °C for 18 h and then, allowed to rt. The mixture was concentrated to dryness in vacuo and purified by flash column chromatography (silica 80 g; dry loaded in silica; MeOH-DCM (10: 1) in DCM 0/100 to 70/30) to afford 4-methyl-lH-pyrazole-5-carbohydrazide 1-120 (4.94 g, yield 96 %) as a white solid.
Synthesis of Intermediate 121 (1-121), 3-methylpyrazolo[l,5-d][l,2,4]triazin-4(5H)- one
1-121
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, consisting of 4-methyl-lH- pyrazole-5-carbohydrazide 1-120 (4.94 g, 35.25 mmol) and DMF (35 mL) at room temperature to give a colourless solution. After sealing the reactor, the mixture was stirred at 165 °C for 16 h. The mixture was allowed to cool to give a brown solution. The mixture was co-distilled wit toluene, 5 times, to give a pale brown slurry. DCM (700 mL), previously cooled to 0 °C, was added to the solid. The mixture was filtrated in a glass Hirsch funnel and dried in vacuo to afford 3-methylpyrazolo[l,5- d][l,2,4]triazin-4(5H)-one 1-121 (3.85 g, yield 72 %) as a white solid.
Synthesis of Intermediate 122 (1-122), 7-bromo-3-methylpyrazolo[l,5- dj[l, 2, 4 ]triazin-4(5H)-one benzyltrimethylammonium tribromide K2CO3, DMF
O
1-122
K2CO3 (4.6 g, 33.1 mmol) was added to a 10 mL round-bottomed flask containing a solution, consisting of 3-methylpyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-121 (3.8 g, 25.5 mmol) and DMF (69 mL) at room temperature. The suspension was stirred at rt for 10 min. Then benzyltrimethylammonium tribromide [111865-47-5] (12.9 g, 33.1 mmol) was added. The reaction mixture was stirred at 40 °C for 16 h. A saturated aq. Na2S2C>3 solution was added and the mixture was diluted with EtOAc (x8). A solid was precipitated and then was filtered and dried under high vacuum to afford 7-bromo-3- methylpyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-122 (4.1 g, yield 67 %) as a white solid.
Synthesis of Intermediate 123 (1-123), tert-butyl (R)-(l-methylpiperidin-3- yl)carbamate formaldehyde (37% in water)
2-m ethy Itetra hyd rof u ra n
1-123
Formaldehyde solution [50-00-0] (4 mL, 53.73 mmol) followed by formic acid [64-18- 6] (2 mL, 53.03 mmol) were added to a solution of tert-butyl (R)-piperi din-3 - ylcarbamate [309956-78-3] (5 g, 24.97 mmol) in 2-methyltetrahydrofuran (50 mL). The resulting solution was stirred 1 h at rt, then Ih at 80 °C. Upon cooling to room temperature, the mixture was concentrated and the residue was directly to column chromatography (silica 80 g; DCM/MeOH (1 :0 to 9:1)) to give tert-butyl (R)-(l- methylpiperi din-3 -yl)carbamate 1-123 (5.63 g, yield: 100%) as a clear oil that crystalized upon standing.
1-124
(R)-(l-methylpiperi din-3 -yl)carbamate 1-123 (14.2 g, 66.26 mmol) was dissolved in 1,4-dioxane (53 mL). The reaction was cooled at 0 °C and HC1 4N in 1,4-dioxane (332 mL, 1327.8 mmol) was added. The mixture was stirred at rt for 1 h and 30 min. The solvent was evaporated in vacuo to give (R)-l-methylpiperi din-3 -amine dihydrochloride 1-124 (12.9 g, yield 100 %) as a white solid.
7-bromo-3-methylpyrazolo[l,5-d][l,2,4]triazin-4(5H)-one 1-122 (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 consisting of (R)-l-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 N2 atmosphere. The mixture was sparged with N2 for 10 min, and then stirred at 60 °C for 7 h. The vial was cooled to rt and the mixture therein concentrated to dryness in vacuo, diluted with water (150 mL) and extracted with DCM (150 mL). The organic layer was separated, dried (MgSCU), filtered and the solvents evaporated 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 afford (7?)-3-methyl-7-((l-methylpiperidin-3-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4(5H)-one 1-125 (420 mg, yield: 24 %) as a yellow oil.
Phosphorus oxychloride [10025-87-3] (1.2 mL, 12.84 mmol) were added to a solution consisting of (R)-3-methyl-7-((l-methylpiperidin-3-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4(5H)-one 1-125 (420 mg, 1.6 mmol)and acetonitrile anhydrous (12 mL) at rt in a 35 mL screw-cap vial under N2 atmosphere. The mixture was sparged with N2 for 10 min and then stirred at 90 °C for 18 h. The vial was cooled to rt and the mixture therein concentrated to dryness in vacuo, diluted with NaHCCL (aq) (50 mL) and extracted with EtOAc (50 mL x 3). The organic layer was separated, dried (MgSCU), filtered and the solvents evaporated in vacuo to afford (/?)-4-chl oro-3 - methyl-N-(l-methylpiperidin-3-yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-126 (260 mg, yield: 51 %) as a yellow sticky solid. The crude product was used as such in the next step.
Synthesis of Intermediate 127 (1-127), (R)-4-(2-(benzyloxy)-4- (trifluoromethyl)phenyl)-3-methyl-N-(l-methylpiperidin-3-yl)pyrazolo[l,5- d][l, 2, 4 ]tri a zin- 7-amine
1-127
(7?)-4-chloro-3-methyl-N-(l-methylpiperidin-3-yl)pyrazolo[l,5-d][l,2,4]triazin-7- amine 1-126 (260 mg, 0.93 mmol), 2-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)- 4,4,5,5-tetramethyl-l,3,2-dioxaborolane 1-3 (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) that had been sparged with nitrogen for 5 min were added to a 35 mL screw-cap vial to give a heterogeneous mixture. Pd(dppf)C12 DCM [95464-05-4] (91 mg, 0.11 mmol) was then added to the vial, which was subsequently capped and heated at 90 °C for 18 h. The vial was cooled to rt and then diluted with water (50 mL) and extracted with DCM/MeOH (50 mL x 2). The combined extracts were dried over anhydrous MgSCU, 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 soliud ; 0-10% MeOH/DCM) to afford (A’)-4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-3-methyl-N- (l-methylpiperidin-3-yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-127 (230 mg, yield: 35%) as a yellow oil.
POCL [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 (relative stereochemistry) 1-61 (520 mg, 1.87 mmol) in acetonitrile (20 mL). The resulting reaction mixture was stirred at 80 °C until complete conversion (2 hours). The reaction mixture was concentrated to dryness and triturated in saturated aqueous solution of NaHCCh at 0°C and saturated with solid NaCl. The resulting mixture was extracted with 2-Me-THF (8 x 20 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated to afford 4-chloro-N-((8S,8aR)-octahydroindolizin-8- yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine (relative stereochemistry) 1-128 (350 mg, yield 64 %).
Preparation of Final Compounds
Sodium triacetoxyborohydride [56553-60-7] (330 mg, 1.51 mmol) was added to a stirred solution of (R)-2-(4-(piperi din-3 -ylamino)pyrrolo[l,2-d][ 1,2, 4]triazin-l-yl)-5 - (trifluoromethyl)phenol trifluoroacetic acid salt 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) at 0 °C under nitrogen atmosphere. The mixture was stirred at rt for 16 h. Then was diluted with sat. aqueous NaHCCL and extracted with EtOAc (x3). The combined organic layers were dried (MgSCU), filtered and concentrated. The crude product was purified by flash column chromatography (methanol/dichloromethane 0/100 to 6/94). The fractions containing the desired product were collected and concentrated. The residue was further purified by preparative HPLC (Column: Phenomenex Gemini C18 30x100 mm 5 pm; gradient water 25mM NH4HCO3/MeCN 70/30 to 27/73). The desired fractions were collected and freeze-dried to yield compound X, (R)-2-(4-((l-methylpiperi din-3 - yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifhioromethyl)phenol FC-1 (265 mg, yield: 50%) as a yellow solid. Additional final compounds were synthesized in an analogous manner using the
10% Palladium on carbon [7440-05-3] (20 mg, 0.02 mmol) was added to a stirred solution of (lR,2R)-2-((l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)pyrrolo[l,2- d][l,2,4]triazin-4-yl)amino)cyclohexan-l-ol 1-34 (40 mg, 0.06 mmol) in methanol (3 mL) at 0 °C under nitrogen atmosphere. The nitrogen atmosphere was replaced by hydrogen (1 atm) and the reaction mixture was stirred at room temperature for 4 h. The mixture was filtered through a celite pad. 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 30x100 mm 5 pm; gradient water 25mM NH4HCO3/acetonitrile 70/30 to 27/73). The fractions containing the desired product were collected and freeze-dried to yield 2-(4-(((lR,2R)- 2-hydroxycyclohexyl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5- (trifhioromethyl)phenol FC-8 (10 mg, yield: 41%) as a yellow solid.
Additional final compounds were synthesized in an analogous manner using the reagents as appropriate:
The 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)C12- dichloromethane [95464-05-4] (94.4 mg, 0.12 mmol, 12 mol%) and K2CO3 [584-08-7] (399 mg, 2.89 mmol, 3 equiv) and flushed with nitrogen (3 vacuum/nitrogen cycles). 7-(((3R,5R)-l- ethyl-5-fluoropiperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl trifluoromethanesulfonate 1-84 (397 mg, 0.96 mmol, 1 equiv) in 1,4-dioxane (6.7 mL) and water (1.5 mL) was added into and the resulting reaction mixture was stirred at 90 °C for 1.5 h. The reaction mixture was quenched with a saturated NaHCO3 aqueous solution and extracted with EtOAc three times. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (0 to 2% methanol :di chloromethane). The residue so obtained was recrystallized in methanol yielding 2-(7-(((3R,5R)-l-ethyl-5- fluoropiperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol FC-14 (16.8 mg, yield 4%) as a yellow solid.
Additional final compounds were synthesized in an analogous manner using the reagents as appropriate:
A reaction flask was charged with N-(4-chloropyrazolo[l,5-d][l,2,4]triazin-7-yl)-4- methyl-l,4-oxazepan-6-amine 1-74 (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)C12- di chloromethane [95464-05-4] ( (51 mg, 0.06 mmol), 1,4-dioxane (4.3 mL) and water (0.4 mL). The resulting mixture was purged with nitrogen and stirred at 90 °C for 3 hours. The mixture was purified by solid phase extraction using Si-Propylsulfonic acid SCX-2 resin (SiliCycle). The crude reaction mixture was transfered to a column loaded with Si-Propylsulfonic acid SCX-2 resin (SiliCycle). The column was first eluted with MeOH after which the desired product was released by elution with ammoniated methanol (7 N). Tubes containing the product were concentrated under reduced pressure. The crude product was purified using by silica gel column chromatography (gradient elution: 0 to 4% methanol in dichloromethane) to give 2-(7-((4-methyl-l,4- oxazepan-6-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC- 16 (155 mg, yield 60%).
Additional final compounds were synthesized in an analogous manner using the reagents as appropriate:
4 M HC1 in 1,4-dioxane [7647-01-0] (0.89 mL, 4 M, 3.54 mmol) was added to a solution of (R)-N-(l-(2-fluoroethyl)piperidin-3-yl)-4-(2-(methoxymethoxy)-4- (trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-amine 1-115 (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 loaded with Si- Propylsulfonic acid SCX-2 resin (SiliCycle). The column was first eluted with methanol. The desired product was released by elution with 7 N NH3/methanol. Tubes containing the desired product were concentrated under reduced pressure. The residue was purified using by silica gel column chromatography (gradient elution: 0 to 3% methanol in dichloromethane) to give (R)-2-(7-((l-(2-fluoroethyl)piperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol FC-21 (85 mg, yield 56%).
Additional final compounds were synthesized in an analogous manner using the reagents as appropriate:
- I l l - 4 M HC1 in 1,4-dioxane [7647-01-0] (2.4 mL, 9.6 mmol) was added to a solution of
(R)-4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-N-(l-methylpiperidin- 3-yl)pyraz°l°[l,5-d][l,2,4]triazin-7-amine 1-119 (235 mg, 0.51 mmol) in 1,4-dioxane (2.42 mL) and the mixture was stirred at room temperature for 2 hr. The mixture was poured in a NaHCO3 saturated solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried on MgSCU, filtered, and evaporated. A purification was performed by preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10pm,30xl50mm, Mobile phase: 0.25% NH4HCO3 solution in water, CH3CN). The pure fractions were concentrated and co-evaporated twice with MeOH. The residue was freeze-dried, yielding (R)-3-methyl-2-(7-((l-methylpiperidin- 3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (130 mg, yield 63.22%) FC-27 as a white foam.
Additional final compounds were synthesized in an analogous manner using the reagents as appropriate:
3-Oxetanone [6704-31-0] (0.020 mL, 0.31 mmoL) was added to a mixture of (R)-2-(4- (piperidin-3-ylamino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifluoromethyl)phenol dihydrochloride I-44b (95 mg, 0.21 mmol) and triethylamine [121-44-8] (0.116 ml, 0.835 mmol) in di chloroethane (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 16 h. The mixture was diluted with sat NaHCCL and then extracted with di chloromethane (x3). The combined organic layers were separated, dried with MgSCU, filtered and concentrated. The crude product was purified by flash column chromatography (dichloromethane/methanol (9: 1) in dichloromethane from 0/100 to 100/0). The material obtained was further purified by reverse phase chromatography (Phenom enex Gemini Cl 8 30x100 mm 5pm Column; from 47% [25mM NH4HCO3] - 53% acetonitrile:methanol (1 : 1) to 18% [25mM NH4HCO3] -
82% acetonitrile:methanol (1 : 1)) to obtain (R)-2-(4-((l-(oxetan-3-yl)piperi din-3 - yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifhioromethyl)phenol FC-31 (38 mg; yield: 41 %) as a yellow foam.
Sodium triacetoxyborohydride [56553-60-7] (112 mg, 0.51 mmol, 1.5 eq.) was added to a stirred solution of 2-(4-((l,4-oxazepan-6-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l- yl)-5-(trifluoromethyl)phenol hydrochloride 1-46 (146.93 mg, 0.34 mmol), triethylamine [121-44-8] (335 μL, 2.39 mmol, 7 eq.) and formaldehyde (37% aqueous solution) [50-00-0] (51 μL, 0.68 mmol, 2 eq.) in methanol (3.4 mL) at 0 °C under nitrogen atmosphere. 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 eq.) and sodium triacetoxyborohydride [56553-60-7] (112 mg, 0.51 mmol, 1.5 eq.). 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: l)/dichloromethane from 0/100 to 100/0) to yield 2-(4- ((4-m ethyl- l,4-oxazepan-6-yl)amino)pyrrolo[l,2-d][ 1,2, 4]tri azin- 1 -yl)-5- (trifluoromethyl)phenol (130 mg) as a yellow solid. 30 mg of the product was repurified by reverse phase chromatography (Phenom enex Gemini C18 3 Ox 100mm 5pm Column; from 59% [25mM NH4HCO3] - 41% [I:MeOH (1 : 1)] to 17% [25mM NH4HCO3] - 83% [I:MeOH (1 : 1)]) to yield 2-(4-((4-m ethyl- l,4-oxazepan-6- yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifhioromethyl)phenol FC-32 (17.2 mg, yield: 12%) as a yellow solid.
Synthesis of Final Compound 33 and Final Compound 34: (R)-2-(4-((4-methyl-l,4- oxazepan-6-yl)anuno)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifluoromethyl)phenol and oxazepan-6-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5- FC-34
2-(4-((4-m ethyl- 1 ,4-oxazepan-6-yl)amino)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin- 1 -y l)-5 - (trifluoromethyl)phenol (racemic) FC-32 (100 mg, 0.25 mmol) was purified by SFC to isolate both chiral products. SFC purification was made using a i-amylose-1 column with ISOC 30% Ethanol + 0.1% diethylamine to collect and concentrate to yield FC-33 (25.9 mg, yield: 25%) as a yellow foam solid and FC-34 (21.4 mg, yield: 21%) as a yellow foam solid. The absolute configuration was not determined.
Sodium cyanoborodeuteride [25895-62-9] (30 mg, 0.45 mmol) was added to a stirred solution of (R)-2-(4-(piperi din-3 -ylamino)pyrrolo[l,2-d][ 1,2, 4]triazin-l-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 (~20 wt. % in D2O, 98 atom % D) [1664-98-8] (86 μL, 0.6 mmol) in methanol-dl (99.5 atom % D) [1455-13-6] (4.2 mL) at 0 °C under nitrogen atmosphere. The mixture was stirred at rt for 16 h. The reaction mixture was concentrated. The residue was purified by reverse phase chromatography (Phenom enex Gemini Cl 8 30x100 mm 5pm Column; from 70% [25mM NH4HCO3] - 30% acetonitrile to 27% [25mM NH4HCO3] - 73% acetonitrile) to yield (R)-2-(4-(( 1 -(methyl-d3)piperi din-3 -yl)amino)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin- 1 - yl)-5-(trifluoromethyl)phenol FC-35 (42 mg, yield: 35%) as a yellow solid.
FC-11 (92 mg, 0.22 mmol) was purified by SFC to isolate both chiral products. SFC purification was made using a i-amylose-1 column with ISOC 25% 2-propanol + 0.1% diethylamine to yield 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 configuration was not determined.
Synthesis of Final Compound 38 (FC-38) and Final Compound 39 (FC-39): 2-(4- (((lR,8aS)-octahydroindolizin-l-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5- (trifluoromethyl)phenol (relative stereochemistry) and 2-(4-(((lS,8aS)-
FC-38 FC-39
Step 1
10% Palladium on carbon [7440-05-3] (180 mg, 0.17 mmol) was added to a stirred solution of l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(octahydroindolizin-l- yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine 1-38 (343 mg, 0.68 mmol) in methanol (10 mL) at 0 °C under nitrogen atmosphere. Then, nitrogen atmosphere was replaced by hydrogen [1333-74-0] (1 atm) and the reaction mixture was stirred at rt for 16 h. The mixture was filtered off over a pad of celite and washed with DCM/MeOH (4: 1). Solvents were evaporated in vacuo to yield 2-(4-((octahydroindolizin-l- yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifhioromethyl)phenol (271 mg, yield: 93%) as a yellow foamy solid.
Step 2
2-(4-((octahydroindolizin- 1 -yl)amino)pyrrolo[ 1 ,2-d] [ 1 ,2,4]triazin- 1 -y l)-5 - (trifluoromethyl)phenol, (270 mg, 0.63 mmol) was separated into its diastereomeric components by preparative HPLC (Column: Phenom enex Gemini Cl 8 30x100 mm 5 pm; gradient water 25mM NFUHCCh/acetonitrile 75/25 to 0/100). The fractions were collected and freeze-dried to yield 2-(4-(((lR,8aS)-octahydroindolizin-l- yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) FC-38 (141 mg, 0.33 mmol) as a yellow solid and 2-(4-(((lS,8aS)- octahydroindolizin-l-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5- (trifluoromethyl)phenol (relative stereochemistry) FC-39 (38 mg, 0.09 mmol) as a yellow solid.
Boron tribromide [10294-33-4] (245 μL, 2.5 mmol) was added dropwise to a stirred solution of 2-(4-(((3R)- l-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)piperi din-3 - yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifluoromethyl)phenol 1-48 (150 mg, 0.25 mmol) in anhydrous dichloromethane (3.7 mL) at -20 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 30 min. Aniline [62-53-3] (45 μL, 0.5 mmol) was added dropwise and carefully. The mixture was poured into an ice/saturated NaHCO3 aqueous solution mixture and extracted with dichloromethane/methanol (9: 1) mixture (x 3). The combined organic layers were dried (MgSCU), filtered and concentrated. The crude was purified by reverse phase (Phenom enex Gemini Cl 8 30x100mm 5pm Column; from 50% [25mM NH4HCO3] - 50% [acetonitrile:methanol (1 : 1)] to 0% [25mM NH4HCO3] - 100% [acetonitrile methanol (1 : 1)]) to afford (R)-2- (4-((l-(2-hydroxyethyl)piperidin-3-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5- (trifhioromethyl)phenol FC-40 (15.5 mg, yield: 14%) as a yellow solid.
Synthesis of Final Compound 41 (FC-41) and Final Compound 42 (FC-42): 2-(4- (((lR,8aR)-octahydroindolizin-l-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-
FC-4-! FC-42
FC-39 (38 mg, 0.09 mmol) was purified by SFC to isolate both chiral products. Stationary phase: Amylose-1 250 x 30 mm 5 pm. Mobile phase: 70% CO2 - 30% EtOH + 0.1 % diethylamine. The fractions were collected and concentrated in vacuo to yield FC-41, first eluting enantiomer, (11 mg, 0.03 mmol) as a dark yellow solid and FC-42, second eluting enantiomer, (12 mg, 0.03 mmol) as a dark yellow solid. The absolute configuration was not determined.
FC-38 (141 mg, 0.33 mmol) was purified by SFC to isolate both chiral components. Stationary phase: Amylose-1 250 x 30 mm 5 pm, Mobile phase: 60% CO2 - 40% MeOH + 0.1 % diethylamine. The fractions were collected, concentrated in vacuo, and triturated with diisopropyl ether to yield FC-43, first eluting enantiomer, (57 mg, 0.13 mmol) as a yellow solid and FC-44, second eluting enantiomer, (57 mg, 0.13 mmol) as a yellow solid. The absolute configuration was not determined.
Boron tribromide [10294-33-4] (114 μL, 1.2 mmol) was added dropwise to a stirred solution of (R)-l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(l-(2- fluoroethyl)piperidin-3-yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine 1-49 (120 mg, 0.23 mmol) in anhydrous di chloromethane (300 pL) at -20 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 30 min. Aniline [62-53-3] (45 μL, 0.5 mmol) was added dropwise and carefully. The mixture was poured into an ice/saturated NaHCCh aqueous solution mixture and extracted with dichloromethane/methanol (9: 1) mixture (x 3). The combined organic layers were dried (MgSCU), filtered and concentrated. The residue was purified by reverse phase (Phenom enex Gemini Cl 8 30x100mm 5pm Column; from 59% [25mM NH4HCO3] - 41% [100% acetonitrile] to 17% [25mM NH4HCO3] - 83% [100% acetonitrile) to yield (R)-2-(4-((l-(2- fhioroethyl)piperidin-3-yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5- (trifhioromethyl)phenol FC-45 (14.7 mg, yield: 15%) as a yellowish solid.
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[l,5-d][l,2,4]triazin- 4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry) 1-108 (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) at 0 °C under nitrogen atmosphere. The mixture was stirred at rt for 16 h. The reaction was recharged with triethylamine [121-44-8] (3 eq, 114 μL, 0.81 mmol), Formaldehyde (37% aqueous solution) [50-00-0] (2 eq, 34 μL, 0.46 mmol) and sodium triacetoxyborohydride [56553-60-7] (1.5 eq, 76 mg, 0.35 mmol) and it was stirred at rt for 32 h more. The mixture was diluted with NaHCCL (sat. aq) and extracted with DCM. The organic layer was extracted, dried over MgSCU and concentrated in vacuo to recover the starting material as free base (71 mg, 77%) as a pale-yellow solid. Sodium cyanoborohydride [25895-60-7] (18 mg, 0.27 mmol) was added to a stirred solution of the recovered 2-(7-(((3R,5R)-5-fluoropiperidin-3-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) (71 mg, 0.18 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 at 0 °C under nitrogen atmosphere. The mixture was stirred at rt for 16 h. The mixture was concentrated. The residue was purified by reverse phase chromatography (Phenomenex Gemini C18 30x100mm 5pm Column; from 59% [25mM NH4HCO3] - 41% [acetonitrile:methanol (1 : 1)] to 17% [25mM NH4HCO3] - 83% [acetonitrile:methanol (1 : 1)]). The relevant fractions were collected and the solvent was freeze-dried to yield 2-(7-(((3 R,5R)-5-fluoro-l-methylpiperidin-3-yl)amino)pyrazolo[l,5-d][l, 2, 4]tri azin-4- yl)-5-(trifluoromethyl)phenol (relative stereochemistry) FC-46 (18.7 mg, yield: 25%) as a white solid. FC-47 FC-48
2-(7-((4-methyl-l,4-oxazepan-6-yl)amino)pyrazolo[l,5-d][l,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 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2. FC-47, first eluting enantiomer, (58 mg, yield: 77%) and FC-48, second eluting enantiomer, (57 mg, yield: 76%) were obtained.
The absolute configuration was not determined.
Synthesis of Final Compound 49 (FC-49), Final Compound 50 (FC-50), Final Compound 51 (FC-51) and Final Compound 52 (FC-52): 2-(7-(((lS,8aR)- octahydroindolizin-l-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol, 2-(7-(((lR,8aS)-octahydroindolizin-l- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol, 2-(7- (((lR,8aR)-octahydroindolizin-l-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-
FC-49 FC-50 FC-51 FC-52
Step 1 A solution of 4-chloro-N-(octahydroindolizin-l-yl)pyrazolo[l,5-d][l,2,4]triazin-7- amine, (200 mg, 0.68 mmol) 1-76 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 potassium phosphate tribasic [7778-53-2] (204 mg, 0.96 mmol) and Pd(dppf)C12- dichloromethane [95464-05-4] (26.22 mg, 0.032 mmol). The reaction was heated at 110°C for 3 hours under microwave irradiation. The reaction mixture was poured into saturated aqueous ammonium chloride solution. The organics were extracted with ethyl acetate (5 x 10 mL). The combined organic layers were dried (Na2SO4) and concentrated to dryness to afford 2-(7-((octahydroindolizin-l-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol (300 mg, yield 85%) as brown solid as a mixture of two racemic diastereomers.
Step 2 The residue was purified by preparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-lOpm, 50x150mm, Mobile phase: 0.25% NH4HCO3 solution in water, CH3CN) to yield the two diastereomers. The first eluting diastereomer was further purified by preparative chiral SFC (Stationary phase: Chiralcel Diacel OD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to yield the optically pure TRANS enantiomers 2-(7-(((lS,8aR)-octahydroindolizin-l-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((lR,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 configuration was not determined. 7.3 mg (10% yield) were obtained for the first eluting enantiomer and 7.9 mg (11% yield) were obtained for the second eluting enantiomer. The second eluting diastereomer was further purified by preparative chiral SFC (Stationary phase: Chiralcel Diacel OD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to yield the optically pure CIS enantiomers 2-(7-(((lR,8aR)-octahydroindolizin-l- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7- (((lS,8aS)-octahydroindolizin-l-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol (FC-51 and FC-52). The absolute configuration was not determined. 4.2 mg (6% yield) were obtained for the first eluting enantiomer and 4.3 mg (6% yield) were obtained for the second eluting enantiomer.
Synthesis of Final Compound 53 (FC-53) and Final Compound 54 (FC-54): 2-(7- (((8S,8aR)-octahydroindolizin-8-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-
FC-53 FC-54
A reaction flask was charged with 4-chloro-N-((8S,8aR)-octahydroindolizin-8- yl)pyrazolo[l,5-d][l,2,4]triazin-7-amine (relative stereochemistry) 1-128 (200 mg, 0.68 mmol), (2-hydroxy-4-(trifluoromethyl)phenyl)boronic acid [1072951-50-8] (155 mg, 0.75mmol), 1,4-dioxane (5 mL) and water (0.5 mL). The resulting mixture was purged with nitrogen. Pd(dppf)C12- di chloromethane [95464-05-4] (56 mg, 0.068 mmol) and potassium phosphate tribasic [7778-53-2] (435 mg, 2.05 mmol) were added and the mixture was heated at 90 °C for 18 hours. The crude reaction mixture was diluted with dichloromethane. The water layer was separated, and the organic layer was filtered over decalite, dried (Na2SO4) and concentrated. The crude product was purified using by flash column chromatography (gradient elution: 0 to 10% MeOH in dichloromethane) to give the racemic mixture 2-(7-(((8S,8aR)-octahydroindolizin-8- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol ( relative stereochemistry) (128 mg, 45% yield). The compound was purified by preparative chiral SFC (Stationary phase: Chiralcel Diacel OD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to yield 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[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-53 and FC- 54). The absolute configuration was not determined. 51 mg (80% yield, for the separation) were obtained for the first eluting enantiomer and 51 mg (80% yield, for the separation) were obtained for the second eluting enantiomer.
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[l,5-d][l,2,4]triazin-7-amine 1-102 (300 mg, 0.71 mmol) in isopropanol (4 mL). The reaction mixture was stirred at 95 °C in a closed reaction vessel for 16 hours. The reaction was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (gradient elution: 0 to 10% MeOH in di chloromethane) to give (1S,2S - relative stereochemistry)-2-((R)-3-((4-(2-(methoxymethoxy)-4- (trifluoromethyl)phenyl)pyrazolo[l,5-d][l,2,4]triazin-7-yl)amino)piperidin-l- yl)cyclopentan-l-ol (198 mg, yield 54%). 41 mg of 2-(7-(((R)-l-((l S,2S - relative stereochemistry)-2 -hydroxy cyclopentyl)piperidin-3-yl)amino)pyrazolo[l, 5- d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (loss of methoxymethyl protecting group) were obtained as a byproduct.
Step 2:
4 M HC1 in 1,4-di oxane [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[l,5-d][l,2,4]triazin-7-yl)amino)piperidin-l- yl)cyclopentan-l-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 loaded with Si-Propyl sulfonic acid SCX-2 resin (SiliCycle) and eluted with MeOH. The product compound was released by elution with 7 N NH3/MeOH. The tubes containing the product compound were combined and concentrated under reduced pressure. The crude product was combined with the 41 mg of 2-(7-(((R)-l- ((1 S,2S - relative stereochemistry)-2 -hydroxy cyclopentyl)piperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol obtained from step 1 as byproduct and purified by silica gel column chromatography (gradient elution: 0 to 8% MeOH in DCM) to obtain 191 mg of product compound (58% over 2 steps). Step 3:
2-(7-(((R)-l-((lS,2S - relative stereochemistry)-2 -hydroxy cy cl opentyl)piperi din-3 - yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifhioromethyl)phenol (191 mg, 0.41 mmol) was separated by preparative SFC (Stationary phase: Chiralpak Diacel AD 20 x 250 mm, Mobile phase: CO2, iPrOH + 0.4 iPrNH2) to obtain 2-(7-(((R)-l-((l S,2S)-2- hydroxycyclopentyl)piperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol and 2-(7-(((R)-l-((lR,2R)-2-hydroxycyclopentyl)piperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-55 and FC- 56). The absolute configuration was not determined. 78 mg (24% yield over three steps) were obtained for the first eluting diastereomer, and 72 mg (24% yield over two steps) were obtained for the second eluting diastereomer.
Synthesis of Final Compound 57 (FC-57) and Final Compound 58 (FC-58): 2-(7- (((R)-l-((lR,2R)-2-hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[l,5- d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol and 2-(7-(((R)-l-((lS,2S)-2- hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- -
Step 1
4 M HC1 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[l,5-d][l,2,4]triazin-7-yl)amino)piperidin-l- yl)cyclobutan-l-ol 1-118 (0.17 mmol) in methanol (3 mL). The reaction mixture was stirred at room temperature overnight. The crude reaction mixture was transferred to a column loaded with Si-Propyl sulfonic acid SCX-2 resin (SiliCycle) and eluted with MeOH after which the reaction product was released by elution with 7 N NFL/MeOH. The tubes containing the reaction product were concentrated under reduced pressure. The residue was purified by silica gel column chromatography (gradient elution: 0 to 5% MeOH in di chloromethane) to give 2-(7-(((R)-l-((lR,2R - relative stereochemistry)-2 -hydroxy cy cl obutyl)piperi din-3 -yl)amino)pyrazolo[l, 5- d][l,2,4]triazin-4-yl)-5-(trifhioromethyl)phenol (48 mg, yield 61%).
Step 2
2-(7-(((R)-l-((lR,2R - relative stereochemistry)-2-hydroxycyclobutyl)piperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifhioromethyl)phenol (48 mg, 0.11 mmol) was separated by preparative SFC (Stationary phase: Chiralpak Diacel AD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to obtain 2-(7-(((R)-l-((lR,2R)-2- hydroxycyclobutyl)piperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol and 2-(7-(((R)-l-((l S,2S)-2 -hydroxy cy cl obutyl)piperi din-3 - yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-57 and FC- 58). The absolute configuration was not determined. 8.3 mg (11% yield over three steps) were obtained for the first eluting diastereomer, and 9.7 mg (13% yield over two steps) were obtained for the second eluting diastereomer. Step 1
Formaldehyde [50-00-0] 37% in H2O (133 μL, 0.82 g/mL, 1.33 mmol, 3.1 equivalents) was added to a stirred solution of 2-(7-(((3R,5R)-5-methylpiperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol dihydrochloride (relative stereochemistry) 1-110 (198 mg, 0.43 mmol, 1 equivalent) in MeOH [67-56-1] (5.9 mL). The resulting mixture was stirred 1 h at room temperature. The mixture was cooled to 0 °C before portion-wise addition of sodium triacetoxyborohydride [56553- 60-7] (225.5 mg, 1.06 mmol, 2.5 equivalents). The mixture was allowed to warm to room temperature and further stirred for 1 h. The mixture was diluted with saturated aqueous NaHCCh and extracted with EtOAc three times. The combined organic layers were dried over MgSCU, filtered off and concentrated under reduced pressure The residue was purified by SiCL flash column chromatography (0 to 6% MeOH: di chloromethane) yielding 2-(7-(((3R,5R)-l,5-dimethylpiperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) (200 mg, ).
Step 2
A purification was performed on 2-(7-(((3R,5R)-l,5-dimethylpiperidin-3- yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (relative stereochemistry) via preparative SFC (Stationary phase: Chiralcel Diacel OJ 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) to yield 2-(7-(((3R,5R)-l,5- dimethylpiperidin-3-yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5- (trifluoromethyl)phenol and 2-(7 -(((3 S, 5 S)- 1 , 5 -dimethylpiperi din-3 - yl)amino)pyrazolo[l,5-d][l,2,4]triazin-4-yl)-5-(trifluoromethyl)phenol (FC-59 and FC- 60). The absolute configuration was not determined. 30 mg (35% yield over two steps) were obtained for the first eluting enantiomer (grey solid), and 29 mg (34% yield over two steps) were obtained for the second eluting enantiomer (yellow solid).
Boron tribromide [10294-33-4] (0.14 mL, 1.40 mmol) was added dropwise to a mixture of ((R)-l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-N-(l-cyclopropylpiperi din-3 - yl)pyrrolo[l,2-d][l,2,4]triazin-4-amine) 1-50 (203 mg, 0.40 mmol) in dichloromethane (4 mL) at -20 °C. The resulting mixture was stirred at 0 °C for 2 h. Aniline [62-53-3] (0.15 mL, 1.60 mmol) was added and the reaction mixture was stirred for 10 min. The mixture was concentrated to dryness. The residue was purified by preparative HPLC (Phenom enex, Gemini 5 pm Cl 8, 30 X 100 mm column, gradient 51-94% (v/v) MeCN/MeOH (1 : l)/water (25 mM NH4HCO3). The product-containing fractions were concentrated to dryness in vacuo to yield (R)-2-(4-((l-cy cl opropylpiperi din-3 - yl)amino)pyrrolo[l,2-d][l,2,4]triazin-l-yl)-5-(trifhioromethyl)phenol FC-61 (77.8 mg, 44%) as a yellow foam solid. Analytical Data - LC-MS
LCMS: [M+H]+ means the protonated mass of the free base of the compound, Rt means retention time (in minutes), method refers to the method used for LCMS.
NMR
1 H NMR spectra were recorded on Bruker Avance III and Avance NEO spectrometers.
The chemical shifts are expressed in ppm relative to tetramethylsilane.
Example B - Pharmaceutical Compositions
A compound of the invention (for instance, a compound of the examples) is brought into association with a pharmaceutically acceptable carrier, thereby providing a pharmaceutical composition comprising such active compound. A therapeutically effective amount of a compound of the invention (e.g. a compound of the examples) is intimately mixed with a pharmaceutically acceptable carrier, in a process for preparing a pharmaceutical composition. Example C - Biological Examples
The activity of a compound according to the present invention can be assessed by in vitro methods. A compound the invention exhibits valuable pharmacological properties, e.g. properties susceptible to inhibit NLRP3 activity, for instance as indicated the following test, and are therefore indicated for therapy related to NLRP3 inflammasome activity.
PBMC assay
Peripheral venous blood was collected from healthy individuals and human peripheral blood mononuclear cells (PBMCs) were isolated from blood by Ficoll-Histopaque (Sigma- Aldrich, A0561) density gradient centrifugation. After isolation, PBMCs were stored in liquid nitrogen for later use. Upon thawing, PBMC cell viability was determined in growth medium (RPMI media supplemented with 10% fetal bovine serum, 1% Pen- Strep and 1% L-glutamine). Compounds were spotted in a 1 :3 serial dilution in DMSO and diluted to the final concentration in 30 pl medium in 96 well plates (Falcon, 353072). PBMCs were added at a density of 7.5 x 104 cells per well and incubated for 30 min in a 5% CO2 incubator at 37 °C. LPS stimulation was performed by addition of 100 ng/ml LPS (final concentration, Invivogen, tlrl-smlps) for 6 hrs followed by collection of cellular supernatant and the analysis of IL-ip (pM), IL6 and TNFa cytokines levels (pM) via MSD technology according to manufacturers’ guidelines (MSD, K151A0H).
The IC50 values (for IL-ip) and EC 50 values (IL6 and TNFa) were obtained on compounds of the invention/examples, and are depicted in the following table:
Example D - Efflux ratio
The objective of this assay is to measure the permeability and efflux of test compounds, using MDCK cells transfected with the P-glycoprotein (MDR1). Two control compounds are screened alongside the test compounds, propranolol (highly permeable) and prazosin (a substrate for P-glycoprotein). MDCK cells are an epithelial cell line of canine kidney origin. These cells can be stably transfected to express active P-glycoprotein (MDR1- MDCK) and are ideal for studying drug efflux due to P-gp. Test compound is added to either the apical or basolateral side of a confluent monolayer of MDR1-MDCK cells and permeability in the apical to basolateral (A-B) and basolateral to apical (B-A) direction is measured by monitoring the appearance of the test compound on the opposite side of the membrane using LCMS/MS. Efflux ratios (B-A permeability over A-B permeability) are calculated to determine if the test compound is subject to P-gp efflux. We deliver an apparent permeability (Papp) coefficient and efflux ratio. We also deliver an experimental recovery value. More details can be found on the Cyprotex website, at https://www.cyprotex.com/admepk/in-vitro-permeability-and-drug-transporters/mdrl- mdck-permeability.
Example E - Further Testing
One or more compound(s) may be tested in a number of other assays to evaluate, amongst other properties, permeability, stability (including metabolic stability and blood stability) and solubility. hERG inhibition
The whole cell patch clamp technique on transfected cells allows the study of ion channels with no - or limited interference from other ion-channels. The effect of compounds on the hERG current are studied with an automated planar patch clamp system, SyncroPatch 384PE (as described in Obergrussberger, A., Briiggemann, A., Goetze, T.A., Rapedius, M., Haarmann, C., Rinke, I., Becker, N., Oka, T., Ohtsuki, A., Stengel, T., Vogel, M., Steindl, J.,
Muller, M., Stiehler, J., George, M. & Fertig, N. (2016). Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module. Journal of Laboratory Automation 21 (6) 779-793). All cells are recorded in the whole cell mode of the patch clamp technique. The SyncroPatch 384PE is an automated patch clamp system which allows to conduct parallel recordings from 384 wells. The module is incorporated in a liquid handling pipetting robot system, Biomek FXP, for application of cells and compounds. On the SyncroPatch 384PE, voltage protocols are constructed, and data acquired using PatchControl384 and analyzed using DataControl384 (both Nanion Technologies).
Different screening approaches are applied to create e.g. two concentrations relationships or up to four concentrations relationships per compound. The different concentrations are applied either in single dose or two cumulatively increasing concentrations. The hERG current is determined as the maximal tail current at -30 mV and percent inhibition upon compound addition as well as pICso are reported below.
Metabolic stability test
In liver microsomes
The metabolic stability of a test compound is tested by using liver microsomes (0.5 mg/ml protein) from human and preclinical species incubated up to 60 minutes at 37°C with 1 pM test compound.
The in vitro metabolic half-life (ti/2 ) is calculated using the slope of the log- linear regression from the percentage parent compound remaining versus time relationship (K), ti/2 = - ln(2)/ K.
The in vitro intrinsic clearance (Clint) (ml/min/mg microsomal protein) is calculated using the following formula:
0.693 Vinc
Cli InHtC = - . x - T/IZ — -
Ll/2 vvmic prot,inc
Where: Fine = incubation volume,
Wmic prot,inc weight of microsomal protein in the incubation.
In hepatocytes
The metabolic stability of a test compound is tested using liver hepatocytes (1 milj cells) from human and preclinical species incubated up to 120 minutes at 37°C with 1 pM test compound.
The in vitro metabolic half-life (ti/2) is calculated using the slope of the log-linear regression from the percentage parent compound remaining versus time relationship (K), ti/2 = - ln(2)/ K. The in vitro intrinsic clearance (Clint) (pl/min/million cells) is calculated using the following formula:
0.693 Vinc
% 1000
Where: Vine = incubation volume,
# cellS/nC = number of cells (xlO6) in the incubation
Plasma and brain tissue binding
Plasma Protein Binding
1. Protocol Summary
Test compound is prepared in species specific plasma (diluted to 25% plasma in buffer). The plasma solution is added to one side of the membrane in an equilibrium dialysis system while buffer (pH 7.4) is added to the other side. The system is allowed to reach equilibrium at 37 °C. Compound on both sides of the membrane is measured by LC-MS/MS and the fraction of unbound compound is calculated. We deliver the fraction unbound in plasma (fu) for each test compound, along with percentage recovery.
2. Objective
To determine the extent of plasma protein binding of the test compound.
3. Customer Provides
• Compound identifier, molecular formula.
• 25 pL of 10 mM or 50 pL or 5 mM test compound in DMSO per species.
4. Materials
Plasma from the following strains and species combinations will be used:
• Human (male and female mix - collected into tubes (not bags)) from ethnically diverse donors
• Rat, male SD
• Mouse, male CD
• Dog, male Beagle
• Monkey, male Cyno
• Guinea Pig, male Dunkin Hartley 5. Experimental Procedure
Solutions of test compound (1 pM test compound concentration; 0.5 % final DMSO concentration) are prepared in species specific plasma diluted to 25% plasma with buffer. The experiment is performed using equilibrium dialysis with the two compartments separated by a semi-permeable membrane. 500 pL of buffer (pH 7.4) is added to one side of the membrane and 300 pL of the plasma solution containing the test compound is added to the other side. After equilibration for 6 hr at 37°C in an incubator with 5% CO2 and agitation at 250 rpm on an orbital shaker, samples are taken from both sides of the membrane.
Samples are matrix matched by addition of either buffer or diluted plasma to relevant samples (i.e. 45 pL of buffer is added to 45 pL of the plasma samples and 45 pL of diluted plasma (25%) is added to 45 pL of the buffer samples). Protein is then precipitated from the matrix-matched samples by addition of 180 pL of methanol containing internal standard followed by centrifugation at 4 °C at 2500 rpm for 30 min. Supernatant (20 pL per compound x 4 compounds) is then diluted with water (100 pL) prior to analysis. Test compound incubations are performed in triplicate. Two control compounds, as specified in the guidance to vendor document, are included in each experiment.
6. Quantitative Analysis
The solutions for each batch of compounds are combined into cassettes of up to 4 compounds prior to cassette analysis by LC-MS/MS. Cyprotex generic LC- MS/MS conditions are used.
7. Data Analysis
The fraction unbound in 25 % plasma (fu25%) is calculated using the following equation: fu25%=Peak area ratio buffer/Peak area ratio plasma
The calculated fu at 25 % plasma (fu25%) is converted to fu at 100 % plasma (ful00%) using the following equation: ful00%= fu25%/(4- (3fu25%))
The % recovery is calculated using the following equation:
% Recovery=100 x ((BufferF x VB)+(PlasmaF x VP)/(PlasmaI x VP)) Where:
BufferF = Final Buffer compartment concentration (after dialysis) PlasmaF = Final Plasma compartment concentration (after dialysis) Plasmal = Initial concentration in plasma VB = volume in the buffer compartment VP = volume in the plasma compartment 8. Deliverables
The fraction unbound in plasma (fu) and percent recovery is returned in the form of an Excel spreadsheet. In addition, the sheet will contain an indication whether the data should be further scrutinized by an internal Janssen Reviewer (based on pre-defined rules supplied in the Janssen guidance document) along with any relevant comments.
Brain Tissue Binding
1. Purpose
The objective of this study is to determine brain tissue bindings of test compound(s) in rat and mouse brain tissue using Equilibrium Dialysis Method. The peak area ratios of test compound(s) in brain tissue homogenate and buffer are evaluated by LC-MS/MS.
2. Materials and reagents
Sponsor provides test compound(s). Control compounds verapamil and fluoxetine are purchased from Sigma Chemical Co. Control compound venlafaxine is purchased from MedChemExpress LLC.
Na2HPO4, NaH2PO4 and NaCl are purchased from local supplier. Acetonitrile and methanol are purchased from Merck (Darmstadt, Germany). Other reagents are purchased from local supplier.
Single-Use RED Plate with Inserts (90006BLCS) are purchased from Thermo. Brain tissue homogenate is prepared by diluting one volume of the whole brain tissue with nine volumes of buffer (PBS, pH 7.4), and the mixture is homogenized using a tissue homogenate machine. Brain tissue homogenate is frozen at -80 °C prior to use. Usually, the brain tissues from three or more individual animals are pooled.)
Brain tissue homogenate Species
Rat SD; Pooled; Male
Mouse CD-I; Pooled; Male
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 pH on the day of experiment and adjust if outside specification of 7.4 ± 0.1.
Thaw the frozen brain tissue homogenate (stored at -80°C) Thaw the frozen brain tissue homogenate immediately in a 37°C water bath. Preparation of stock solutions and working solutions
Prepare the stock solutions of test compound(s) and control compounds verapamil, fluoxetine and venlafaxine in DMSO at the concentration of 10 mM. Dilute 2 pL of stock solution (10 mM) with 198 pL DMSO to obtain working solution (100 pM). And then remove 12 pL of working solution to mix with 1200 pL of brain tissue homogenate to achieve final concentration of 1 pM (1% DMSO). Mix the spiked brain tissue homogenate with pipette 5-6 times and vortex thoroughly.
Procedure for equilibrium dialysis
Assemble the 48-well RED device apparatus. Add 500 pL of PBS to the buffer side of the designated wells. Add 300 uL of spiked brain homogenate immediately to the opposite sides of the designated wells. The assay was performed triplicate. Seal the RED device and place the device in an incubator at 37°C with 5% CO2 at 150 RPM for 6 hours. At the end of incubation, remove the seal and pipette 50 pL of samples from both buffer and brain tissue homogenate chambers into separate wells of a new 96-well plate.
Preparation of equilibrium dialysis samples
Add 50 pL of blank brain tissue homogenate to the buffer samples, and an equal volume of PBS to the collected brain tissue homogenate samples. Add 400 pL of room temperature quench solution (acetonitrile containing internal standards (IS, 200 nM Labetalol, 100 nM Alprazolam, 200 nM Imipramine and 2 pM Ketoprofen)) to precipitate protein. Vortex for 5 minutes. Samples in plate are centrifuged at 3,220 g for 30 minutes at room temperature. Transfer 100 pL of the supernatant to a new plate. The supernatant may be diluted with 100 pL or 200 pL water according to the LC/MS signal response and peak shape. Mix well and analyze samples using LC/MS/MS.
Preparation of stability samples
For time 0 samples, transfer 50 pL of the spiked brain tissue homogenate sample to a new plate containing with 50 pL PBS, and then add 400 pL of acetonitrile containing internal standards (IS, 200 nM Labetalol, 100 nM Alprazolam, 200 nM Imipramine and 2 pM Ketoprofen) to precipitate protein. Vortex for 5 minutes. Transfer 50 pL of the spiked brain tissue homogenate sample to a new plate and incubate the plate for 6 hours at 37°C with 5% CO2. After the incubation, add 50 pL PBS and 400 pL of acetonitrile containing internal standards (IS, 200 nM Labetalol, 100 nM Alprazolam, 200 nM Imipramine and 2 pM Ketoprofen) to precipitate protein. Vortex for 5 minutes. Centrifuge all stability samples at 3,220 g for 30 minutes at room temperature. Transfer 100 pL of the supernatant to a new plate. The supernatant may be diluted with 100 pL or 200 pL water according to the LC/MS signal response and peak shape. Mix well and analyze samples using LC/MS/MS.
4. Data analysis
All calculations are carried out using Microsoft Excel.
Determine the peak area ratios of test compound(s) and control compound in the buffer and brain tissue homogenate chambers from peak area ratios. Calculate the percentages of test compound(s) and control compound bound as follows:
2*Peak Area Ratiobuffer chamber Peak Area Ratiobuffer chamber
FUann= - -
2 Peak Area Ratiobssue bomogenate cbamber Peak Area Ratiobssue bomogenate cbamber
Fu =■ FUapp brain D+Fuapp-(D*Fuapp)
Bound %=100%-Fubrain*100%
Recovery % = (Peak Area Ratio buffer chamber * V buffer chamber+ Peak Area Ratio tissue homogenate chamber*V tissue homogenate chamber) / Peak Area Ratio T=0 sample* V tissue homogenate chamber* 100 %
Fuapp = apparent unbound fraction measured with brain tissue homogenate
D = the dilution factor of brain tissue
% Bound = Brain tissue binding
Data
Pharmacokinetics
Dosing
Test System Mouse: Swiss Crl:CDl
Balb/cAnNCrl
NOD Cg-Prkdcscidll2rgtmWjl/SzJ-NSG C57BL/6JRj
Rat: Sprague Dawley Wistar Supplier: Charles River Germany
Age: 6-8 weeks
Acclimatization period: min. 3 days
Diet and feeding: SAFE A04 maintenance diet and water ad libitum
Dosing: PO Orally by gavage
IV Intravenous tail vein
SC at the back
Dose volume: PO 10 ml/kg
IV 2 ml/kg
SC 10 ml/kg
According to good practice guide for administration volumes
N=3 per time point
Blood sampling
Sampling site: At each time point, animals are sacrificed by decapitation and blood is collected by exsanguination into capillaries in case of micro sampling and otherwise in BD vacutainers. Blood samples are placed immediately on melting ice and plasma is obtained following centrifugation at 4° C for 10 minutes at approximately 1900 x g.
Decapitation under isoflurane anesthesia Induction: 4 % (02 and room air)
Amount: Micro sampling: 32 pl on EDTA
Collection tubes: EDTA coated 75 mm capillaries, Vitrex® Capillaries
EDTA, Cat.No.164113
BD Vacutainer 2 mL K2E (EDTA) 3.6 mg. BD (REF.
368841)
Sampling times: flexible ; depending on dose route and expected PK profile
Suggested for PO : 1 h, 4 h, 7 h, 24 h post-dose
For rat serial blood sampling is performed through the tail vein. Selection of sampling method (Microvette tubes or capillary) depends of the amount of plasma needed for bioanalysis.
In mice serial blood sampling is normally performed via puncturing the saphenic vein. Occasionally, blood sampling in mice may be performed via the tail vein. Sample volumes may not exceed the recommended maximal blood sample volume from the animal.
According to Guidelines for blood collection for common Laboratory animals
For tissue or terminal blood sampling: Animals are anesthetized with Isoflurane mixture. Blood sampling is performed through decapitation and tissues can be collected after bleeding the animal.
Anesthesia Isoflurane :
Induction: 4 % (02 and room air)
Maintenance: 2 % (02 and room air)
Tissue sampling
Sampling: After bleeding, individual samples of brain are dissected and weighed. Collection tubes: Super Polyethylene vial 20 ml Perkin Elmer (REF.
6008117) (Polytron)
Lysing Matrix D tube ImL MP Biomedicals (REF. 6913- 500) (Fast Prep)
Instrument: Polytron PT3100
Fast prep sample preparation instrument
Homogenization tissue: Tissue samples were homogenised in demineralised water (1/9 w/v or + 3 ml if tissue weight < 0.33 g). Homogenisation is carried out under dimmed light conditions.
Plasma preparation
Centrifugation: Start within 1 h after sampling
Centrifugation conditions: 4 °C, 1900 x g, ± 10 min
Collection method: Collect 10 -pL plasma with VitJ. ex® end-to-end pipettes
(Cat. No.174313) in 96-well format holder. In the event that less than 10 pL of plasma can be collected, 4 -pL of plasma will be collected (using Vitrex® end-to-end 4-tL, Cat. No. 174213). If less than 4-pL can be collected, no sample will be transferred. Storage: All samples are shielded from daylight and stored at -18
°C prior to analysis.
Sample transfer Frozen to the Department of Bioanalysis Animals are observed during the experiment. During the acclimatization period (after transfer), animals are monitored by the LAM personal daily.
During the experiment, animals are observed visually by the lab staff conducting the animal study, after dosing and at each sample time point. Appearance, behavior, potential side effects. Abnormalities will be registered in the remark sheet of the study protocol.
Body weight loss > 20 %, body temperature is checked when animals are in sub optimal condition, mobility, changed behavior, pain expression. When the body temperature is < 33 °C animals will be euthanized and excluded from the experiment. In case of doubt the veterinarian physician will be consulted and he/she decide of the fate of these animals. Deviations will be registered in the amendments of the study file.
Determination of partition coefficient kpuu.brain
Kpuu, brain was calculated as follows:
Kpuu,brain=(AUC, last, brain*BTB,r)/(AUC, last, plasma*PPB,m) Where AUC,last is defined as the area under the concentration-time curve from dosing (time 0) to the time of the last measured concentration respectively in the brain and in the plasma
BTB,r is the brain tissue binding, as defined above, in rat
PPB,m is the plasma protein binding, as defined above, in mouse Human whole blood assay
For the human whole blood assay, 125 pl of undiluted blood was added to each well of a 96-well plate, followed by the administration of 25 pl of lipopolysaccharide (LPS E. Coli, L4130, Sigma-Aldrich) at a concentration of 30 ng/ml. Blood was primed with LPS for one hour at 37°C before compound dilutions (dose-response, 25 pl/well) were added for 30 minutes at 37°C. Subsequently, the NLRP3 pathway was activated by adding 25 pl of BzATP (A-385, Alomone Labs) to each well at a concentration of ImM. After 1,5 hours at 37°C, plates were centrifuged (2000 rpm, 5 minutes), supernatant was collected and stored at -80°C before analysis of ILip using MSD (V-PLEX Human IL-ip Kit, K151QPD-2, Meso Scale) according to manufacturer’s instructions. Data were analyzed in GraphPad. Potencies are expressed as IC50 (concentration necessary to inhibit 50% of the effect). If a compound is tested multiple times, the potency is reported as the geometrical mean of the different repeats. Free potencies were determined by multiplying the whole blood potency by the free fraction in plasma (free potency = whole blood potency x free fraction in plasma), assuming a blood/plasma ratio of 1. The free fraction in plasma is defined as follows: fu,p = PPB (% free)/100. The determination of the PPB (% free) is described in the plasma protein binding section.
Mouse whole blood assay
For the mouse whole blood assay, blood of several mice was pooled (approximately 300 pl) before adding 75 pl of undiluted mouse blood to each well of a 96-well plate. The NLRP3 pathway was primed by adding 25 pl of LPS (1 pg/ml) to each well for a duration of three hours at 37°C. Compounds were added at different concentrations (dose- response) and incubated for 30 minutes at 37°C before activation of the pathway with BzATP (5 mM, 25 pl/well) for 1 hour. At the end of the experiment, plates were centrifuged at 2000 rpm for 5 minutes, supernatant was collected and stored at -80°C before ILip analysis using MSD (V-PLEX Mouse IL-ip Kit, K152QPD, Meso Scale) according to manufacturer’s instructions. Data were analyzed in GraphPad. Potencies are expressed as IC50 (concentration necessary to inhibit 50% of the effect). If a compound is tested multiple times, the potency is reported as the geometrical mean of the different repeats. Free potencies were determined by multiplying the whole blood potency by the free fraction in plasma (free potency = whole blood potency x free fraction in plasma), assuming a blood/plasma ratio of 1. The free fraction in plasma is defined as follows: fu,p = PPB (% free)/100. The determination of the PPB (% free) is described in the plasma protein binding section.
Solubility Assay An aliquot of a DMSO solution containing the test compound is dispensed in a 96-well plate, the DMSO is evaporated, and the pellet is re-dissolved by adding the buffer. The compound solubility in pH 2.0 or 7.0 buffer is measured after three days of agitation at 25 °C. The samples are centrifuged, and the super-natant is filtered. The filtrates are pooled, and the concentration is measured by liquid chromatography/tandem mass spectroscopy (LC-MS/MS). An assessment of the solid-state character of the residues is conducted by polarized light microscopy (PLM).
Phospholipidosis Assay
The phospholipidogenic potential of the compounds was assessed according to a reported procedure (Mesens, N.; Steemans, M.; Hansen, E.; Peters, A.; Verheyen, G.; Vanparys, P. A 96-well flow cytometric screening assay for detecting in vitro phospholipidosisinduction in the drug discovery phase, Toxicology in Vitro 23, (2009), 217-226. Data are reported as concentration showing a 2-fold increase in fluorescence.
Data:
Chromatography Hydrophobicity Index (CHI) CHI LogD, also referred as ChromLogD in the literature, values were determined for the compounds of the invention. For a description of an assay, see for example, Rombouts et al. , J. Med. Chem. 2021, 64, 19, 14175-14191.

Claims

Claims
1. A compound of formula (I), Or a pharmaceutically acceptable salt thereof, wherein
2. The compound of claim 1, wherein R1 is
3. The compound of claim 1 wherein wherein R6 is CH3 or
CH2CH3; R7 is H, CH3 or F, and R8 is H, or both R7 and R8 are F.
4. The compound of claim 1 wherein
5. The compound of claim 1 wherein R2, R4 and R5 are hydrogen.
6. The compound of claim 1 wherein R3 is CF3.
7. The compound of claim 1 wherein R6 is CH3, CH2CH3, CH(CH3)2, CH2CH2OH, CH2CH2F or CD3.
8. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in any one of claims 1 to 7 and a pharmaceutically acceptable carrier.
9. A process for preparing a pharmaceutical composition as defined in claim 8, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound as defined in any one of claims
EP24702714.7A 2023-01-31 2024-01-26 Pyrrolo[1,2-d][1,2,4]triazines and pyrazolo[1,5-d] [1,2,4]triazines as nlrp3 inhibitors Pending EP4658654A1 (en)

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