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WO2025097247A1 - Method of treatment of fibrosis with inverse agonist of rar related orphan receptors (rors) - Google Patents

Method of treatment of fibrosis with inverse agonist of rar related orphan receptors (rors) Download PDF

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Publication number
WO2025097247A1
WO2025097247A1 PCT/CA2024/051468 CA2024051468W WO2025097247A1 WO 2025097247 A1 WO2025097247 A1 WO 2025097247A1 CA 2024051468 W CA2024051468 W CA 2024051468W WO 2025097247 A1 WO2025097247 A1 WO 2025097247A1
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compound
carbonyl
thiophen
tetrahydrobenzo
dimethylpiperidine
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Ahmed FOUDA
Jean Tchervenkov
Steven PARASKEVAS
Sarita NEGI
Jonathan COOLS-LARTIGUE
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11949098 Canada Inc
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11949098 Canada Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • 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

Definitions

  • This invention relates to a method of treatment of fibrosis by administering to a patient in need thereof an inverse agonist of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORa or RORA) and gamma (RORC, RORy or RORyt).
  • RORs retinoic-acid-receptor-related orphan nuclear receptors alpha
  • RORC gamma
  • Fibrosis is a pathological condition present in most chronic inflammatory diseases. Fibrosis happens when connective tissue replaces normal parenchymal tissue, which can lead to organ malfunction and death.
  • Liver fibrosis due to viral or metabolic liver diseases is a major global health problem.
  • Liver fibrosis is the over-accumulation of extracellular matrix proteins, such as collagen, that occurs in most types of chronic liver diseases, among which is nonalcoholic fatty liver disease (NAFLD).
  • NAFLD nonalcoholic fatty liver disease
  • liver lesions from steatosis to cirrhosis, via non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • Liver fibrosis increases the risk for liver-related complications and other co-morbidities such as hepatocellular carcinoma, insulin resistance, diabetes, and cardiovascular events.
  • NASH NASH is a powerful driving force for the progression of fibrosis. It has been found that patients with NASH have higher levels of inflammatory cytokines. Immune response has an important driver of the progression of the disease. Immune imbalances, reflected at the cellular level through the level of pro-inflammatory cytokines including those involved in Th17 differentiation (IL-6, IL-21 , IL-23 and transforming growth factor-beta) and those released by Th17 cells (IL-17A, IL-17F, IL-21 , and IL-22) have been studied.
  • IL-17A a type 3 inflammatory cytokine either produced by Th17, CD4+ T cells or neutrophils
  • IL-17A a type 3 inflammatory cytokine either produced by Th17, CD4+ T cells or neutrophils
  • Liver infiltration by Th17 cells have also shown to be a critical element for NASH initiation and development of fibrosis and this infiltration was accompanied by an increase in the production of pro-inflammatory cytokines.
  • the activation of the IL-17 axis is key in disease progression since IL-17 exacerbates the liver tissue inflammation and has a pro-fibrotic effect.
  • ROR retinoic acid receptor-related
  • RORyt is exclusively expressed in cells of the immune system including CD4 + CD8 + double positive thymocytess, Th17, Tc17, and y6 T cells, as well as a subset of innate lymphoid cells (ILCs) and regulatory T cells (Tregs).
  • RORyt is a key transcription factor driving Th17 cell differentiation, and production of IL-17A, IL-17F and IL-22 in innate and adaptive immune cells, also termed “type 17” cells.
  • Th17 cytokines, IL-17A, IL-17F, and IL-22 stimulate tissue cells to produce a panel of inflammatory chemokines, cytokines and metalloproteases, resulting in the recruitment of granulocytes to sites of inflammation.
  • Th17 cell subset has been shown to be the major pathogenic population in several models of autoimmune inflammation, including collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE).
  • CIA collagen-induced arthritis
  • EAE experimental autoimmune encephalomyelitis
  • RORyt deficient mice show impaired Th17 cell differentiation in vitro, significantly reduced Th17 cell populations in vivo, and decreased susceptibility to EAE and intestinal inflammation.
  • RORyt-deficient T cells fail to induce colitis in the mouse T cell transfer model.
  • a method of treatment of fibrosis which comprises administering to a patient in need thereof a therapeutically effective amount of an inverse agonist of RORs or a silencing RNA that inhibits protein synthesis of RORs.
  • the fibrosis is pulmonary fibrosis, retroperitoneal fibrosis (RPF), hepatic fibrosis and/or cirrhosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, intestinal fibrosis, muscle fibrosis, ocular fibrosis, fibrosis in the central nervous system, renal fibrosis, and pancreatic fibrosis, autoimmune fibrotic diseases, fibrosis associated with infectious diseases, fibrosis associated with metabolic diseases, fibrosis associated with cancer, fibrosis associated with environmental exposures, or a combination thereof.
  • RPF retroperitoneal fibrosis
  • the inverse agonist of RORs is a compound of Formula (I), (II), (III), or (IV), a pharmaceutically acceptable salts thereof, or stereoisomers thereof: [0015] wherein,
  • R1 , R2, R3, and/or R4 group(s) is/are H, halogen, NO2, 1-6 alkoxy, OH, NH2, 1 -6 alkyl, 1-6 alkenyl, 1-6 haloalkyl, N-dialkyl, haloalkoxy, 1-6 hydroxyalkyl, and/or -CO2(1-6 alkyl);
  • R5 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, aryl, alkylarene, halo aryl, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, haloaryl, benzene, phenyl, benzyl, pyridine, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, haloalkyl, cyanoalkyl, methylpyrimidine, toluene, methyl
  • R6 is H, 1-6 alkyl, or may form a five or six ring structure with R5;
  • R7 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, substituted haloaryl, substituted benzene, substituted phenyl, benzyl, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, aryl, halo aryl, alkylarene, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methyl
  • the inverse agonist of ROR may be:
  • Izumerogant (IMU-935);
  • Cedirogant (ABBV-157);
  • Compound 48 -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 49 -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
  • Compound 50 -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • the inverse agonist of ROR may be:
  • Compound 7 N-benzyl-2-[(trifluoroacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
  • Compound 15 N-(3-(3-ethylpyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
  • Compound 17 N-(3-(3-(hydroxymethyl)pyrrolidine-1-carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 18 N-(6,6-dimethyl-3-(morpholine-4-carbonyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2-carboxamide;
  • Compound 20 (S)-N-(3-((1-cyanoethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
  • Compound 42 -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 43 -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 44 -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 48 -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 50 -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • the fibrosis is associated with Liver diseases, Liver Failure, Acute- and-Chronic Liver Failure, Chemical and Drug Induced Liver Injury, Liver Fibrosis, Fatty Liver, viral Hepatitis, Non-viral hepatitis, Liver diseases, Liver Failure, Hemangioma of liver, Non-alcoholic Fatty Liver Disease, Acute-On-Chronic Liver Failure, Liver diseases, Liver neoplasms, Chemical and Drug Induced Liver Injury, Alcoholic Liver Diseases, Liver and Intrahepatic Biliary Tract Carcinoma, Chronic Hepatitis, Autoimmune hepatitis, Metabolic dysfunction-Associated Fatty Liver Disease’ (MAFLD), alcohol induced liver conditions, metabolic associated steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), liver fibrosis, liver failure, non-
  • a pharmaceutical composition for treatment of fibrosis comprising an inverse agonist of RORs and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising:
  • a method of treatment of a vascular injury or protecting against a vascular injury by reducing circulating levels of IL- 17, which comprises administering to a patient in need thereof an inverse agonist of RORs.
  • the injury may be the injury is a brain injury.
  • FIG. 1 A-1 B illustrate novel compounds of the present invention.
  • Fig. 2 illustrates an experiment set up and timeline of mice treated with CCI4 to induce liver fibrosis that are administered GSK805 for positive control, TF-1 (compound 10) and TF-2 (compound 14) and a negative control.
  • Fig. 3 illustrates the gating strategy for myeloid cells, monocytes, neutrophils and macrophages.
  • Fig. 4 illustrates the gating strategy for T cells, both for un-stimulated lymphocytes and for stimulated lymphocytes.
  • Fig. 5 illustrates a reduction in stimulated lymphocytes consisting of leukocytes, T cells, and NK cells, after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 15 mice/group, 6X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 6 illustrates a reduction in stimulated cytokines after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group, 4X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 7 illustrates a reduction in stimulated cytokines after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group, 4X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 8 illustrates a reduction in granulocytes consisting of granulocytes, myeloid, monocytes and neutrophils, after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 15 mice/group, 6X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 9 illustrates a reduction in granulocytes consisting of macrophages, eosinophil, dendritic cells and MHC II+ presenting cells, after IP injection of GSK805, CCI4, TF1-S10 (compound 10) and TF2-S14 (Compound 14), N: 15 mice/group, 6X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 10 illustrates a reduction in stimulated lymphocytes consisting of leukocytes, T cells and NK cells after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group, 4X each 2- 3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 11 illustrates a reduction in unstimulated lymphocytes consisting of T cells and NK cells after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group (4X each 2-3 mice/group), using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 12 illustrates a reduction in granulocytes consisting of granulocytes, myeloid, monocytes and neutrophils, after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group (4X each 2-3 mice/group), using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 13 illustrates a reduction in granulocytes consisting of macrophages, dendritic cells, eosinophils and MHC II+ presenting cells, after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group (4X each 2-3 mice/group), using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
  • ANOVA analysis of variance
  • Fig. 14 illustrates significantly reduced ALT and AST after IP injection of CCI4, GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14) for 4 weeks (8 CCI4 injections), N: 7-8 mice/group for AST, 10 mice/group for ALT.
  • Fig. 15 illustrates a reduction in collagen deposition through Sirius red staining after injection of CCI4, GSK805, TF1 -S10 (compound 10), TF2-S14 (Compound 14) and com oil.
  • Fig. 16 illustrates a reduction in the infiltration of immune cells through hematoxylin and eosin staining of liver tissue following IP injection of CCI4, GSK805, TF1 - S10 (compound 10), TF1 -S14 (compound 14) and com oil.
  • Fig. 17 illustrates a reduction in the infiltration of immune cells through hematoxylin and eosin staining of liver tissue following SC injection of CCI4, GSK805, TF1 -S10 (compound 10), TF1 -S14 (compound 14) and com oil.
  • Fig. 18 illustrates a hydroxyproline assay to evaluate liver hydroxyproline content following injection with CCI4, GSK805, TF1-S10 (compound 10), TF2-S14 (Compound 14) and oil.
  • Fig. 19 illustrates the anti-fibrotic effects of pharmacological inhibitors of IL- 17 (10 mg/kg) were evaluated in CCI4-induced liver fibrosis of C57BL/6 mice model.
  • FIG. 19A illustrates schematic diagram of the experimental design for C57BL/6 mice intraperitoneally treated with 0.5 ml/kg CCI4 in com oil for 4 weeks. Mice were intraperitoneally administered with inhibitors (10 mg/kg) for 2 weeks, starting at week 3 post initiation of CCI4 challenge.
  • Fig. 19B illustrates representative images of H&E staining of liver tissues indicated necrosis and numerous inflammatory cells immersed in mice dosed with CCI4 and in vivo shoot of liver tissues.
  • Fig. 20 illustrates pharmacological inhibitors of IL-17 developed a reduction in the absolute number of innate immune cells, but not neutrophils, in the mice liver compared with CCI4 liver injury at 4 weeks. IHLs were extracted from livers and analyzed by flow cytometry following injected with CCI4 (0.5 ml/kg) and treated with inhibitors (10 mg/kg).
  • Fig. 20A illustrates representative zebra plots showing an outline for the gating strategy of granulocytes (CD45+ CD11 b+), neutrophils (CD45+ CD11 b+ Ly6Cint Ly6G+), monocytes (CD45+ CD11 b+ Ly6Chi Ly6G-), macrophages (CD11 b+ Ly6C- Ly6G- F4/80+), and eosinophils (CD11 b+ Ly6C- Ly6G- CD170+).
  • Fig. 20B illustrates that the indicated numbers of cell subsets of IHLs represent the cell number/gram of liver. Data are expressed as means ⁇ SEM for 15 mice per group (data are combined from 3 individual experiments). Vehicle group contains 5 mice. ANOVA test. *P ⁇ .05, **P ⁇ .01 , and ***P ⁇ .001 . SSC, side scatter characteristics. IHL, intrahepatic lymphocytes.
  • Fig. 21 illustrates a reduction in the absolute number of adaptive immune cells in the livers of inhibitors treated mice compared with the CCI4-only treated at 4 weeks. IHLs were extracted from injected livers and analyzed by flow cytometry.
  • Fig. 21 A illustrates representative FACS plots showing an outline for the gating strategy of T cells (CD45+CD3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), T cell receptors y5 T cells (CD3+T cell receptor y5 +).
  • SSC side scatter characteristics. IHL, intrahepatic lymphocytes. SSC, side scatter characteristics. IHL, intrahepatic lymphocytes.
  • Fig. 22 illustrates inhibitors selectively block the secretion of IL-17 but not IFNy and IL-22.
  • the intrahepatic lymphocytes were extracted from livers of inhibitors treated liver fibrosis vs CCI4-treated liver fibrosis as well as vehicle control mice receiving only corn oil at 4 weeks, and then stimulated with/without PMA/ionomycin (PMA/lono) for 5 hours at 37°C.
  • SSC side scatter characteristics.
  • Fig. 23 illustrates IP injection of RORyt inhibitors result in significant reduction of hepatic inflammatory genes in CCI4-induced fibrotic mice compare with the CCI4 only treated mice.
  • C57BL/6 mice were treated for 4 weeks with CCI4 and then subjected to the inhibitors.
  • TF-S10/S14 or GSK805 (10 mg/kg daily) were administered daily for 2 weeks as of week 3.
  • Fig. 23A illustrates bar graphs representing proinflammatory genes expression (normalized to r28s) as indicated and represented as expression level of specific genes.
  • Fig. 23B illustrates quantification of collagen deposition by PSR.
  • Representative microscopic of liver sections stained with PSR (collagen shown in red) show reduction in collagen deposition following administration of inhibitors.
  • Scale bars 100 pm and original magnification of PSR+ area in liver with/without inhibitors.
  • ANOVA test *P ⁇ .05, **P ⁇ .01 , and ***P ⁇ .001.
  • Col1a1 collagen type 1 alpha 1. Acta2, actin alpha 2. Tgf
  • Fig. 24 illustrates RORyt inverse agonists alleviated liver damage/fibrosis parameters in chronically CCk-injured mice.
  • Fig. 24A illustrates a schematic diagram of the experimental design. Briefly, C57BL/6 mice were treated (i.p) with 0.5 ml/kg CCk in com oil for 4 weeks. Mice then received intraperitoneally administration of RORyt inverse agonists (10 mg/kg) daily for 2 weeks, starting at week 3 post initiation of CCk challenge. [00186] Fig. 24B and Fig. 24C illustrate representative images of H&E and IF staining of liver tissue sections. Scale bars: 50 and 100 (second line) pm. Black arrows represent red spots and immune cells infiltration, respectively
  • CCL carbon tetrachloride
  • H&E hematoxylin-eosin
  • IF immunofluorescence ALT, Alanine amino transferase
  • AST aspartate amino transferase.
  • Fig. 25 illustrates RORyt inverse agonists reduced the absolute number of intrahepatic myeloid cells, mainly eosinophils, in mice with chronic CCk-induced liver injury.
  • IHLs were extracted from livers of CCL-injured mice no-inhibitor or treated with RORyt inverse agonists as well as livers of vehicle control mice. Cells were then stained for various markers of different leukocytic populations and analyzed by flow cytometry.
  • Fig. 25A illustrates representative zebra plots showing an outline for the gating strategy of granulocytes (CD45 + CD11 b + ), neutrophils (CD45 + CD11 b + Ly6C int Ly6G + ), monocytes (CD45 + CD11 b + Ly6C hl Ly6G'), macrophages (CD11 b + Ly6C'Ly6G' F4/80 + ), and eosinophils (CD11b + Ly6C’Ly6G’CD170 + ).
  • Fig. 25B illustrates graphs showing the cell number of the different subsets of IHLs per gram of liver (cell number/gram of liver). Data are expressed as means ⁇ SEM for 15 mice per group (data are combined from 3 individual experiments). Vehicle group contains 5 mice. One-way ANOVA test. * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001. IHL, intrahepatic lymphocytes.
  • Fig. 26 illustrates RORyt inverse agonists reduced the absolute number of adaptive immune cells.
  • IHLs were extracted from livers of CCL-injured mice no-inhibitor or treated with RORyt inverse agonists as well as from livers of vehicle control mice. Cells were then stained for various markers of different leukocytic populations and analyzed by flow cytometry.
  • Fig. 26A illustrates representative FACS plots showing an outline for the gating strategy of T cells (CD45 + CD3 + ), CD4 + T cells(CD3+CD4+), CD8+ T cells (CD3+CD8+), TCR yb-T cells (CD3+TCR yb+).
  • Fig. 27 illustrates RORyt inverse agonists block the secretion of IL-17 and to a lesser extent IFNy but not IL-22.
  • Representative flow cytometry plots showing intrahepatic IL-17- IFNy-, or IL- 22-producing cells, yb-T cells (IL-17+, IFNy+ or IL-22+ yb TCR+/CD3+), CD4+ T cells (IL-17+, IFNy+, or IL-22+CD4+/CD3+), CD8+ T cells (IL- 17-*-, IFNy+, or IL-22+CD8+/CD3+).
  • Fig. 28 illustrates RORyt inverse agonists significantly decrease the expression of hepatic profibrogenic genes in CCI4-injured mice. Livers were extracted from CCI4-injured mice no- inhibitor or treated with RORQt inverse agonists as well as livers of vehicle control mice, their total RNA extracted and analyzed by RT-qPCR, or fixed and sectioned for PSR staining.
  • Fig. 28A illustrates bar graphs represent expression of profibrogenic genes (normalized to r28s)
  • Fig. 29 illustrates that siRNA knockdown of RORC reduces RORC Expression and Reduces Cell Viability.
  • Fig. 30 illustrates that SiRNA knockdown of RORC Increases Caspase 3/7 Expression and Increases Cell Death.
  • the present invention relates to a method of treatment of fibrosis or acute and/or chronic liver conditions by administering to a patient in need thereof an inverse agonist of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORa or RORA) and gamma (RORC, RORy or RORyt) or a silencing RNA that inhibits protein synthesis of RORs.
  • RORs retinoic-acid-receptor-related orphan nuclear receptors alpha
  • RORC gamma
  • the fibrosis is pulmonary fibrosis, retroperitoneal fibrosis (RPF), hepatic fibrosis and/or cirrhosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, intestinal fibrosis, muscle fibrosis, ocular fibrosis, fibrosis in the central nervous system, renal fibrosis, and pancreatic fibrosis, autoimmune fibrotic diseases, fibrosis associated with infectious diseases, fibrosis associated with metabolic diseases, fibrosis associated with cancer, fibrosis associated with environmental exposures, or a combination thereof.
  • RPF retroperitoneal fibrosis
  • RORs retinoic-acid-receptor-related orphan nuclear receptors
  • RORC retinoic-acid-receptor-related orphan nuclear receptors
  • R1 , R2, R3, and/or R4 group(s) is/are H, halogen, NO2, 1-6 alkoxy, OH, NH2, 1 -6 alkyl, 1-6 alkenyl, 1-6 haloalkyl, N-dialkyl, haloalkoxy, 1-6 hydroxyalkyl, and/or
  • R5 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, aryl, alkylarene, halo aryl, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, haloaryl, benzene, phenyl, benzyl, pyridine, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, haloalkyl, cyanoalkyl, methylpyrimidine, toluene, methyl
  • R6 is H, 1-6 alkyl, or may form a five or six ring structure with R5;
  • R7 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, substituted haloaryl, substituted benzene, substituted phenyl, benzyl, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, aryl, halo aryl, alkylarene, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methyl
  • the invention includes the compounds as shown, and also includes (where possible) individual diastereomers, enantiomers, and epimers of the compounds, and mixtures of diastereomers and/or enantiomers thereof including racemic mixtures. Although the specific stereochemistries disclosed herein are preferred, other stereoisomers, including diastereomers, enantiomers, epimers, and mixtures of these may also be useful. Inactive or less active diastereoisomers and enantiomers are useful for scientific studies relating to the nuclear receptor targeting and the mechanism of activation. [00209]
  • the compounds disclosed herein may be used in pharmaceutical compositions comprising (a) the compound(s) or pharmaceutically acceptable salts thereof, and (b) a pharmaceutically acceptable carrier.
  • the compounds may be used in pharmaceutical compositions that include one or more other active pharmaceutical ingredients.
  • the compounds may also be used in pharmaceutical compositions in which the compound of Formula I or a pharmaceutically acceptable salt thereof is the only active ingredient.
  • the inverse agonist of ROR may be:
  • Izumerogant IMU-935
  • ESR-114 ESR-114
  • Cedirogant (ABBV-157);
  • Compound 46 -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 47 -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; [00234] Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • the inverse agonist of ROR may be:
  • Compound 4 N-(6-ethyl-3- ⁇ [(2-methylphenyl)amino]carbonyl ⁇ -4, 5,6,7- tetrahydro-1 -benzothien-2-yl)-1 -methyl-1 H-pyrazole-5-carboxam ide;
  • Compound 5 N- ⁇ 6-tert-butyl-3-[(4-methyl-1-piperazinyl)carbonyl]-4,5,6,7- tetrahydro-1-benzothien-2-yl ⁇ -2 -fluorobenzamide;
  • Compound 7 N-benzyl-2-[(trifluoroacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
  • Compound 15 N-(3-(3-ethylpyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
  • Compound 17 N-(3-(3-(hydroxymethyl)pyrrolidine-1-carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 18 N-(6,6-dimethyl-3-(morpholine-4-carbonyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2-carboxamide;
  • Compound 20 (S)-N-(3-((1-cyanoethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
  • Compound 43 -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 44 -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • Compound 48 -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
  • the fibrosis is associated with Liver diseases, Liver Failure, Acute- and-Chronic Liver Failure, Chemical and Drug Induced Liver Injury, Liver Fibrosis, Fatty Liver, viral Hepatitis, Non-viral hepatitis, Liver diseases, Liver Failure, Hemangioma of liver, Non-alcoholic Fatty Liver Disease, Acute-On-Chronic Liver Failure, Liver diseases, Liver neoplasms, Chemical and Drug Induced Liver Injury, Alcoholic Liver Diseases, Liver and Intrahepatic Biliary Tract Carcinoma, Chronic Hepatitis, Autoimmune hepatitis, Metabolic dysfunction-Associated Fatty Liver Disease’ (MAFLD), alcohol induced liver conditions, metabolic associated steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), liver fibrosis, liver failure, non
  • a pharmaceutical composition for treatment of fibrosis comprising an inverse agonist of RORs and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising:
  • a method of treatment of a vascular injury or protecting against a vascular injury by reducing circulating levels of IL- 17, which comprises administering to a patient in need thereof an inverse agonist of RORs.
  • the injury may be the injury is a brain injury.
  • the compounds of the present invention may be given directly to a patient in need of such treatment, using oral, intravenous, topical, intranasal, intrapulmonary, subcutaneous (slow release implant or patch), sublingual, inhalation or intramuscular administration.
  • ROR gamma t is expressed in lymphocytes during maturation. Irregularities in the expressions of ROR genes may lead to cancer, autoimmune disease.
  • RORyt is the master regulator is Th17.
  • Th17 cells produce many proinflammatory and pleiotropic cytokines IL17A, IL17F (can be also anti-inflammatory), IL21 , IL22, IL24, IL26.
  • Th17 cells secrete GM-CSF and TNF alpha.
  • T cytotoxic 17 is also a type of lymphocyte that secrete all the aforementioned cytokines.
  • T follicular helper cells secrete IL21 and are involved in many inflammatory diseases and types of cancers.
  • the invention presented herein binds to ROR gamma and block the production of IL17A, IL21 , IL17F, IL24, IL26 and other cytokines produced by either T helper 17, T cytotoxic 17, T folicular helper cells, and those produced by all variants or cytotoxic T cells, and Helper T cells as well as Variants of B cells that produce the aforementioned cytokines.
  • Th17 cells have a role in macrophage and leukocytes recruitment to cause inflammation.
  • RORC is highly expressed in fibrosis, certain types of cancer and in autoimmune diseases. Patients waitlisted on organ transplantation lists and who are categorized as Highly sensitized patients have a high activity of T helper 17, T follicular cells, and all T cell variants that produce the aforementioned cytokines. The invention would confer a therapeutic benefit to this category and to all other people who receive allogenic transplantation.
  • profibrotic genes in liver fibrosis include ADAMTS2, ADH7, ADRB3, AFP, AGTR1A, AHR, AKT1 , ALDH2, ANGPT1 , ANXA1 , AOC3, AQP1 , AREG, ATP8B1 , BCHE, BCL2L1 , BMP6, C5, CASP8, CBS, CCL2, CCL21 , CCL25, CCL5, CCR1 , CCR5, CCR6, CCR9, CD163, CD40LG, CD44, CDKN2A, CLDN1 , CNR1 , CNR2, C0L1A1, CPT1A, CSF1, CTGF, CTSB, CTSD, CX3CR1, CXCL10, CXCL9, CXCR3, CXCR4, CYBB, CYLD, DDX5, DGAT1, DKK1, ELAVL1, EL0VL6, ENPP1, EZH2, F5, FABP1, FAS, FGF
  • the current invention is useful in treatment of acute and/or chronic liver conditions involving the immune system whenever any of the aforementioned cytokines is involved either directly or indirectly in the activation of the immune system.
  • Ac is acetyl [CH3C(O)-], AC2O is acetic anhydride; APC is antigen- presenting cell; 9-BBN is 9-borabicyclo[3.3.1]nonane; Bn is benzyl; BOC is tert Butyloxycarbonyl; DIAD is diisopropylazodicarboxylate; DIBAL is diisobutylaluminum hydride; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; EDAC (or EDC) is 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide HCI; EtsN is triethylamine; Et is ethyl; EtOAc is ethyl acetate; EtOH is ethanol; 3-F-Ph is 3-fluorophenyl, HCI is hydrochloric acid; HOBt is 1 -hydroxybenzotriazole
  • alkyl as well as other groups having the prefix "alk”, such as alkoxy and alkanoyl, means carbon chains which may be linear or branched, and combinations thereof, unless the carbon chain is defined otherwise.
  • alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like.
  • alkyl also includes cycloalkyl groups, and combinations of linear or branched alkyl chains combined with cycloalkyl structures.
  • C1 -6 is intended.
  • Cycloalkyl is a subset of alkyl and means a saturated carbocyclic ring having a specified number of carbon atoms.
  • Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • a cycloalkyl group generally is monocyclic unless stated otherwise. Cycloalkyl groups are saturated unless otherwise defined.
  • alkoxy refers to straight or branched chain alkoxides of the number of carbon atoms specified (e.g., C1 -6 alkoxy), or any number within this range [i.e. , methoxy (MeO-), ethoxy, isopropoxy, etc.].
  • alkylthio refers to straight or branched chain alkylsulfides of the number of carbon atoms specified (e.g., C1 -6 alkylthio), or any number within this range [i.e., methylthio (MeS-), ethylthio, isopropylthio, etc.].
  • alkylamino refers to straight or branched alkylamines of the number of carbon atoms specified (e.g., C1-6 alkylamino), or any number within this range [i.e., methylamino, ethylamino, isopropylamino, t-butylamino, etc.].
  • alkylsulfonyl refers to straight or branched chain alkylsulfones of the number of carbon atoms specified (e.g., C1 -6 alkylsulfonyl), or any number within this range [i.e., methylsulfonyl (MeSO2’), ethylsulfonyl, isopropylsulfonyl, etc.].
  • alkylsulfinyl refers to straight or branched chain alkylsulfoxides of the number of carbon atoms specified (e.g., C1-6 alkylsulfinyl), or any number within this range [i.e., methylsulfinyl (MeSO-), ethylsulfinyl, isopropylsulfinyl, etc.].
  • alkyloxycarbonyl refers to straight or branched chain esters of a carboxylic acid derivative of the present invention of the number of carbon atoms specified (e.g., C1-6 alkyloxycarbonyl), or any number within this range [i.e., methyloxycarbonyl (MeOCO-), ethyloxycarbonyl, or butyloxycarbonyl].
  • Aryl means a mono- or polycyclic aromatic ring system containing carbon ring atoms.
  • the preferred aryls are monocyclic or bicyclic 6-10 membered aromatic ring systems. Phenyl and naphthyl are preferred aryls. The most preferred aryl is phenyl.
  • Heterocyclyl refer to saturated or unsaturated non-aromatic rings or ring systems containing at least one heteroatom selected from O, S and N, further including the oxidized forms of sulfur, namely SO and SO2.
  • heterocycles include tetrahydrofuran (THF), dihydrofuran, 1 ,4-dioxane, morpholine, 1 ,4-dithiane, piperazine, piperidine, 1 ,3-dioxolane, imidazolidine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1 ,3-dioxane, 1 ,3-dithiane, oxathiane, thiomorpholine, 2-oxopiperidin-1 -yl, 2-oxopyrrolidin-1 -yl, 2-oxoazetidin-1 -
  • Heteroaryl means an aromatic or partially aromatic heterocycle that contains at least one ring heteroatom selected from 0, S and N. Heteroaryls thus include heteroaryls fused to other kinds of rings, such as aryls, cycloalkyls and heterocycles that are not aromatic.
  • heteroaryl groups include: pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl (in particular, 1 ,3,4-oxadiazol-2-yl and 1 ,2,4- oxadiazol-3-yl), thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furyl, triazinyl, thienyl, pyrimidyl, benzisoxazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, dihydrobenzofuranyl, indolinyl, pyridazinyl, indazolyl, isoindolyl, dihydrobenzothienyl, indolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, naph
  • Halogen refers to fluorine, chlorine, bromine and iodine. Chlorine and fluorine are generally preferred. Fluorine is most preferred when the halogens are substituted on an alkyl or alkoxy group (e.g. CF3O and CF3CH2O).
  • composition « as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • Such term in relation to pharmaceutical composition is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable or “acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • Compounds of structural Formula I may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of structural Formula I.
  • Compounds of structural Formula I may be separated into their individual diastereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase.
  • Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.
  • any stereoisomer of a compound of the general structural Formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known absolute configuration.
  • racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated.
  • the separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography.
  • the coupling reaction is often the formation of salts using an enantiomerically pure acid or base.
  • the diasteromeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue.
  • racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art.
  • Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
  • Some of the compounds described herein may exist as tautomers, which have different points of attachment of hydrogen accompanied by one or more double bond shifts.
  • a ketone and its enol form are keto-enol tautomers.
  • the individual tautomers as well as mixtures thereof are encompassed with compounds of the present invention.
  • the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.
  • the present invention is meant to include all suitable isotopic variations of the compounds of generic Formula I.
  • different isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2 H).
  • Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
  • Isotopically-enriched compounds within generic Formula I can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
  • references to the compounds of structural Formula I are meant to also include the pharmaceutically acceptable salts, and also salts that are not pharmaceutically acceptable when they are used as precursors to the free compounds or their pharmaceutically acceptable salts or in other synthetic manipulations.
  • pharmaceutically acceptable salt refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term “pharmaceutically acceptable salt” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.
  • Representative salts of basic compounds of the present invention include, but are not limited to, the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N- methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate
  • suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, and basic ion-exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
  • basic ion-exchange resins such as arginine, betaine, caffeine, choline, N,N-
  • esters of carboxylic acid derivatives such as methyl, ethyl, or pivaloyloxymethyl
  • acyl derivatives of alcohols such as acetyl, pivaloyl, benzoyl, and aminoacyl
  • esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.
  • the compounds of structural Formula I may be included in various formulations for use as medicaments.
  • Formulations for oral use may be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • an oil medium for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxypropylmethy-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example poly
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., sodium EDTA
  • suspending agent e.g., sodium EDTA
  • preservatives e.g., sodium EDTA, sodium bicarbonate, sodium bicarbonate
  • the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavouring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenteral ly-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol.
  • a non-toxic parenteral ly-acceptable diluent or solvent for example as a solution in 1 ,3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomiser (preferably an I atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1 , 1 ,1 ,2-tetrafluoroethane or 1 , 1 ,1 , 2, 3,3,3- heptafluoropropane.
  • the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
  • the pressurized container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
  • a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
  • the drug product Prior to use in a dry powder or suspension formulation, the drug product is micronized to a size suitable for delivery by inhalation (typically less than 5 microns).
  • This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
  • Capsules made, for example, from gelatin or HPMC
  • blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate.
  • the lactose may be anhydrous or in the form of the monohydrate, preferably the latter.
  • Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
  • a suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from log to 20mg of the compound of the invention per actuation and the actuation volume may vary from 11 to 1001.
  • a typical formulation may comprise a compound of formula I, propylene glycol, sterile water, ethanol and sodium chloride.
  • Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
  • Suitable flavors such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.
  • sweeteners such as saccharin or saccharin sodium
  • Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, poly(DL-lactic-coglycolic acid (PGLA).
  • Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • the dosage unit is determined by means of a valve which delivers a metered amount.
  • Units in accordance with the invention are typically arranged to administer a metered dose or "puff" containing from 1 ng to 10 mg of the compound of Formula I.
  • the overall daily dose will typically be in the range 1 ng to 10 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
  • Compounds of Formula I may also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed.
  • topical application shall include mouth washes and gargles.
  • the inhibitors of RORyt may improve and may have utility in preventing or treating fibrosis and acute and/or chronic liver conditions.
  • One aspect of the invention provides a method for the treatment and control of fibrosis and acute and/or chronic liver conditions, which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula I.
  • mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent, such as a mouse, species can be treated.
  • the method can also be practiced in other species, such as avian species (e.g., chickens).
  • Activity of the compounds of this application may be evaluated using the following assays for RORyt -inhibiting activity.
  • Compounds of Formula I will have activities of ⁇ 10 pM in this assay, and preferably, activity of ⁇ 1 pM.
  • PBMCs were treated with CD3/CD28 for 14 days in presence of IL-6 10ng/ml, IL-1 B 10ng/ml, TGF-B 10ng/ml, and IL-23 10ng/ml.
  • NASH mice model and the effects of IL17 inhibitors on several inflammation markers for liver NASH activity
  • mice were induced liver fibrosis by being treated with 0.1 ml of a 0.5 mg/kg dose of CCI4 twice per week.
  • the mice were given 0.1 ml of GSK805, 0.1 ml of Transcription Factor 1 (S10) (Compound 10) and 0.1 ml of Transcription Factor 2 (S14) (Compound 14) each at a daily dose of 10 mg/kg in 4 weeks.
  • mice/group or 10 male mice/group the mice were treated daily with ip injection for both CCI4 and the inhibitors or with ip injection for CCI4 and with SC injection for inhibitors.
  • the CCL4 induced fibrosis can be acute that is induced over a short period of time (days to weeks) with high doses. Fibrosis can be chronic and manifest over a few weeks or months following small doses of CCL4 administered over a long period of time.
  • the hepatitis was reduced as evidenced by the reduction of the infiltrating IL17A+ lymphocytes which promote neutrophilic infiltration to and secrete cytokines.
  • the compounds also reduce CD8+ cells infiltration which produces interferon gamma and increase hepatitis and inflammation.
  • the compounds reduce the expression of fibrotic and pro fibrotic genes in liver hepatocytes such as IL17A, IL22, RORA and RORC, TNF-beta, lipoxygenases, actins, and collagens (Figs. 1 -23).
  • RORs retinoic-acid-receptor-related orphan nuclear receptors alpha (RORg or RORA) and gamma (RORC, RORy or RORyt) in Promoting Fibrosis in the Liver, and Fibrosis in General
  • liver disease and inflammation is a serious global health problem occurs in response to multiple insults such as alcohol exposure, obesity, diabetes, chronic viral hepatitis, autoimmunity, chronic inflammatory conditions, and drug toxicity which can lead to progressive fibrosis.
  • Perpetuation of the fibrotic process commonly leads to cirrhosis and Hepatocellular carcinoma (HCC), which are leading causes of morbidity and mortality worldwide.
  • Liver fibrosis characterized by the proliferation and trans differentiation of quiescent hepatic stellate cells (HSCs) into myofibroblasts which are principal executive and responsible for collagen and extracellular matrix deposition (ECM) leading to tissue scarring.
  • HSCs quiescent hepatic stellate cells
  • ECM extracellular matrix deposition
  • CCI4 carbon tetrachloride
  • RORyt is induced during the transition from double negative to double positive thymocytes, allowing for maturation into single positive T cells.
  • Expression of RORyt and its targeted cytokines is important for differentiation of other cell types such as CD8 + Tc17 cells, invariant natural killer T cells and yb T cells.
  • IL-17 plays a crucial role in liver fibrosis as well as multiple human autoimmune diseases.
  • IL-17A a hallmark of the Th17 subset, secreted by infiltrating Th17 cells in the liver, promotes the synthesis of type I collagen and a-smooth muscle actin (SMA) in HSCs and participates in initiation and progression of hepatic fibrosis.
  • SMA smooth muscle actin
  • RORyt is expressed in a variety of tissues and is a highly attractive drug target in Th17 cell mediated diseases especially due to its involvement in IL17/IL23 axis and because its activity can be modulated by applying small-molecular weight inverse agonist by RORyt ligand-binding pocket. Deficiency of RORyt in mice leads to the profound deficiency in Th17 differentiation and protection against many diseases such as encephalomyelitis (EAE). Several X-ray crystallography and NMR structural studies have identified numerous small-molecular-weight inhibitors including ligands, agonists, inverse agonists, and antagonists targeting RORyt capable to alleviate proinflammatory diseases.
  • Conformational changes in H3 and H5 helix can interface with ligand-binding pocket and the back wall of coactivator-binding pocket, which can disrupt protein-coactivator interactions and reduce or completely loss RORyt activity.
  • Th17-type cytokine RORyt inhibitor (GSK805) resulted in reduced fibrosis and cause reduction in neutrophil recruitment to the liver.
  • GSK805 is an orally available inhibitor of RORyt-mediated transcription, able to supresses Th17 response and cause reduction in frequency and total number of IL17 producing Th17 cells.
  • mice treated with CCI4 ⁇ Carbon tetrachloride) (Cat. No. 270652-100 ML; Sigma-Aldrich) resuspended in com oil (Cat. No. C8267-500 ML; Sigma-Aldrich) (0.5 ml/kg two intraperitoneal (i.p.) injections per week) for four weeks.
  • Vehicle control mice received com oil instead of CCI4 throughout the period of the experiment.
  • mice were treated with the three different RORpt antagonists including GSK805, TF1 -S10 and TF2-S14 (10 mg/kg i.p. dissolved in com oil) starting as of week three daily injection (during weeks 3 through 4). All the mice were euthanized 24 hrs after the final injection of CCI4 by using sodium pentobarbital (400 mg/kg). After dissection, the liver, spleen, and heart blood samples were harvested. All the mice were reared and housed under specific pathogen free conditions (20 ⁇ 2°C; 12 hr light/12 hr dark) with free access to food (standard chow diet) and water. All the animal experimental procedures were approved by the Centre de mecanic du Centre Hospitalier de I’Universite de Montreal (CRCHUM) animal Ethics Committee, Comite Institutionel de Protection des Animaux (CIPA) (protocol IP22059NSs).
  • CIPA Comite Institutionel de Protection des Animaux
  • IHLs Intrahepatic leukocytes
  • mice were harvested from mice and was transferred into a sterile petri dish.
  • Intrahepatic leukocytes IHLs were isolated from mice livers using a Percoll gradient (Cat. No. 17089101 ; Cytiva) in isotonic solution. Briefly, the liver tissues were gently minced into small pieces and then were digested in collagenase D (0.025 lU/mL, Cat. No. 11088866001 ; Roche) and benzonase (10 lU/mL, Cat. No. 70664-10KUN; EMD Millipore) at 37°C with rotation for 30 minutes. The liver and the cell suspension then were passed through a 70- pm cell strainer (Cat. No.
  • IHLs were washed, and the remaining RBCs were lysed using ACK (Ammonium-Chloride-Potassium) lysing buffer (Cat. No. A10492-01 ; Thermo Fisher Scientific). IHLs were directly stained for surface markers or stimulated with PMA/ionomycin (50 ng/mL, Cat. No. P1585 and 1 pg/mL, Cat. No. I-0634; Sigma-Aldrich, respectively) in the presence of brefeldin A (5 pg/mL, Cat. No. B6542; Sigma-Aldrich) and monensin (5 pg/mL, Cat. No. M5273; Sigma-Aldrich) in a cell incubator with 10 % CO2 at 37°C for 5 hours before intracellular cytokine staining (ICS).
  • ICS cytokine staining
  • RNA of the frozen liver samples was extaracted and purified from mouse livers using the RNeasy Plus Mini kit (Cat. No. 74134; QIAGEN) and quantified according to the manufacturer’s instructions.
  • Complementary DNAs cDNAs
  • cDNAs were generated from 1 mg RNA of total liver RNA using the Transcriptor Universal cDNA Master (Cat. No. 05893151001 ; Roche) kit.
  • Produced cDNAs were then diluted 1 :10 with ultrapure water and the relative expression of the mRNA was measured using real-time qPCR with the Light Cycler 480 SYBR Green I system (Cat. No. 04707516001 ; Roche).
  • the sections then were incubated with the primary antibodies (Table III) diluted in the blocking buffer without human serum at 4°C.
  • the sections were then washed in PBS-Tween (0.05 % v/v) and then incubated with the appropriate secondary antibodies (Table III) diluted in blocking buffer containing the 10 % of donkey serum for 1 hour at RT in dark.
  • the sections were mounted in Slow Fade Gold antifade reagent wit DAP I (Cat. No. S36938; Invitrogen).
  • the images were acquired at the CRCHUM molecular pathology platform using a wholeslide scanner (BX61 VS; Olympus).
  • RORyt inhibitors reduced the infiltration of immune cells around the portal and central veins in CCI4-induced liver fibrosis in mice
  • liver fibrosis group no-inhibitor
  • vehicle control group com oil
  • Inhibitors were therefore applied (TF1 -S10, TF2-S14 and GSK805 as our internal control) to attenuate liver fibrosis in mice with CCI4-induced hepatic fibrosis. H&E staining revealed that CCI4 administration alone leads to the remarkable increase in the size of the portal and central veins. Inhibitor-treated mice showed much smaller infiltration of immune cells around liver veins than CCI4-induced mice (Fig. 20B). More specifically, the fibrotic areas and infiltration of immune cells around the portal and central veins were reduced in inhibitor-treated groups.
  • RORyt inverse agonists reduce accumulation of myeloid cells in CCI4- induced liver fibrosis
  • neutrophils level (CD45 + CD11 b + Ly6C int+ Ly6G hi+ ) did not affected in the livers of inhibitors treated mice compared with the CCI4-treated control mice.
  • GSK805 and TF1 -S10 are the most efficient RORyt inhibitors applied to exacerbate liver injury and liver inflammation (Fig- 20B). Additionally, our data suggesting that neutrophils are not the cellular target of RORyt inhibitors applied in our study.
  • RORyt inverse agonists decreased levels of IL-17 and reduced infiltration of IL-17 producing cells in the livers of CCI4-treated mice
  • IL-17 level were significantly reduced in CCI4 + inhibitor -treated mice in comparison with CCI4 only treated animals, suggesting the importance of RORpt- mediated inhibitors in attenuation of IL-17-driven liver fibrosis.
  • flow cytometry analysis of liver infiltrated lymphocytes was performed.
  • CD4 + T p ⁇ 0.05 following applying all the inhibitors
  • yb-TCR p ⁇ 0.01 by administering GSK805 and TF2-S14 and P ⁇ 0.074 following application of TF1 -S10
  • these cell populations did not significantly affect by administration of these inhibitors.
  • the inhibitors are not effective at inhibiting the intracellular secretion of IL-22, another member of TH17 T cell family which suggest the inhibitors specifically target IL-17 producing TH17 T cells (Fig. 22A-C).
  • TH1 T cell family could be another population affected by the administration of the inhibitors as there is a remarkable reduction in the frequency of CD4 + IFN-y + (P ⁇ 0.05) and CD8 + IFN-y + (P ⁇ 0.05) cytokine secretion level by applying GSK805 and TF2-S14, respectively (Fig. 22B-C).
  • inhibitors may relieve liver fibrosis through regulating pro inflammatory cytokines.
  • RORs retinoic-acid-receptor-related orphan nuclear receptors alpha (RORg or RORA) and gamma (RORC, RORy or RORyt) in Promoting Fibrosis in the Liver, and Fibrosis in General #2
  • mice Male wildtype C57BL/6 mice (6-8 weeks old) were purchased from Charles River Laboratories (Montreal, CA). To induce chronic liver fibrosis, mice were treated with CCk (Carbon tetrachloride) (Sigma-Aldrich, Oakland, ON, Canada) resuspended in com oil (Sigma-Aldrich, Oakland, ON, Canada) (0.5 ml/kg two intraperitoneal (i.p.) injections per week for four weeks). Vehicle control mice received corn oil instead of CCL throughout the period of the experiment.
  • CCk Carbon tetrachloride
  • com oil Sigma-Aldrich, Oakland, ON, Canada
  • mice were treated daily for two weeks with the two novel RORyt inverse agonists TF-S10 and TF- S14, or GSK805 as a control (10 mg/kg i.p. in corn oil) (Fig. 24A).
  • mice were euthanized 24 hrs after the final injection of CCk by using sodium pentobarbital (400 mg/kg). All the mice were reared and housed under specific pathogen free conditions (20 ⁇ 2°C; 12 hrs light/12 hrs dark) with free access to food (standard chow diet) and water. All animal experimental procedures were approved by the Centre de mecanic du Centre hospitalier de I’Universite de Montreal (CRCHUM) animal Ethics Committee, Comite Institutionel de Protection des Animaux (CIPA) (protocol IP22059NSs).
  • CIPA Comite Institutionel de Protection des Animaux
  • Mouse livers were harvested, fixed in Tissue Fix (Chaptec, Montreal, Quebec, Canada) for 24 hrs at 4°C, then embedded in paraffin for sectioning and histological analysis (Pathology department, CHUM, Montreal, Quebec, Canada).
  • the 4- pm -thick Formalin-Fixed Paraffin- Embedded (FFPE) tissue sections were stained with H&E (Hematoxylin and eosin) (Varistain XY, Shandon) by the pathology platform of the CRCHUM using standard procedures.
  • H&E Hematoxylin and eosin
  • FFPE sections were deparaffinized and rehydrated, then stained with PSR (Picrosirius Red) with Fast green (Sigma-Aldrich, Oakland, ON, Canada) in Picric acid solution (Sigma-Aldrich, Oakland, ON, Canada). Collagen fibers were shown in red by PSR staining. Sirius red positive area was calculated by threshold settings based on pixel value in the green channel using FIJI (Image J) image analysis software.
  • liver was harvested from mice and was transferred into a sterile petri dish.
  • Intrahepatic leukocytes IHLs were isolated using a Percoll gradient (Cytiva, Uppsala, Sweden) in isotonic solution. Briefly, liver tissues were gently minced into small pieces then digested in collagenase D (0.025 lll/mL, Roche, Laval, Quebec, Canada) and benzonase (10 lll/mL, EMD Millipore, Darmstadt, Germany) at 37°C with rotation for 30 minutes.
  • the liver and the cell suspension were then passed through a 70- pm cell strainer (Thermo Fisher Scientific, Fremont, CA) and centrifuged at 500 g to remove hepatocytes. The supernatant was transferred to a new tube and centrifuged at 1000 g for 10 minutes at 4°C.
  • the cell pellet was then resuspended in Percoll 40% in 1 % Hank’s balanced salt solution in sterile water (vol/vol) and layered over Percoll 80% in 1 % Hank’s balanced salt solution in sterile water (vol/vol), followed by centrifugation without brakes for 25 minutes at 4°C. IHLs were collected at the interface, washed, and the remaining RBCs were lysed using ACK (Ammonium-Chloride- Potassium) lysing buffer (Thermo Fisher Scientific, Fremont, CA).
  • ACK Ammonium-Chloride- Potassium
  • IHLs were stimulated with PMA/ionomycin (50 ng/mL, and 1 pg/mL; Sigma-Aldrich, respectively) in the presence of brefeldin A (5 pg/mL; Sigma-Aldrich) and monensin (5 pg/mL; Sigma-Aldrich) in a cell incubator with 10 % CO2 at 37°C for 5 hrs then stained for surface markers.
  • Cells were then fixed and permeabilized using FOXP3 fixation buffer (eBioscience), washed twice with permeabilization wash buffer (eBioscience), and incubated with antibodies for intracellular cytokines at 4°C for 30 minutes in the dark.
  • RNA level was measured using real-time qPCR with the Light Cycler 480 SYBR Green I system (Roche, Germany).
  • r28S was used as the standard housekeeping gene and the 2’ AACt method was applied to calculate the mRNA level.
  • the 28S primers sequences are: forward, 5’-3’(CGAGATTCCCACTGTCCCTA) and reverse, 5’- 3’(GGGGCCTCCCACTTATTCTA).
  • the primers for Tgf-/31, Col1a1, Acta2, and Loxl2 genes were purchased from QIAGEN.
  • Thermo Fisher Scientific rabbit polyclonal desmin antibody (Thermo Fisher Scientific, Massachusetts, US) mouse monoclonal a-SMA antibody (Clone 1A4, Sigma-Aldrich) antibodies followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) diluted in the blocking buffer containing the 10 % of donkey serum for 1 hour at room temperature in dark.
  • the sections were mounted in Slow Fade Gold antifade reagent with DAPI (Thermo Fisher Scientific). Finally, the images were acquired at the CRCHUM molecular pathology platform using a whole slide scanner (BX61 VS; Olympus).
  • RORyt inhibitors reduced the hepatic infiltration of immune cells and ameliorated liver damage in chronic CCI4-injured liver injury in mice
  • mice were treated daily (i.p.) with either one of the RORyt inverse agonists, GSK805, TF-S10 ( co m p o u n d 1 0 ) and TF-S14 (compound 13), or vehicle as described in materials and methods.
  • Fig. 24A Mice were sacrificed and their liver processed for analysis 24 hrs after the last injection of CCk to evaluate hepatic fibrosis and efficacy of treatments.
  • Fig. 24B Red spots of irregular shape were visible on the surface of CCk-injured livers as compared to the smooth uniform surface of livers from vehicle control mice. These spots, probably originating from hemorrhaged damaged vessel, decreased in frequency in the RORyt inverse agonists + CCk mice (Fig. 24B).
  • RORyt inverse agonists reduce accumulation of myeloid cells in CCI4- induced liver injury
  • FIG. 25A We observed an overall increase in infiltration of CD45 + cells in mice receiving CCI4. Treating CCL-injured mice with GSK805 resulted in a significant reduction in the total number of leukocytes (CD45 + ) and myeloid cells (CD45 + CD11 b + ) in the liver (p ⁇ 0.01 and p ⁇ 0.05, respectively) (Fig. 25B).
  • RORyt inverse agonists reduce hepatic accumulation of T cells and the infiltration of IL-17-producing cells in livers of CCI4-injured mice
  • Treatment of CCI4-injured mice with our RORyt inverse agonists decreased the number of IL-17-producing CD4+ and yb-T cells as compared to no-inhibitor animals (Fig. 27A, 4B).
  • Fig. 27C there was no significant difference in the total number of hepatic infiltrating IL-17-producing CD8+ T cells between all experimental mice groups (control, CCI4-injured or RORyt inverse agonists + CCI4- injured mice) (Fig. 27C).
  • IL-22 another Th17 cytokine which is only partially controlled by RORyt.
  • IL-22 expression by the different T cell populations (CD4 + , CD8 + and yb-T cells) was generally low following CCL injury.
  • RORyt inverse agonists examined did not alter the frequency of IL-22 -producing cells (Fig. 27A-C).
  • type 1 cytokines mainly IFNy are of critical importance in liver disease and inflammation and maybe increased as a mechanism of rebound inflammation.
  • intracellular staining for IFNy in intrahepatic T cells revealed upregulation of IFNy- producing CD4 + and CD8 + cells in CCL-injured mice as compared to the vehicle-injected group (p ⁇ 0.01 and p ⁇ 0.001 , respectively).
  • This increase of IFNy-producing CD4 + or CD8 + was significantly reversed upon treatment of CCL-injured mice with the control inhibitor, GSK805 and TF-S10 (p ⁇ 0.05), respectively.
  • profibrogenic genes type 1 collagen, a-smooth muscle actin, TGF-01 , and lysyl oxidase like 2 enzymes (Col1a1, Acta2, Tgf-/31 and Loxl2) by quantitative RT-PCR in treated and no-inhibitor/CCh-injured mice.
  • Expression of theses profibrogenic genes was increased in livers of CCk-injured mice compared to control (no CCk) mice (p ⁇ 0.001) and was significantly reduced upon treatment with GSK805 or TF-S10 (p ⁇ 0.001 ) (Fig. 28A).
  • eosinophils can be important mediators of liver inflammation. Intrahepatic eosinophils are detectable in halothane-induced liver injury and are the pathogenic drivers of inflammation in this model. Liver eosinophilic infiltrate was a significant finding in patients with chronic hepatitis C who consume drugs ⁇ Tarantino, 2008 #151 ⁇ . We have also reported increasing eosinophilic type 2 liver inflammation in mice and humans with Metabolic dysfunction-associated steatohepatitis (MASH).
  • MASH Metabolic dysfunction-associated steatohepatitis
  • Eosinophils can release eosinophilic extracellular traps (EETs), considered as damage-associated molecular patterns (DAMPs), important activators of hepatic injury.
  • EETs extracellular traps
  • DAMPs damage-associated molecular patterns
  • IL-17 was produced by other leukocytes such as neutrophils and macrophages derived from splenocytes of sensitized mice (BALB/C-sensitized C57BL/6 mouse).
  • yb- T cells were the main producers of IL-17 and the ones most affected by treatment with RORyt inverse agonists.
  • Our results agree with several studies that showed that IL-17- producing yb-T cells are one of the key mediators of liver inflammation and fibrosis.
  • the inverse agonists used in our study did not reduce the frequency of IL- 22-producing cells, underscoring their specificity for the IL-17 pathway.
  • IL-22 plays a critical hepatoprotective role by inducing anti-apoptotic and mitogenic processes in hepatocytes. It also exerts anti-fibrogenic functions, by inactivating HSCs and inducing their senescence.
  • siRNA was tested using pancreatic cell lines HPAF-II and Miapaca-ll for Cell Viability, % Caspase activity and Cell death.
  • HPAF-II peritoneal ascitic fluid with primary pancreatic adenocarcinoma and metastases to the liver, diaphragm and lymph nodes.
  • Miapaca-ll from tumor tissue of the pancreas
  • MERS-CoV infection in humans is associated with a pro-inflammatory Th1 and Th17 cytokine profile. Cytokine. 2018 Apr; 104:8-13.

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Abstract

This invention relates to a method of treatment of fibrosis which comprises administering to a patient in need thereof a therapeutically effective amount of an inverse agonist of retinoic-acid-receptor-related orphan nuclear receptors (RORs).

Description

METHOD OF TREATMENT OF FIBROSIS WITH INVERSE AGONIST OF RAR RELATED ORPHAN RECEPTORS (RORS)
FIELD OF THE INVENTION
[0001] This invention relates to a method of treatment of fibrosis by administering to a patient in need thereof an inverse agonist of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORa or RORA) and gamma (RORC, RORy or RORyt).
BACKGROUND
[0002] Fibrosis is a pathological condition present in most chronic inflammatory diseases. Fibrosis happens when connective tissue replaces normal parenchymal tissue, which can lead to organ malfunction and death.
[0003] Liver fibrosis due to viral or metabolic liver diseases is a major global health problem. Liver fibrosis is the over-accumulation of extracellular matrix proteins, such as collagen, that occurs in most types of chronic liver diseases, among which is nonalcoholic fatty liver disease (NAFLD). Histologically, NAFLD can encompass a spectrum of liver lesions, from steatosis to cirrhosis, via non-alcoholic steatohepatitis (NASH). Liver fibrosis increases the risk for liver-related complications and other co-morbidities such as hepatocellular carcinoma, insulin resistance, diabetes, and cardiovascular events.
[0004] Inflammation plays an important role in the development and progression of (NASH), and NASH is a powerful driving force for the progression of fibrosis. It has been found that patients with NASH have higher levels of inflammatory cytokines. Immune response has an important driver of the progression of the disease. Immune imbalances, reflected at the cellular level through the level of pro-inflammatory cytokines including those involved in Th17 differentiation (IL-6, IL-21 , IL-23 and transforming growth factor-beta) and those released by Th17 cells (IL-17A, IL-17F, IL-21 , and IL-22) have been studied. Particularly, it has been found that IL-17A, a type 3 inflammatory cytokine either produced by Th17, CD4+ T cells or neutrophils, are critical in fibrogenesis and steatohepatitis. Liver infiltration by Th17 cells have also shown to be a critical element for NASH initiation and development of fibrosis and this infiltration was accompanied by an increase in the production of pro-inflammatory cytokines. The activation of the IL-17 axis is key in disease progression since IL-17 exacerbates the liver tissue inflammation and has a pro-fibrotic effect.
[0005] Currently, there are no licensed therapies for NASH. The majority of drugs in development aim at insulin and lipids. However, while a number of drugs are in development, drug candidates face many challenges because of the complexity of the disease. There is a need for more accurate biomarkers to be used as therapeutic targets.
[0006] Therapies targeted to reverse the immune imbalance have shown potential to alleviate steatosis and the progression of NASH. Inhibitors of the IL-17 axis are new therapeutic targets in the treatment of liver fibrosis. Blocking IL-17A production will limit fibrosis progression by reducing recruitment of inflammatory cells and blocking their profibrogenic effect.
[0007] The retinoic acid receptor-related (ROR) sub-family of orphan nuclear receptors was initially identified on the basis of sequence similarities to the retinoic acid and retinoid X receptor families. Through alternative promoter usage and exon splicing, the ROR genes encode different isoforms of RORa, p and y, which exhibit differential tissue expression and functions. RORyt is a differentially spliced isoform of RORy, that differs only in the N-terminus by the presence of 21 additional amino acids in RORy. The endogenous physiological ligands for RORyt have recently been identified as 7p-27-dihydroxy cholesterol, and two other cholesterol biosynthetic intermediates.
[0008] RORyt is exclusively expressed in cells of the immune system including CD4+ CD8+ double positive thymocytess, Th17, Tc17, and y6 T cells, as well as a subset of innate lymphoid cells (ILCs) and regulatory T cells (Tregs). RORyt is a key transcription factor driving Th17 cell differentiation, and production of IL-17A, IL-17F and IL-22 in innate and adaptive immune cells, also termed “type 17” cells. Th17 cytokines, IL-17A, IL-17F, and IL-22, stimulate tissue cells to produce a panel of inflammatory chemokines, cytokines and metalloproteases, resulting in the recruitment of granulocytes to sites of inflammation. The Th17 cell subset has been shown to be the major pathogenic population in several models of autoimmune inflammation, including collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). RORyt deficient mice show impaired Th17 cell differentiation in vitro, significantly reduced Th17 cell populations in vivo, and decreased susceptibility to EAE and intestinal inflammation. RORyt-deficient T cells fail to induce colitis in the mouse T cell transfer model.
[0009] Human genetic studies have shown association of polymorphisms in the genes for Th17 cell-surface receptors, IL-23R and CCR6, with susceptibility to inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA) ankylosing spondylitis (AS) and psoriasis. Clinical modulation of the IL-23/IL-17 pathway through biologies targeting IL-12/23, IL-23, IL-17A or IL-17RA has provided validation of its critical role in human autoimmune diseases. RORyt is a nuclear receptor target in the IL-23/IL-17 pathway and has been shown to be tractable to modulation by oral small molecules. Indeed, other nuclear receptors have been successfully targeted by orally available small molecules that are now marketed drugs.
[0010] Traditional treatments of most fibrosis involve the administration of corticosteroids, such as prednisone, and/or other medications that suppress the body's immune system. Additionally, patient response to such treatment is variable, and the toxicity and side effects can be important.
[0011] There is a need to have modulators that inhibit the activity of RORs to control fibrosis.
SUMMARY
[0012] According to an embodiment, there is provided a method of treatment of fibrosis which comprises administering to a patient in need thereof a therapeutically effective amount of an inverse agonist of RORs or a silencing RNA that inhibits protein synthesis of RORs.
[0013] The fibrosis is pulmonary fibrosis, retroperitoneal fibrosis (RPF), hepatic fibrosis and/or cirrhosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, intestinal fibrosis, muscle fibrosis, ocular fibrosis, fibrosis in the central nervous system, renal fibrosis, and pancreatic fibrosis, autoimmune fibrotic diseases, fibrosis associated with infectious diseases, fibrosis associated with metabolic diseases, fibrosis associated with cancer, fibrosis associated with environmental exposures, or a combination thereof.
[0014] The inverse agonist of RORs is a compound of Formula (I), (II), (III), or (IV), a pharmaceutically acceptable salts thereof, or stereoisomers thereof:
Figure imgf000005_0001
[0015] wherein,
[0016] R1 , R2, R3, and/or R4 group(s) is/are H, halogen, NO2, 1-6 alkoxy, OH, NH2, 1 -6 alkyl, 1-6 alkenyl, 1-6 haloalkyl, N-dialkyl, haloalkoxy, 1-6 hydroxyalkyl, and/or -CO2(1-6 alkyl);
[0017] R5 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, aryl, alkylarene, halo aryl, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, haloaryl, benzene, phenyl, benzyl, pyridine, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, haloalkyl, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methylimidazole, methyldiazole, methyltriazole, methylthiadiazole, methylimidazolidine, methylthizolidine, methylpyrrolidine, methylpiperazine, methylpiperidine, methylpyridazine, methylpyrazine, methyltriazine, methylpyrrole, methylpyrroline, methylpyrazolidine, methylpyrazoline, methylthiazole, methylisothiazole, methylisoxazole, or arylalkyl.
[0018] R6 is H, 1-6 alkyl, or may form a five or six ring structure with R5; and
[0019] R7 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, substituted haloaryl, substituted benzene, substituted phenyl, benzyl, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, aryl, halo aryl, alkylarene, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methylimidazole, methyldiazole, methyltriazole, methylthiadiazole, methylimidazolidine, methylthizolidine, methylpyrrolidine, methylpiperazine, methylpiperidine, methylpyridazine, methylpyrazine, methyltriazine, methylpyrrole, methylpyrroline, methylpyrazolidine, methylpyrazoline, methylthiazole, methylisothiazole, methylisoxazole, or arylalkyl.
[0020] The inverse agonist of ROR may be:
[0021] N-[2,6-dichloro-2'-(trifluoromethoxy)[1 ,T-biphenyl]-4-yl]-4-(ethylsulfonyl)- benzeneacetamide (GSK805) or analogs thereof;
[0022] N-[[(2S)-1-[[5-(4-fluorophenyl)-2-methyl-4-thiazolyl]carbonyl]-2- piperidinyl]methyl]-4-benzofurancarboxamide (SB-649868);
[0023] 3-isoxazolebutanoic acid (JTE-151 );
[0024] 5H-Pyrrolo[3,4-b]pyridine-3-carboxamide, N-[[5-(ethylsulfonyl)-2- pyridinyl]methyl]-6,7-dihydro7-(1-methylethyl)-6-[[trans-4- (trifluoromethyl)cyclohexyl]methyl]- hydrochloride (VTP-43742);
[0025] (1 R,3S,4R)-4-((3aR,9bR)-9b-((4-Fluorophenyl)sulfonyl)-7-
(perfluoropropan-2-yl)-2,3,3a,4,5,9b-hexahydro-1 H-benzo(E)indole-3-carbonyl)-3- methylcyclohexane-1 -carboxylic acid (BMS-986251 );
[0026] 2-((1 S,3S)-3-((R)-5-((7-fluoro-1 , 1 -dimethyl-2,3-dihydro-1 H-inden-5- yl)carbamoyl)-2-methoxy-5,6,7,8-tetrahydro-1 ,6-naphthyridine-6- carbonyl)cyclobutyl)acetic acid (TAK-828);
[0027] N-(5-(N-(4-(1 ,1 ,1,3,3,3-hexafluoro-2-hydroxypropan-2- yl)phenyl)sulfamoyl)-4-methylthiazol-2-yl)acetamide (SR1001 );
[0028] AUR-101 ;
[0029] Izumerogant (IMU-935);
[0030] ESR-114;
[0031] Cedirogant (ABBV-157);
[0032] analogs thereof; or
[0033] combinations thereof. [0034] The preferred compound of Formula (I), (II), (III), or (IV) pharmaceutically acceptable salts thereof, or stereoisomers thereof is selected from the group consisting of:
[0035] Compound 39: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4,5,6,7 tetrahydrobenzo[b]thiophen-2-yl) nicotinamide;
[0036] Compound 40: -rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]-
4.5.6.7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
[0037] Compound 41 : -N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6,7- tetrahydro-1 -benzothiophen-2-yl} pyridine-3-carboxamide;
[0038] Compound 42: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0039] Compound 43: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0040] Compound 44: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0041] Compound 45: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[0042] Compound 46: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0043] Compound 47: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0044] Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0045] Compound 49: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide; [0046] Compound 50: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0047] Compound 51 : -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0048] Compound 52: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamidel; and
[0049] Compound 53: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.
[0050] The inverse agonist of ROR may be:
Figure imgf000009_0001
N-(3-(2-methylpyrrolidine- 1 -carbonyl)- 4, 5 , 6, 7 -tetrahydrobenzo [b] thiophen-2 -
[0051] yl)nicotinamide
Figure imgf000010_0001
A'-(3-((2/?.4/?)-2.4-dimethylpiperidine- l-carbonyl)-4, 5,6,7- tetrahydrobenzo [6] th iophen-2-
[0052] yl)nicotinamide
Figure imgf000010_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7-
[0053] tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
Figure imgf000011_0001
N-(3 -(3 -ethylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[b]thiophen-2-
[0054] yl)nicotinamide
Figure imgf000011_0002
7V-benzyl-2-(2-(( 1 -methyl- 1/f-imidazol- 2-yl)thio)acetamido)-4, 5,6,7- tetrahy drobenzo [6] thiophene-3 -
[0055] carboxamide
Figure imgf000011_0003
N-(3 -(3 -(hydroxymethyl)pyrrolidine- 1 - carbonyl)-4, 5,6,7- tetrahy drobenzo [/?] thi ophcri-2-
[0056] yl)nicotinamide
Figure imgf000012_0001
Figure imgf000013_0001
7V-(3-benzoyl-4, 5,6,7- tetrahy drobenzo [6] thiophen-2-y 1) - 5 - methyl-2- (methylsulfonyl)pyrimidine-4-
[0060] carboxamide
Figure imgf000014_0002
N-(3 -((( 1 -methyl- 177-1 ,2,4-triazol-3 - yl)methyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo [7>] th iophen-2-
[0061] yl)nicotinamide , and
Figure imgf000014_0001
7V-(6,6-dimethyl-3-(morpholine-4-carbonyl)-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-
[0062] yl)pyrazine-2-carboxamide
[0063] The preferred compound of Formula (I), pharmaceutically acceptable salts thereof, or stereoisomers thereof is selected from the group consisting of:
Figure imgf000015_0001
N-benzyl-2-(2-(4-methylpipeiazin-lyl)acetamido)-4.5.6,7- tenaliydrobenzo[b]thiophene-3-
- carboxamide
[0064]
Figure imgf000015_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7-
[0065] tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
Figure imgf000015_0003
N-(3 -(3 -ethylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[6]thiophen-2-
[0066] yl)nicotinamide
Figure imgf000016_0001
N -benzyl-2-(2-(( 1 -methyl- 17/-i m idazol- 2-yl)thio)acetamido)-4,5,6,7- tetrahydrobenzo [6] thiophene-3 -
[0067] carboxamide
Figure imgf000016_0002
N-(3 -(3 -(hydroxymethyl)pyrrolidine- 1 - carbonyl)-4, 5,6,7- tetrahydrobenzo [/?] thi ophcri-2-
[0068] yl)nicotinamide
Figure imgf000017_0001
Figure imgf000018_0001
7V-(3-benzoyl-4, 5,6,7- tetrahy drobenzo [6] thiophen-2-y 1) - 5 - methyl-2- (methylsulfonyl)pyrimidine-4-
[0072] carboxamide
Figure imgf000019_0002
N-(3 -((( 1 -methyl- 177-1 ,2,4-triazol-3 - yl)methyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo [b] th iophen-2-
[0073] yl)nicotinamide
Figure imgf000019_0001
?/-(6,6-dimethyl-3-(morpholine-4-carbonyl)- 4,5,6,7-tetrahydrobenzo[6]thiophen-2- [0074] yl)pyrazine-2-carboxamide
[0075] Compound 1 : N-(3-(4-benzylpiperazine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)-2 -fluorobenzamide;
[0076] Compound 2: N-(3-(benzylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-
2-yl)-5-chloro-2-(methylthio)pyrimidine-4-carboxamide;
[0077] Compound 3: 5-chloro-N-(3-{[(1 ,1-dioxidotetrahydro-3-thienyl)amino]- carbonyl}-6-methyl-4,5,6,7-tetrahydro-1 -benzothien-2-yl)-2-(methylthio)-4-pyrimidine- carboxamide;
[0078] Compound 4: N-(6-ethyl-3-{[(2-methylphenyl)amino]carbonyl}-4, 5,6,7- tetrahydro-1 -benzothien-2-yl)-1 -methyl-1 H-pyrazole-5-carboxam ide; [0079] Compound 5: N-{6-tert-butyl-3-[(4-methyl-1-piperazinyl)carbonyl]-4,5,6,7- tetrahydro-1-benzothien-2-yl}-2 -fluorobenzamide;
[0080] Compound 6: 2-fluoro-N-{6-methyl-3-[(4-methyl-1 -piperazinyl)carbonyl]-
4.5.6.7-tetrahydro-1-benzothien-2-yl}benzamide;
[0081] Compound 7: N-benzyl-2-[(trifluoroacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
[0082] Compound 8: N-benzyl-2-({[(4-methyl-2-pyrimidinyl)thio]acetyl}amino)-
4.5.6.7-tetrahydro-1-benzothiophene-3-carboxamide;
[0083] Compound 9: N-benzyl-2-[(1-piperidinylacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
[0084] Compound 10: N-benzyl-2-{[(4-methyl-1-piperazinyl)acetyl]amino}-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
[0085] Compound 11 : N-benzyl-2-{[(4-methyl-1-piperidinyl)acetyl]amino}-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
[0086] Compound 12: N-(3-(2-methylpyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[0087] Compound 13: N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0088] Compound 14: N-(3-(((1 r,4r)-4-hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0089] Compound 15: N-(3-(3-ethylpyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[0090] Compound 16: N-benzyl-2-(2-((1-methyl-1 H-imidazol-2-yl)thio)acetamido)-
4.5.6.7-tetrahydrobenzo[b]thiophene-3-carboxamide;
[0091] Compound 17: N-(3-(3-(hydroxymethyl)pyrrolidine-1-carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; [0092] Compound 18: N-(6,6-dimethyl-3-(morpholine-4-carbonyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2-carboxamide;
[0093] Compound 19: N-(3-(((1-methyl-1 H-1 ,2,4-triazol-3-yl)methyl)carbamoyl)-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[0094] Compound 20: (S)-N-(3-((1-cyanoethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[0095] Compound 21 : N-(3-((piperidin-4-ylmethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[0096] Compound 22: N-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)pyrazine-2 -carboxamide; and
[0097] Compound 23: N-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5- methyl-2-(methylsulfonyl)pyrimidine-4-carboxamide.
[0098] Compound 24: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl) nicotinamide
[0099] Compound 25: rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]-
4.5.6.7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide
[00100] Compound 26: N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6,7- tetrahydro-1 -benzothiophen-2-yl} pyridine-3-carboxamide
[00101] Compound 27: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00102] Compound 28: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00103] Compound 29: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00104] Compound 30: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide [00105] compound 31 : N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00106] Compound 32: N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00107] Compound 33: N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00108] Compound 34: N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide
[00109] Compound 35: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00110] Compound 36: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00111] Compound 37: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00112] Compound 38: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.
[00113] Compound 39: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-4,5,6,7 tetrahydrobenzo[b]thiophen-2-yl) nicotinamide
[00114] Compound 40: -rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1-carbonyl]-
4,5,6,7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
[00115] Compound 41 : -N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6,7- tetrahydro-1 -benzothiophen-2-yl} pyridine-3-carboxamide;
[00116] Compound 42: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00117] Compound 43: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; [00118] Compound 44: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00119] Compound 45: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[00120] Compound 46: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00121] Compound 47: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00122] Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00123] Compound 49: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[00124] Compound 50: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00125] Compound 51 : -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00126] Compound 52: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamidel; and
[00127] Compound 53: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.N-(3-benzoyl-4, 5, 6, 7 - tetrahydrobenzo[b]thiophen-2-yl)-5-methyl-2 (methylsulfonyl)pyrimidine-4-carboxamide;
[00128] N-(4-ethylphenyl)-3-(hydroxymethyl)-N-isobutyl-4-((tetrahydro-2H-pyran-4- yl)methoxy)benzenesulfonamide;
[00129] (7S)-N-{[5-(Ethylsulfonyl)-2-pyridinyl]methyl}-7-isopropyl-6-{[trans-4-
(trifluoromethyl)cyclohexyl]methyl}-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3- carboxamide; [00130] N-{3-[(3-methylbut-2-en-1 -yl){methyl[trans-4-(pyridin-4- yl)cyclohexyl]carbamoyl}amino]phenyl}benzamide;
[00131] 2-(1-(2,4-dichloro-3-((7-chloro-5-(trifluoromethyl)-1 H-indol-1 - yl)methyl)benzoyl)piperidin-4-yl)acetic acid;
[00132] (S)-2-(4-cyclopropyl-6-methylpyrimidin-5-yl)-8-(1-cyclopropylethyl)-6-(((5-
(methylsulfonyl)pyridin-2-yl)methyl)amino)pteridin-7(8H)-one;
[00133] (S)-6-(2,6-dimethylpyrimidin-4-yl)-N-(4-(ethylsulfonyl)benzyl)-6-methyl-5- oxo-5, 6, 7, 8-tetrahydroquinoline-2 -carboxamide;
[00134] 2-(2-((S)-(3,5-dimethylisoxazol-4-yl)(hydroxy)methyl)benzofuran-5-yl)-N-
((S)-(2,4-dimethylphenyl)(phenyl)methyl)acetamide;
[00135] N-(4-(1 ,1 ,1 ,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-N-(2,2,2- trifluoroethyl)benzenesulfonamide;
[00136] 1 ,1 ,1 ,3,3,3-hexafluoro-2-(2-fluoro-4'-((4-(pyridin-4-ylmethyl)piperazin-1 - yl)methyl)-[1 , 1 '-biphenyl]-4-yl)propan-2-ol;
[00137] (1 R,3aS,5aR,5bR,7aR,9S,11aR,11 bR,13aR,13bR)-N-(3-
(dimethylamino)propyl)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-1 -(prop-1 -en-2- yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxamide; and
[00138]
Figure imgf000024_0001
[00139] or a pharmaceutically acceptable salts thereof, and stereoisomers thereof. [00140] According to an embodiment, there is provided the method of treatment of acute and/or chronic liver conditions by administering an inverse agonist, wherein the inverse agonist are the following compounds:
Figure imgf000025_0002
N-beiizyl-2-(2-(4-niethylpiperazni-lyl)acetamido)-4.5.6:7- tetiahydrobenzo[b]thiaphene-3- carboxamide
[00141]
Figure imgf000025_0001
7V-(3-((2R ,4R )-2,4-dimethylpiperidine- 1 -carbonyl)-4, 5,6,7- tetrahydrobenzo [b ]thiophen-2-
[00142] yl)nicotinamide
24
Figure imgf000026_0001
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4,5, 6, 7 -
[00143] tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00144] or a pharmaceutically acceptable salts thereof, and stereoisomers thereof.
[00145] According to an embodiment, there is provided the method of treatment of acute and/or chronic liver conditions, the fibrosis is associated with Liver diseases, Liver Failure, Acute- and-Chronic Liver Failure, Chemical and Drug Induced Liver Injury, Liver Fibrosis, Fatty Liver, viral Hepatitis, Non-viral hepatitis, Liver diseases, Liver Failure, Hemangioma of liver, Non-alcoholic Fatty Liver Disease, Acute-On-Chronic Liver Failure, Liver diseases, Liver neoplasms, Chemical and Drug Induced Liver Injury, Alcoholic Liver Diseases, Liver and Intrahepatic Biliary Tract Carcinoma, Chronic Hepatitis, Autoimmune hepatitis, Metabolic dysfunction-Associated Fatty Liver Disease’ (MAFLD), alcohol induced liver conditions, metabolic associated steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), liver fibrosis, liver failure, non-alcoholic fatty liver disease (NAFLD), alcohol-driven liver hepatits, liver cirrhosis, hepatocellular carcinoma and liver cancer.
[00146] According to an embodiment, there is provided a pharmaceutical composition for treatment of fibrosis comprising an inverse agonist of RORs and a pharmaceutically acceptable carrier. [00147] According to an embodiment, there is provided a pharmaceutical composition comprising:
(1 ) a first compound chosen from an inverse agonist of RORs or a pharmaceutically acceptable salt thereof;
(2) one or more additional compounds selected from the group consisting of:
(a) a GLP-1 analog;
(b) Ozempic/Liraglutide;
(c) Resmetirom and
(3) a pharmaceutically acceptable carrier.
[00148] According to an embodiment, there is provided a method of treatment of a vascular injury or protecting against a vascular injury by reducing circulating levels of IL- 17, which comprises administering to a patient in need thereof an inverse agonist of RORs.
[00149] The injury may be the injury is a brain injury.
[00150] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[00151] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which: [00152] Figs. 1 A-1 B illustrate novel compounds of the present invention.
[00153] Fig. 2 illustrates an experiment set up and timeline of mice treated with CCI4 to induce liver fibrosis that are administered GSK805 for positive control, TF-1 (compound 10) and TF-2 (compound 14) and a negative control.
[00154] Fig. 3 illustrates the gating strategy for myeloid cells, monocytes, neutrophils and macrophages.
[00155] Fig. 4 illustrates the gating strategy for T cells, both for un-stimulated lymphocytes and for stimulated lymphocytes.
[00156] Fig. 5 illustrates a reduction in stimulated lymphocytes consisting of leukocytes, T cells, and NK cells, after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 15 mice/group, 6X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00157] Fig. 6 illustrates a reduction in stimulated cytokines after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group, 4X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00158] Fig. 7 illustrates a reduction in stimulated cytokines after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group, 4X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00159] Fig. 8 illustrates a reduction in granulocytes consisting of granulocytes, myeloid, monocytes and neutrophils, after IP injection of GSK805, CCI4, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 15 mice/group, 6X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00160] Fig. 9 illustrates a reduction in granulocytes consisting of macrophages, eosinophil, dendritic cells and MHC II+ presenting cells, after IP injection of GSK805, CCI4, TF1-S10 (compound 10) and TF2-S14 (Compound 14), N: 15 mice/group, 6X each 2-3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00161] Fig. 10 illustrates a reduction in stimulated lymphocytes consisting of leukocytes, T cells and NK cells after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group, 4X each 2- 3/group, using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00162] Fig. 11 illustrates a reduction in unstimulated lymphocytes consisting of T cells and NK cells after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group (4X each 2-3 mice/group), using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00163] Fig. 12 illustrates a reduction in granulocytes consisting of granulocytes, myeloid, monocytes and neutrophils, after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group (4X each 2-3 mice/group), using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00164] Fig. 13 illustrates a reduction in granulocytes consisting of macrophages, dendritic cells, eosinophils and MHC II+ presenting cells, after IP injection of CCI4 and SC injection of GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14), N: 10 mice/group (4X each 2-3 mice/group), using a one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test.
[00165] Fig. 14 illustrates significantly reduced ALT and AST after IP injection of CCI4, GSK805, TF1 -S10 (compound 10) and TF2-S14 (Compound 14) for 4 weeks (8 CCI4 injections), N: 7-8 mice/group for AST, 10 mice/group for ALT.
[00166] Fig. 15 illustrates a reduction in collagen deposition through Sirius red staining after injection of CCI4, GSK805, TF1 -S10 (compound 10), TF2-S14 (Compound 14) and com oil. [00167] Fig. 16 illustrates a reduction in the infiltration of immune cells through hematoxylin and eosin staining of liver tissue following IP injection of CCI4, GSK805, TF1 - S10 (compound 10), TF1 -S14 (compound 14) and com oil.
[00168] Fig. 17 illustrates a reduction in the infiltration of immune cells through hematoxylin and eosin staining of liver tissue following SC injection of CCI4, GSK805, TF1 -S10 (compound 10), TF1 -S14 (compound 14) and com oil.
[00169] Fig. 18 illustrates a hydroxyproline assay to evaluate liver hydroxyproline content following injection with CCI4, GSK805, TF1-S10 (compound 10), TF2-S14 (Compound 14) and oil.
[00170] Fig. 19 illustrates the anti-fibrotic effects of pharmacological inhibitors of IL- 17 (10 mg/kg) were evaluated in CCI4-induced liver fibrosis of C57BL/6 mice model.
[00171] Fig. 19A illustrates schematic diagram of the experimental design for C57BL/6 mice intraperitoneally treated with 0.5 ml/kg CCI4 in com oil for 4 weeks. Mice were intraperitoneally administered with inhibitors (10 mg/kg) for 2 weeks, starting at week 3 post initiation of CCI4 challenge.
[00172] Fig. 19B illustrates representative images of H&E staining of liver tissues indicated necrosis and numerous inflammatory cells immersed in mice dosed with CCI4 and in vivo shoot of liver tissues.
[00173] Fig. 19C liver function assessment by serum levels of ALT and AST to track liver damage caused by CCI4 and evaluate effectiveness of inhibitors to prevent CCI4- induced liver fibrosis to the normal condition. (n=12 mice per group, n=5 in vehicle group). Results are meant SEM, * p<0.05, ** p<0.01 , *** p<0.001 , as assessed with ANOVA. CCI4, carbon tetrachloride. H&E, hematoxylin-eosin. ALT, Alanine amino transferase. AST, aspartate amino transferase.
[00174] Fig. 20 illustrates pharmacological inhibitors of IL-17 developed a reduction in the absolute number of innate immune cells, but not neutrophils, in the mice liver compared with CCI4 liver injury at 4 weeks. IHLs were extracted from livers and analyzed by flow cytometry following injected with CCI4 (0.5 ml/kg) and treated with inhibitors (10 mg/kg).
[00175] Fig. 20A illustrates representative zebra plots showing an outline for the gating strategy of granulocytes (CD45+ CD11 b+), neutrophils (CD45+ CD11 b+ Ly6Cint Ly6G+), monocytes (CD45+ CD11 b+ Ly6Chi Ly6G-), macrophages (CD11 b+ Ly6C- Ly6G- F4/80+), and eosinophils (CD11 b+ Ly6C- Ly6G- CD170+).
[00176] Fig. 20B illustrates that the indicated numbers of cell subsets of IHLs represent the cell number/gram of liver. Data are expressed as means ± SEM for 15 mice per group (data are combined from 3 individual experiments). Vehicle group contains 5 mice. ANOVA test. *P < .05, **P < .01 , and ***P < .001 . SSC, side scatter characteristics. IHL, intrahepatic lymphocytes.
[00177] Fig. 21 illustrates a reduction in the absolute number of adaptive immune cells in the livers of inhibitors treated mice compared with the CCI4-only treated at 4 weeks. IHLs were extracted from injected livers and analyzed by flow cytometry.
[00178] Fig. 21 A illustrates representative FACS plots showing an outline for the gating strategy of T cells (CD45+CD3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), T cell receptors y5 T cells (CD3+T cell receptor y5 +).
[00179] Fig. 21 B illustrates total cell number of several T cells populations in C57BL/6 liver mononuclear during 4 weeks of CCI4 infection. (n=15 mice per group, n=5 vehicle group). ANOVA test. *P < .05, **P < .01 , and ***P < .001. SSC, side scatter characteristics. IHL, intrahepatic lymphocytes. SSC, side scatter characteristics. IHL, intrahepatic lymphocytes.
[00180] Fig. 22 illustrates inhibitors selectively block the secretion of IL-17 but not IFNy and IL-22. Representative flow cytometry plots showing intrahepatic IL17/I FN y/l L22- producing cells: yb-T cells (IL17+/IFNY+/IL22+CD3+T cell receptor Y6+), CD4+ T cells (IL17+/IFNY+/IL22+CD3+ CD4+ T cell), CD8+ T cells (IL17+/IFNY+/IL22+CD3+CD8+ T cell). The intrahepatic lymphocytes were extracted from livers of inhibitors treated liver fibrosis vs CCI4-treated liver fibrosis as well as vehicle control mice receiving only corn oil at 4 weeks, and then stimulated with/without PMA/ionomycin (PMA/lono) for 5 hours at 37°C. The frequency quantification of IL17/IFNy/IL22-producing y5+-T cells, CD4+ T cells, CD8+-T cells, in lymphocytes panel. Data are expressed as means ± SD (n =15 mice per group, n=5 for vehicle group). ANOVA test. *P < .05, **P < .01 , and ***P < .001. SSC, side scatter characteristics.
[00181] Fig. 23 illustrates IP injection of RORyt inhibitors result in significant reduction of hepatic inflammatory genes in CCI4-induced fibrotic mice compare with the CCI4 only treated mice. C57BL/6 mice were treated for 4 weeks with CCI4 and then subjected to the inhibitors. TF-S10/S14 or GSK805 (10 mg/kg daily) were administered daily for 2 weeks as of week 3.
[00182] Fig. 23A illustrates bar graphs representing proinflammatory genes expression (normalized to r28s) as indicated and represented as expression level of specific genes.
[00183] Fig. 23B illustrates quantification of collagen deposition by PSR. Representative microscopic of liver sections stained with PSR (collagen shown in red) show reduction in collagen deposition following administration of inhibitors. Scale bars: 100 pm and original magnification of PSR+ area in liver with/without inhibitors. Data are expressed as means ± SD (n= 15 mice per group, n=5 mice in vehicle group). ANOVA test. *P < .05, **P < .01 , and ***P < .001. Col1a1 , collagen type 1 alpha 1. Acta2, actin alpha 2. Tgf|3, transforming growth factor beta. Loxl2, lysyl oxidase-like 2.
[00184] Fig. 24 illustrates RORyt inverse agonists alleviated liver damage/fibrosis parameters in chronically CCk-injured mice.
[00185] Fig. 24A illustrates a schematic diagram of the experimental design. Briefly, C57BL/6 mice were treated (i.p) with 0.5 ml/kg CCk in com oil for 4 weeks. Mice then received intraperitoneally administration of RORyt inverse agonists (10 mg/kg) daily for 2 weeks, starting at week 3 post initiation of CCk challenge. [00186] Fig. 24B and Fig. 24C illustrate representative images of H&E and IF staining of liver tissue sections. Scale bars: 50 and 100 (second line) pm. Black arrows represent red spots and immune cells infiltration, respectively
[00187] Fig. 24D illustrates liver function assessment by evaluating serum levels of ALT and AST. (n=15 mice per group, n=5 in vehicle group). Results are represented as meant SEM, * P<0.05, ** P<0.01 , *** P<0.001 , as assessed with one-way ANOVA. CCL, carbon tetrachloride; H&E, hematoxylin-eosin; IF, immunofluorescence ALT, Alanine amino transferase; AST, aspartate amino transferase.
[00188] Fig. 25 illustrates RORyt inverse agonists reduced the absolute number of intrahepatic myeloid cells, mainly eosinophils, in mice with chronic CCk-induced liver injury. IHLs were extracted from livers of CCL-injured mice no-inhibitor or treated with RORyt inverse agonists as well as livers of vehicle control mice. Cells were then stained for various markers of different leukocytic populations and analyzed by flow cytometry.
[00189] Fig. 25A illustrates representative zebra plots showing an outline for the gating strategy of granulocytes (CD45+CD11 b+), neutrophils (CD45+CD11 b+Ly6Cint Ly6G+), monocytes (CD45+CD11 b+Ly6ChlLy6G'), macrophages (CD11 b+Ly6C'Ly6G' F4/80+), and eosinophils (CD11b+Ly6C’Ly6G’CD170+).
[00190] Fig. 25B illustrates graphs showing the cell number of the different subsets of IHLs per gram of liver (cell number/gram of liver). Data are expressed as means ± SEM for 15 mice per group (data are combined from 3 individual experiments). Vehicle group contains 5 mice. One-way ANOVA test. * P<0.05, ** P<0.01 , *** P<0.001. IHL, intrahepatic lymphocytes.
[00191] Fig. 26 illustrates RORyt inverse agonists reduced the absolute number of adaptive immune cells. IHLs were extracted from livers of CCL-injured mice no-inhibitor or treated with RORyt inverse agonists as well as from livers of vehicle control mice. Cells were then stained for various markers of different leukocytic populations and analyzed by flow cytometry. [00192] Fig. 26A illustrates representative FACS plots showing an outline for the gating strategy of T cells (CD45+CD3+), CD4+ T cells(CD3+CD4+), CD8+ T cells (CD3+CD8+), TCR yb-T cells (CD3+TCR yb+).
[00193] Fig. 26B illustrates graphs showing the cell number of the different populations of T cells per gram of liver (cell number/gram of liver). (n=15 mice per group, n=5 vehicle group). One-way ANOVA test. * P<0.05, ** P<0.01 , *** P<0.001. SSC, side scatter characteristics. IHL, intrahepatic lymphocytes.
[00194] Fig. 27 illustrates RORyt inverse agonists block the secretion of IL-17 and to a lesser extent IFNy but not IL-22. Representative flow cytometry plots showing intrahepatic IL-17- IFNy-, or IL- 22-producing cells, yb-T cells (IL-17+, IFNy+ or IL-22+ yb TCR+/CD3+), CD4+ T cells (IL-17+, IFNy+, or IL-22+CD4+/CD3+), CD8+ T cells (IL- 17-*-, IFNy+, or IL-22+CD8+/CD3+). IHLs were extracted from livers of CCL-injured mice no-inhibitor or treated with RORyt inverse agonists as well as livers of vehicle control mice. Cells were then stimulated with/without PMA/ionomycin (PMA/lono) for 5 hrs at 37°C. The quantification (frequency) of IL-17-, IFNy-, or IL-22- producing yb+, CD4+, or CD8+ T cells is presented. Data are expressed as means ± SEM (n =15 mice per group, n=5 for vehicle group). One-way ANOVA test. * P<0.05, ** P<0.01 , *** P<0.001.
[00195] Fig. 28 illustrates RORyt inverse agonists significantly decrease the expression of hepatic profibrogenic genes in CCI4-injured mice. Livers were extracted from CCI4-injured mice no- inhibitor or treated with RORQt inverse agonists as well as livers of vehicle control mice, their total RNA extracted and analyzed by RT-qPCR, or fixed and sectioned for PSR staining.
[00196] Fig. 28A illustrates bar graphs represent expression of profibrogenic genes (normalized to r28s)
[00197] Fig. 28B illustrates quantification of collagen deposition by PSR. Representative microscopic images of liver sections stained with PSR (collagen shown in red). Scale bars: 100 pm and original magnification of PSR+ area in liver with/without inhibitors. Data are expressed as means ± SEM (n= 15 mice per group, n=5 mice in vehicle group). One-way ANOVA test. * P<0.05, ** P<0.01 , *** P<0.001 . Coll a1 , collagen type 1 alpha 1 ; Acta2, actin alpha 2; Tgf|3, transforming growth factor beta; Loxl2, lysyl oxidase-like 2.
[00198] Fig. 29 illustrates that siRNA knockdown of RORC reduces RORC Expression and Reduces Cell Viability.
[00199] Fig. 30 illustrates that SiRNA knockdown of RORC Increases Caspase 3/7 Expression and Increases Cell Death.
DETAILED DESCRIPTION
[00200] The present invention relates to a method of treatment of fibrosis or acute and/or chronic liver conditions by administering to a patient in need thereof an inverse agonist of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORa or RORA) and gamma (RORC, RORy or RORyt) or a silencing RNA that inhibits protein synthesis of RORs.
[00201] The fibrosis is pulmonary fibrosis, retroperitoneal fibrosis (RPF), hepatic fibrosis and/or cirrhosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, intestinal fibrosis, muscle fibrosis, ocular fibrosis, fibrosis in the central nervous system, renal fibrosis, and pancreatic fibrosis, autoimmune fibrotic diseases, fibrosis associated with infectious diseases, fibrosis associated with metabolic diseases, fibrosis associated with cancer, fibrosis associated with environmental exposures, or a combination thereof.
[00202] The inverse agonists of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORa or RORA) and gamma (RORC, RORy or RORyt) includes a compound of Formula (I), (II), (III), or (IV), a pharmaceutically acceptable salts thereof, or stereoisomers thereof:
Figure imgf000036_0001
[00203] wherein,
[00204] R1 , R2, R3, and/or R4 group(s) is/are H, halogen, NO2, 1-6 alkoxy, OH, NH2, 1 -6 alkyl, 1-6 alkenyl, 1-6 haloalkyl, N-dialkyl, haloalkoxy, 1-6 hydroxyalkyl, and/or
-CO2(1-6 alkyl);
[00205] R5 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, aryl, alkylarene, halo aryl, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, haloaryl, benzene, phenyl, benzyl, pyridine, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, haloalkyl, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methylimidazole, methyldiazole, methyltriazole, methylthiadiazole, methylimidazolidine, methylthizolidine, methylpyrrolidine, methylpiperazine, methylpiperidine, methylpyridazine, methylpyrazine, methyltriazine, methylpyrrole, methylpyrroline, methylpyrazolidine, methylpyrazoline, methylthiazole, methylisothiazole, methylisoxazole, or arylalkyl.
[00206] R6 is H, 1-6 alkyl, or may form a five or six ring structure with R5; and
[00207] R7 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, substituted haloaryl, substituted benzene, substituted phenyl, benzyl, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, aryl, halo aryl, alkylarene, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methylimidazole, methyldiazole, methyltriazole, methylthiadiazole, methylimidazolidine, methylthizolidine, methylpyrrolidine, methylpiperazine, methylpiperidine, methylpyridazine, methylpyrazine, methyltriazine, methylpyrrole, methylpyrroline, methylpyrazolidine, methylpyrazoline, methylthiazole, methylisothiazole, methylisoxazole, or arylalkyl.
[00208] The invention includes the compounds as shown, and also includes (where possible) individual diastereomers, enantiomers, and epimers of the compounds, and mixtures of diastereomers and/or enantiomers thereof including racemic mixtures. Although the specific stereochemistries disclosed herein are preferred, other stereoisomers, including diastereomers, enantiomers, epimers, and mixtures of these may also be useful. Inactive or less active diastereoisomers and enantiomers are useful for scientific studies relating to the nuclear receptor targeting and the mechanism of activation. [00209] The compounds disclosed herein may be used in pharmaceutical compositions comprising (a) the compound(s) or pharmaceutically acceptable salts thereof, and (b) a pharmaceutically acceptable carrier. The compounds may be used in pharmaceutical compositions that include one or more other active pharmaceutical ingredients. The compounds may also be used in pharmaceutical compositions in which the compound of Formula I or a pharmaceutically acceptable salt thereof is the only active ingredient.
[00210] The inverse agonist of ROR may be:
[00211] N-[2,6-dichloro-2'-(trifluoromethoxy)[1 , 1 '-biphenyl]-4-yl]-4-(ethylsulfonyl)- benzeneacetamide (GSK805) or analogs thereof;
[00212] N-[[(2S)-1 -[[5-(4-fluorophenyl)-2-methyl-4-thiazolyl]carbonyl]-2- piperidinyl]methyl]-4-benzofurancarboxamide (SB-649868);
[00213] 3-isoxazolebutanoic acid ( JTE-151 );
[00214] 5H-Pyrrolo[3,4-b]pyridine-3-carboxamide, N-[[5-(ethylsulfonyl)-2- pyridinyl]methyl]-6,7-dihydro7-(1-methylethyl)-6-[[trans-4- (trifluoromethyl)cyclohexyl]methyl]- hydrochloride (VTP-43742);
[00215] (1 R,3S,4R)-4-((3aR,9bR)-9b-((4-Fluorophenyl)sulfonyl)-7- (perfluoropropan-2-yl)-2,3,3a,4,5,9b-hexahydro-1 H-benzo(E)indole-3-carbonyl)-3- methylcyclohexane-1 -carboxylic acid (BMS-986251 );
[00216] 2-((1 S,3S)-3-((R)-5-((7-fluoro-1 , 1 -dimethyl-2,3-dihydro-1 H-inden-5- yl)carbamoyl)-2-methoxy-5,6,7,8-tetrahydro-1 ,6-naphthyridine-6- carbonyl)cyclobutyl)acetic acid (TAK-828);
[00217] N-(5-(N-(4-(1 ,1 ,1 ,3,3,3-hexafluoro-2-hydroxypropan-2- yl)phenyl)sulfamoyl)-4-methylthiazol-2-yl)acetamide (SR1001 );
[00218] AUR-101 ;
[00219] Izumerogant (IMU-935); [00220] ESR-114;
[00221] Cedirogant (ABBV-157);
[00222] analogs thereof; or
[00223] combinations thereof.
[00224] The preferred compound of Formula (I), (II), (III), or (IV) pharmaceutically acceptable salts thereof, or stereoisomers thereof is selected from the group consisting of:
[00225] Compound 39: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4,5,6,7 tetrahydrobenzo[b]thiophen-2-yl) nicotinamide;
[00226] Compound 40: -rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]-
4.5.6.7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
[00227] Compound 41 : -N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6,7- tetrahydro-1 -benzothiophen-2-yl} pyridine-3-carboxamide;
[00228] Compound 42: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00229] Compound 43: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00230] Compound 44: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00231] Compound 45: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[00232] Compound 46: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00233] Compound 47: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; [00234] Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00235] Compound 49: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[00236] Compound 50: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00237] Compound 51 : -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00238] Compound 52: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamidel; and
[00239] Compound 53: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.
[00240] The inverse agonist of ROR may be:
Figure imgf000040_0001
N-(3-(2-methylpyrrolidine- 1 -carbonyl)- 4, 5 , 6, 7 -tetrahydrobenzo [b] thiophen-2 -
[00241] yl)nicotinamide
Figure imgf000041_0001
A'-(3-((2/?.4/?)-2.4-dimethylpiperidine- l-carbonyl)-4, 5,6,7- tetrahydrobenzo [6] th iophen-2-
[00242] yl)nicotinamide
Figure imgf000041_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7-
[00243] tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
Figure imgf000042_0001
N-(3 -(3 -ethylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[b]thiophen-2-
[00244] yl)nicotinamide
Figure imgf000042_0002
7V-benzyl-2-(2-(( 1 -methyl- 1/f-imidazol- 2-yl)thio)acetamido)-4, 5,6,7- tetrahy drobenzo [6] thiophene-3 -
[00245] carboxamide
Figure imgf000042_0003
N-(3 -(3 -(hydroxymethyl)pyrrolidine- 1 - carbonyl)-4, 5,6,7- tetrahy drobenzo [/?] thi ophcri-2-
[00246] yl)nicotinamide
Figure imgf000043_0001
Figure imgf000044_0001
7V-(3-benzoyl-4, 5,6,7- tetrahy drobenzo [6] thiophen-2-y 1) - 5 - methyl-2- (methylsulfonyl)pyrimidine-4-
[00250] carboxamide
Figure imgf000045_0002
N-(3 -((( 1 -methyl- 177-1 ,2,4-triazol-3 - yl)methyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo [b] th iophen-2-
[00251] yl)nicotinamide , and
Figure imgf000045_0001
7V-(6,6-dimethyl-3-(morpholine-4-carbonyl)-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-
[00252] yl)pyrazine-2-carboxamide
[00253] The preferred compound of Formula (I), pharmaceutically acceptable salts thereof, or stereoisomers thereof is selected from the group consisting of:
[00254]
Figure imgf000046_0001
N-benzyl-2-(2-(4-methylpipeiaziii-lyl)acetamido)-4.5.6,7- teualiydrobenzo[b]thiophene-3- carboxamide
[00255]
Figure imgf000046_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7-
[00256] tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
Figure imgf000046_0003
N-(3 -(3 -ethylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[6]thiophen-2-
[00257] yl)nicotinamide
Figure imgf000047_0001
7V-benzyl-2-(2-(( 1 -methyl- 17/-i m idazol- 2-yl)thio)acetamido)-4,5,6,7- tetrahydrobenzo [6] thiophene-3 -
[00258] carboxamide
Figure imgf000047_0002
N-(3 -(3 -(hydroxymethyl)pyrrolidine- 1 - carbonyl)-4, 5,6,7- tetrahydrobenzo [/?] thi ophcri-2-
[00259] yl)nicotinamide
Figure imgf000048_0001
[00262]
[00263]
Figure imgf000049_0001
Figure imgf000050_0002
N-(3 -((( 1 -methyl- 1 H- 1 ,2,4-triazol-3 - yl)methyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo [6] th iophen-2-
[00264] yl)nicotinamide
Figure imgf000050_0001
?/-(6,6-dimethyl-3-(morpholine-4-carbonyl)- 4,5,6,7-tetrahydrobenzo[6]thiophen-2- [00265] yl)pyrazine-2-carboxamide
[00266] Compound 1 : N-(3-(4-benzylpiperazine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)-2 -fluorobenzamide;
[00267] Compound 2: N-(3-(benzylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-
2-yl)-5-chloro-2-(methylthio)pyrimidine-4-carboxamide;
[00268] Compound 3: 5-chloro-N-(3-{[(1 ,1-dioxidotetrahydro-3-thienyl)amino]- carbonyl}-6-methyl-4,5,6,7-tetrahydro-1 -benzothien-2-yl)-2-(methylthio)-4-pyrimidine- carboxamide;
[00269] Compound 4: N-(6-ethyl-3-{[(2-methylphenyl)amino]carbonyl}-4, 5,6,7- tetrahydro-1 -benzothien-2-yl)-1 -methyl-1 H-pyrazole-5-carboxam ide; [00270] Compound 5: N-{6-tert-butyl-3-[(4-methyl-1-piperazinyl)carbonyl]-4,5,6,7- tetrahydro-1-benzothien-2-yl}-2 -fluorobenzamide;
[00271] Compound 6: 2-fluoro-N-{6-methyl-3-[(4-methyl-1 -piperazinyl)carbonyl]-
4.5.6.7-tetrahydro-1-benzothien-2-yl}benzamide;
[00272] Compound 7: N-benzyl-2-[(trifluoroacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
[00273] Compound 8: N-benzyl-2-({[(4-methyl-2-pyrimidinyl)thio]acetyl}amino)-
4.5.6.7-tetrahydro-1-benzothiophene-3-carboxamide;
[00274] Compound 9: N-benzyl-2-[(1-piperidinylacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
[00275] Compound 10: N-benzyl-2-{[(4-methyl-1-piperazinyl)acetyl]amino}-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
[00276] Compound 11 : N-benzyl-2-{[(4-methyl-1-piperidinyl)acetyl]amino}-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
[00277] Compound 12: N-(3-(2-methylpyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[00278] Compound 13: N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00279] Compound 14: N-(3-(((1 r,4r)-4-hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00280] Compound 15: N-(3-(3-ethylpyrrolidine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[00281] Compound 16: N-benzyl-2-(2-((1-methyl-1 H-imidazol-2-yl)thio)acetamido)-
4.5.6.7-tetrahydrobenzo[b]thiophene-3-carboxamide;
[00282] Compound 17: N-(3-(3-(hydroxymethyl)pyrrolidine-1-carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; [00283] Compound 18: N-(6,6-dimethyl-3-(morpholine-4-carbonyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2-carboxamide;
[00284] Compound 19: N-(3-(((1-methyl-1 H-1 ,2,4-triazol-3-yl)methyl)carbamoyl)-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00285] Compound 20: (S)-N-(3-((1-cyanoethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[00286] Compound 21 : N-(3-((piperidin-4-ylmethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
[00287] Compound 22: N-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)pyrazine-2 -carboxamide; and
[00288] Compound 23: N-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5- methyl-2-(methylsulfonyl)pyrimidine-4-carboxamide.
[00289] Compound 24: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl) nicotinamide
[00290] Compound 25: rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]-
4.5.6.7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide
[00291] Compound 26: N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6,7- tetrahydro-1 -benzothiophen-2-yl} pyridine-3-carboxamide
[00292] Compound 27: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00293] Compound 28: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00294] Compound 29: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00295] Compound 30: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide [00296] compound 31 : N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00297] Compound 32: N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00298] Compound 33: N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00299] Compound 34: N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide
[00300] Compound 35: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00301] Compound 36: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00302] Compound 37: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00303] Compound 38: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.
[00304] Compound 39: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-4,5,6,7 tetrahydrobenzo[b]thiophen-2-yl) nicotinamide
[00305] Compound 40: -rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1-carbonyl]-
4,5,6,7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
[00306] Compound 41 : -N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6,7- tetrahydro-1 -benzothiophen-2-yl} pyridine-3-carboxamide;
[00307] Compound 42: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00308] Compound 43: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; [00309] Compound 44: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00310] Compound 45: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[00311] Compound 46: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00312] Compound 47: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00313] Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00314] Compound 49: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
[00315] Compound 50: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00316] Compound 51 : -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
[00317] Compound 52: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamidel; and
[00318] Compound 53: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.N-(3-benzoyl-4, 5, 6, 7 - tetrahydrobenzo[b]thiophen-2-yl)-5-methyl-2 (methylsulfonyl)pyrimidine-4-carboxamide;
[00319] N-(4-ethylphenyl)-3-(hydroxymethyl)-N-isobutyl-4-((tetrahydro-2H-pyran-4- yl)methoxy)benzenesulfonamide;
[00320] (7S)-N-{[5-(Ethylsulfonyl)-2-pyridinyl]methyl}-7-isopropyl-6-{[trans-4-
(trifluoromethyl)cyclohexyl]methyl}-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3- carboxamide; [00321] N-{3-[(3-methylbut-2-en-1-yl){methyl[trans-4-(pyridin-4- yl)cyclohexyl]carbamoyl}amino]phenyl}benzamide;
[00322] 2-(1 -(2,4-dichloro-3-((7-chloro-5-(trifluoromethyl)-1 H-indol-1 - yl)methyl)benzoyl)piperidin-4-yl)acetic acid;
[00323] (S)-2-(4-cyclopropyl-6-methylpyrimidin-5-yl)-8-(1-cyclopropylethyl)-6-(((5-
(methylsulfonyl)pyridin-2-yl)methyl)amino)pteridin-7(8H)-one;
[00324] (S)-6-(2,6-dimethylpyrimidin-4-yl)-N-(4-(ethylsulfonyl)benzyl)-6-methyl-5- oxo-5, 6, 7, 8-tetrahydroquinoline-2 -carboxamide;
[00325] 2-(2-((S)-(3,5-dimethylisoxazol-4-yl)(hydroxy)methyl)benzofuran-5-yl)-N-
((S)-(2,4-dimethylphenyl)(phenyl)methyl)acetamide;
[00326] N-(4-(1 ,1 ,1 ,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-N-(2,2,2- trifluoroethyl)benzenesulfonamide;
[00327] 1 ,1 ,1 ,3,3,3-hexafluoro-2-(2-fluoro-4'-((4-(pyridin-4-ylmethyl)piperazin-1 - yl)methyl)-[1 , 1 '-biphenyl]-4-yl)propan-2-ol;
[00328] (1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR,13aR,13bR)-N-(3-
(dimethylamino)propyl)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-1 -(prop-1 -en-2- yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxamide; and
[00329]
Figure imgf000055_0001
[00330] or a pharmaceutically acceptable salts thereof, and stereoisomers thereof. [00331] According to an embodiment, there is provided the method of treatment of acute and/or chronic liver conditions by administering an inverse agonist, wherein the inverse agonist are the following compounds:
Figure imgf000056_0001
N-benzyl-2-(2-(4-methylpiperazni-lyl)acetaiindo)-4,5.6:7- tetialiydrobenzo[b]tliiophene-3- caiboxamide
[00332]
Figure imgf000057_0001
A'-(3-((2/?.4/?)-2.4-dimethylpiperidine- l-carbonyl)-4, 5,6,7- tetrahydrobenzo [6] th iophen-2-
Figure imgf000057_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
[00334] or a pharmaceutically acceptable salts thereof, and stereoisomers thereof.
[00335] According to an embodiment, there is provided the method of treatment of acute and/or chronic liver conditions, the fibrosis is associated with Liver diseases, Liver Failure, Acute- and-Chronic Liver Failure, Chemical and Drug Induced Liver Injury, Liver Fibrosis, Fatty Liver, viral Hepatitis, Non-viral hepatitis, Liver diseases, Liver Failure, Hemangioma of liver, Non-alcoholic Fatty Liver Disease, Acute-On-Chronic Liver Failure, Liver diseases, Liver neoplasms, Chemical and Drug Induced Liver Injury, Alcoholic Liver Diseases, Liver and Intrahepatic Biliary Tract Carcinoma, Chronic Hepatitis, Autoimmune hepatitis, Metabolic dysfunction-Associated Fatty Liver Disease’ (MAFLD), alcohol induced liver conditions, metabolic associated steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), liver fibrosis, liver failure, non-alcoholic fatty liver disease (NAFLD), alcohol-driven liver hepatits, liver cirrhosis, hepatocellular carcinoma and liver cancer.
[00336] According to an embodiment, there is provided a pharmaceutical composition for treatment of fibrosis comprising an inverse agonist of RORs and a pharmaceutically acceptable carrier.
[00337] According to an embodiment, there is provided a pharmaceutical composition comprising:
(1 ) a first compound chosen from an inverse agonist of RORs or a pharmaceutically acceptable salt thereof;
(2) one or more additional compounds selected from the group consisting of:
(a) a GLP-1 analog;
(b) Ozempic/Liraglutide;
(c) Resmetirom and
(3) a pharmaceutically acceptable carrier.
[00338] According to an embodiment, there is provided a method of treatment of a vascular injury or protecting against a vascular injury by reducing circulating levels of IL- 17, which comprises administering to a patient in need thereof an inverse agonist of RORs.
[00339] The injury may be the injury is a brain injury. [00340] According to another embodiment, the compounds of the present invention may be given directly to a patient in need of such treatment, using oral, intravenous, topical, intranasal, intrapulmonary, subcutaneous (slow release implant or patch), sublingual, inhalation or intramuscular administration.
[00341] ROR gamma t is expressed in lymphocytes during maturation. Irregularities in the expressions of ROR genes may lead to cancer, autoimmune disease. RORyt is the master regulator is Th17. Th17 cells produce many proinflammatory and pleiotropic cytokines IL17A, IL17F (can be also anti-inflammatory), IL21 , IL22, IL24, IL26. In addition, Th17 cells secrete GM-CSF and TNF alpha. T cytotoxic 17 is also a type of lymphocyte that secrete all the aforementioned cytokines. T follicular helper cells secrete IL21 and are involved in many inflammatory diseases and types of cancers.
[00342] The invention presented herein, binds to ROR gamma and block the production of IL17A, IL21 , IL17F, IL24, IL26 and other cytokines produced by either T helper 17, T cytotoxic 17, T folicular helper cells, and those produced by all variants or cytotoxic T cells, and Helper T cells as well as Variants of B cells that produce the aforementioned cytokines.
[00343] Th17 cells have a role in macrophage and leukocytes recruitment to cause inflammation.
[00344] RORC is highly expressed in fibrosis, certain types of cancer and in autoimmune diseases. Patients waitlisted on organ transplantation lists and who are categorized as Highly sensitized patients have a high activity of T helper 17, T follicular cells, and all T cell variants that produce the aforementioned cytokines. The invention would confer a therapeutic benefit to this category and to all other people who receive allogenic transplantation.
[00345] Examples of profibrotic genes in liver fibrosis include ADAMTS2, ADH7, ADRB3, AFP, AGTR1A, AHR, AKT1 , ALDH2, ANGPT1 , ANXA1 , AOC3, AQP1 , AREG, ATP8B1 , BCHE, BCL2L1 , BMP6, C5, CASP8, CBS, CCL2, CCL21 , CCL25, CCL5, CCR1 , CCR5, CCR6, CCR9, CD163, CD40LG, CD44, CDKN2A, CLDN1 , CNR1 , CNR2, C0L1A1, CPT1A, CSF1, CTGF, CTSB, CTSD, CX3CR1, CXCL10, CXCL9, CXCR3, CXCR4, CYBB, CYLD, DDX5, DGAT1, DKK1, ELAVL1, EL0VL6, ENPP1, EZH2, F5, FABP1, FAS, FGF19, FGF21, FMOD, FOSL1, FOXO1, GATA4, GFER, GLIS3, GLT8D2, GNMT, GP1BA, GT(ROSA)26SOR, HABP2, HBB, HDAC7, HFE, HFIB1, HGF, HLA- DPA1, HLA-DPB1, HMOX1, HNF4A, HTR2B, ID1, IFNAR1, IFNG, IFNGR2, IFNL3, IFNL4, IKBKG, IL13, IL13RA2, IL17A, IL1B, IL1RL1, IL22, IL33, IL34, IL4, IL4RA, ILK, IRS1, ITGAM, ITGAV, JAK2, JUND, KLF2, KLRK1, KRT18, KRT8, LEP, LGALS7, LINC01508, LTB, LTBR, LYZ2, MAP3K7, MAPK1, MAPK14, MAPK3, MAPK8, MAPK9, MCL1, MDM2, MECP2, MEF2D, MERTK, MET, MIR-29, MIR132, MIR214, MIR221, MIR222, MMP2, MMP7, MTHFR, MYOD1, NCR1, NFKB1, NGFR, NLRP3, NOS2, NPHP3, NR0B2, NR1D1, NR1H2, NR1H3, NR1H4, NR1I2, NRP1, PARP1, PDGFA, PDGFB, PF4, PIK3R1, PKD1L1, PNPLA3, PPARA, PPARG, PPP1R13L, PRKCZ, PROM1, RAC1, RAG1, RNF7, RORA, RPS5, RTN4, SELP, SIRT1, SLC11A1, SMAD2, SMAD3, SMAD7, SPP1, SRSF3, STAT1, STAT3, TGFBR2, TIMP1, TIMP3, TLR2, TLR7, TLR9, TNFRI, TNFRSF1A, TNFRSF1B, TP53, TPH1, TRP53, TULP1, VDR, VEGFA.
[00346] The current invention is useful in treatment of acute and/or chronic liver conditions involving the immune system whenever any of the aforementioned cytokines is involved either directly or indirectly in the activation of the immune system.
Abbreviations
[00347] Abbreviations and terms that are commonly used in the fields of organic chemistry, medicinal chemistry, pharmacology, and medicine and are well known to practitioners in these fields are used herein. Representative abbreviations and definitions are provided below:
[00348] Ac is acetyl [CH3C(O)-], AC2O is acetic anhydride; APC is antigen- presenting cell; 9-BBN is 9-borabicyclo[3.3.1]nonane; Bn is benzyl; BOC is tert Butyloxycarbonyl; DIAD is diisopropylazodicarboxylate; DIBAL is diisobutylaluminum hydride; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; EDAC (or EDC) is 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide HCI; EtsN is triethylamine; Et is ethyl; EtOAc is ethyl acetate; EtOH is ethanol; 3-F-Ph is 3-fluorophenyl, HCI is hydrochloric acid; HOBt is 1 -hydroxybenzotriazole; HPLC is high performance liquid chromatography; LCMS is HPLC with mass Spectral detection; LG is leaving group; M is molar; mmol is millimole; Me is methyl; MeOH is methanol; MsCI methanesulfonyl chloride; N is normal; NaHMDS is sodium hexamethyldisiliazide; NaOAc is sodium acetate; NaOtBu is sodium tert-butoxide; NMO is N-methylmorpholine N oxide; NMP is N Methyl pyrrolidinone; Pd(dba)2 is tris(dibenzylideneacetone)dipalladium; PdCl2(Ph3P)2 is dichlorobis- (triphenylphosphene) palladium; PG Denotes an unspecified protecting group; Ph is phenyl; PhMe is toluene; PPhs is triphenylphosphine; PMB is para-methoxybenzyl; RT is room temperature; TBAF is tetrabutyl ammonium fluoride; TBS is tert-butyldimethylsilyl; tBu is tert-butyl; Tf is triflate; TFA is trifluoroacetic acid; THF is tetrahydrofuran; TLC is thin layer chromatography; TMS is trimethylsilyl; TPAP is tetrapropylammonium perruthenate.
Definitions
[00349] 'Alkyl", as well as other groups having the prefix "alk", such as alkoxy and alkanoyl, means carbon chains which may be linear or branched, and combinations thereof, unless the carbon chain is defined otherwise. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like. Where the specified number of carbon atoms permits, e.g., from C3- 10, the term alkyl also includes cycloalkyl groups, and combinations of linear or branched alkyl chains combined with cycloalkyl structures. When no number of carbon atoms is specified, C1 -6 is intended.
[00350] "Cycloalkyl" is a subset of alkyl and means a saturated carbocyclic ring having a specified number of carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. A cycloalkyl group generally is monocyclic unless stated otherwise. Cycloalkyl groups are saturated unless otherwise defined. [00351] The term “alkoxy” refers to straight or branched chain alkoxides of the number of carbon atoms specified (e.g., C1 -6 alkoxy), or any number within this range [i.e. , methoxy (MeO-), ethoxy, isopropoxy, etc.].
[00352] The term “alkylthio” refers to straight or branched chain alkylsulfides of the number of carbon atoms specified (e.g., C1 -6 alkylthio), or any number within this range [i.e., methylthio (MeS-), ethylthio, isopropylthio, etc.].
[00353] The term “alkylamino” refers to straight or branched alkylamines of the number of carbon atoms specified (e.g., C1-6 alkylamino), or any number within this range [i.e., methylamino, ethylamino, isopropylamino, t-butylamino, etc.].
[00354] The term “alkylsulfonyl” refers to straight or branched chain alkylsulfones of the number of carbon atoms specified (e.g., C1 -6 alkylsulfonyl), or any number within this range [i.e., methylsulfonyl (MeSO2’), ethylsulfonyl, isopropylsulfonyl, etc.].
[00355] The term “alkylsulfinyl” refers to straight or branched chain alkylsulfoxides of the number of carbon atoms specified (e.g., C1-6 alkylsulfinyl), or any number within this range [i.e., methylsulfinyl (MeSO-), ethylsulfinyl, isopropylsulfinyl, etc.].
[00356] The term “alkyloxycarbonyl” refers to straight or branched chain esters of a carboxylic acid derivative of the present invention of the number of carbon atoms specified (e.g., C1-6 alkyloxycarbonyl), or any number within this range [i.e., methyloxycarbonyl (MeOCO-), ethyloxycarbonyl, or butyloxycarbonyl].
[00357] "Aryl" means a mono- or polycyclic aromatic ring system containing carbon ring atoms. The preferred aryls are monocyclic or bicyclic 6-10 membered aromatic ring systems. Phenyl and naphthyl are preferred aryls. The most preferred aryl is phenyl.
[00358] “Heterocyclyl" refer to saturated or unsaturated non-aromatic rings or ring systems containing at least one heteroatom selected from O, S and N, further including the oxidized forms of sulfur, namely SO and SO2. Examples of heterocycles include tetrahydrofuran (THF), dihydrofuran, 1 ,4-dioxane, morpholine, 1 ,4-dithiane, piperazine, piperidine, 1 ,3-dioxolane, imidazolidine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1 ,3-dioxane, 1 ,3-dithiane, oxathiane, thiomorpholine, 2-oxopiperidin-1 -yl, 2-oxopyrrolidin-1 -yl, 2-oxoazetidin-1 -yl, 1 ,2,4-oxadiazin-5(6H)-one-3-yl, and the like.
[00359] "Heteroaryl" means an aromatic or partially aromatic heterocycle that contains at least one ring heteroatom selected from 0, S and N. Heteroaryls thus include heteroaryls fused to other kinds of rings, such as aryls, cycloalkyls and heterocycles that are not aromatic. Examples of heteroaryl groups include: pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl (in particular, 1 ,3,4-oxadiazol-2-yl and 1 ,2,4- oxadiazol-3-yl), thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furyl, triazinyl, thienyl, pyrimidyl, benzisoxazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, dihydrobenzofuranyl, indolinyl, pyridazinyl, indazolyl, isoindolyl, dihydrobenzothienyl, indolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, carbazolyl, benzodioxolyl, quinoxalinyl, purinyl, furazanyl, isobenzylfuranyl, benzimidazolyl, benzofuranyl, benzothienyl, quinolyl, indolyl, isoquinolyl, dibenzofuranyl, and the like. For heterocyclyl and heteroaryl groups, rings and ring systems containing from 3-15 atoms are included, forming 1 -3 rings.
[00360] "Halogen" refers to fluorine, chlorine, bromine and iodine. Chlorine and fluorine are generally preferred. Fluorine is most preferred when the halogens are substituted on an alkyl or alkoxy group (e.g. CF3O and CF3CH2O).
[00361] The term « composition » as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to pharmaceutical composition is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" or “acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[00362] Compounds of structural Formula I may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of structural Formula I.
[00363] Compounds of structural Formula I may be separated into their individual diastereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase. Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.
[00364] Alternatively, any stereoisomer of a compound of the general structural Formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known absolute configuration.
[00365] If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diasteromeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art. [00366] Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
[00367] Some of the compounds described herein may exist as tautomers, which have different points of attachment of hydrogen accompanied by one or more double bond shifts. For example, a ketone and its enol form are keto-enol tautomers. The individual tautomers as well as mixtures thereof are encompassed with compounds of the present invention.
[00368] In the compounds of generic Formula I, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds of generic Formula I. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically-enriched compounds within generic Formula I can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
Salts and formulations
[00369] It will be understood that, as used herein, references to the compounds of structural Formula I are meant to also include the pharmaceutically acceptable salts, and also salts that are not pharmaceutically acceptable when they are used as precursors to the free compounds or their pharmaceutically acceptable salts or in other synthetic manipulations. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term "pharmaceutically acceptable salt" refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts of basic compounds of the present invention include, but are not limited to, the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N- methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, and basic ion-exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
[00370] Also, in the case of a carboxylic acid (-COOH) or alcohol group being present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic acid derivatives, such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of alcohols, such as acetyl, pivaloyl, benzoyl, and aminoacyl, can be employed. Included are those esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.
[00371] Solvates, in particular hydrates, of the compounds of structural Formula I are included in the present invention as well.
[00372] According to an embodiment, the compounds of structural Formula I may be included in various formulations for use as medicaments. Formulations for oral use may be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
[00373] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxypropylmethy-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame. [00374] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
[00375] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
[00376] The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.
[00377] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenteral ly-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
[00378] The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomiser (preferably an I atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1 , 1 ,1 ,2-tetrafluoroethane or 1 , 1 ,1 , 2, 3,3,3- heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.
[00379] The pressurized container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.
[00380] Prior to use in a dry powder or suspension formulation, the drug product is micronized to a size suitable for delivery by inhalation (typically less than 5 microns).
[00381] This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.
[00382] Capsules (made, for example, from gelatin or HPMC), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.
[00383] A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from log to 20mg of the compound of the invention per actuation and the actuation volume may vary from 11 to 1001. A typical formulation may comprise a compound of formula I, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.
[00384] Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.
[00385] Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, poly(DL-lactic-coglycolic acid (PGLA). Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
[00386] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or "puff" containing from 1 ng to 10 mg of the compound of Formula I. The overall daily dose will typically be in the range 1 ng to 10 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.
[00387] All the molecules are effective starting at nano molar concentrations and they don't cause cell death in culture in high micro-molar concentrations.
[00388] Compounds of Formula I may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.
[00389] For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed. (For purposes of this application, topical application shall include mouth washes and gargles.)
Utilities
[00390] The compounds specifically exemplified herein exhibit good efficacy in modulating RORyt, as shown by their in vitro assays.
[00391] According to an embodiment, the inhibitors of RORyt may improve and may have utility in preventing or treating fibrosis and acute and/or chronic liver conditions.
[00392] One aspect of the invention provides a method for the treatment and control of fibrosis and acute and/or chronic liver conditions, which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula I.
[00393] In addition to primates, such as humans, a variety of other mammals can be treated according to the method of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent, such as a mouse, species can be treated. However, the method can also be practiced in other species, such as avian species (e.g., chickens).
ASSAYS FOR MEASURING BIOLOGICAL ACTIVITY
[00394] Activity of the compounds of this application may be evaluated using the following assays for RORyt -inhibiting activity. Compounds of Formula I will have activities of <10 pM in this assay, and preferably, activity of <1 pM.
[00395] PBMCs were treated with CD3/CD28 for 14 days in presence of IL-6 10ng/ml, IL-1 B 10ng/ml, TGF-B 10ng/ml, and IL-23 10ng/ml.
[00396] Culture medium was changed every other day for the whole duration. [00397] Drugs were added to the polarized cells at day 14 for 48 hours.
[00398] PMA/lonomycin/Monensin cocktail was added for 6 hours to the cells at the end of 48 hours incubation.
[00399] Live/dead (455 UV), cell surface (CD4, CD3, CD8), and intracellular nuclear staining (IL-17, IL-21 , IL-22 and ROR gamma)
[00400] The following Examples are provided to illustrate the invention and are not to be construed as limiting the invention in any manner. The scope of the invention is defined by the appended claims.
EXAMPLE 1
NASH mice model and the effects of IL17 inhibitors on several inflammation markers for liver NASH activity
[00401] C57BL/6 male mice were induced liver fibrosis by being treated with 0.1 ml of a 0.5 mg/kg dose of CCI4 twice per week. Pharmacological inhibitors of IL17 including GSK805, a potent RORyt inverse agonist, was tested in parallel to the two novel pharmacological compounds and thus served as a positive control. The mice were given 0.1 ml of GSK805, 0.1 ml of Transcription Factor 1 (S10) (Compound 10) and 0.1 ml of Transcription Factor 2 (S14) (Compound 14) each at a daily dose of 10 mg/kg in 4 weeks. In a 15 male mice/group or 10 male mice/group, the mice were treated daily with ip injection for both CCI4 and the inhibitors or with ip injection for CCI4 and with SC injection for inhibitors. The IL17A+ cells were characterized and quantified in liver tissue sections by using flowcytometry. Statistical analysis of stimulated lymphocytes, stimulated cytokines, granulocytes and unstimulated lymphocytes. Results are shown in Fig. 5 to Fig. 13. Inhibitors reduced the number of infiltrating intrahepatic lymphocytes (CD3+CD4+and CD8+, y5, p<0.05) and myeloid (CD11 b+) cells (p=0.04) with a trend in reduction of neutrophils (p=0.09). Significant reduction in secretion of IL-17A produced from CD4+ and TCRyb was observed by flow cytometry (p<0.01 and p<0.001 , respectively). [00402] The ALT and AST serum levels were evaluated. Results are shown in Fig. 14. Treatment with GSK805 and the new compounds significantly reduced ALT and AST levels as compared to the untreated mice.
[00403] Hematoxylin and eosin staining and Piero Sirius red staining (PSR) were used to evaluate severity of injury. Results are shown in Fig. 15 to Fig. 17. The results show reduction in the infiltration of immune cells around the portal and central veins. Inhibitors also significantly reduced collagen deposition as measured by PSR+ area (p<0.05).
[00404] A hydroxyproline test was performed. Results are shown in Fig. 18. The results show a trend towards reduction.
EXAMPLE 2
Therapeutic effect of three compounds on mice
[00405] Methods: Three compounds of 2, 3 derivatives of 4, 5, 6, 7 tetra- hydrobenzothiophene were assessed for their therapeutic effect on CCL4 liver injury. The compounds were injected daily to mice at several dose levels 1 -10, 10-100 mg/kg after the induction of CCL4 liver hepatitis. Liver inflammation was assessed by measuring liver enzymes, histology and by flow-cytometry. CCL4 causes generalized toxicity to liver hepatocytes and induces severe irreversible cirrhotic changes that ultimately lead to liver fibrosis. CCL4 dosing frequency and length of exposure determines whether the liver disease model mimics either hepatitis, cirrhosis, or fibrosis. The CCL4 induced fibrosis can be acute that is induced over a short period of time (days to weeks) with high doses. Fibrosis can be chronic and manifest over a few weeks or months following small doses of CCL4 administered over a long period of time.
[00406] The compounds tested:
[00407] N-(3-(Benzylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5- chloro-2-(methylthio) Pyrimidine-4-carboxamide [00408] N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide
[00409] N-benzyl-2-{[(4-methyl-1 -piperazinyl)acetyl]amino}-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamideResults: The compounds reduced the liver injury markers ALT and AST. The compounds also reduced the infiltration of IL17A+ lymphocytes to the liver. The compounds reduced liver hepatitis, cirrhosis, and fibrosis. Liver sections histology was assessed, and the compounds reduced the areas of hepatitis, cirrhosis, and fibrosis. The hepatitis was reduced as evidenced by the reduction of the infiltrating IL17A+ lymphocytes which promote neutrophilic infiltration to and secrete cytokines. The compounds also reduce CD8+ cells infiltration which produces interferon gamma and increase hepatitis and inflammation. In addition, the compounds reduce the expression of fibrotic and pro fibrotic genes in liver hepatocytes such as IL17A, IL22, RORA and RORC, TNF-beta, lipoxygenases, actins, and collagens (Figs. 1 -23).
EXAMPLE 3
Role of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORg or RORA) and gamma (RORC, RORy or RORyt) in Promoting Fibrosis in the Liver, and Fibrosis in General
[00410] Chronic liver disease and inflammation is a serious global health problem occurs in response to multiple insults such as alcohol exposure, obesity, diabetes, chronic viral hepatitis, autoimmunity, chronic inflammatory conditions, and drug toxicity which can lead to progressive fibrosis. Perpetuation of the fibrotic process commonly leads to cirrhosis and Hepatocellular carcinoma (HCC), which are leading causes of morbidity and mortality worldwide. Liver fibrosis characterized by the proliferation and trans differentiation of quiescent hepatic stellate cells (HSCs) into myofibroblasts which are principal executive and responsible for collagen and extracellular matrix deposition (ECM) leading to tissue scarring.
[00411] In animals, various causes of fibrogenesis have been studied, but the main hepatic fibrosis model is based on repeated application of carbon tetrachloride (CCI4) over a period of several weeks. CCI4 is a hepatotoxin that causes lobular central hepatic necrosis, proinflammatory and profibrotic cytokine release, and the metabolic activation in the liver, consequently, results in liver fibrosis and even cirrhosis after long-term exposure.
[00412] CD4+ Th17 cells are characterized by the production of effector cytokines such as Interleukin-17 (IL-17A), IL-17F, IL-22, GM-CSF, and, to a lesser extent, tumor necrosis factor (TNF) and IL-6. In addition to promoting autoimmune inflammation, Th17 cells are critical for host immunity against fungi and extracellular bacteria. Differentiation and functionality of Th17 cells require the expression of the master transcription factor, retinoic acid receptor-related orphan receptor gamma t (RORyt), the T-cell-specific RORyt isoform, which is induced upon stimulation of naive CD4+ T-cells by TGF-[3 and IL-6. RORyt is induced during the transition from double negative to double positive thymocytes, allowing for maturation into single positive T cells. Expression of RORyt and its targeted cytokines is important for differentiation of other cell types such as CD8+ Tc17 cells, invariant natural killer T cells and yb T cells.
[00413] In addition, different studies have suggested that IL-17 plays a crucial role in liver fibrosis as well as multiple human autoimmune diseases. IL-17A, a hallmark of the Th17 subset, secreted by infiltrating Th17 cells in the liver, promotes the synthesis of type I collagen and a-smooth muscle actin (SMA) in HSCs and participates in initiation and progression of hepatic fibrosis. Thus, antagonizing this pro-inflammatory pathway by applying antibodies against involved cytokines such as IL-17 have demonstrated variably beneficial effects.
[00414] RORyt is expressed in a variety of tissues and is a highly attractive drug target in Th17 cell mediated diseases especially due to its involvement in IL17/IL23 axis and because its activity can be modulated by applying small-molecular weight inverse agonist by RORyt ligand-binding pocket. Deficiency of RORyt in mice leads to the profound deficiency in Th17 differentiation and protection against many diseases such as encephalomyelitis (EAE). Several X-ray crystallography and NMR structural studies have identified numerous small-molecular-weight inhibitors including ligands, agonists, inverse agonists, and antagonists targeting RORyt capable to alleviate proinflammatory diseases. Most of the clinical and preclinical ligands were rejected to apply due to the safety and their side effects during process of drug development. Recently, 2,3 derivatives of 4, 5,6,7- tetrahydro-benzothiophene modulators of RORs were discovered following performing “in silico/in vitro” screening, molecular docking, and molecular dynamic simulation studies in comparison with selected reference RORyt modulators from different classes. The range of synthetic RORyt agonist/inverse agonist is huge and includes sulfonamides, pyrazole amides, biaryl amides, and many others. Conformational changes in H3 and H5 helix, can interface with ligand-binding pocket and the back wall of coactivator-binding pocket, which can disrupt protein-coactivator interactions and reduce or completely loss RORyt activity. We have demonstrated that treatment with the Th17-type cytokine RORyt inhibitor (GSK805) resulted in reduced fibrosis and cause reduction in neutrophil recruitment to the liver. GSK805 is an orally available inhibitor of RORyt-mediated transcription, able to supresses Th17 response and cause reduction in frequency and total number of IL17 producing Th17 cells.
[00415] There is described in vitro and in vivo two potent and selective new small- molecular weight RORyt inverse agonists. The compounds effectively reduced IL-17A production by polarized mice CD4+ T cells and y5+ T cells and attenuate severity of CCI4- induced liver fibrosis. The inhibitors reduced the frequencies of IL-17A producing cells making them an attractive target for IL-17-mediated disorders.
[00416] Materials and Methods
[00417] Mice
[00418] The 6- to 8-week-old male wildtype C57BL/6 mice were purchased from
Charles River Laboratories (Montreal, CA). To induce chronic liver fibrosis, mice treated with CCI4 {Carbon tetrachloride) (Cat. No. 270652-100 ML; Sigma-Aldrich) resuspended in com oil (Cat. No. C8267-500 ML; Sigma-Aldrich) (0.5 ml/kg two intraperitoneal (i.p.) injections per week) for four weeks. Vehicle control mice received com oil instead of CCI4 throughout the period of the experiment. To investigate the therapeutic effects of inhibitors, a subset of CCI4 treated mice were treated with the three different RORpt antagonists including GSK805, TF1 -S10 and TF2-S14 (10 mg/kg i.p. dissolved in com oil) starting as of week three daily injection (during weeks 3 through 4). All the mice were euthanized 24 hrs after the final injection of CCI4 by using sodium pentobarbital (400 mg/kg). After dissection, the liver, spleen, and heart blood samples were harvested. All the mice were reared and housed under specific pathogen free conditions (20 ± 2°C; 12 hr light/12 hr dark) with free access to food (standard chow diet) and water. All the animal experimental procedures were approved by the Centre de Recherche du Centre Hospitalier de I’Universite de Montreal (CRCHUM) animal Ethics Committee, Comite Institutionel de Protection des Animaux (CIPA) (protocol IP22059NSs).
[00419] Histopathological analysis
[00420] Mouse livers were harvested, and a section was fixed in Tissue Fix (Cat. No. T-50; Chaptec) for 24h at 4°C, and finally embedded in paraffin for sectioning for histologic analysis (BZ-Histo Services, Inc., Montreal, Quebec, Canada). Some portions of the liver were stored at -80 until use. The 4- pm -thick Formalin-Fixed Paraffin- Embedded (FFPE) tissue sections were stained with H&E (Hematoxylin and eosin) (Varistain XY, Shandon, CR-CHUM). FFPE sections were deparaffinized and rehydrated, then stained with PSR (Picrosirius Red) with Fast green (Cat. No. F7252-5G; Sigma) in Picric acid solution (Cat. No. P67744-1 GA; Sigma). Collagen fibres were shown in red by PSR staining. Sirius red positive area was calculated by threshold settings based on pixel value in the green channel using FIJI (Image J) image analysis software.
[00421] Detection of Serum ALT and AST
[00422] Blood samples were drawn using cardiac puncture of the mice applying 23- gauge needle and the serum ALT and AST levels were measured at the OPTILAB of the CHUM.
[00423] Mouse Intrahepatic Leukocyte Isolation
[00424] The liver was harvested from mice and was transferred into a sterile petri dish. Intrahepatic leukocytes (IHLs) were isolated from mice livers using a Percoll gradient (Cat. No. 17089101 ; Cytiva) in isotonic solution. Briefly, the liver tissues were gently minced into small pieces and then were digested in collagenase D (0.025 lU/mL, Cat. No. 11088866001 ; Roche) and benzonase (10 lU/mL, Cat. No. 70664-10KUN; EMD Millipore) at 37°C with rotation for 30 minutes. The liver and the cell suspension then were passed through a 70- pm cell strainer (Cat. No. 22363548; Thermo Fisher Scientific) and centrifuged at 500 g to remove hepatocytes. The supernatant was transferred to a new tube and centrifuged at 1000 g for 10 min at 4°C. To isolate leukocytes, the cell pellet then was resuspended in Percoll 40% in 1 % Hank’s balanced salt solution in sterile water (vol/vol) and layered it over Percoll 80% in 1 % Hank’s balanced salt solution in sterile water (vol/vol), followed by centrifugation without brakes for 25 minutes at 4°C. Following collection of IHLs, they were washed, and the remaining RBCs were lysed using ACK (Ammonium-Chloride-Potassium) lysing buffer (Cat. No. A10492-01 ; Thermo Fisher Scientific). IHLs were directly stained for surface markers or stimulated with PMA/ionomycin (50 ng/mL, Cat. No. P1585 and 1 pg/mL, Cat. No. I-0634; Sigma-Aldrich, respectively) in the presence of brefeldin A (5 pg/mL, Cat. No. B6542; Sigma-Aldrich) and monensin (5 pg/mL, Cat. No. M5273; Sigma-Aldrich) in a cell incubator with 10 % CO2 at 37°C for 5 hours before intracellular cytokine staining (ICS). [00425] Flow Cytometry
[00426] For cell surface staining, freshly isolated IHLs from mice were washed with flow cytometry buffer (1 % heat inactivated fetal bovine serum and 0.01 % azide in PBS), followed by incubation protected from light with mix of primary antibodies (Table I) at 4°C for 30 minutes for the labelling of the surface markers. The cells then were washed with FACS buffer and fixed with PBS 1 % PFA (Paraformaldehyde). For ICS (intracellular staining), first we perform surface staining and cells were fixed and permeabilized using FOXP3 fixation buffer (Cat. No. 00-5523-00; Invitrogen). Then, cells were washed twice with permeabilization wash buffer (Cat. No. 00-8333-56; Invitrogen) and incubated with antibodies for intracellular antigen staining at 4°C for 30 minutes in dark. For detection of live cells, the Aqua Live/Dead Fixable Aqua Dead Cell Stain kit was used (Cat. No. L34966; Invitrogen). Data were acquired using a multicolor HTC FORTESSA A cytometer (BD Bioscience) equipped with FACS DIVA software version 9.2 (BD Bioscience) and analyzed using FlowJo software, version 10.8.1 (BD Bioscience).
[00427] Table I. List of Flow cytometry antibodies
Figure imgf000079_0001
[00428] Real-Time qPCR
[00429] Total RNA of the frozen liver samples was extaracted and purified from mouse livers using the RNeasy Plus Mini kit (Cat. No. 74134; QIAGEN) and quantified according to the manufacturer’s instructions. Complementary DNAs (cDNAs) were generated from 1 mg RNA of total liver RNA using the Transcriptor Universal cDNA Master (Cat. No. 05893151001 ; Roche) kit. Produced cDNAs were then diluted 1 :10 with ultrapure water and the relative expression of the mRNA was measured using real-time qPCR with the Light Cycler 480 SYBR Green I system (Cat. No. 04707516001 ; Roche). We used the r28S as the standard housekeeping gene and the 2-AACt method was applied to calculate the mRNA level. After the reaction was completed, we verified specificity by melting curve analysis. The list of the applied primers and their sequence used for realtime qPCR are summarized in Table II. The primers for Tgf-|31 , Col1 a1 , Acta2, and Loxl2 genes were purchased from QIAGEN.
[00430] Table II. List of IF primary and secondary antibodies
Figure imgf000080_0001
[00431] IF Staining and Image Analysis
[00432] 4- pm FFPE mice liver sections were deparaffinized and rehydrated. Heat- induced antigen retrieval was performed by immersing the dewaxed slides in sodium citrate solution (pH 6) for 10 minutes in an electric high-pressure cooker (Salton) containing the tap water. At the next step, immerse the slides in a Coplin jar containing 0.1 mol/L glycine for 15 minutes at room temperature to reduce autofluorescence, followed by applying blocking buffer (10% filtered human serum, 1 % bovine serum albumin, 0.1 % Tween 20, and 0.3% Triton-X 100 in phosphate buffered saline) for 30 minutes at RT. The sections then were incubated with the primary antibodies (Table III) diluted in the blocking buffer without human serum at 4°C. The sections were then washed in PBS-Tween (0.05 % v/v) and then incubated with the appropriate secondary antibodies (Table III) diluted in blocking buffer containing the 10 % of donkey serum for 1 hour at RT in dark. The sections were mounted in Slow Fade Gold antifade reagent wit DAP I (Cat. No. S36938; Invitrogen). Finally, the images were acquired at the CRCHUM molecular pathology platform using a wholeslide scanner (BX61 VS; Olympus).
[00433] Table III. List of Primer Sequences
Figure imgf000081_0001
[00434] Statistical Analysis
[00435] Data were analyzed using Prism version 10 software (GraphPad Software) and as indicated in the figure legends. One-way anova analysis test was used to determine the differences between different groups for comparing the effect of different inhibitors on mice.
[00436] Results
[00437] RORyt inhibitors reduced the infiltration of immune cells around the portal and central veins in CCI4-induced liver fibrosis in mice [00438] We used a CCI4-induced liver fibrosis mice model. Administration of CCI4 in 4 weeks resulted in liver fibrosis, while co-administration of CCI4 with RORyt inhibitors, three pharmacological inhibitors GSK805, TF1 -S10 and TF2-S14, suppressed such liver injuries. Mice were sacrificed 24 h after the last injection of CCI4 to evaluate hepatic fibrosis and efficacy of the treatments (Fig. 19A). Liver sections of mice were fixed in formalin and underwent histopathological analysis to investigate the degree of hepatic fibrosis in CCI4-induced hepatic fibrosis. 65 mice were participated in our study and were divided into liver fibrosis group (no-inhibitor) (n = 15), treated with inhibitor groups (n=15 in each inhibitor group) and the vehicle control group (com oil) (n = 5). After the experiment was completed, and mice were euthanized, we examined the potential effects of pharmacological inhibitors and pathogenesis of chronic liver fibrosis. H&E staining demonstrated that CCI4 injection caused significant liver fibrosis damage including inflammatory cell infiltration and changes in the overall morphology of the liver compared with the vehicle control group. In comparison, no evidence of liver injury was observed in the vehicle control group (Fig. 19B). Inhibitors were therefore applied (TF1 -S10, TF2-S14 and GSK805 as our internal control) to attenuate liver fibrosis in mice with CCI4-induced hepatic fibrosis. H&E staining revealed that CCI4 administration alone leads to the remarkable increase in the size of the portal and central veins. Inhibitor-treated mice showed much smaller infiltration of immune cells around liver veins than CCI4-induced mice (Fig. 20B). More specifically, the fibrotic areas and infiltration of immune cells around the portal and central veins were reduced in inhibitor-treated groups. Liver morphology seemed more stable in mice treated with GSK805 and TF1 -S10 (Fig. 20B). Additionally, several irregular red dots were found on the surface of the livers of CCI4-treated mice. On contrary, livers obtained from vehicle control mice had a smooth surface with uniform and soft textures. Livers of CCI4+inhibitors-treaed mice had fewer red dots compared with thee CCI4-induced mice. These results indicated that the mouse model of CCI4-induced liver fibrosis was successfully established, and treatment protected the mice from CCI4- induced hepatic fibrosis. [00439] Pharmacological inhibitors of IL-17 ameliorate liver injury in CCI4- induced liver fibrosis mice
[00440] To examine the effect of inhibitors on CCI4-induced liver fibrosis, the serum of mice in all the groups were collected. According to the Fig. 19C, we found that CCI4 administration mice significantly increased the levels of serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST), markers of liver injury compared with vehicle control (p<0.01 ). Interestingly, treated mice with the pharmacological compounds developed less pronounced liver injury compared with CCI4-induced mice, as shown by Fig. 19C. We observed significant reduction in serum ALT and AST levels by administering TF1-S10 compared with the no-inhibitor mice (p<0.05). Significant reduction in AST level by applying TF2-S14 (p<0.05) and reduction in ALT level (p= 0.064) by applying TF1 -S10 compared with the no-inhibitor group. Surprisingly, GSK805 was not effective enough to significantly reduced the levels of the liver enzymes, however the increase of serum ALT and AST levels was partially reversed by treatment with GSK805 inhibitor (Fig. 19C).
[00441] RORyt inverse agonists reduce accumulation of myeloid cells in CCI4- induced liver fibrosis
[00442] Hepatic infiltration of myeloid cells was obtained from mice following sacrifice of the animal and analyzed by flow cytometry. Using CD11 b+ gate, we investigated recruitment of myeloid cells to the site of injury and fibrosis (Fig. 20A). We found a significant reduction in the number of leukocytes (CD45+) and myeloid cells (CD45+ CD11 b+) by applying GSK805 compared with the no-inhibitor control mice (p<0.01 and p<0.05, respectively). The number of monocytes (CD45+ CD11 b+ Ly6Chi Ly6G_) reduced by administering GSK805 and TF2-S14 compared with the no-inhibitor mice, although these changes are not significant. Intriguingly, immunostaining against the eosinophils surface marker (CD11 b+ Ly6O Ly6G_ F4/80’ CD170+) demonstrated that the numbers of CD170+ (SiglecF+) cells were markedly and significantly reduced following administration of GSK805 and TF1 -S10 (p<0.05) compared with the CCI4-treated control mice and there is an observed trend in the total infiltratory cell counts by administration of TF2-S14. We observed the reduction trend in the accumulation of macrophages (CD45+ CD11 b+ Ly6O Ly6G’ F4/80+) specially following administration of GSK805 and TF2-S14 compared with the CCI4-treated control mice. Apparently, neutrophils level (CD45+ CD11 b+ Ly6Cint+ Ly6Ghi+) did not affected in the livers of inhibitors treated mice compared with the CCI4-treated control mice. Collectively, these findings suggest that GSK805 and TF1 -S10 are the most efficient RORyt inhibitors applied to exacerbate liver injury and liver inflammation (Fig- 20B). Additionally, our data suggesting that neutrophils are not the cellular target of RORyt inhibitors applied in our study.
[00443] RORyt inverse agonists decreased levels of IL-17 and reduced infiltration of IL-17 producing cells in the livers of CCI4-treated mice
[00444] At the next step, we explored whether RORyt inhibitors would inhibits or at least decrease IL-17 secretion. For this purpose, hepatic-infiltration mononuclear cells were obtained from mice 24 hrs after sacrifice and IL-17 producing cells were analysed by flow cytometry following 5 hrs incubation at 37°C for cytokine stimulation (Figs. 21 -22). Using CD3+ gate, we investigated IL-17 expression in different T cell populations after liver fibrosis treatment. An attenuated liver fibrosis, noticed in CCI4+inhibitor treated mice, correlated with the decreased levels of inflammatory and pro-fi brogen ic IL-17 cytokine. Interestingly, IL-17 level were significantly reduced in CCI4+inhibitor -treated mice in comparison with CCI4 only treated animals, suggesting the importance of RORpt- mediated inhibitors in attenuation of IL-17-driven liver fibrosis. To dissect out cellular target of pharmacological inhibitors modulation of liver fibrosis, flow cytometry analysis of liver infiltrated lymphocytes was performed. Intracellular flowcytometry demonstrated that GSK805 and TF1-S10 were the most effective inhibitors able to significantly reduce infiltration of lymphocytes CD45+(p<0.01 ), CD45+CD3+(p<0.05), and CD45+CD3+CD8+ (p<0.01 ) T cells compared with the CCI4-only treated mice in which the level of infiltration is significantly higher. Treatment with the inhibitors lead to the obvious reduction trend of CD45+CD3+CD4+ and CD45+CD3+ yb-TCR+ T compared with the CCI4-only treated mice while the reduction is not significant (Fig. 21 B). [00445] In the line with these finding, significant reduction in the number of IL-17- producing CD4+ and yb-TCR+ T cells following administration of IL-17 targeting inhibitors (Fig. 22A-C) compared to animals treated with CCI4 only. On contrary, there was no significant difference in the total number of liver infiltrating IL-17-producing CD8+ T lymphocytes between these groups although the inhibitors were able to significantly be reduced the infiltration of CD3+CD8+ T cells. Here we reported that CD4+ T (p<0.05 following applying all the inhibitors) and yb-TCR (p<0.01 by administering GSK805 and TF2-S14 and P<0.074 following application of TF1 -S10) are the major contribution on IL- 17 production following liver fibrosis. Interestingly, as we previously showed, these cell populations did not significantly affect by administration of these inhibitors. Interestingly, the inhibitors are not effective at inhibiting the intracellular secretion of IL-22, another member of TH17 T cell family which suggest the inhibitors specifically target IL-17 producing TH17 T cells (Fig. 22A-C). TH1 T cell family could be another population affected by the administration of the inhibitors as there is a remarkable reduction in the frequency of CD4+ IFN-y+ (P<0.05) and CD8+ IFN-y+ (P<0.05) cytokine secretion level by applying GSK805 and TF2-S14, respectively (Fig. 22B-C).
[00446] RORyt inverse agonists regulate the mRNA expression and ameliorate fibrosis
[00447] To investigate the involvement of the inhibitors in protecting against CCI4- induced liver fibrosis, first, we measured the mRNA expression levels of the profibrogenic genes Col1 a1 , Acta2, Tgf-|31 and Loxl2 by quantitative RT-PCR in the treated and not- treated mice. As shown in Fig. 23A and consistent with the histologic data the expression of proinflammatory genes were increased in the liver of CCI4-treated mice, while these were significantly decreased by applying GSK805 and TF1 -S10 (p<0.0001 ) in all the cases except for Acta2 mRNA expression level in mice received TF1 -S10 (p<0.01 ), however, the expression levels of genes were not significantly changed following TF2- S14 treatment (Fig. 23A). Expression level of vehicle control is significantly lower in all the cases compared with the no-inhibitor group (p<0.0001 ). The data indicated that TF1 - S10 and GSK805 are the most efficient inhibitors to inhibit inflammatory genes expression induced by CCI4 injection.
[00448] Next, we investigated weather the pharmacological inhibitors of IL-17 could relieve liver fibrosis and protect the liver damage from CCI4 toxicity. Sirius red staining of liver tissues obtained from CCI4-treated mice revealed extensive collagen deposition suggesting development of liver fibrosis. Treatment with the pharmacological compounds resulted in drastically reduced CCI4-induced fibrosis as illustrated by collagen type I deposition measured by picrosirius red (PSR) staining (Fig. 23B). All three inhibitors were able to drastically and significant reduction in the size of stained area of fibrous dense tissue as shown by percentage of PSR positive area (p<0.0001 and p<0.001 for GSK805, TF1 -S10 and TF2-S14, respectively).
[00449] These findings collectively demonstrated that inhibitors may relieve liver fibrosis through regulating pro inflammatory cytokines.
EXAMPLE 4
Role of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORg or RORA) and gamma (RORC, RORy or RORyt) in Promoting Fibrosis in the Liver, and Fibrosis in General #2
[00450] Materials and Methods
[00451] Mice
[00452] Male wildtype C57BL/6 mice (6-8 weeks old) were purchased from Charles River Laboratories (Montreal, CA). To induce chronic liver fibrosis, mice were treated with CCk (Carbon tetrachloride) (Sigma-Aldrich, Oakland, ON, Canada) resuspended in com oil (Sigma-Aldrich, Oakland, ON, Canada) (0.5 ml/kg two intraperitoneal (i.p.) injections per week for four weeks). Vehicle control mice received corn oil instead of CCL throughout the period of the experiment. To investigate the therapeutic effects of inhibitors, as of week 3, groups of CCL-injured mice were treated daily for two weeks with the two novel RORyt inverse agonists TF-S10 and TF- S14, or GSK805 as a control (10 mg/kg i.p. in corn oil) (Fig. 24A). At the end of week 4, mice were euthanized 24 hrs after the final injection of CCk by using sodium pentobarbital (400 mg/kg). All the mice were reared and housed under specific pathogen free conditions (20 ± 2°C; 12 hrs light/12 hrs dark) with free access to food (standard chow diet) and water. All animal experimental procedures were approved by the Centre de Recherche du Centre hospitalier de I’Universite de Montreal (CRCHUM) animal Ethics Committee, Comite Institutionel de Protection des Animaux (CIPA) (protocol IP22059NSs).
[00453] Histopathological analysis
[00454] Mouse livers were harvested, fixed in Tissue Fix (Chaptec, Montreal, Quebec, Canada) for 24 hrs at 4°C, then embedded in paraffin for sectioning and histological analysis (Pathology department, CHUM, Montreal, Quebec, Canada). The 4- pm -thick Formalin-Fixed Paraffin- Embedded (FFPE) tissue sections were stained with H&E (Hematoxylin and eosin) (Varistain XY, Shandon) by the pathology platform of the CRCHUM using standard procedures. FFPE sections were deparaffinized and rehydrated, then stained with PSR (Picrosirius Red) with Fast green (Sigma-Aldrich, Oakland, ON, Canada) in Picric acid solution (Sigma-Aldrich, Oakland, ON, Canada). Collagen fibers were shown in red by PSR staining. Sirius red positive area was calculated by threshold settings based on pixel value in the green channel using FIJI (Image J) image analysis software.
[00455] Detection of Serum ALT and AST
[00456] Blood samples were drawn using cardiac puncture of the mice applying 23- gauge needle and the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured at the clinical laboratory (OPTILAB) of the CHUM.
[00457] Mouse Intrahepatic Leukocyte Isolation
[00458] The liver was harvested from mice and was transferred into a sterile petri dish. Intrahepatic leukocytes (IHLs) were isolated using a Percoll gradient (Cytiva, Uppsala, Sweden) in isotonic solution. Briefly, liver tissues were gently minced into small pieces then digested in collagenase D (0.025 lll/mL, Roche, Laval, Quebec, Canada) and benzonase (10 lll/mL, EMD Millipore, Darmstadt, Germany) at 37°C with rotation for 30 minutes. The liver and the cell suspension were then passed through a 70- pm cell strainer (Thermo Fisher Scientific, Fremont, CA) and centrifuged at 500 g to remove hepatocytes. The supernatant was transferred to a new tube and centrifuged at 1000 g for 10 minutes at 4°C. To isolate leukocytes, the cell pellet was then resuspended in Percoll 40% in 1 % Hank’s balanced salt solution in sterile water (vol/vol) and layered over Percoll 80% in 1 % Hank’s balanced salt solution in sterile water (vol/vol), followed by centrifugation without brakes for 25 minutes at 4°C. IHLs were collected at the interface, washed, and the remaining RBCs were lysed using ACK (Ammonium-Chloride- Potassium) lysing buffer (Thermo Fisher Scientific, Fremont, CA).
[00459] Flow Cytometry
[00460] For cell surface staining, freshly isolated IHLs from mice were washed with flow cytometry buffer (1 % heat inactivated fetal bovine serum (FBS) and 0.01 % azide in PBS) and incubated in the dark with the mix of surface antibodies (Table I) at 4°C for 30 minutes. Cells were then washed with FACS buffer and fixed with PBS 1 % PFA (Paraformaldehyde). For intracellular cytokine staining (ICS), IHLs were stimulated with PMA/ionomycin (50 ng/mL, and 1 pg/mL; Sigma-Aldrich, respectively) in the presence of brefeldin A (5 pg/mL; Sigma-Aldrich) and monensin (5 pg/mL; Sigma-Aldrich) in a cell incubator with 10 % CO2 at 37°C for 5 hrs then stained for surface markers. Cells were then fixed and permeabilized using FOXP3 fixation buffer (eBioscience), washed twice with permeabilization wash buffer (eBioscience), and incubated with antibodies for intracellular cytokines at 4°C for 30 minutes in the dark. For detection of live cells, the Aqua Live/Dead Fixable Aqua Dead Cell Stain kit was used (Life Technologies, Burlington, Ontario, Canada). Data were acquired using a multicolor BD LSRFortessa instrument equipped with five lasers (UV [355 nm], violet [450 nm], blue [488 nm], yellow-green [561 nm] and red [640 nm]) equipped with FACS DIVA software version 9.2 (BD Bioscience) and analyzed using FlowJo software, version 10.8.1 (BD Bioscience). [00461] Real-Time qPCR
[00462] Real time qPCR was performed as previously described. Briefly, total RNA of the frozen liver samples was extracted, purified using the RNeasy Plus Mini kit (QIAGEN, Germany) and quantified according to the manufacturer’s instructions. Complementary DNAs (cDNAs) were generated from 1 pg RNA of total liver RNA using the Transcriptor Universal cDNA Master (Roche, Germany) kit. Produced cDNAs were then diluted 1 :10 with ultrapure water and the relative expression of the mRNA was measured using real-time qPCR with the Light Cycler 480 SYBR Green I system (Roche, Germany). We used the r28S as the standard housekeeping gene and the 2’ AACt method was applied to calculate the mRNA level. After the reaction was completed, we verified specificity by melting curve analysis. The 28S primers sequences are: forward, 5’-3’(CGAGATTCCCACTGTCCCTA) and reverse, 5’- 3’(GGGGCCTCCCACTTATTCTA). The primers for Tgf-/31, Col1a1, Acta2, and Loxl2 genes were purchased from QIAGEN.
[00463] Immunofluorescence Staining and Image Analysis
[00464] Four pm FFPE mice liver sections were deparaffinized and rehydrated. Heat-induced antigen retrieval was performed by immersing the dewaxed slides in sodium citrate solution (pH 6) for 10 minutes in an electric high-pressure cooker (Salton, China) containing tap water. Next, slides were immersed in a Coplin jar containing 0.1 mol/L glycine for 15 minutes at room temperature to reduce autofluorescence, followed by applying blocking buffer (10% filtered human serum, 1 % bovine serum albumin,0.1 % Tween 20, and 0.3% Triton-X 100 in phosphate buffered saline) for 30 minutes at RT. The sections were then incubated with rabbit polyclonal desmin antibody (Thermo Fisher Scientific, Massachusetts, US) mouse monoclonal a-SMA antibody (Clone 1A4, Sigma-Aldrich) antibodies followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) diluted in the blocking buffer containing the 10 % of donkey serum for 1 hour at room temperature in dark. The sections were mounted in Slow Fade Gold antifade reagent with DAPI (Thermo Fisher Scientific). Finally, the images were acquired at the CRCHUM molecular pathology platform using a whole slide scanner (BX61 VS; Olympus).
[00465] Statistical Analysis
[00466] Data were analyzed using Prism version 10 software (GraphPad Software) and as indicated in the figure legends. One-way ANOVA test was used to determine the differences between different groups for comparing the effect of different inhibitors on mice.
[00467] Results
[00468] RORyt inhibitors reduced the hepatic infiltration of immune cells and ameliorated liver damage in chronic CCI4-injured liver injury in mice
[00469] To determine the effect of RORyt inverse agonists on liver fibrosis, we used the CCk-injured liver fibrosis mouse model. Mice (n=15/group) were given two injections of CCk per week (0.5 ml/kg body weight), or vehicle (corn oil, n=5) as control, for a period of four weeks. At week 3 post-CCk initiation, mice were treated daily (i.p.) with either one of the RORyt inverse agonists, GSK805, TF-S10 ( co m p o u n d 1 0 ) and TF-S14 (compound 13), or vehicle as described in materials and methods. Mice were sacrificed and their liver processed for analysis 24 hrs after the last injection of CCk to evaluate hepatic fibrosis and efficacy of treatments (Fig. 24A). Red spots of irregular shape were visible on the surface of CCk-injured livers as compared to the smooth uniform surface of livers from vehicle control mice. These spots, probably originating from hemorrhaged damaged vessel, decreased in frequency in the RORyt inverse agonists + CCk mice (Fig. 24B). Histological examination using hematoxylin- eosin (H&E) staining of liver sections revealed significant liver damage post-CCk, that involved infiltration of inflammatory cells and changes in the overall morphology of the liver such as a remarkable increase in the size of portal and central veins as compared with the vehicle control group (Fig. 24C, top panels). On the other hand, all RORyt inverse agonists + CCk-injured mice exhibited reduced infiltration of immune cells around liver veins as compared to the no-inhibitor CCk-injured group (Fig. 24C, top panels). Immunofluorescence (IF) analysis of parallel sections showed that all RORyt inverse agonists + CCL-injured livers exhibited decreased expression of desmin and o-SMA suggesting reduced activation of HSCs, as compared to the no-inhibitor group (Fig 24C, lower panels). Desmin staining also showed that bridging fibrosis, visible in livers of the CCL-injured mice, was diminished upon treatment with the RORyt inverse agonists, GSK805, TF-S10 or TF-S14 (Fig. 24C, lower panels).
[00470] To examine the therapeutic efficacy of RORyt inhibitors on CCL-induced liver injury, at the time of sacrifice, we measured serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of hepatocyte damage (Fig. 24D). CCL administration significantly increased ALT and AST serum levels as compared to vehicle control mice (p<0.01 and p<0.05, respectively). There was a trend towards reduction in serum ALT and AST levels in mice treated with the RORyt inverse agonists but it was only significant for AST levels in TF- S14 treated mice (p<0.05) (Fig. 24D). Altogether these results demonstrate that RORyt inverse agonists reduced CCI4-injured liver damage.
[00471] RORyt inverse agonists reduce accumulation of myeloid cells in CCI4- induced liver injury
[00472] Next, we evaluated the immune cell subsets infiltrating the injured liver by analyzing intrahepatic leukocytes (IHLs) using flow cytometry. Representative gating strategy is presented in Fig. 25A. We observed an overall increase in infiltration of CD45+ cells in mice receiving CCI4. Treating CCL-injured mice with GSK805 resulted in a significant reduction in the total number of leukocytes (CD45+) and myeloid cells (CD45+CD11 b+) in the liver (p<0.01 and p<0.05, respectively) (Fig. 25B). When this effect was stratified by the different myeloid cell subsets, there was mild reduction in monocyte recruitment (CD45+CD11 b+Ly6ChlLy6G') in GSK805- or TF-S14-treated mice as compared to no-inhibitor/CCL-injured mice. CCL-induced injury increased levels of intrahepatic eosinophils (CD11 b+Ly6C'Ly6GT4/80'CD170 (SiglecF)+) as compared to control mice (p<0.05). Administration of GSK805 or TF-S10 to CCL-injured mice significantly decreased eosinophil numbers in the liver (p<0.05) (Fig. 25B). The trend towards reduced infiltration of eosinophils was also observed in TF-S14-treated mice but was not statistically significant. Macrophages (CD45+CD11 b+Ly6C'Ly6G'F4/80+) also showed a trend towards increased accumulation in the liver upon CCL-induced injury, that was slightly reversed upon administration of GSK805 and TF-S14. Intrahepatic neutrophils (CD45+CD11 b+Ly6CintLy6G+) were not altered in CCk-injured mice as compared to controls, and their level was not affected by treatment of mice with any of the RORyt inverse agonists (Fig. 25B). Altogether, these findings suggest that GSK805 and TF-S10 are the most efficient RORyt inverse agonists in decreasing hepatic recruitment of total myeloid cells, especially eosinophils, thus reducing liver inflammation.
[00473] RORyt inverse agonists reduce hepatic accumulation of T cells and the infiltration of IL-17-producing cells in livers of CCI4-injured mice
[00474] Next, we assessed the effect of RORyt inverse agonists on the intrahepatic recruitment of T cells. IHLs were stained for various phenotypic markers of T cell (CD3+) populations, namely, CD4+, CD8+ and yb -T cells (Fig. 26A). CCI4-injured mice exhibited increased numbers of infiltrating T cells (CD45+CD3+, p<0.05), and more specifically the CD4+ and CD8+ T cells (CD45+CD3+CD4+ and CD8+, p<0.05, Fig. 26B). Treatment of CCI4-injured mice with GSK805 or TF-S10 significantly reduced the intrahepatic recruitment of total T cells (CD45+CD3+, p<0.05), and CD45+CD3+CD4+ and CD8+ cells (p<0.01 and p<0.05, respectively) as compared to the no-inhibitor/CCI4-injured group (Fig. 26B). There was also a trend towards reduced infiltration of the various T cell populations (CD45+, CD4+, and CD8+ T cells) in the TF-S14-treated CCI4- injured mice (Fig. 26B). Finally, infiltration of yb-T cells (CD45+CD3+yb TCR+, p<0.05) was significantly decreased in GSK805 treated CCI4-injured mice (Fig. 26B).
[00475] Next, we evaluated the effect of RORyt inverse agonists on the production of IL-17, given the role of RORyt in the production of this cytokine and the implication of IL-17 itself in liver fibrosis. For this purpose, IHLs derived from the three experimental groups of mice were stimulated with PMA/lonomycin and assessed for IL-17A production using intracellular cytokine staining (ICS). By gating on CD3 and different phenotypic T cell markers (CD4, CD8, and y6 TCR), we investigated IL-17 production by the different T cell populations (Fig. 27 A-C). IL-17-producing yb-T cells significantly increased upon CCI4-induced injury (p<0.001 ) (Fig. 27A). IL- 17-producing CD4+ T cells from CCI4- injured mice also showed a close to significant increased frequency as compared to control (no CCI4) mice (p=0.088) (Fig. 27B). Treatment of CCI4-injured mice with our RORyt inverse agonists, decreased the number of IL-17-producing CD4+ and yb-T cells as compared to no-inhibitor animals (Fig. 27A, 4B). However, there was no significant difference in the total number of hepatic infiltrating IL-17-producing CD8+ T cells between all experimental mice groups (control, CCI4-injured or RORyt inverse agonists + CCI4- injured mice) (Fig. 27C).
[00476] In the same IHLs, we simultaneously evaluated production of IL-22, another Th17 cytokine which is only partially controlled by RORyt. IL-22 expression by the different T cell populations (CD4+, CD8+ and yb-T cells) was generally low following CCL injury. RORyt inverse agonists examined did not alter the frequency of IL-22 -producing cells (Fig. 27A-C). In addition to the type 3 cytokines, type 1 cytokines, mainly IFNy are of critical importance in liver disease and inflammation and maybe increased as a mechanism of rebound inflammation. Indeed, intracellular staining for IFNy in intrahepatic T cells revealed upregulation of IFNy- producing CD4+ and CD8+ cells in CCL-injured mice as compared to the vehicle-injected group (p<0.01 and p<0.001 , respectively). This increase of IFNy-producing CD4+ or CD8+ was significantly reversed upon treatment of CCL-injured mice with the control inhibitor, GSK805 and TF-S10 (p<0.05), respectively.
[00477] Altogether, our data show an increase in liver-infiltrating T cells during short term chronic CCL-injured mice. yb-T cells were the major IL-17 producers followed by CD4+ T cells. Treatment with RORyt inverse agonists reduced the frequency of IL-17- producing cells, especially yb-T cells highly suggesting that a decreased expression of IL-17 is implicated in the attenuation of CCL-induced inflammation.
[00478] RORyt inverse agonists regulate mRNA expression of fibrogenic factors and ameliorate fibrosis
[00479] To investigate the effect of the RORyt inverse agonists on liver fibrosis in CCL-induced liver injury, we first evaluated expression of profibrogenic genes, type 1 collagen, a-smooth muscle actin, TGF-01 , and lysyl oxidase like 2 enzymes (Col1a1, Acta2, Tgf-/31 and Loxl2) by quantitative RT-PCR in treated and no-inhibitor/CCh-injured mice. Expression of theses profibrogenic genes was increased in livers of CCk-injured mice compared to control (no CCk) mice (p<0.001) and was significantly reduced upon treatment with GSK805 or TF-S10 (p<0.001 ) (Fig. 28A). These data indicate that TF- S10 and GSK805 are highly efficient in inhibiting the fibrogenic process induced upon CCI4 injection in mice.
[00480] Next, we investigated whether RORyt inverse agonists could reduce collagen deposition, important marker of liver fibrosis. By evaluating collagen content in liver sections, using the Sirius red staining, we observed that treatment with the RORyt inverse agonists, GSK805, TF- S10 and TF-S14 significantly reduced collagen deposition as measured by percentage of PSR positive area (p< 0.01 ) (Fig. 28B). Consistent with observations using desmin staining (Fig 24C, lower panels), we also noted a reduction in fibrogenic cells with a bridging pattern. These findings collectively demonstrate that RORyt inverse agonists alleviate liver fibrosis through downmodulating key profibrogenic factors.
[00481] We provide proof of concept that targeting RORyt transcriptional activity can reduce immune infiltration and production of IL-17 by infiltrating lymphocytes, with concomitant reduction in profibrogenic gene expression and fibrosis in CCL-induced liver injury. The RORyt inverse agonists tested in our study were well tolerated by mice and partially lowered serum ALT and AST levels. Recruitment of immune cells including monocytes/macrophages, T cells and neutrophils into the liver is one of drivers of tissue damage and subsequent inflammatory events. In our study, TF-S10 significantly inhibited T cell recruitment, more specifically that of CD3+, CD4+ and CD8+ T cells into injured liver, while TF-S14 only slightly reduced hepatic infiltration of T cells.
[00482] Increased infiltration of eosinophils in the CCk injured mice was significantly reduced by TF-S10. Indeed, eosinophils can be important mediators of liver inflammation. Intrahepatic eosinophils are detectable in halothane-induced liver injury and are the pathogenic drivers of inflammation in this model. Liver eosinophilic infiltrate was a significant finding in patients with chronic hepatitis C who consume drugs {Tarantino, 2008 #151}. We have also reported increasing eosinophilic type 2 liver inflammation in mice and humans with Metabolic dysfunction-associated steatohepatitis (MASH). Eosinophils can release eosinophilic extracellular traps (EETs), considered as damage-associated molecular patterns (DAMPs), important activators of hepatic injury. Hence, RORyt inverse agonists effect on blocking hepatic eosinophils recruitment could be one of the mechanisms underlying their protective action against liver inflammation and fibrosis.
[00483] Fouda et al recently reported the efficacy of TF-S14 in improving skin graft survival by blocking leukocytes recruitment into skin grafts, more specifically that of neutrophils. Indeed, IL-17 inflammatory properties are partly mediated through recruitment of neutrophils. We did not observe alterations in neutrophil infiltrates in our CCL-injured mice treated or with RORyt inverse agonists which is possibly due to the short duration of the injury, the small dose of CCI4 used and/or low expression level of the receptor for IL-17 on liver neutrophils.
[00484] Fouda et al., also showed that in addition to T cells, IL-17 was produced by other leukocytes such as neutrophils and macrophages derived from splenocytes of sensitized mice (BALB/C-sensitized C57BL/6 mouse). In our model, yb- T cells were the main producers of IL-17 and the ones most affected by treatment with RORyt inverse agonists. Our results agree with several studies that showed that IL-17- producing yb-T cells are one of the key mediators of liver inflammation and fibrosis. Interestingly, the inverse agonists used in our study did not reduce the frequency of IL- 22-producing cells, underscoring their specificity for the IL-17 pathway. This is important because IL-22 plays a critical hepatoprotective role by inducing anti-apoptotic and mitogenic processes in hepatocytes. It also exerts anti-fibrogenic functions, by inactivating HSCs and inducing their senescence.
[00485] We observed reduced infiltration of IFN-y- producing CD8+ T cells upon administration of TF-S10 to CCL-injured mice. Although an effect on IFNy-producing cells was not expected with RORyt inverse agonists, this was also reported in previous studies by other groups where SR1001 , a RORay inverse agonist, reduced IFNy production and the frequency of IFNy- producing CD4+ and CD8+ T cells in the non- obese diabetic (NOD) mice. It is possible that Th17 cells could convert to Th1 cells in an inflammatory environment such as NOD mice (60, 61 ), and that SR1001 might be inhibiting such Th17/Th1 conversion. Moreover, the RORyt inhibitor TMP778 exhibited inhibitory activities against IFNy-producing cells in experimental autoimmune uveitis, suggesting an effect on Th17 which are switching into Th1 cells. These results outlined additional modes of action of RORyt inhibitors, in addition to their direct effect on Th17 and yb-T cells that merit further investigations.
[00486] We show that expression of the profibrogenic genes, Col1a1, Acta2, TGF- (3 and Loxl2, and the collagen content of injured areas were significantly reduced upon treatment with all RORyt inverse agonists assessed herein. These results further outline the efficiency of these novel RORyt inverse agonists in alleviating some aspects of liver inflammation and fibrosis upon short term chronic toxic injury. Our insights are consistent with previous reports demonstrating that interfering with Th17/IL17 axis, i.e., in mice lacking Th17 cells or IL-17 signaling, protects against liver fibrosis development.
[00487] The clinical application of RORyt inverse agonists has not been widely adopted, probably due to low efficacy or poor safety. Indeed, the initial clinical trials using RORyt inverse agonists involved patients with psoriasis where the topical administration of the drug resulted in inadequate exposure or duration leading to low efficacy. Furthermore, given the role of RORyt in T cell differentiation, its inhibition might limit T cell diversity. In addition, long-term treatment of mice with some of the inverse agonists caused T-cell lymphomas and thymic aberrations. Therefore, it may be interesting for future research to investigate the influence of RORyt on IL-17-mediated liver fibrosis in long-term chronic liver injuries induced by CCI4 or other insults such as high fat diet or alcohol. Also, it remains to be seen whether such inhibition will be effective during acute liver injuries and/or will facilitate fibrosis regression upon insult withdrawal. Our study provides evidence for the efficiency of inhibiting RORyt activity in alleviating symptoms of short-term liver fibrosis. These data together with recent advances in clinical development underscore the therapeutic potential of RORyt inverse agonists in chronic liver disease.
EXAMPLE 5
SiRNA knockdown of RORC and reduction of RORC Expression
[00488] siRNA was tested using pancreatic cell lines HPAF-II and Miapaca-ll for Cell Viability, % Caspase activity and Cell death.
[00489] HPAF-II: peritoneal ascitic fluid with primary pancreatic adenocarcinoma and metastases to the liver, diaphragm and lymph nodes.
[00490] Miapaca-ll: from tumor tissue of the pancreas
[00491] SiRNA knockdown of RORC was found to reduce RORC Expression and Reduces Cell Viability (Fig. 29).
[00492] SiRNA knockdown of RORC was found to increase Caspase 3/7 Expression and Increase Cell Death (Fig. 30).
[00493] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure. References
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Claims

CLAIMS:
1 . A method of treatment of fibrosis which comprises administering to a patient in need thereof a therapeutically effective amount of an inverse agonist of RORs or a silencing RNA that inhibits protein synthesis of RORs.
2. The method of claim 1 , wherein the fibrosis is pulmonary fibrosis, retroperitoneal fibrosis (RPF), hepatic fibrosis and/or cirrhosis, kidney fibrosis, cardiac fibrosis, skin fibrosis, intestinal fibrosis, muscle fibrosis, ocular fibrosis, fibrosis in the central nervous system, renal fibrosis, and pancreatic fibrosis, autoimmune fibrotic diseases, fibrosis associated with infectious diseases, fibrosis associated with metabolic diseases, fibrosis associated with cancer, fibrosis associated with environmental exposures, or a combination thereof.
3. The method of any one of claim 1 or 2, wherein the inverse agonist is a compound of structural Formula (I), (II), (III), or (IV), a pharmaceutically acceptable salts thereof, or stereoisomers thereof:
Figure imgf000101_0001
wherein,
R1 , R2, R3, and/or R4 group(s) is/are H, halogen, NO2, 1 -6 alkoxy, OH, NH2, 1 -6 alkyl, 1 -6 alkenyl, 1-6 haloalkyl, N-dialkyl, haloalkoxy, 1 -6 hydroxyalkyl, and/or - CO2(1-6 alkyl);
R5 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, aryl, alkylarene, halo aryl, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, haloaryl, benzene, phenyl, benzyl, pyridine, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, haloalkyl, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methylimidazole, methyldiazole, methyltriazole, methylthiadiazole, methylimidazolidine, methylthizolidine, methylpyrrolidine, methylpiperazine, methylpiperidine, methylpyridazine, methylpyrazine, methyltriazine, methylpyrrole, methylpyrroline, methylpyrazolidine, methylpyrazoline, methylthiazole, methylisothiazole, methylisoxazole, or arylalkyl.
R6 is H, 1 -6 alkyl, or may form a five or six ring structure with R5; and
R7 is a substituted or unsubstituted five or six membered saturated or unsaturated heterocycle, ring substituted alkylarene, ring substituted alkylhexane, ring substituted alkylcyclopentane, substituted haloaryl, substituted benzene, substituted phenyl, benzyl, pyrimidine, pyridine, imidazole, diazole, triazole, thiadiazole, imidazolidine, thizolidine, pyrrolidine, piperazine, aryl, halo aryl, alkylarene, piperidine, pyridazine, pyrazine, triazine, 1 H pyrrole, 2H pyrrole, pyrroline, pyrazolidine, pyrazoline, thiazole, isothiazole, isoxazole, cyanoalkyl, methylpyrimidine, toluene, methylpyridine, methylimidazole, methyldiazole, methyltriazole, methylthiadiazole, methylimidazolidine, methylthizolidine, methylpyrrolidine, methylpiperazine, methylpiperidine, methylpyridazine, methylpyrazine, methyltriazine, methylpyrrole, methylpyrroline, methylpyrazolidine, methylpyrazoline, methylthiazole, methylisothiazole, methylisoxazole, or arylalkyl.
4. The method of claim 1 or 2, wherein the inverse agonist of RORs is
• N-[2,6-dichloro-2'-(trifluoromethoxy)[1 , 1 '-biphenyl]-4-yl]-4-(ethylsulfonyl)- benzeneacetamide (GSK805) or analogs thereof;
• N-[[(2S)-1-[[5-(4-fluorophenyl)-2-methyl-4-thiazolyl]carbonyl]-2- piperidinyl]methyl]-4-benzofurancarboxamide (SB-649868);
• 3-isoxazolebutanoic acid (JTE-151 ); • 5H-Pyrrolo[3,4-b]pyridine-3-carboxamide, N-[[5-(ethylsulfonyl)-2- pyridinyl]methyl]-6,7-dihydro7-(1-methylethyl)-6-[[trans-4- (trifluoromethyl)cyclohexyl]methyl]- hydrochloride (VTP-43742);
• (1 R,3S,4R)-4-((3aR,9bR)-9b-((4-Fluorophenyl)sulfonyl)-7- (perfluoropropan-2-yl)-2,3,3a,4,5,9b-hexahydro-1 H-benzo(E)indole-3- carbonyl)-3-methylcyclohexane-1 -carboxylic acid (BMS-986251);
• 2-((1 S,3S)-3-((R)-5-((7-fluoro-1 , 1 -dimethyl-2,3-dihydro-1 H-inden-5- yl)carbamoyl)-2-methoxy-5,6,7,8-tetrahydro-1 ,6-naphthyridine-6- carbonyl)cyclobutyl)acetic acid (TAK-828);
• N-(5-(N-(4-(1 ,1 , 1 ,3,3, 3-hexafluoro-2-hydroxypropan-2- yl)phenyl)sulfamoyl)-4-methylthiazol-2-yl)acetamide (SR1001 );
• AUR-101 ;
• Izumerogant (IMU-935);
• ESR-114;
• Cedirogant (ABBV-157);
• analogs thereof; or combinations thereof.
5. The method of claim 3, wherein wherein the compound or a pharmaceutically acceptable salt thereof, is selected from the following compounds:
Compound 39: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4,5,6,7 tetrahydrobenzo[b]thiophen-2-yl) nicotinamide;
Compound 40: -rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]- 4,5,6,7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide; Compound 41 : -N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4, 5,6, 7- tetrahydro-1-benzothiophen-2-yl} pyridine-3-carboxamide;
Compound 42: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 43: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 44: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 45: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
Compound 46: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 47: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 49: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
Compound 50: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-6- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; Compound 51 : -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-7- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 52: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-5- hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamidel; and
Compound 53: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.
6. The method of claim 3, wherein the compound or a pharmaceutically acceptable salt thereof, is selected from the following compounds:
Figure imgf000105_0001
N-(3-(2-methylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[b]thiophen-2- yl)nicotinamide
Figure imgf000106_0001
7V-(3-((27?,47?)-2,4-dimethylpiperidine- l-carbonyl)-4, 5,6,7- tetrahydrobenzo [6] th iophcn-2- yl)nicotinamide
Figure imgf000106_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
Figure imgf000107_0001
N-(3 -(3 -ethylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[6]thiophen-2- yl)nicotinamide
Figure imgf000107_0002
7V-benzyl-2-(2-(( 1 -methyl- l /¥-imidazol- 2-yl)thio)acetamido)-4,5,6,7- tetrahy drobenzo [6] thiophene-3 - carboxamide
Figure imgf000107_0003
N-(3 -(3 -(hydroxymethyl)pyrrolidine- 1 - carbonyl)-4, 5,6,7- tetrahy drobenzo [/?] thi opbcri-2- yl)nicotinamide
Figure imgf000108_0001
Figure imgf000109_0002
7V-(3-benzoyl-4, 5,6,7- tetrahydrobenzo [ b\ thiophen-2-yl)- 5 - methyl-2- (methylsulfonyl)pyrimidine-4- carboxamide
Figure imgf000109_0003
V-(3 -((( 1 -methyl- 177-1 ,2,4-triazol-3 - yl)methyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo [/?] th i ophen-2 - l i i id
Figure imgf000109_0001
V-(6,6-dimethyl-3-(morpholine-4-carbonyl)-
4,5,6,7-tetrahydrobenzo[6]thiophen-2- yl)pyrazine-2-carboxamide
7. The method of claim 3, wherein the compound of structural Formula I is selected from the group consisting of:
Figure imgf000110_0001
N-benzyl-2-(2-(4-methylpipera zin-lyl)acetamido)-4.5.6,7- tetialiydrobenzo[b]thiophene-3- caiboxamide
Figure imgf000110_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[6]thiophen-2-yl)nicotinamide
Figure imgf000111_0001
N-(3 -(3 -ethylpyrrolidine- 1 -carbonyl)- 4,5,6,7-tetrahydrobenzo[6]thiophen-2- yl)nicotinamide
Figure imgf000111_0002
7V-benzyl-2-(2-(( 1 -methyl- l /¥-imidazol- 2-yl)thio)acetamido)-4,5,6,7- tetrahy drobenzo [6] thiophene-3 - carboxamide
Figure imgf000111_0003
N-(3 -(3 -(hydroxymethyl)pyrrolidine- 1 - carbonyl)-4, 5,6,7- tetrahy drobenzo [/?] thi opbcri-2- yl)nicotinamide
Figure imgf000112_0001
Figure imgf000113_0001
7V-(3-benzoyl-4, 5,6,7- tetrahydrobenzo [ b\ thiophen-2-yl)- 5 - methyl-2- (methylsulfonyl)pyrimidine-4- carboxamide
Figure imgf000113_0003
N-(3 -((( 1 -methyl- 1 H- 1 ,2,4-triazol-3 - yl)methyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo [/?] th i ophen-2 - yl)nicotinamide
Figure imgf000113_0002
?/-(6,6-dimethyl-3-(morpholine-4-carbonyl)-
4,5,6,7-tetrahydrobenzo[6]thiophen-2- yl)pyrazine-2-carboxamide Compound 1 : N-(3-(4-benzylpiperazine-1-carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)-2 -fluorobenzamide;
Compound 2: N-(3-(benzylcarbamoyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5- chloro-2-(methylthio)pyrimidine-4-carboxamide;
Compound 3: 5-chloro-N-(3-{[(1 ,1-dioxidotetrahydro-3-thienyl)amino]-carbonyl}-6- methyl-4,5,6,7-tetrahydro-1-benzothien-2-yl)-2-(methylthio)-4-pyrimidine- carboxamide;
Compound 4: N-(6-ethyl-3-{[(2-methylphenyl)amino]carbonyl}-4,5,6,7-tetrahydro- 1 -benzothien-2-yl)-1 -methyl-1 H-pyrazole-5-carboxamide;
Compound 5: N-{6-tert-butyl-3-[(4-methyl-1 -piperazinyl)carbonyl]-4,5,6,7- tetrahydro-1-benzothien-2-yl}-2 -fluorobenzamide;
Compound 6: 2-fluoro-N-{6-methyl-3-[(4-methyl-1 -piperazinyl)carbonyl]-4, 5,6,7- tetrahydro-1-benzothien-2-yl}benzamide;
Compound 7: N-benzyl-2-[(trifluoroacetyl)amino]-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide;
Compound 8: N-benzyl-2-({[(4-methyl-2-pyrimidinyl)thio]acetyl}amino)-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
Compound 9: N-benzyl-2-[(1 -piperidinylacetyl)amino]-4,5,6,7-tetrahydro-1 - benzothiophene-3-carboxamide;
Compound 10: N-benzyl-2-{[(4-methyl-1 -piperazinyl)acetyl]amino}-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
Compound 11 : N-benzyl-2-{[(4-methyl-1-piperidinyl)acetyl]amino}-4, 5,6,7- tetrahydro-1-benzothiophene-3-carboxamide;
Compound 12: N-(3-(2-methylpyrrolidine-1 -carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide; Compound 13: N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 14: N-(3-(((1 r,4r)-4-hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 15: N-(3-(3-ethylpyrrolidine-1 -carbonyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;
Compound 16: N-benzyl-2-(2-((1-methyl-1 H-imidazol-2-yl)thio)acetamido)- 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide;
Compound 17: N-(3-(3-(hydroxymethyl)pyrrolidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 18: N-(6,6-dimethyl-3-(morpholine-4-carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2 -carboxamide;
Compound 19: N-(3-(((1-methyl-1 H-1 ,2,4-triazol-3-yl)methyl)carbamoyl)-4,5,6,7- tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 20: (S)-N-(3-((1 -cyanoethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide; and
Compound 21 : N-(3-((piperidin-4-ylmethyl)carbamoyl)-4,5,6,7-tetrahydro- benzo[b]thiophen-2-yl)nicotinamide;N-(3-benzoyl-4, 5, 6, 7- tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2 -carboxamide; and
Compound 22: N-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)pyrazine-2- carboxamide;
Compound 23: N-(3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-5-methyl-2- (methylsulfonyl)pyrimidine-4-carboxamide;
Compound 24: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl) nicotinamide; Compound 25: rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]-4, 5,6,7- tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
Compound 26: N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4,5,6,7-tetrahydro-1- benzothiophen-2-yl} pyridine-3-carboxamide;
Compound 27: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1-carbonyl)-6-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 28: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 29: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 30: rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide; compound 31 : N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-6-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 32: N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 33: N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 34: N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
Compound 35: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-6-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 36: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-7-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 37: N-(3-((2S,4S)-2,4-dimethylpiperidine-1-carbonyl)-5-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; Compound 38: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
Compound 39: N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4,5,6,7 tetrahydrobenzo[b]thiophen-2-yl) nicotinamide;
Compound 40: -rac-N-{3-[(2R,4R)-2,4- dimethylpiperidine-1 -carbonyl]-4, 5,6,7- tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
Compound 41 : -N-{3-[cis-2,4-dimethylpiperidine-1 -carbonyl]- 4,5,6,7-tetrahydro-1 - benzothiophen-2-yl} pyridine-3-carboxamide;
Compound 42: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-6-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 43: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 44: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 45: -rac-N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
Compound 46: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-6-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 47: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-7-hydroxy-
4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 48: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 49: -N-(3-((2R,4R)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide;
Compound 50: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-6-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide; Compound 51 : -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-7-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamide;
Compound 52: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-5-hydroxy-
4.5.6.7-tetrahydrobenzo[b]thiophen-2-yl)nicotinamidel; and
Compound 53: -N-(3-((2S,4S)-2,4-dimethylpiperidine-1 -carbonyl)-4, 5,6,7- tetrahydrobenzo[b]thiophen-2-yl)-6-hydroxynicotinamide.
N-(3-benzoyl-4, 5, 6, 7 -tetrahydrobenzo[b]thiophen-2-yl)-5-methyl-2
(methylsulfonyl)pyrimidine-4-carboxamide.
N-(4-ethylphenyl)-3-(hydroxymethyl)-N-isobutyl-4-((tetrahydro-2H-pyran-4- yl)methoxy)benzenesulfonamide;
(7S)-N-{[5-(Ethylsulfonyl)-2-pyridinyl]methyl}-7-isopropyl-6-{[trans-4- (trifluoromethyl)cyclohexyl]methyl}-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3- carboxamide;
N-{3-[(3-methylbut-2-en-1-yl){methyl[trans-4-(pyridin-4- yl)cyclohexyl]carbamoyl}amino]phenyl}benzamide;
2-(1 -(2,4-dichloro-3-((7-chloro-5-(trifluoromethyl)-1 H-indol-1 - yl)methyl)benzoyl)piperidin-4-yl)acetic acid;
(S)-2-(4-cyclopropyl-6-methylpyrimidin-5-yl)-8-(1-cyclopropylethyl)-6-(((5- (methylsulfonyl)pyridin-2-yl)methyl)amino)pteridin-7(8H)-one;
(S)-6-(2,6-dimethylpyrimidin-4-yl)-N-(4-(ethylsulfonyl)benzyl)-6-methyl-5-oxo-
5.6.7.8-tetrahydroquinoline-2-carboxamide;
2-(2-((S)-(3,5-dimethylisoxazol-4-yl)(hydroxy)methyl)benzofuran-5-yl)-N-((S)-(2,4- dimethylphenyl)(phenyl)methyl)acetamide;
N-(4-(1 ,1 ,1 ,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-N-(2,2,2- trifluoroethyl)benzenesulfonamide; 1 ,1 ,1 ,3,3,3-hexafluoro-2-(2-fluoro-4'-((4-(pyridin-4-ylmethyl)piperazin-1- yl)methyl)-[1 , 1 '-biphenyl]-4-yl)propan-2-ol;
(1 R,3aS,5aR,5bR,7aR,9S,11 aR,11 bR,13aR,13bR)-N-(3-(dimethylamino)propyl)- 9-hydroxy-5a,5b,8,8, 11 a-pentamethyl-1 -(prop-1 -en-2-yl)icosahydro-3aH- cyclopenta[a]chrysene-3a-carboxam ide; and
Figure imgf000119_0001
or a pharmaceutically acceptable salts thereof, and stereoisomers thereof.
8. The method of claim 3, wherein the compound or a pharmaceutically acceptable salt thereof, is selected from the following compounds
Figure imgf000119_0002
Figure imgf000120_0001
JV-(3-((2R ,4R )-2,4-dimethylpiperidine-
1 -carbonyl)-4, 5,6,7- tetrahydrobenzo [ /?] thiophen-2 -
Figure imgf000120_0002
7V-(3-(((lr,4r)-4- hydroxycyclohexyl)carbamoyl)-4, 5,6,7- tetrahydrobenzo[6]thiophen-2-yl)nicotinamide or a pharmaceutically acceptable salts thereof, and stereoisomers thereof.
9. The method of any one of claims 1 to 7, wherein the fibrosis is associated withLiver diseases, Liver Failure, Acute- and-Chronic Liver Failure, Chemical and Drug Induced Liver Injury, Liver Fibrosis, Fatty Liver, viral Hepatitis, Non-viral hepatitis, Liver diseases, Liver Failure, Hemangioma of liver, Non-alcoholic Fatty Liver Disease, Acute-On-Chronic Liver Failure, Liver diseases, Liver neoplasms, Chemical and Drug Induced Liver Injury, Alcoholic Liver Diseases, Liver and Intrahepatic Biliary Tract Carcinoma, Chronic Hepatitis, Autoimmune hepatitis, Metabolic dysfunction-Associated Fatty Liver Disease’ (MAFLD), alcohol induced liver conditions, metabolic associated steatohepatitis (MASH), non-alcoholic steatohepatitis (NASH), liver fibrosis, liver failure, non-alcoholic fatty liver disease (NAFLD), alcohol-driven liver hepatitis, liver cirrhosis, hepatocellular carcinoma and liver cancer.
10. A pharmaceutical composition for treatment of fibrosis comprising an inverse agonist of RORs and a pharmaceutically acceptable carrier.
11. A pharmaceutical composition comprising:
(1 ) a first compound chosen from an inverse agonist of RORs or a pharmaceutically acceptable salt thereof;
(2) one or more additional compounds selected from the group consisting of:
(a) a GLP-1 analog;
(b) Ozempic/Liraglutide;
(c) Resmetirom and
(3) a pharmaceutically acceptable carrier.
12. A method of treatment of a vascular injury or protecting against a vascular injury by reducing circulating levels of IL-17, which comprises administering to a patient in need thereof an inverse agonist of RORs.
13. The method of claim 12, wherein the injury is a brain injury.
PCT/CA2024/051468 2023-11-07 2024-11-06 Method of treatment of fibrosis with inverse agonist of rar related orphan receptors (rors) Pending WO2025097247A1 (en)

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