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WO2024160690A1 - 2-(pyridazin-3-yl)-5-(trifluoromethyl)phenols as nlrp3 inhibitors - Google Patents

2-(pyridazin-3-yl)-5-(trifluoromethyl)phenols as nlrp3 inhibitors Download PDF

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WO2024160690A1
WO2024160690A1 PCT/EP2024/051978 EP2024051978W WO2024160690A1 WO 2024160690 A1 WO2024160690 A1 WO 2024160690A1 EP 2024051978 W EP2024051978 W EP 2024051978W WO 2024160690 A1 WO2024160690 A1 WO 2024160690A1
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mmol
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methyl
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hydrogen
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Inventor
Oscar MAMMOLITI
Soufyan JERHAOUI
Laura PEREZ BENITO
Santiago CAÑELLAS ROMAN
Daniel Oehlrich
Marion Laurence Christiane PRIERI
Michael Eric MURATORE
Riccardo RICCIOLI
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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Priority to AU2024214952A priority Critical patent/AU2024214952A1/en
Priority to CN202480022510.0A priority patent/CN120957980A/en
Priority to KR1020257028703A priority patent/KR20250152598A/en
Priority to EP24702518.2A priority patent/EP4658644A1/en
Publication of WO2024160690A1 publication Critical patent/WO2024160690A1/en
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
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    • 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
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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    • C07—ORGANIC CHEMISTRY
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    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
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    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
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    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring

Definitions

  • NLRP3 NOD-like receptor protein 3
  • 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
  • MOS myelodysplastic syndromes
  • lung cancer colon cancer
  • Described herein are compounds which inhibit the NLRP3 inflammasome pathway.
  • compounds of Formula (I), or pharmaceutically acceptable salts thereof wherein R 1 is hydrogen, methyl or chloro; wherein Y is CH2, NR 21 or O;
  • R 20 is oxetan-3-yl, CD3, C alkyl optionally substituted with halo, hydroxy, or cyano;
  • R 21 is hydrogen or C 1-2 alkyl
  • R 3 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl
  • R 4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; or
  • 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.
  • Figure 1 in vivo Long Term Potentiation (LTP) experiments: measurement of the effect of NLRP3 inhibitor compound 9 on LPS triggered pro-inflammatory cytokine ILip: Figure 1(A) measurement of ILIP; Figure 1(B) measurement of IL6; Figure 1(C) measurement of TNFa.
  • Figure 2 in vivo Long Term Potentiation (LTP) experiments: measurement of the effect of NLRP3 inhibitor compound 51 on LPS triggered pro-inflammatory cytokine ILip: Figure 2(A) measurement of ILIP; Figure 2(B) measurement of IL6; Figure 2(C) measurement of TNFa.
  • R 1 is hydrogen, methyl or chloro; wherein Y is CH2, NR 21 or O;
  • R 20 is oxetan-3-yl, C1-4 alkyl optionally substituted with halo, hydroxy, or cyano;
  • R 21 is hydrogen or C 1-2 alkyl
  • R 3 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl
  • R 4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; or
  • R 1 is hydrogen or methyl.
  • R 2 is wherein R 20 is C1-2 alkyl, CD3, 2-hydroxyethyl, 2-fluoroethyl, 3-fluoropropyl, or cyanomethyl.
  • R 2 is wherein R 20 is methyl.
  • R 21 is hydrogen or methyl.
  • R 3 is hydrogen or methyl.
  • R 4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl.
  • R 1 is hydrogen or methyl; wherein Y is CH2 or O;
  • R 20 is methyl
  • R 3 is hydrogen
  • R 4 is hydrogen
  • R 3 and R 4 together form a bivalent radical -R 3 -R 4 - selected from the following list a) -CH2-CH2-CH2-, e) -CH2-CH2-O-CH2-, and each Z is hydrogen.
  • 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 counterion 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 (zusammeri) 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.
  • stereoisomer 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.
  • 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 2 H, 3 H, n C, 13 C, 14 C , 13 N, 15 O, 17 O, 18 O, and 18 F.
  • Tritiated ( 3 H) and carbon-14 ( 14 C) 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 15 O, 13 N, n C and 18 F 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.
  • Cn q alkyl groups (where q is the upper limit of the range) defined herein may be straight-chain or be branched-chain.
  • Cs-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.
  • Ci-q alkoxy groups (where q is the upper limit of the range) refers to the radical of formula -OR a , where R a is a Cn q alkyl group as defined herein.
  • HaloCi-q alkyl (where q is the upper limit of the range) groups refer to Cn q alkyl groups, as defined herein, where such group is substituted by one or more halo.
  • HydroxyCi-q alkyl (where q is the upper limit of the range) refers to Cn 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.
  • Intermediates of Formula (II) can be prepared by reacting an intermediate of Formula (III) with a suitable organozinc reagent in the presence of a suitable nickel catalyst such as, for example, (l,2-dimethoxyethane)nickel dibromide, in the presence of a suitable ligand such as, for example, 4,4’ -di -tert-butyl-2, 2’ -bipyridine, with a suitable base such as, for example, pyridine, in a suitable solvent such as, for example, DMA, at a suitable temperature such as, for example, 100 °C;
  • a suitable nickel catalyst such as, for example, (l,2-dimethoxyethane)nickel dibromide
  • a suitable ligand such as, for example, 4,4’ -di -tert-butyl-2, 2’ -bipyridine
  • a suitable base such as, for example, pyridine
  • a suitable solvent such as, for example, DMA
  • Intermediates of Formula (III) can be prepared by reaction of an Intermediate of Formula (IV) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium, in the presence of a suitable base such as, for example, sodium carbonate, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C.
  • a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium
  • a suitable base such as, for example, sodium carbonate
  • a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C.
  • Intermediates of Formula (V) can be prepared by reaction of Intermediate of Formula (IV) with a suitable organozinc reagent in the presence of a suitable nickel catalyst such as, for example, (l,2-dimethoxyethane)nickel dibromide, in the presence of a suitable ligand such as, for example, 4,4’ -di -tert-butyl-2, 2’ -bipyridine, with a suitable base such as, for example, pyridine, in a suitable solvent such as, for example, DMA, at a suitable temperature such as, for example, 100 °C;
  • a suitable nickel catalyst such as, for example, (l,2-dimethoxyethane)nickel dibromide
  • a suitable ligand such as, for example, 4,4’ -di -tert-butyl-2, 2’ -bipyridine
  • a suitable base such as, for example, pyridine
  • a suitable solvent such as, for example, DMA
  • Intermediates of Formula (VI) can be prepared by reacting an intermediate of Formula (VII) with a suitable fluorinating agent such as, for example, DAST, in the presence of a suitable supplement such as, for example, triethylamine trishydrofluoride, in a suitable solvent such as, for example, anhydrous DCM, at a suitable temperature such as, for example, 0 °C;
  • a suitable fluorinating agent such as, for example, DAST
  • a suitable supplement such as, for example, triethylamine trishydrofluoride
  • a suitable solvent such as, for example, anhydrous DCM
  • Intermediates of Formula (VII) can be prepared by reacting an intermediate of Formula (VIII) with an appropriate zincate reagent in the presence of a suitable catalyst such as, for example, copper(I) cyanide, in the presence of a suitable supplement such as, for example, anhydrous lithium chloride, in a suitable solvent such as, for example, THF, at a suitable temperature such as, for example, -20 °C.
  • a suitable catalyst such as, for example, copper(I) cyanide
  • a suitable supplement such as, for example, anhydrous lithium chloride
  • a suitable solvent such as, for example, THF
  • Intermediates of Formula (IX) can be prepared by reacting an intermediate of Formula (X) with a suitable fluorinating agent such as, for example, DAST, in the presence of a suitable supplement such as, for example, triethylamine trishydrofluoride, in a suitable solvent such as, for example, anhydrous DCM, at a suitable temperature such as, for example, 0 °C;
  • a suitable fluorinating agent such as, for example, DAST
  • a suitable supplement such as, for example, triethylamine trishydrofluoride
  • a suitable solvent such as, for example, anhydrous DCM
  • Intermediates of Formula (X) can be prepared by reacting an intermediate of Formula (VII) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium, in the presence of a suitable base such as, for example, sodium carbonate, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C;
  • a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium
  • a suitable base such as, for example, sodium carbonate
  • a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C;
  • Intermediates of Formula (XI) can be prepared by saponification of an intermediate of Formula (XII), initially protected by any suitable protecting group PG such as, for example, methyl or ethyl, using a suitable base such as, for example, lithium hydroxide, in a suitable solvent such as, for example, a mixture of THF, water and methanol, at a suitable temperature such as, for example, room temperature or 50 °C;
  • Intermediates of Formula (XII) can be prepared by reacting an intermediate of Formula (IV) in a pressure vessel under carbon monoxide atmosphere, in the presence of a suitable catalyst such as, for example, Pd(dppf)C12, in the presence of a suitable base such as, for example, sodium acetate or triethylamine, in a suitable solvent such as, for example, methanol or ethanol, at a suitable temperature such as, for example, 100 °C, at a suitable pressure such as, for example, 10 bars.
  • a suitable catalyst such as, for example, Pd(dppf)C12
  • a suitable base such as, for example, sodium acetate or triethylamine
  • a suitable solvent such as, for example, methanol or ethanol
  • a suitable temperature such as, for example, 100 °C
  • a suitable pressure such as, for example, 10 bars.
  • R 2 , R 3 and/or R 4 contain a protecting group such as, for example, Boc, deprotection of intermediates of Formula (la) or (lb) will afford the deprotected compound of Formula (la) or (lb).
  • a reductive agent such as, for example, sodium triacetoxyborohydride
  • a suitable solvent such as, for example, methanol or dichloromethane, at a suitable temperature such as, for example, 0 °C.
  • the instant compounds are potent, brain-penetrant, have low cardiovascular liability 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.
  • 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.
  • 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.
  • Lithium hydroxide hydrate (2.8 g, 65.24 mmol) was added to a stirred solution of Tris(dihenzylideneacetone)dipalladium(0) [CAS 51364-51-3] (616 mg, 0.652 mmol) and BippyPhos [CAS 894086-00-1] (681 mg, 1.31 mmol) in 50 mL of 1,4-dioxane and 5 ml of distilled water (previously bubbled with nitrogen for 5 min). The mixture was stirred at rt for 5 min, then 3-bromo-5-methylbenzotrifluoride [CAS 86845-28-5] (5.25 g, 21.74 mmol) was added.
  • Ethylene glycol [CAS 107-21-1] (1.49 mL, 26.72 mmol) and sodium hydride (1.1 g, 27.5 mmol) were added to a solution of perchloropyridazine [CAS 20074-67-3] (5 g, 22.95 mmol) in 130 mL of anhydrous DMF at 0 °C.
  • the reaction mixture was stirred at room temperature for 18 h.
  • more sodium hydride (1.1 g, 27.5 mmol) was added and the mixture was stirred at 60 °C for 3 h. Solvent was evaporated in vacuo.
  • Oxalyl chloride [CAS 79-37-8] (1.0 mL, 12.05 mmol) was added dropwise to a stirred solution of 6-chloropyridazine-3 -carboxylic acid [CAS 5096-73-1] (1.5 g, 9.27 mmol) and anhydrous DMF (75 pL, 0.97 mmol) in 32 mL of anhydrous DCM at 0 °C under nitrogen atmosphere.
  • the suspension was stirred at 0 °C for 10 min, then was stirred at rt for 2 h until a clear brownish solution was obtained.
  • Solvent was evaporated in vacuo to yield Intermediate 13 (1.65 g, assumed quant, yield) as a pale brown solid which was used in the next step without further purification.
  • Triethylammonium fluoride [CAS 73602-61-6] (77 pL, 0.46 mmol) and DAST [CAS 38078- 09-0] (171 pL, 1.23 mmol) were added dropwise sequentially to a stirred solution of Intermediate 14 (100 mg, 0.31 mmol) in 2.5 mL of anhydrous DCM in a PTFE flask at 0 °C under nitrogen atmosphere.
  • the reaction was stirred at rt for 10 min and then at 40 °C for 16 h. Then was cooled down to rt and was poured into a vigorous stirred ice/(sat. NaHCOs aqueous solution) mixture and extracted with DCM (x3).
  • the methyl tert-butyl ether suspension was transferred to a 6 mL syringe under air. Then a syringe filter and new needle were installed on the syringe, before the methyl tert-butyl ether solution was injected through the syringe filter into the dimethylacetamide solution.
  • the reaction mixture was sparged with nitrogen before sealing with parafilm.
  • the vial was stirred at 1500 rpm stir rate and irradiated under 450 nm LED modules at 100% light intensity with maxed fan speed of 1500 rpm stirring rate in a PennOC Integrated Photoreactor for 4 h.
  • the reaction mixture was filtered over a pad of dicalite and the cake was washed with EtOAc (50 mL).
  • 1-pyrrolidino-l -cyclopentene [CAS 7148-07-4] (8 g, 58.3 mmol) was added dropwise to a mixture of tert-butyl (A)-3-((6-chloro-l,2,4,5-tetrazin-3-yl)amino)piperidine-l-carboxylate 125 (16.68 g, 53 mmol) in dry toluene (390.98 mL) in a 1 L four necked reactor. The mixture was stirred at room temperature for 30 min (attention: some nitrogen was released). Than the mixture was heated at 50 °C for 30 min and then the mixture was heated at reflux for 6 h. The first 10 mL reflux effluent were removed.
  • HC1 (4M in 1,4- dioxane) (6.04 mL, 4 M, 24.17 mmol) was added to a soluton of dihydro- 57/-cyclopenta[t/]pyridazin- l -yl)amino)piperidine- l -carboxylate 127 (701 mg, 1.27 mmol) in 1,4-di oxane (6.1 mL) and the mixture was stirred at room temperature for 2h. LC/MS showed complete conversion. The mixture was poured out in sat NaHCOs solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried on MgSO4, filtered and concentrated, yielding the desired product (495 mg, yield 99.08%) as a white solid.
  • Compound 29 was synthesized as comparator with the corresponding 3-piperidinyl (Compound 30) (.S)-2-(4-((4-methylmorpholiii-2-yl)methyl)-7.8-dihydro-5//-pyr:ino
  • Compound 34 was made by analogy with Compound 33, using (R)-Intermediate 18-6 as starting material.
  • HPLC High-Performance Liquid Chromatography
  • MS Mass Spectrometer
  • SQL Single Quadrupole Detector
  • MSD Mass Selective Detector
  • RT room temperature
  • BEH bridged ethylsiloxane/silica hybrid
  • DAD Diode Array Detector
  • HSS High Strength silica
  • a compound for instance, a compound of the examples
  • a pharmaceutically acceptable carrier for instance, a pharmaceutically acceptable carrier
  • a therapeutically effective amount of a compound as provided herein is intimately mixed with a pharmaceutically acceptable carrier, in a process for preparing a pharmaceutical composition.
  • a compound may exhibit 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 inDMSO 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
  • EC50 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
  • Example E - 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 Nani on Technologies).
  • hERG current is determined as the maximal tail current at -30 mV and percent inhibition upon compound addition as well as pIC50 are reported below.
  • Example F Further Testing One or more compound(s) were/may be tested in a number of other assays to evaluate, amongst other properties, permeability, stability (including metabolic stability and blood stability) and solubility.
  • 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.
  • V mc 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: 0.693 V inc x 1000
  • V mc incubation volume
  • 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.
  • Peak Area Ratio bssue homogenate chamber Peak Area Ratio bssue homogenate chamber Fu FUapp bra ' n D+Fu app -(D*Fu app )
  • 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.
  • Sample volumes may not exceed the recommended maximal blood sample volume from the animal.
  • 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)
  • 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
  • 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.
  • mice were treated with NLRP3 inhibitors prior to LPS administration to evaluate the effect of NLRP3 inhibitors on inflammasome activation by measuring ILip.
  • IL6 and TNFa were measured as well.
  • Compounds were administered via oral gavage (PO) 30 minutes before intraperitoneal LPS injection (10 mg/kg) injection (Escherichia coli O11 LB4; L4130, Sigma-Aldrich). Three doses were tested for each compound.
  • Nlrpr3 knockout mice were included as a negative control, i.e., to define endogenous levels of ILip in these experiments.
  • animals were sacrificed by decapitation and plasma samples were collected for bioanalysis and cytokine (ILip, IL6 and TNFa) analysis using ELISA (ILip, Quantikine MLB00C, R&D Systems Minneapolis, Canada) and MSD (IL6 and TNFa, V-Plex K15048D MSD, Meso Scale Diagnostics, Maryland, USA). Plasma samples were diluted 1/20 for ILip and TNFa measurements and further diluted to 1/60 for the analysis of IL6.
  • 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 (doseresponse) and incubated for 30 minutes at 37°C before activation of the pathway with BzATP (5 mM, 25 pl/well) for 1 hour.
  • CHI LogD also referred as ChromLogD in the literature, values were determined for the compounds of the invention.
  • values were determined for the compounds of the invention.
  • Rombouts et al. J. Med. Chem. 2021, 64, 19, 14175-14191.
  • the compounds 5 of the present invention display advantageous properties, such as in terms of brain penetration, phospholipidosis potential and/or polarity.
  • the following compound, with a monocyclic core structure was tested in the same assays as described herein for brain penetration and phospholipidosis potential and can be compared with final compound 9 according to the invention:
  • the following compounds, with a bicyclic core structure were tested in the same assays as described herein for CHI LogD, and can also be compared to final compounds described herein:

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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 and Z 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

2-(PYRID AZIN-3- YL)-5-(TRIFLUOROMETHYL)PHENOLS AS NLRP3 INHIBITORS
TECHNICAL FIELD
Described herein are 2-(pyridazin-3-yl)-5-(trifluoromethyl)phenols 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-2023/003002.
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),
Figure imgf000004_0001
or pharmaceutically acceptable salts thereof, wherein R1 is hydrogen, methyl or chloro;
Figure imgf000004_0002
wherein Y is CH2, NR21 or O;
R20 is oxetan-3-yl, CD3, C alkyl optionally substituted with halo, hydroxy, or cyano;
R21 is hydrogen or C 1-2 alkyl;
R3 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl;
R4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; or
R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) -CH2-CH2-CH2-NH-, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-, f) -O-CH2-CH2-O-, g) -CH=CH-CH=N-, and h) -N=CH-CH=CH-; and each Z independently is hydrogen or fluoro.
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.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : in vivo Long Term Potentiation (LTP) experiments: measurement of the effect of NLRP3 inhibitor compound 9 on LPS triggered pro-inflammatory cytokine ILip: Figure 1(A) measurement of ILIP; Figure 1(B) measurement of IL6; Figure 1(C) measurement of TNFa. Figure 2: in vivo Long Term Potentiation (LTP) experiments: measurement of the effect of NLRP3 inhibitor compound 51 on LPS triggered pro-inflammatory cytokine ILip: Figure 2(A) measurement of ILIP; Figure 2(B) measurement of IL6; Figure 2(C) measurement of TNFa.
DETAILED DESCRIPTION
Provided herein are compounds of formula (I),
Figure imgf000005_0001
and pharmaceutically acceptable salts thereof, wherein: wherein R1 is hydrogen, methyl or chloro;
Figure imgf000006_0001
wherein Y is CH2, NR21 or O;
R20 is oxetan-3-yl, C1-4 alkyl optionally substituted with halo, hydroxy, or cyano;
R21 is hydrogen or C 1-2 alkyl;
R3 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl;
R4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; or
R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) -CH2-CH2-CH2-NH-, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-, f) -O-CH2-CH2-O-, g) -CH=CH-CH=N-, and h) -N=CH-CH=CH-; and each Z independently is hydrogen or fluoro.
In an embodiment R1 is hydrogen or methyl.
In an embodiment R2 is
Figure imgf000006_0002
wherein R20 is C1-2 alkyl, CD3, 2-hydroxyethyl, 2-fluoroethyl, 3-fluoropropyl, or cyanomethyl.
In an embodiment R2 is
Figure imgf000006_0003
wherein R20 is methyl. In an embodiment R21 is hydrogen or methyl.
In an embodiment R3 is hydrogen or methyl.
In an embodiment R4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl.
In an embodiment R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) -CH2-CH2-CH2-NH-, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-, f) -O-CH2-CH2-O-, g) -CH=CH-CH=N-, and h) -N=CH-CH=CH-.
In an embodiment
R1 is hydrogen or methyl;
Figure imgf000007_0001
wherein Y is CH2 or O;
R20 is methyl;
R3 is hydrogen;
R4 is hydrogen; or
R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2-, e) -CH2-CH2-O-CH2-, and each Z is hydrogen.
Compounds of particular interest are
(S)-2-(4-((l-methylpiperidin-3-yl)methyl)-6,7-dihydro-5H-cyclopenta[d]pyridazin-l- yl)-5-(trifluoromethyl)phenol,
(S)-3-methyl-2-(6-((l-methylpiperi din-3 -yl)methyl)pyridazin-3-yl)-5-
(trifluoromethyl)phenol, and
(S)-2-(4-((4-methylmorpholin-2-yl)methyl)-7,8-dihydro-5H-pyrano[3,4-d]pyridazin-l- yl)-5-(trifluoromethyl)phenol. In another aspect, provided herein is the following compound
Figure imgf000008_0001
, or a pharmaceutically acceptable salt thereof.
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 counterion 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 (zusammeri) 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, nC, 13C, 14C , 13N, 15O, 17O, 18O, and 18F. Tritiated (3H) and carbon-14 (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, nC 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, Cnq alkyl groups (where q is the upper limit of the range) defined herein may be straight-chain or be branched-chain.
Cs-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.
Ci-q alkoxy groups (where q is the upper limit of the range) refers to the radical of formula -ORa, where Ra is a Cnq alkyl group as defined herein.
HaloCi-q alkyl (where q is the upper limit of the range) groups refer to Cnq alkyl groups, as defined herein, where such group is substituted by one or more halo. HydroxyCi-q alkyl (where q is the upper limit of the range) refers to Cnq 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
Final compounds according to Formula (la), wherein each Z represents H, can be prepared:
Figure imgf000011_0001
- By deprotecting an intermediate of Formula (II) in a suitable acidic medium such as, for example, hydrochloric acid solution in 1,4-di oxane, at a suitable temperature such as, for example, room temperature; or by deprotecting an intermediate of Formula (II) in suitable hydrogenative conditions such as, for example, Pd/C in an hydrogen atmosphere, in a suitable solvent such as, for example, ethyl acetate or ethanol, at a suitable temperature such as, for example, room temperature;
Intermediates of Formula (II) can be prepared by reacting an intermediate of Formula (III) with a suitable organozinc reagent in the presence of a suitable nickel catalyst such as, for example, (l,2-dimethoxyethane)nickel dibromide, in the presence of a suitable ligand such as, for example, 4,4’ -di -tert-butyl-2, 2’ -bipyridine, with a suitable base such as, for example, pyridine, in a suitable solvent such as, for example, DMA, at a suitable temperature such as, for example, 100 °C;
Alternatively, by reacting an intermediate of Formula (III) with a suitable organozinc reagent via Negishi coupling in the presence of a suitable palladium catalyst such as, for example, cataCXium Pd G4, in a suitable solvent such as, for example, THF, at a suitable temperature such as, for example, 50 °C;
Alternatively, by reacting an intermediate of Formula (III) with a suitable alcohol derivative in the presence of a suitable photocatalyst such as, for example, Ir[ppy]2(dtbbpy)PF6, with a suitable catalyst such as, for example, NiBr2(dtbbpy), with a suitable photoactivator such as, for example, 5, 7-di -tert-butyl -3 -phenyl -1,3- benzoxazol-3-ium, with a suitable base or mixture of bases such as, for example, pyridine and quinuclidine, in a suitable solvent or mixture of solvents such as, for example, MTBE and DMA, under LED irradiation at 450 nm with 100% light intensity; Alternatively, by reacting an intermediate of Formula (III) with a suitable vinylboronic acid or vinyl boronate ester via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, dichlorobis(triphenylphosphine)palladium(II), in the presence of a suitable base such as, for example, potassium phosphate tribasic, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C; followed by reduction of the double bond by hydrogenation with Pd/C under hydrogen atmosphere in a suitable solvent, such as, for example, ethanol or ethyl acetate, at a suitable temperature such as, for example, room temperature or via reduction using mild sodium borohydride, sodium triacetoxyborohydride or related agents in a suitable solvent such as, for example, methanol, at a suitable temperature such as, for example, 0 °C or room temperature;
Intermediates of Formula (III) can be prepared by reaction of an Intermediate of Formula (IV) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium, in the presence of a suitable base such as, for example, sodium carbonate, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C.
Alternatively, intermediates according to Formula (II), can be prepared:
Figure imgf000012_0001
- By reacting an Intermediate of Formula (V) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium, in the presence of a suitable base such as, for example, sodium carbonate, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C;
Intermediates of Formula (V) can be prepared by reaction of Intermediate of Formula (IV) with a suitable organozinc reagent in the presence of a suitable nickel catalyst such as, for example, (l,2-dimethoxyethane)nickel dibromide, in the presence of a suitable ligand such as, for example, 4,4’ -di -tert-butyl-2, 2’ -bipyridine, with a suitable base such as, for example, pyridine, in a suitable solvent such as, for example, DMA, at a suitable temperature such as, for example, 100 °C;
Alternatively, by reacting an intermediate of Formula (IV) with a suitable organozinc reagent via Negishi coupling in the presence of a suitable palladium catalyst such as, for example, cataCXium Pd G4, in a suitable solvent such as, for example, THF, at a suitable temperature such as, for example, 50 °C; Alternatively, by reacting an intermediate of Formula (IV) with a suitable alcohol derivative in the presence of a suitable photocatalyst such as, for example, Ir[ppy]2(dtbbpy)PF6, with a suitable catalyst such as, for example, NiBr2(dtbbpy), with a suitable photoactivator such as, for example, 5, 7-di -tert-butyl -3 -phenyl -1,3- benzoxazol-3-ium, with a suitable base or mixture of bases such as, for example, pyridine and quinuclidine, in a suitable solvent or mixture of solvents such as, for example, MTBE and DMA, under LED irradiation at 450 nm with 100% light intensity; Alternatively, by reacting an intermediate of Formula (IV) with a suitable vinylboronic acid or vinyl boronate ester via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, dichlorobis(triphenylphosphine)palladium(II), in the presence of a suitable base such as, for example, potassium phosphate tribasic, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C; followed by reduction of the double bond by hydrogenation with Pd/C under hydrogen atmosphere in a suitable solvent, such as, for example, ethanol or ethyl acetate, at a suitable temperature such as, for example, room temperature or via reduction using mild sodium borohydride, sodium triacetoxyborohydride or related agents in a suitable solvent such as, for example, methanol, at a suitable temperature such as, for example, 0 °C or room temperature;
The skilled person will understand that when R2 bears a heteroatom in beta-position to the reactive site, such as in 2-(bromomethyl)morpholino derivatives, neither Negishi nor Suzuki coupling can be performed, and MacMillan chemistry (as described in Dong, Z., MacMillan, D.W.C. Metallaphotoredox-enabled deoxygenative arylation of alcohols. Nature 598, 451— 456 (2021). https://doi.org/10.1038/s41586-021-03920-6) is the best option.
Final compounds according to Formula (lb), wherein each Z represents F, can be prepared:
Figure imgf000013_0001
- By reacting an Intermediate of Formula (VI) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium, in the presence of a suitable base such as, for example, sodium carbonate, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C;
Intermediates of Formula (VI) can be prepared by reacting an intermediate of Formula (VII) with a suitable fluorinating agent such as, for example, DAST, in the presence of a suitable supplement such as, for example, triethylamine trishydrofluoride, in a suitable solvent such as, for example, anhydrous DCM, at a suitable temperature such as, for example, 0 °C;
Intermediates of Formula (VII) can be prepared by reacting an intermediate of Formula (VIII) with an appropriate zincate reagent in the presence of a suitable catalyst such as, for example, copper(I) cyanide, in the presence of a suitable supplement such as, for example, anhydrous lithium chloride, in a suitable solvent such as, for example, THF, at a suitable temperature such as, for example, -20 °C.
Alternatively, final compounds according to Formula (lb), wherein each Z represents F, can be prepared:
Figure imgf000014_0001
- By deprotecting an intermediate of Formula (IX) in a suitable acidic medium such as, for example, hydrochloric acid solution in 1,4-di oxane, at a suitable temperature such as, for example, room temperature; or by deprotecting an intermediate of Formula (XI) in suitable hydrogenative conditions such as, for example, Pd/C in an hydrogen atmosphere, in a suitable solvent such as, for example, ethyl acetate or ethanol, at a suitable temperature such as, for example, room temperature;
Intermediates of Formula (IX) can be prepared by reacting an intermediate of Formula (X) with a suitable fluorinating agent such as, for example, DAST, in the presence of a suitable supplement such as, for example, triethylamine trishydrofluoride, in a suitable solvent such as, for example, anhydrous DCM, at a suitable temperature such as, for example, 0 °C;
Intermediates of Formula (X) can be prepared by reacting an intermediate of Formula (VII) with an appropriate boronic acid or boronic ester derivative via Suzuki coupling in the presence of a suitable palladium catalyst such as, for example, tetrakis triphenylphosphine palladium, in the presence of a suitable base such as, for example, sodium carbonate, in a suitable solvent such as, for example, a mixture of 1,4-di oxane and water, at a suitable temperature such as, for example, 100 °C;
Intermediates of Formula (VIII) can be prepared:
Figure imgf000014_0002
- By reacting an Intermediate of Formula (XI) with a suitable activating agent such as, for example, oxalyl chloride, in the presence of a suitable catalyst such as, for example, DMF, in a suitable solvent such as, for example, DCM, at a suitable temperature such as, for example, 0 °C; Intermediates of Formula (XI) can be prepared by saponification of an intermediate of Formula (XII), initially protected by any suitable protecting group PG such as, for example, methyl or ethyl, using a suitable base such as, for example, lithium hydroxide, in a suitable solvent such as, for example, a mixture of THF, water and methanol, at a suitable temperature such as, for example, room temperature or 50 °C;
Intermediates of Formula (XII) can be prepared by reacting an intermediate of Formula (IV) in a pressure vessel under carbon monoxide atmosphere, in the presence of a suitable catalyst such as, for example, Pd(dppf)C12, in the presence of a suitable base such as, for example, sodium acetate or triethylamine, in a suitable solvent such as, for example, methanol or ethanol, at a suitable temperature such as, for example, 100 °C, at a suitable pressure such as, for example, 10 bars.
The skilled chemist will understand that, in the case that R2, R3 and/or R4 contain a protecting group such as, for example, Boc, deprotection of intermediates of Formula (la) or (lb) will afford the deprotected compound of Formula (la) or (lb). Further functionalization of those nor-compounds is possible using an aldehyde coupling partner in the presence of a reductive agent such as, for example, sodium triacetoxyborohydride, in a suitable solvent such as, for example, methanol or dichloromethane, at a suitable temperature such as, for example, 0 °C.
PHARMACOLOGY
The instant compounds are potent, brain-penetrant, have low cardiovascular liability 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
Several methods for preparing the Compounds of this disclosure 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
Figure imgf000019_0001
Figure imgf000020_0002
Preparation of intermediates
For intermediates that were used in a next reaction step as a crude or as a partially purified intermediate, either 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.
2-iodo-5-(trifluoromethyl)phenol [CAS 102771-00-6] Il
Figure imgf000020_0001
Sodium hydride [CAS 7646-69-7] (2.47 g, 61.69 mmol) was suspended in anhydrous toluene (92 mL) at 0 °C under nitrogen atmosphere. Then 3 -trifluoromethylphenol [CAS 98-17-9] (3.8 mL, 30.84 mmol) was added dropwise. The mixture was stirred at 0 °C for 30 min. Then iodine [7553-56-2] (7.83 g, 30.84 mmol) was added portionwise and the mixture was stirred from 0°C to rt for 3 h. The mixture was acidified at 0 °C with 37% HC1 sol. in water until pH reached 4-5, then was extracted with EtOAc and washed twice with brine. The organic layer was separated, dried (MgSO4), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g; EtOAc in heptane 0/100 to 10/90). The desired fractions were collected and concentrated in vacuo to yield Intermediate 1 (6.5 g, yield 72 %) as a colourless oil. l-iodo-2-(methoxymethoxy)-4-(trifluoromethyl)benzene 12
Figure imgf000021_0001
Intermediate 1 (6.5 g, 22.6 mmol) and K2CO3 [CAS 584-08-7] (5.3 g, 38.4 mmol) were dissolved in anhydrous DMF (95 mL). The reaction mixture was stirred at rt for 30 minutes. Then chloromethyl methyl ether [CAS 107-30-2] (2.4 mL, 29.3 mmol) was added dropwise and the reaction mixture was stirred at rt for 16 h. The reaction mixture was diluted with water and extracted with EtOAc. The organic layers were combined, dried (MgSO4), filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica 80 g; heptane 100%). The desired fractions were collected and concentrated in vacuo to yield the Intermediate 2 (5.5 g, yield 72%) as a colorless oil.
2-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2- dioxaborolane 13
Figure imgf000021_0002
Isopropyl magnesium chloride (2 M in THF) [CAS 1068-55-9] (7.2 mL, 14.46 mmol) was added dropwise to a stirred solution of Intermediate 2 (4 g, 12.05 mmol) in anhydrous THF (96 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 2 h. Afterwards, 2-isopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaborolane [CAS 61676-62-8] (3.7 mL, 18.07 mmol) was added dropwise to the mixture. The reaction mixture was slowly warmed up to rt and stirred for 18 h. The reaction was quenched with NH4C1 (saturated in water) and extracted with EtOAc. The organic layer was separated, dried (MgSO4), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 80 g; EtOAc in heptane 0/100 to 20/80). The desired fractions were collected and concentrated in vacuo to yield Intermediate 3 (2.6 g, yield 63 %) as a colorless oil. 3-methyl-5-(tnfluoromethyl)phenol [CAS 934180-46-8] 14
Figure imgf000022_0001
Lithium hydroxide hydrate (2.8 g, 65.24 mmol) was added to a stirred solution of Tris(dihenzylideneacetone)dipalladium(0) [CAS 51364-51-3] (616 mg, 0.652 mmol) and BippyPhos [CAS 894086-00-1] (681 mg, 1.31 mmol) in 50 mL of 1,4-dioxane and 5 ml of distilled water (previously bubbled with nitrogen for 5 min). The mixture was stirred at rt for 5 min, then 3-bromo-5-methylbenzotrifluoride [CAS 86845-28-5] (5.25 g, 21.74 mmol) was added. The reaction mixture was stirred at 100 °C for 16 h. The mixture was filtered over a pad of CCelite and was washed with AcOEt. The filtrate was washed with HC1 (2 M in water). The organic layer was separated, dried (MgSO4), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120g; AcOEt in heptane 0/100 to 2/98). The desired fractions were collected and concentrated in vacuo to yield Intermediate 4 (3.4 g, yield 80%) as a yellow oil.
2-iodo-3-methyl-5-(trifluoromethyl)phenol 15
Figure imgf000022_0002
Sodium hydride (60% dispersion in mineral oil, 1.59 g, 39.7 mmol) was added to a stirred solution of Intermediate 4 (3.4 g, 19.9 mmol) in 60 mL of anhydrous toluene at 0 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 30 min. Then iodine (5.05 g, 19.87 mmol) was added portionwise and the mixture was stirred from 0 °C for 3 h. The mixture was acidified at 0 °C with cone. HC1 until pH 4-5, then was extracted with AcOEt and washed twice with brine. The organic layer was separated, dried (MgSO4), filtered and the solvents evaporated in vacuo to yield Intermediate 5 (6.5 g, yield 74%) as a brown oil which was used as such without further purification.
2-iodo-l-(methoxymethoxy)-3-methyl-5-(trifluoromethyl)benzene 16
Figure imgf000022_0003
Intermediate 5 (9.05 g, 29.96 mmol) was dissolved in DCM (300 mL) and cooled to 0°C. To this solution was added N,N-diisopropylethylamine [CAS 7087-68-5] (6.34 mL, 35.96 mmol) followed by dropwise addition of chloromethyl methyl ether [CAS 107-30-2] (2.8 mL, 35.96 mmol). This was allowed to gradually warm to rt and stirred for 16 h. The mixture was concentrated and the residue was purified by flash column chromatography (dry load in silica 120g; DCM in heptane from 0/100 to 3/97). The desired fractions were collected and concentrated in vacuo to yield Intermediate 6 (7.39 g, yield 71%) as a yellowish oil.
2-(2-(methoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2- dioxaborolane 17
Figure imgf000023_0001
Intermediate 6 (7.4 g, 21.34 mmol) was added dropwise to a stirred solution of Palladium(II) acetate [CAS 3375-31-3] (489 mg, 2.13 mmol), CyJohnPhos [CAS 247940-06-3] (787 mg, 0.57 mmol) and triethylamine (15 mL, 106.72 mmol) and anhydrous 1,4-dioxane (92 mL) (previously bubbled with nitrogen for 5 min) in a sealed tube. The mixture was stirred at rt for 5 min, then 4,4,5,5-tetramethyl[l,3,2]dioxaborolane [CAS 25015-63-8] (16 mL, 106.72 mmol) was added. The reaction mixture was stirred at 100°C for 16h. The mixture was filtered over a pad of Celite and was washed with EtOAc. The filtrate was washed with sat. aqueous NH4C1. The organic layer was separated, dried (MgSO4), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120g; EtOAc in heptane 0/100 to 1/99). The desired fractions were collected and concentrated in vacuo to yield Intermediate 7 (6.87 g, yield 90%) as an orange solid.
2-(2-chloro-6-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2- dioxaborolane 18
Figure imgf000023_0002
Intermediate 8 was prepared by a similar sequence as Intermediate 7, using 3-bromo-5- chlorobenzotrifluoride as starting material. l-(benzyloxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene 19
Figure imgf000024_0001
Intermediate 5 (9.457 g, 24.11 mmol) and K2CO3 [584-08-7](5.049 g, 36.16 mmol) were dissolved in acetone (120 mL). The reaction mixture was stirred at rt for 15 minutes. Then benzyl bromide [ 100-39-0](3.3 mL, 26.52 mmol) was added dropwise and the reaction mixture was stirred at reflux for 16 h. The reaction mixture was diluted with water and extracted with EtOAc. The organic layers were combined, dried over MgSO4, filtered off and concentrated in vacuo. The crude product was purified by flash column chromatography (silica; 120g; Heptane 100%). The desired fractions were collected and concentrated in vacuo to give a pinkish solid (6.44 g, purity: 66%). The impure product was dissolved in EtOAc and washed with NH3 (5% in water). The organic layer was separated, dried over MgSO4, filtered off and concentrated in vacuo. The solid was purified by flash column chromatography (silica; 120g; Heptane 100%). The desired fractions were collected and concentrated in vacuo to give Intermediate 9 (4.61 g, yield 46%) as a white solid.
2-(2-(benzyloxy)-6-methyl-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-l,3,2- dioxaborolane 110
Figure imgf000024_0002
Intermediate 9 (4.575 g, 11.08 mmol) was added portionwise to a stirred solution of palladium(II) acetate [3375-31-3] (254 mg, 1.11 mmol) and CyJohnPhos [247940-06-3] (409 mg, 1.11 mmol) in triethylamine [121-44-8] (7.8 mL, 55.42 mmol) and anhydrous 1,4- dioxane (48 mL) (previously bubbled with nitrogen for 5 min). The mixture was stirred at rt for 5 min, then 4,4,5,5-tetramethyl[l,3,2]dioxaborolane [25015-63-8] (8.5 mL, 55.42 mmol) was added. The reaction mixture was stirred in a sealed tube at 100 °C for 16 h. The mixture was filtered over a pad of Celite and was washed with EtOAc. The filtrate was washed with NH4C1 (saturated in water). The organic layer was separated, dried (MgSO4), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120 g; EtOAc in heptane 0/100 to 3/97). The desired fractions were collected and concentrated in vacuo to yield Intermediate 10 (3345 mg, yield 73%) as a pale brown solid. l,4-dichloro-6,7-dihydro-5H-cyclopenta[d]pyridazine [CAS 5466-43-3] Ill
Figure imgf000025_0001
l-(trimethylsilyoxy)cyclopentene [CAS 19980-43-9] (17 mL, 0.878 g/mL, 95.496 mmol) was added to a stirred solution of 3,6-dichloro-l,2,4,5-tetrazine [CAS 106131-61-7] (10 g, 66.245 mmol) in 250 mL of toluene. The resulting mixture was heated at reflux for 5 h. Upon completion, observed by disappearance of red color, the mixture was cooled to room temperature, evaporated under reduced pressure and dried in vacuo at room temperature to afford Intermediate 11 (12.67 g, assumed quant, yield) as a brown solid.
5,8-dichloro-2,3-dihydro-[l,4]dioxino[2,3-d]pyridazine [CAS 1313733-23-1] 112
Figure imgf000025_0002
Ethylene glycol [CAS 107-21-1] (1.49 mL, 26.72 mmol) and sodium hydride (1.1 g, 27.5 mmol) were added to a solution of perchloropyridazine [CAS 20074-67-3] (5 g, 22.95 mmol) in 130 mL of anhydrous DMF at 0 °C. The reaction mixture was stirred at room temperature for 18 h. Then more sodium hydride (1.1 g, 27.5 mmol) was added and the mixture was stirred at 60 °C for 3 h. Solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (silica 120 g, EtOAc in Heptane, 0/100 to 50/50). The desired fractions were collected and concentrated under reduced pressure to yield Intermediate 12 (315 mg, yield 7%) as a white solid.
6-chloropyridazine-3-carbonyl chloride [CAS 6531-04-0] 113
O
Hi ci cr N
Oxalyl chloride [CAS 79-37-8] (1.0 mL, 12.05 mmol) was added dropwise to a stirred solution of 6-chloropyridazine-3 -carboxylic acid [CAS 5096-73-1] (1.5 g, 9.27 mmol) and anhydrous DMF (75 pL, 0.97 mmol) in 32 mL of anhydrous DCM at 0 °C under nitrogen atmosphere. The suspension was stirred at 0 °C for 10 min, then was stirred at rt for 2 h until a clear brownish solution was obtained. Solvent was evaporated in vacuo to yield Intermediate 13 (1.65 g, assumed quant, yield) as a pale brown solid which was used in the next step without further purification. tert-butyl 4-(6-chloropyridazine-3-carbonyl)piperidine-l-carboxylate 114
Figure imgf000026_0001
A freshly prepared solution of (l-(tert-butoxycarbonyl)piperidin-4-yl)zinc(II) iodide (0.42 M in THF) (23.3 mL, 9.79 mmol, made from tert-butyl 4-iodopiperidine-l -carboxylate) was added dropwise to a stirred solution of copper(I) cyanide [CAS 544-92-3] (970 mg, 10.72 mmol) and anhydrous lithium chloride [CAS 7447-41-8] (910 mg, 21.44 mmol) in 40 mL of anhydrous THF at -20 °C under nitrogen atmosphere. The mixture was stirred at -20 °C for 30 min. Then, Intermediate 13 (1.65 g, 9.32 mmol) diluted in 10 mL of anhydrous THF was added dropwise. The reaction mixture was stirred at -20 °C for 30 min, at rt for 16 h, then diluted sat. NH4CI aqueous solution (50 mL) and extracted with EtOAc (x3). The combined organic layers were dried (MgSO4), filtered over a pad of Celite and solvents from filtrate were evaporated in vacuo. The crude was purified by flash column chromatography (silica 80 g; dry load in silica; EtOAc in heptane 0/100 to 28/72). The desired fractions were collected and concentrated in vacuo to yield Intermediate 14 (1.52 g, yield 50%) as a beige solid. tert-butyl 4-((6-chloropyridazin-3-yl)difluoromethyl)piperidine-l-carboxylate 115
Figure imgf000026_0002
Triethylammonium fluoride [CAS 73602-61-6] (77 pL, 0.46 mmol) and DAST [CAS 38078- 09-0] (171 pL, 1.23 mmol) were added dropwise sequentially to a stirred solution of Intermediate 14 (100 mg, 0.31 mmol) in 2.5 mL of anhydrous DCM in a PTFE flask at 0 °C under nitrogen atmosphere. The reaction was stirred at rt for 10 min and then at 40 °C for 16 h. Then was cooled down to rt and was poured into a vigorous stirred ice/(sat. NaHCOs aqueous solution) mixture and extracted with DCM (x3). The combined organic layers were dried (MgSO4), filtered and the solvents evaporated in vacuo. The crude product was purified by flash column chromatography (silica; EtOAc in heptane 0/100 to 25/75) to yield Intermediate 15 (92 mg, yield 71%) as a colorless sticky solid (18% impure with starting material). tert-butyl 3-(6-chloropyridazine-3-carbonyl)piperidine-l-carboxylate 116
Figure imgf000027_0001
Intermediate 16 was made by analogy with Intermediate 14, using tert-butyl 3-iodopiperidine- 1-carboxylate [CAS 850761-36-3] as coupling partner in the previous step. tert-butyl 3-((6-chloropyridazin-3-yl)difluoromethyl)piperidine-l-carboxylate 117
Figure imgf000027_0002
Intermediate 17 was made by analogy with Intermediate 15, using Intermediate 16 as starting material. l-chloro-4-(2-(methoxymethoxy)-4-(trifluoromethyl)phenyl)-6,7-dihydro-5Z/- cyclopenta [djpyridazine 118-1
Figure imgf000027_0003
2 batches: A 20 mL microwave vial was charged with Intermediate 11 (680 mg, 3.597 mmol), Intermediate 3 (1.428 g, 4.085 mmol) and potassium phosphate tribasic [CAS 7778-53-2] (2.312 g, 10.892 mmol). 15 mL of 1,4-dioxane and 5 mL of water were added. The mixture was degassed with nitrogen for 5 min, before addition of dichlorobis(triphenylphosphine)palladium(II) [CAS 13965-03-2] (122.4 mg, 0.174 mmol). The vial was sealed and heated under microwave irradiation at 100 °C for 15 min. Upon completion, the vial were combined and the reaction mixture was diluted with water and extracted with EtOAc (x2). The combined organic layers were dried (Na2SO4), filtered off and concentrated under reduced pressure. The crude was purified by flash column chromatography with Hept/EtOAc (1 :0 to 4: 1) yielding Intermediate 18-1 (745 mg, yield 58%) as a pale brownish powder.
Alternatively, Intermediate 11 (1.5 g, 10.58 mmol), Intermediate 3 (1.8 g, 8.741 mmol) and sodium carbonate (1.6 g, 15.096 mmol) were dissolved in 45 mL of 1,4-dioxane and 15 mL of water. The mixture was degassed with nitrogen for 5 min, before addition Pd(PPh3)4 [CAS 14221-01-3] (500 mg, 0.433 mmol). The vial was heated at 110 °C for 2 h. Upon completion, the reaction mixture was diluted with water and extracted with EtOAc (x2). The combined organic layers were dried (Na2SO4), filtered off and concentrated under reduced pressure. The crude was purified by Prep HPLC (Stationary phase: RP XBridge Prep C18 OBD-lOpm, 50x150mm, Mobile phase: 0.25% NH4HCO3 solution in water, CH3CN). The purest fractions were collected, evaporated under reduced pressure and coevaporated with MeOH (x3) to Intermediate 18-1 (1.3 g, yield 46%) as a pale brown oil that crystallized upon standing. The following intermediates were obtained using Suzuki cross-coupling, typically with PdCl2(PPh3)2 [CAS 13965-03-2], Pd(PPh3)4 [CAS 14221-01-3] or cataCXium Pd G4 [CAS 2230788-67-5] as catalysts.
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0002
tert-butyl (5)-3-((6-chloropyridazin-3-yl)methyl)piperidine-l-carboxylate 119-1
Figure imgf000031_0001
General procedure A via Negishi coupling: Activated Zinc (233.4 mg, 3.57 mmol), magnesium chloride (25.49 mg, 0.27 mmol), (l,2-dimethoxyethane)nickel dibromide (110.16 mg, 0.36 mmol), 3,6-dichloropyridazine [CAS 141-30-0] (0.7 g, 1.96 mmol), 4,4'-di-tert- butyl-2,2'-bipyridine (95.8 mg, 0.36 mmol) and tert-butyl 3-(hydroxymethyl)piperidine-l- carboxylate [CAS 116574-71-1] (500 mg, 1.78 mmol) were mixed as solids. Then DMA (6.26 mL) and pyridine (244.39 pL, 3.03 mmol) were added. The vial was flushed with nitrogen, sealed and stirred at 100 °C for 2 hours. Then, aqueous LiCl (5%) was added and the product was extracted in EtOAc (x 3). The combined organic layers were washed with brine, dried on MgSO4, filtered and the solvent was evaporated. The residue was purified on a column with silica gel, eluent: MeOH in DCM, from 0 to 5 %. The fractions containing product were evaporated. A purification was performed via Prep HPLC (Stationary phase: RP XBridge Prep C18 OBD-10pm,30xl50mm, Mobile phase: 0.25% NH4HCO3 solution in water, CH3CN). The pure fractions were evaporated and coevaoporated twice with MeOH, yielding Intermediate 19-1 (130 mg, yield 14%) as a sticky oil.
General procedure B via MacMillan coupling: In a 20 mL vial (oven-dried overnight), 8 mL of anhydrous MTBE was added to a mixture, previously flushed 3 times with nitrogen/vacuum, of tert-butyl 3 -(hydroxymethyl)piperidine-l -carboxylate [CAS 116574-71- 1] (380.9 mg, 1.769 mmol) and 5,7-di-tert-butyl-3-phenyl-l,3-benzoxazol-3-ium (498.3 mg, 1.616 mmol). The suspension (white) was stirred 5 min at rt. Then, a solution of pyridine [CAS 110-86-1] (0.13 mL, 1.611 mmol) in 1 mL of anhydrous MTBE [was added to the previous solution. The reaction mixture was stirred 30 min. at rt. In a 40 mL vial (oven-dried overnight), 10 mL of anhydrous DMA was added to a mixture, previously flushed 3 times with nitrogen/vacuum, of 3,6-dichloropyridazine [CAS 141-30-0] (151.4 mg, 1.016 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine nickel(II) bromide (25 mg, 0.0513 mmol), (4,4'-di-tert-butyl- 2,2'-bipyridine)bis[(2-pyridinyl)phenyl]iridium(III) hexafluorophosphate [CAS 676525-77-2] (15 mg, 0.0164 mmol) and quinuclidine [CAS 100-76-5] (196.4 mg, 1.766 mmol). The methyl tert-butyl ether suspension was transferred to a 6 mL syringe under air. Then a syringe filter and new needle were installed on the syringe, before the methyl tert-butyl ether solution was injected through the syringe filter into the dimethylacetamide solution. The reaction mixture was sparged with nitrogen before sealing with parafilm. The vial was stirred at 1500 rpm stir rate and irradiated under 450 nm LED modules at 100% light intensity with maxed fan speed of 1500 rpm stirring rate in a PennOC Integrated Photoreactor for 4 h. The reaction mixture was filtered over a pad of dicalite and the cake was washed with EtOAc (50 mL). The filtrate was quenched with water (50 mL). The product was extracted with DCM (~50 mL) and again EtOAc (50 mL). The different organic layers were combined, dried over MgSO4, filtered and concentrated under reduced pressure at 40 °C. The crude was purified by column chromatography on a 25 g Sfar Silica HC in a Biotage system using gradient from 0 till 60% EtOAc in heptane (25 CV). The different product fractions were combined and concentrated under reduced pressure at 40 °C to afford Intermediate 19-1 (135 mg, yield 14 %) as an orange oil. The following intermediates were obtained using General procedure A and/or B as precised in the table.
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0002
2-(6-(piperidin-3-ylmethyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol hydrochloride salt
Figure imgf000036_0001
HC1 (4M in 1,4-di oxane) [CAS 7647-01-0] (2 mL, 8 mmol) was added to a solution of Intermediate 19-7 (172.1 mg, 0.33 mmol) in 4 mL of THF at rt. The colourless solution was stirred at rt overnight. The reaction mixture was concentrated in vacuo yielding Intermediate 20-1 (124 mg, assumed quant, yield) as a yellow residue which was used without further purification in the next step. Alternatively, Intermediate 19-7 (43.7 mg, 0.1 mmol) was dissolved in DCM (0.5 mL) then TFA [76-05-1] (0.084 mL, 1.49 g/mL, 1.1 mmol) was added and the mixture was stirred at room temperature for 1 h. The solvent was evaporated, the residue redissolved in MeOH and purified by a SCX2 column eluting with MeOH/NH4. The fractions were evaporated to yield Intermediate 20-1 (32 mg, assumed quant, yield) as a yellow residue which was used without further purification in the next step.
The following intermediates were obtained accordingly:
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
(5)-l-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-4-((4-methylmorpholin-2-yl)methyl)-7,8- d ihy d ro-5//-py ra no [3,4- ] pyridazine 121
Figure imgf000041_0001
Sodium triacetoxyborohydride (25 mg, 0.4 mmol) was added to a 10 ml round-bottom flask containing a stirred solution, consisting of 120-21 (140 mg, 0.3 mmol), triethylamine (0.26 mL, 1.8 mmol) and Formaldehyde solution (40 pL, 0.5 mmol) in Methanol (2.7 mL) at 0 °C under nitrogen atmosphere. The mixture was stirred at rt for 16 h. The mixture was concentrated and the residue was subjected to silica gel chromatography (12 g irregular 40-60 um; dry loaded in silica; 0-8% MeOH/DCM) to give Intermediate 21 as a sticky solid (33 mg, 24 %). benzyl (5)-2-((4-(2-(benzyloxy)-4-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrophthalazin- l-yl)methyl)-4-methylpiperazine-l-carboxylate 122
Figure imgf000041_0002
Intermediate 22 was synthesized by analogy with Intermediate 21, using Intermediate 20-22 as starting material. l-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene 123
Figure imgf000041_0003
A solution of 2-iodo-3-methyl-5-(trifluoromethyl)phenol (71 g, 235.07 mmol, 1.00 equiv), CS2CO3 (153.18 g, 470.14 mmol, 2.00 equiv) in DMF (0.71 L) under nitrogen atmosphere followed by the addition of chloromethyl ethyl ether (44.45 g, 470.14 mmol, 2.00 equiv) in dropwise at 0°C. The solution was stirred for overnight at room temperature. The resulting mixture was diluted with ice water (2 L). The resulting mixture was extracted with EA (2x3 L). The combined organic layers were washed with brine (2x3 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 1- (ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene (42 g, 49.61%) as a yellow oil.
2-[2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-l,3,2- dioxaborolane 124
Figure imgf000042_0001
A solution of l-(ethoxymethoxy)-2-iodo-3-methyl-5-(trifluoromethyl)benzene (42 g, 116.62 mmol, 1.00 equiv), [l,l'-biphenyl]-2-yldicyclohexylphosphane (4.0 g, 0.10 equiv), TEA (70.81 g, 699.7 mmol, 6.00 equiv), Pd(AcO)2 (1.31 g, 5.83 mmol, 0.05 equiv) in dioxane (0.42 L), then added 4,4,5,5-tetramethyl-l,3,2-dioxaborolane (59.70 g, 466.51 mmol, 4.00 equiv) under nitrogen atmosphere. The solution was stirred for 2 h at 100°C. The mixture was allowed to cool down to r.t. The resulting mixture was diluted with ice water (2 L). The resulting mixture was extracted with EA (2x2 L). The combined organic layers were washed with brine (2x2 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 as a white solid.
1HNMR: (300 MHz, CDCh, ppm) 5 7.13-7.03 (m, 2H), 5.23 (s, 2H), 3.74 (q, J= 7.1 Hz, 2H), 2.41 (s, 3H), 1.41 (s, 12H), 1.29-1.18 (m, 3H).
/‘cr/’-butyl (l?)-3-((6-chloro-l,2,4,5-tetrazin-3-yl)amino)piperidine-l-carboxylate 125
Figure imgf000042_0002
A solution of (/ )-/c/7-butyl 3 -aminopiperidine- 1 -carboxylate [CAS 188111-79-7] (10.61 g, 53 mmol) and TEA (8.82 mL, 63.6 mmol) in 80 ml DCM was added via a syringe pump to a stirring solution of 3,6-dichloro-l,2,4,5-tetrazine [CAS 106131-61-7] (8 g, 53 mmol) in DCM, dry (400 mL) over a 15 min period at a temp between -5°C and 0 °C in a Optimax reactor. The reaction mixture was stirred at 0°c for 30 min. LC /MS showed formation of the desired product. The solution was washed with a solution of 10 g K2CO3 in 300 ml water. The organic layer was separated, dried on MgSO4 and evaporated (25°C), yielding the desired product as a red solid (16.7 g, yield 100%). This was used immediately in the next step without furtrher purification. tert-butyl (/?)-3-((4-chloro-6.7-dihydro-5//-cyclopenta| |pyridazin-l- yl)amino)piperidine-l-carboxylate 126
Figure imgf000043_0001
1-pyrrolidino-l -cyclopentene [CAS 7148-07-4] (8 g, 58.3 mmol) was added dropwise to a mixture of tert-butyl (A)-3-((6-chloro-l,2,4,5-tetrazin-3-yl)amino)piperidine-l-carboxylate 125 (16.68 g, 53 mmol) in dry toluene (390.98 mL) in a 1 L four necked reactor. The mixture was stirred at room temperature for 30 min (attention: some nitrogen was released). Than the mixture was heated at 50 °C for 30 min and then the mixture was heated at reflux for 6 h. The first 10 mL reflux effluent were removed. After 6 h , LC/MS showed no SM or intermediate anymore. The reaction mixture was cooled, washed with water and 8 g K2CO3. The organic layer was separated, dried on MgSCU, filtered and concentrated. The residue was purified on a column with silicagel, eluent : EtOAc in Heptane, from 0 to 100 %. The pure fractions were evaporated, yielding the desired product (9.09 g, yield 48.6%) as a pink foam. tert-butyl (31?)-3-((4-(2-(ethoxymethoxy)-6-methyl-4-(trifluoromethyl)phenyl)-6,7- yl)ainino)piperidine-l -carboxylate 127
Figure imgf000043_0002
In a pressure tube, 1,4-dioxane (9.06 mL) and water, deionized (2.56 mL) was degassed with nitrogen for 5 min. Then tert-butyl (3R)-3-((4-(2-(ethoxymethoxy)-6-methyl-4- (trifluoromethyl)phenyl)-6,7-dihydro-5H-cyclopenta[d]pyridazin-l-yl)amino)piperidine-l- carboxylate 127 (500 mg, 1.42 mmol), 2-[2-(ethoxymethoxy)-6-methyl-4- (trifluoromethyl)phenyl]-4,4,5,5-tetramethyl-l,3,2-dioxaborolane 124 (612.44 mg, 1.7 mmol) and potassium phosphate tribasic [CAS 7778-53-2] (932.41 mg, 4.39 mmol) were added. methanesulfonato(2-dicyclohexylphosphino-2',4',6'-tri-i-propyl-l, T biphenyl)(2'-amino-l, T- biphenyl-2-yl)palladium(II) [CAS 1445085-55-1] (239.88 mg, 0.28 mmol) was added and the tube was flushed with nitrogen and then closed. The reaction mixture was heated in an for 2 h at 105 °C. LC/MS showed complete conversion. The reaction mixture was cooled; poured out in sat. NaHCOs solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried on MgSCU, filtered and concentrated. The residue was purified on a column with silicagel, eluent : EtOAc in Heptane, from 0 to 80%. The pure fractions were combined and concentrated, yielding the desired product (703 mg, yield 90.1%) as a whithe foam.
3-methyl-2-(4-(((R)-piperidin-3-yl)amino)-6,7-dihydro-5H-cyclopenta[d]pyridazin-l-yl)-
5-(trifluoromethyl)phenol 128
Figure imgf000044_0001
HC1 (4M in 1,4- dioxane) (6.04 mL, 4 M, 24.17 mmol) was added to a soluton of dihydro- 57/-cyclopenta[t/]pyridazin- l -yl)amino)piperidine- l -carboxylate 127 (701 mg, 1.27 mmol) in 1,4-di oxane (6.1 mL) and the mixture was stirred at room temperature for 2h. LC/MS showed complete conversion. The mixture was poured out in sat NaHCOs solution and extracted three times with EtOAc. The combined organic layers were washed with brine, dried on MgSO4, filtered and concentrated, yielding the desired product (495 mg, yield 99.08%) as a white solid.
Preparation of final compounds
Generally, the preparation of the final compounds was done using racemic mixture separated via preparative SFC. Absolute configurations were attributed by synthesizing the enantiopure compounds starting from chiral intermediates. (l?)-2-(6-((l-methylpiperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 1 and ( )-2-(6-((l-methylpiperidin-3-yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 2
Compound 1 Compound 2 Formalin 37% sol. in water [CAS 50-00-0] (600 pL, 0.815 g/mL, 6.026 mmol, 4.2 equiv.) was added to a stirred solution of Intermediate 20-1 (577 mg, 1.406 mmol, 1 equiv.) and EtsN (500 pL, 0.728 g/mL, 3.597 mmol, 2.5 equiv.) in 20 mL of MeOH and the resulting mixture was stirred 3 h at room temperature. Then, sodium tri acetoxyborohy ride [CAS 56553-60-7] (600 mg, 2.831 mmol, 2 equiv.) was added portionwise and the mixture was further stirred overnight. The mixture was diluted with sat. NaHCOs sol. and extracted with EtOAc. The organic layer was separated, washed with brine, dried (Na2SO4), filtered off and evaporated under reduced pressure. The crude was purified by short column chromatography on silica gel with DCM/7N NH3 in MeOH (1 :0 to 9: 1) then by Prep SFC (Stationary phase: Chiralpak Daicel IC 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrlME) to yield Compounds 1 (57 mg, yield 23%) and 2 (56 mg, yield 23%) as pale-yellow solids.
The following final compounds were synthesized accordingly:
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
1 Compound 29 was synthesized as comparator with the corresponding 3-piperidinyl (Compound 30) (.S)-2-(4-((4-methylmorpholiii-2-yl)methyl)-7.8-dihydro-5//-pyr:ino|3.4- |pyridazin- 1 - yl)-5-(trifluoromethyl)phenol 24 and (5)-2-(l-((4-methylmorpholin-2-yl)methyl)-7,8- dihydro-5H- l)-5-(trifluoromethyl)phenol 25
Figure imgf000050_0001
Figure imgf000050_0002
Compound 24 Compound 25
10% Palladium on carbon (0.028 g, 0.026 mmol) was added to a 10 mL round-bottomed flask containing a stirring solution, consinting of Intermediate 21 (0.066 g, 0.132 mmol) and MeOH (2.6 mL), under a nitrogen atmosphere to give black heteregeneous mixture (s/1). Then, nitrogen atmosphere was replaced by hydrogen (1 atm, balloon) and the resulting mixture was stirred at rt for 16 h. The heterogeneous mixture was filtered through Celite pad, rinsing with MeOH (3 x 5 ml). Solvents were concentrated to dryness in vacuo. The crude was subjected to SFC (Phenomenex Lux Amylose-1 250 x 30mm 5um); ISOC 20 % Ethanol + 0.1 % DEA) to collect and concentrate to yield Compounds 24 (14.4 mg, 26 %) and 25 (8.3 mg, 15 %) as off-white solids.
(R)-2-(4-((4-methylpiperazin-2-yl)methyl)-5,6,7,8-tetrahydrophthalazin-l-yl)-5- (trifluoromethyl)phenol 26 and (S)-2-(4-((4-methylpiperazin-2-yl)methyl)-5, 6,7,8- tetrahydrophthalazin-l-yl)-5-(trifluoromethyl)phenol 27
Figure imgf000050_0003
Compound 26 Compound 27
Compound 26 (resp. 27) were synthesized by analogy with Compound 24 (resp. 25), using Intermediate 22 as starting material.
(5)-2-(8-((l-methylpiperidin-3-yl)methyl)-l,2,3,4-tetrahydropyrido[2,3-</|pyridazin-5- yl)-5-(trifluoromethyl)phenol 31 and (l?)-2-(8-((l-methylpiperidin-3-yl)methyl)-l,2,3,4- tetrahydropyrido [2,3- ] pyridazin-5-yl)-5-(trifluoromethyl)phenol 32
Figure imgf000051_0001
A racemic mixture of Compounds 5 and 6 (60 mg, 0.15 mmol) and Pd/C (10%) (10 mg, 0.0095 mmol) in 6 mL of EtOH was stirred under 15 bar EE at 50 °C for 16 h. The mixture was filtered with a syringe with 45 pm filter, washed with EtOH and the solvent was evaporated under vacuum. A purification was performed via Prep SFC (Stationary phase: Chiralpak Daicel IC 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrlME) to yield Compounds 31 (18 mg, yield 30%) and 32 (15 mg, yield 25%) as white solids.
(5)-3-methyl-2-(6-((l-(oxetan-3-yl)piperidin-3-yl)methyl)pyridazin-3-yl)-5- (trifluoromethyl)phenol 33
Figure imgf000051_0002
MeOH (1.5 mL) was added to a mixture of (^-Intermediate 20-6 (106.8 mg, 0.275 mmol) and 3-oxetanone [6704-31-0] (83.4 mg, 1.157 mmol). The mixture was stirred 15 min at room temperature. Sodium cyanoborohydride [25895-60-7] (58.7 mg, 0.934 mmol) was added to the previous solution. The reaction was stirred 6 h at room temperature. An extra amount of 3- oxetanone [6704-31-0] (58.3 mg, 0.809 mmol) was added and the reaction mixture was stirred 30 min at room temperature. The reaction mixture was quenched with NaHCO3 sat. in water and diluted with EtOAc. The product was extracted with EtOAc (x3) and DCM. The combined organic layers were dried (MgSO4), filtered off and concentrated under reduced pressure. The crude was purified by column chromatography on a 10 g Sfar Silica HC in a Biotage system using a gradient from 0 till 10 % NH3 in MeOH (3.5 M) in DCM. The purest fractions were combined and concentrated under reduced pressure. The product was precipitated with DIPE and dried in vacuo to afford Compound 33 (42 mg, yield 37%) as a white powder. (l?)-3-methyl-2-(6-((l-(oxetan-3-yl)piperidin-3-yl)methyl)pyridazin-3-yl)-5-
(trifluoromethyl)phenol 34
Figure imgf000052_0001
Compound 34 was made by analogy with Compound 33, using (R)-Intermediate 18-6 as starting material.
(5)-3-methyl-2-(6-((l-(methyl-d5)piperidin-3-yl)methyl)pyridazin-3-yl)-5-
(trifluoromethyl)phenol 35 and (l?)-3-methyl-2-(6-((l-(methyl-</5)piperidin-3- yl)methyl)pyridazin-3-yl)-5-(trifluoromethyl)phenol 36
Figure imgf000052_0002
Formaldehyde-t/? [1664-98-8] (38 pL, 0.261 mmol) was added to a solution of Intermediate 20-6 (49.8 mg, 0.128 mmol) in dry DCM (1.4 mL) and dry DMF (0.5 mL). The mixture was stirred 30 min. at R.T. An extra amount of sodium cyanoborodeuteride [25895-62-9] (20 mg, 0.304 mmol) was added at R.T. and the reaction mixture was stirred 2 h 30 min at room temperature. The reaction mixture was quenched with a sat. NaHCO3 sol. and DCM and extracted with DCM (x3). The combined organic layers were dried (MgSO4), filtered off and concentrated under reduced pressure. The crude was purified by column chromatography on a 10 g Sfar Silica HC in a Biotage sytem using a gradient from 0 till 10% NH3 in MeOH (3.5 M) in DCM (15 CV) and 10% NH3 in MeOH (3.5 M) in DCM (15 CV) then the enantiomers were separated by prep SFC. (Stationary phase: Chiralpak Daicel IG 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4 iPrNH2) The purest fractions were combined, concentrated under reduced pressure and dried in vacuo to afford Compound 35 (9 mg, yield 18%) and Compound 36 (15 mg, yield 32%) as white solids.
(l?)-2-(6-((l-ethylpiperidin-3-yl)methyl)pyridazin-3-yl)-3-methyl-5- (trifluoromethyl)phenol 37
Figure imgf000052_0003
A solution of iodoethane [75-03-6] (35 pL, 0.435 mmol) and DIPEA [7087-68-5] (0.25 mL, 1.451 mmol) in 2 mL of anhydrous DMF was added to ( ’)-Intermediate 20-6 (107.5 mg, 0.277 mmol). The reaction mixture was stirred overnight at R.T. The reaction mixture was diluted with EtOAc and washed with a sat. NaHCO3 sol. The aqueous layer was back- extracted with EtOAc (x2) and DCM. The combined organic layers were dried (MgSO4), filtered off and concentrated under reduced pressure. The crude was purified by column chromatography on 10 g SfAr Silica HC in a Biotage sytem using a gradient from 0 till 10 % DCM in MeOH (3.5 M) in DCM (15 CV) and 10 % DCM in MeOH (3.5 M) in DCM (10 CV). The purest fractions were collected and evaporated under reduced pressure. The product was precipitated with DIPE and dried in vacuo to afford Compound 37 (56 mg, yield 53%) as a beige solid.
The following final compounds were synthesized accordingly:
Figure imgf000053_0001
Figure imgf000054_0002
3-niethyl-2-(4-(((/?)-l-niethylpiperidin-3-yl)ainino)-6.7-dihydro-5//- cyclopenta[J|pyridazin-l-yl)-5-(trifluoromethyl)phenol 51
Figure imgf000054_0001
Formalin (0.39 mL, 0.82 g/mL, 3.96 mmol) was added to a stirred solution of 3-methyl-2-(4- (((A)-piperidin-3-yl)amino)-6,7-dihydro-5J/-cyclopenta[t ]pyridazin-l-yl)-5- (trifluoromethyl)phenol 128 (495 mg, 1.26 mmol) in MeOH (19.7 ml) and the resulting mixture was stirred 1 h at room temperature. The mixture was cooled to 0 °C before portionwise addition of sodium triacetoxyborohydride [CAS 56553-60-7] (0.67 g, 3.15 mmol). Then, the mixture was allowed to warm to room temperature and further stirred 1 h. LC/MS showed complete conversion. The reaction mixture was poured in sat NaHCCh, extracted with EtOAc, washed with brine, driedon MgSCU and concentrated. The residue was purified on a column with silicagel, eluent MeOH/NH3 7 N in DCM from 0 to 10 %. The pure fractions were combined and concentrated. The residue was crystalized from ACN, yielding 3-methyl-2-(4-(((A)-l-methylpiperidin-3-yl)amino)-6,7-dihydro-5JT- cyclopenta[d]pyridazin-l-yl)-5-(trifluoromethyl)phenol 51 (437 mg, yield 99.28%) as a white solid. The following final compounds were synthesized accord to the procedures described for compounds 1-50:
Figure imgf000055_0001
Analytical and biological data
Example A - Analytical data
LCMS
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.
Figure imgf000056_0001
Figure imgf000057_0001
LCMS data for all final compounds are depicted below.
Figure imgf000057_0002
Figure imgf000058_0001
NMR
1 H NMR spectra were recorded on Bruker Avance III and Avance NEO spectrometers. CDCh was used as solvent, unless otherwise mentioned. The chemical shifts are expressed in ppm relative to tetramethylsilane. In CDCh, OH signal may be missing.
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Example B - Pharmaceutical Compositions
A compound (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 as provided herein (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 can be assessed by in vitro methods. A compound may exhibit 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 inDMSO 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 EC50 values (IL6 and TNFa) were obtained on compounds of the invention/examples, and are depicted in the following table:
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
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.
Figure imgf000066_0001
Figure imgf000067_0001
Example E - 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 Nani on 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 pIC50 are reported below.
Figure imgf000068_0001
Figure imgf000068_0002
Figure imgf000069_0002
Figure imgf000069_0001
Example F - Further Testing One or more compound(s) were/may be tested in a number of other assays to evaluate, amongst other properties, permeability, stability (including metabolic stability and blood stability) and solubility.
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
Figure imgf000070_0001
Where: Vmc = incubation volume,
Wmic prot,inc weight of microsomal protein in the incubation.
Figure imgf000070_0002
Figure imgf000071_0001
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 x 1000
Figure imgf000072_0001
Where: Vmc = incubation volume,
# cells;nc = number of cells (xlO6) in the incubation
Figure imgf000072_0002
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:
P > 2 Peak Area Ratio uffer chamber > Peak Area Ratiobuffer chamber
2 Peak Area Ratiobssue homogenate chamber Peak Area Ratiobssue homogenate chamber Fu FUapp bra'n 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
Figure imgf000077_0001
Figure imgf000078_0001
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
Figure imgf000081_0001
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).
Data:
Figure imgf000082_0002
Figure imgf000082_0001
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:
Figure imgf000082_0003
Figure imgf000082_0004
In vivo LPS experiments: measurement of the effect of NLRP3 inhibitors on LPS triggered pro-inflammatory cytokine ILip
Animals were treated with NLRP3 inhibitors prior to LPS administration to evaluate the effect of NLRP3 inhibitors on inflammasome activation by measuring ILip. To exclude an effect on NF-kB signaling induced by LPS, IL6 and TNFa were measured as well. Compounds were administered via oral gavage (PO) 30 minutes before intraperitoneal LPS injection (10 mg/kg) injection (Escherichia coli O11 LB4; L4130, Sigma-Aldrich). Three doses were tested for each compound. The control groups, wild type and NLRP3 knockout mice, received vehicle PO. Nlrpr3 knockout mice were included as a negative control, i.e., to define endogenous levels of ILip in these experiments. Each treatment group included 8 animals. In certain experiments, however, the Nlrp3 knockout group included less animals (usually n = 4). Four hours after LPS injection, animals were sacrificed by decapitation and plasma samples were collected for bioanalysis and cytokine (ILip, IL6 and TNFa) analysis using ELISA (ILip, Quantikine MLB00C, R&D Systems Minneapolis, Canada) and MSD (IL6 and TNFa, V-Plex K15048D MSD, Meso Scale Diagnostics, Maryland, USA). Plasma samples were diluted 1/20 for ILip and TNFa measurements and further diluted to 1/60 for the analysis of IL6. Plates were read using a SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA, USA) or the MSD reader (Meso Scale reader sector S600) for the Quantikine and MSD assays respectively. Data were further analyzed in Excel and GraphPad Prism, including statistical analysis (one-way ANOVA). Concentrations in plasma were determined according to the procedures described in the pharmacokinetic section. Free concentrations were determined by multiplying the plasma concentration by the free fraction in plasma (free concentration = plasma concentration x fu,p). The free fraction in plasma is defined as follows: fu,p = PPB (% free)/100. The determination of the PPB (% free) is described in the plasma protein binding section.
Examples:
Compound 9
Figure imgf000083_0001
Compound 51
Figure imgf000084_0001
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
Figure imgf000084_0002
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 (doseresponse) 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.
Figure imgf000085_0001
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.
Figure imgf000085_0002
Figure imgf000085_0003
Figure imgf000086_0001
Figure imgf000086_0002
Without wishing to be bound by theory, as already indicated hereinabove, the compounds 5 of the present invention display advantageous properties, such as in terms of brain penetration, phospholipidosis potential and/or polarity.
For example, the following compound, with a monocyclic core structure, was tested in the same assays as described herein for brain penetration and phospholipidosis potential and can be compared with final compound 9 according to the invention:
Figure imgf000086_0003
The following compounds, with a bicyclic core structure, were tested in the same assays as described herein for CHI LogD, and can also be compared to final compounds described herein:
Figure imgf000087_0001
Replacement of a -NH- for a -CH2- spacer in these compounds unexpectedly increased the polarity in the compounds.

Claims

Claims
1. A compound of formula (I),
Figure imgf000088_0001
or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, methyl or chloro;
Figure imgf000088_0002
wherein Y is CH2, NR21 or O;
R20 is oxetan-3-yl, CD3, C alkyl optionally substituted with halo, hydroxy, or cyano;
R21 is hydrogen or C 1-2 alkyl;
R3 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl;
R4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl; or
R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2-, b) -CH2-CH2-CH2-CH2-, c) -CH2-CH2-CH2-NH-, d) -CH2-O-CH2-CH2-, e) -CH2-CH2-O-CH2-,
0 -O-CH2-CH2-O-, g) -CH=CH-CH=N-, h) -N=CH-CH=CH-; and each Z independently is hydrogen or fluoro.
2. The compound of claim 1, wherein R1 is hydrogen or methyl.
3. The compound of claim 1 wherein R2 is
Figure imgf000089_0001
wherein R20 is C1-2 alkyl, CD3, 2-hydroxyethyl, 2-fluoroethyl, 3 -fluoropropyl, or cyanomethyl.
4. The compound of claim 1 wherein R2 is
Figure imgf000089_0002
wherein R20 represents methyl.
5. The compound according to claim 1 wherein R4 is hydrogen, methyl, methoxy, azetidinyl, or morpholinyl.
6. The compound according to claim 1 wherein R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2- b) -CH2-CH2-CH2-CH2- c) -CH2-CH2-CH2-NH- d) -CH2-O-CH2-CH2- e) -CH2-CH2-O-CH2- f) -O-CH2-CH2-O- g) -CH=CH-CH=N- h) -N=CH-CH=CH-.
7. The compound according to claim 1 wherein each Y represents hydrogen.
8. The compound according to claim 1
R1 is hydrogen or methyl;
Figure imgf000090_0001
wherein Y is CH2 or O;
R20 is methyl;
R3 is hydrogen;
R4 is hydrogen; or
R3 and R4 together form a bivalent radical -R3-R4- selected from the following list a) -CH2-CH2-CH2-, e) -CH2-CH2-O-CH2-, and each Z is hydrogen.
9. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
10. A process for preparing a pharmaceutical composition as defined in claim 9, 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 1 to 8.
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