Pyridine sulfonamide compounds for treating interleukin 1β-related disorders

Abstract

The present invention relates to a new class of pyridine-sulfonamide compounds and compositions containing the same. The compounds and compositions of the present invention can be used as drugs for treating diseases that respond to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF).

Description

Technical Field

[0001] This invention relates to a new class of pyridine-sulfonamide compounds and compositions comprising them. The compounds and compositions of this invention (e.g., pharmaceutical compositions) can be used as remedies for treating interleukin-1β (IL-1β)-related diseases such as inflammatory and fibrotic diseases. Background Art

[0002] IL-1 has been shown to be associated with a wide range of human pathologies, from autoinflammatory diseases to rheumatoid arthritis. IL-1 blockers (IL-1Ra, Anakinra; anti-IL-1b monoclonal antibody [mAb], Canakinumab; and anti-IL-1a, MABp1) have been approved for clinical use or are being evaluated in some of these diseases (Dinarello, 2009; Gabay et al., 2010; Garlanda et al., 2013; Udalova et al., 2016).

[0003] IL-1 has long been associated with inflammation and innate immunity. It is now clear that this cytokine plays diverse roles in shaping and directing innate immunity and inflammation in response to different microbial or environmental challenges. Furthermore, preclinical studies on the role of IL-1β have, over the past decade, extended beyond classical inflammation to understand its role in immunopathology, fibrotic diseases, degenerative diseases, cardiovascular diseases, and cancer. In addition, clinical studies on the effects of blocking IL-1 (Aaron et al., 2018; Trankle CR et al., 2018; Ridker PM., 2018; Ridker PM., 2017) have shown that in over 10,000 patients, blocking IL-1 not only prevented atherosclerosis-driven cardiovascular death but also prevented a range of diseases, including lung cancer, osteoarthritis, and gout. This finding reveals the diversity and commonalities of disease mechanisms, suggesting that IL-1 represents a paradigm of inflammation and immunity and a promising drug target (Montovani et al., 2019).

[0004] The production and subsequent secretion of IL-1β primarily depend on the activation of Toll-like receptor 4 (TLR4) and the inflammasome. First, inflammatory stimuli or infection signal via the TLR4 receptor, triggering the production of pro-IL-1β. Second, caspase-1 activated by the inflammasome releases IL-1β into the bloodstream via proteolytic activity. This cytokine is responsible for important functions such as T cell activation and antigen recognition. Interestingly, IL-1β production depends on the action of mitogen-activated protein kinases (MAPKs), a group of proteins in the TLR4 receptor signaling pathway. MAPKs (e.g., p38, JNK, and ERK) activate nuclear factors that bind to promoters of genes associated with IL-1β production. Therefore, inhibiting MAPKs is an effective way to prevent IL-1β production in the context of inflammatory diseases and conditions.

[0005] Nonalcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF) are particularly important among IL-1β-driven inflammatory diseases and conditions. These diseases represent unmet clinical needs, with only a few treatment options primarily focused on symptomatic relief.

[0006] Nonalcoholic steatohepatitis (NASH)

[0007] In particular, non-alcoholic fatty liver disease (NAFLD) is a common liver disease that exhibits histological features of alcohol-induced fatty liver disease in individuals who drink little or no alcohol (Yeh M et al., 2007; Marchesini G et al., 2003). NAFLD is caused by abnormal retention of intracellular lipids (often defined as steatosis), an event that is more common in the liver because the organ is primarily responsible for lipid metabolism. NAFLD has a histological profile that includes hepatic steatosis and non-alcoholic steatohepatitis (NASH), characterized by liver inflammation, steatosis, necrosis, and fibrosis due to hepatocyte destruction.

[0008] Liver imaging systems can also be used to assess liver structure and the presence of steatosis. However, liver biopsy remains the gold standard for assessing liver fibrosis, but due to its invasiveness, this analytical approach is not suitable for every study. Non-invasive assessments of liver biochemistry and metabolism are commonly used to define liver diseases such as NAFLD and NASH (Gressner A et al., 2009, World J Gastroenterol; 15:2433-2440; Vuppalanchi R and Chalasani N, 2009, Hepatology; 49:306-317). In addition to serum glucose and insulin resistance parameters, plasma is typically used to measure the presence of high levels of enzymes (e.g., alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (AP), and / or gamma-glutamyl transferase (GGT)) and other liver-derived proteins (including haptoglobin, total bilirubin, α-2-microglobulin, resistin, and cleaved or intact cytokeratin-18).

[0009] Effective treatments for liver fibrosis, particularly NAFLD and NASH, remain inadequate. No treatment has been established for NASH patients, although several treatment options have been tested in clinical trials (Vuppalanchi R and Chalasani N, 2009, Hepatology; 49:306-317; Dowman JK et al., 2009, QJ Med; 103:71-83). These studies have involved the use of many different classes of chemical compounds (fibrates, thiazolidinediones, biguanides, statins, cannabinoids) and therapeutic targets (nuclear receptors, angiotensin receptors, cannabinoid receptors, HMG-CoA reductase).

[0010] It has been shown that c-Jun expression is associated with disease progression from steatosis to NASH (Schulien et al., 2019, Cell Death & Differentiation; 26; 1688-1699). It has also been shown that JNK1 knockout mice are resistant to diet-induced steatohepatitis and liver fibrosis, and that JNK1 promotes the development of liver fibrosis by inducing chronic inflammation (Kodama et al., 2009, Gastroenterology; 137(4); 1467-1477).

[0011] Mouse animal models have been developed as preclinical in vivo models of NASH (Hansen H et al., 2017, DrugDiscoveR). yToday, 22:1707-1718). C57BL / 6 mice are the most commonly used because they are sensitive to a high-fat diet and exhibit many of the same symptoms observed in human NASH. Furthermore, it is known that injecting streptozotocin into a mouse model of NASH development susceptes it to a high-fat diet.

[0012] Idiopathic pulmonary fibrosis (IPF)

[0013] Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation and subsequent progressive scarring of the lungs. Interstitial lung disease (ILD) is a heterogeneous group of solid lung diseases characterized by varying degrees of inflammation and fibrosis. Some may be secondary to known precipitating factors, such as medications, autoimmune connective tissue diseases, hypersensitivity to inhaled organic antigens, or sarcoidosis, while others, namely idiopathic interstitial pneumonia (IIP), have no identifiable cause. Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of IIP, characterized by chronic, progressive fibrosis associated with an uncontrollable decline in lung function, progressive respiratory failure, and high mortality (Shaney et al., 2018).

[0014] In recent years, two novel antifibrotic therapies, pirfenidone and nintedanib, have been developed, providing treatment for many IPF patients. Unfortunately, their properties as disease modulators are poor, necessitating new treatment options.

[0015] Elevated IL-1β levels are known to contribute to a pro-inflammatory and pro-fibrotic environment in the lungs of patients with idiopathic pulmonary fibrosis (IPF) (Barlo et al., 2011). In preclinical studies, animal models of pulmonary fibrosis have shown that transient IL-1β expression induces acute lung injury and chronic repair, leading to pulmonary fibrosis (Kolb M., 2001; Gasse P et al., 2011). Furthermore, bleomycin-induced IPF lung injury models have extensively demonstrated the role of IL-1β in pulmonary fibrosis (Hoshino et al., 2009; Burgy et al., 2016).

[0016] The role of MAPK in IPF has been a focus of animal and human studies, with findings showing a significant increase in activated MAPK in lung homogenates from IPF patients compared to controls (Yoshida et al., 2002). Furthermore, JNK inhibition reduces systemic markers of lung remodeling and pulmonary fibrosis in IPF animal models (Van del Velden JL et al., 2016). IPF findings point to activated MAPK, and subsequently, the production of IL-1β, as one of the major drivers of IPF occurrence and progression.

[0017] There remains a clear and urgent need for new treatment options for managing liver diseases and other fibrotic conditions, particularly those involving liver and lung fibrosis. Summary of the Invention

[0018] This invention provides a new class of compounds having formula I and / or formula Ia, said compounds comprising their pharmaceutically acceptable salts. The compounds of this invention are intended to treat diseases responsive to inhibition of IL-1β, such as nonalcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF).

[0019] The compounds of this invention are compounds of formula I or pharmaceutically acceptable salts thereof:

[0020] Formula I

[0021]

[0022] in

[0023] R 1 It is an aromatic or heteroaromatic ring system, wherein the aromatic or heteroaromatic ring system is optionally substituted with one or two substituents selected from the group consisting of: C 1-4 Alkyl-Y 1 -、C 2-4 alkenyl-Y 1 -、C 2-4 alkynyl-Y 1 - C substituted with halogens such as fluorine 1-4 Alkyl-Y 1 - C replaced by halogens 2-4 alkenyl-Y 1 - C replaced by halogens 2-4 alkynyl-Y 1 HO-C 1-4 Alkyl-Y 1 -、HO-C 2-4 alkendiyl-Y 1 -、HO-C 2-4 ynedi-Y 1 -、HO-C 1-4 Alkyl-, HO-C 2-4 alkenadiyl-, HO-C 2-4 yndiyl-, C 1-4 Alkyl-C 2-4 alkenyl-, C 2-4 Alkyne-, halogenated C 1-4 Alkyl groups, halogenated C 2-4 Alkenyl-, halogenated C 2-4 Alkynes and halogens;

[0024] Y 1Choose the group consisting of O, S, NH, C(O), C(O)O, C(O)NH, O(CO) and NHC(O);

[0025] X 1 It is NH, O, or CH2;

[0026] n1 is either 0 or 1;

[0027] X 3 Not present or NR y ;

[0028] R x and R y Independently C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkyne group or H, such as CH3 or H;

[0029] L is O, S, S(O), S(O)2, NH, C(O) or CH2;

[0030] Z 1 , Z 2 and Z 3 Independently selected from N and CH;

[0031] R 3 Choose from the following groups: H, halogens, C(O)NR 2a R 2b 、C(O)OR 2a , OR 2a NR 2a R 2b 、OC(O)R 2a NR 2a C(O)R 2b C that is optionally substituted by one or more halogens 1-4 Alkyl groups, C substituted with one or more halogens (optionally) 2-4 Alkenyl groups and C groups optionally substituted with one or more halogens 2-4 alkynyl group, wherein R 2a and R 2b Independently select H and C 1-4 Alkyl, C 2-4 alkenyl and C 2-4 A group composed of alkynyl groups.

[0032] In another respect, the present invention relates to pharmaceutical compositions comprising compounds according to the invention and pharmaceutically acceptable excipients or carriers.

[0033] On the other hand, the present invention relates to compounds or compositions according to the invention used as pharmaceuticals.

[0034] In another aspect, the present invention relates to compounds or compositions according to the invention for the treatment of diseases that respond to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF). Attached Figure Description

[0035] Figure 1 Effects of compounds 5 and 7 on NAFLD activity score (NAS).

[0036] Figure 2 Serum AST (U / L) levels 28 days after treatment.

[0037] Figure 3 Effects of compounds 5 and 7 on liver body weight ratio.

[0038] Figure 4 The effect of compound 7 on pulmonary hydroxyproline levels in the treatment of bleomycin-induced IPF.

[0039] Figure 5A Total cell count in BALF.

[0040] Figure 5B White blood cell differential count in BALF.

[0041] Figure 6A H&E Ashcroft rating.

[0042] Figure 6B .% Collagen percentage area (CPA). DETAILED DESCRIPTION

[0043] definition

[0044] In this article, the term "C" 1-4 "Alkyl" is intended to represent a straight-chain or branched hydrocarbon group having 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0045] Similarly, the term "C" 2-4 The term "alkenyl" is intended to encompass straight-chain or branched hydrocarbon groups having 2 to 4 carbon atoms and containing double bonds. Examples of alkenyl groups are vinyl, allyl, and butenyl. Preferred examples of alkenyl groups are vinyl and allyl, especially allyl.

[0046] In the context, the term "C" 2-4 "Alkyne" is intended to represent a straight-chain or branched hydrocarbon group having 2 to 4 carbon atoms and containing a triple bond. 2-4Illustrative examples of alkynyl groups include acetylene, propynyl, butynyl, and their branched forms. The unsaturated (triple bond) position can be anywhere along the carbon chain. More than one bond can be unsaturated, in which case "C 2-4 "Alkyne" is a diyne known to those skilled in the art.

[0047] In this document, the term "halo / halogen" includes fluorine, chlorine, bromine, and iodine, and more specifically, fluorine, chlorine, and bromine.

[0048] In this context, the term "aromatic ring or ring system" is intended to refer to a complete or partial aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracene, phenanthryl, pyrene, benzo[a]pyrene, fluorenyl, and xanthonyl.

[0049] The term "heteroaromatic ring or cyclic system" is intended to refer to a complete or partial aromatic carbon ring or cyclic system in which one or more carbon atoms have been replaced by heteroatoms such as nitrogen (=N- or -NH-), sulfur, and / or oxygen atoms. Examples of such heteroaromatic ring or cyclic system groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrroleyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, triazinyl, coumarinyl, furanyl, thiopheneyl, quinolinyl, benzothiazolyl, benzotriazolyl, benzooxazolyl, phthalazinyl, phthaloyl, triazolyl, tetrazolyl, isoquinolinyl, acridineyl, carbazoleyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl, benzofuranyl, dihydrobenzofuranyl, and benzodioxazolyl.

[0050] In this context, the term "heterocyclic or cyclic system" is intended to refer to a non-aromatic carbon ring or cyclic system in which one or more carbon atoms have been replaced by heteroatoms such as nitrogen (=N- or -NH-), sulfur, and / or oxygen. Examples of such heterocyclic groups are: imidazolides, piperazines, hexahydropyridazines, hexahydropyrimidines, diazacycloheptanes, diazacyclooctanes, pyrrolidines, piperidines, azacycloheptanes, azacyclooctanes, acridines, aziridines, acridines, pyroline, scopolanes, oxazines (morpholine), aziridines, dihydroaziridines, tetrahydroaziridines, hexahydroaziridines, oxazolane, oxazolane, oxazolidinanes, oxazolidinanes, oxazolidinanes, thiazolides. olane), thiazinane, thiazine, thiazetane, oxazetane, diazetane, thiazetane, tetrahydrofuran, tetrahydropyran, oxepane, tetrahydrothiophene, tetrahydrothiaran, thiazetane, dithiazine, dithiazetane, dioxane, dioxane, oxathiane, and oxathiane.

[0051] In this context, the term "optionally substituted" is intended to mean that the group in question can be substituted once or more, preferably once or twice. Furthermore, the term "optionally substituted" can also mean that the group in question is not substituted.

[0052] The compounds of the present invention may be in free form or in pharmaceutically acceptable salt form. In the context of the present invention, the term "pharmaceutically acceptable salt" should be understood as a salt formed with a base or acid, wherein the resulting counterion does not significantly increase the toxicity of the compounds of the present invention.

[0053] Examples of pharmaceutically acceptable salts include: inorganic acid salts, such as hydrochlorides, sulfates, nitrates, phosphates, or hydrobroms; and organic acid salts, such as acetates, fumarates, oxalates, citrates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, or maleates. Additionally, when a compound has a substituent such as a carboxyl group, salts formed with a base (e.g., alkali metal salts such as sodium or potassium salts, or alkaline earth metal salts such as calcium salts) may be mentioned.

[0054] Compound

[0055] The compounds of this invention are compounds of formula I or pharmaceutically acceptable salts thereof:

[0056] Formula I

[0057]

[0058] in

[0059] R 1 It is an aromatic or heteroaromatic ring system, wherein the aromatic or heteroaromatic ring system is optionally substituted with one or two substituents selected from the group consisting of: C 1-4 Alkyl-Y 1 -、C 2-4 alkenyl-Y 1 -、C 2-4 alkynyl-Y 1 - C substituted with halogens such as fluorine 1-4 Alkyl-Y 1 - C replaced by halogens 2-4 alkenyl-Y 1 - C replaced by halogens 2-4 alkynyl-Y 1 HO-C 1-4 Alkyl-Y 1 -、HO-C 2-4 alkendiyl-Y 1 -、HO-C 2-4 ynedi-Y 1 -、HO-C 1-4 Alkyl-, HO-C 2-4 alkenadiyl-, HO-C 2-4 yndiyl-, C 1-4 Alkyl-C 2-4 alkenyl-, C 2-4 Alkyne-, halogenated C 1-4 Alkyl groups, halogenated C 2-4 Alkenyl-, halogenated C 2-4 Alkynes and halogens;

[0060] Y 1 Choose the group consisting of O, S, NH, C(O), C(O)O, C(O)NH, O(CO) and NHC(O);

[0061] X 1 It is NH, O, or CH2;

[0062] n1 is either 0 or 1;

[0063] X 3 Not present or NR y ;

[0064] R x and R y Independently C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkyne group or H, such as CH3 or H;

[0065] L is O, S, S(O), S(O)2, NH, C(O) or CH2;

[0066] Z 1 , Z 2 and Z 3 Independently selected from N and CH;

[0067] R 3 Choose from the following groups: H, halogens, C(O)NR 2a R 2b 、C(O)OR 2a , OR 2a NR 2a R 2b 、OC(O)R 2a NR 2a C(O)R 2b C that is optionally substituted by one or more halogens 1-4 Alkyl groups, C substituted with one or more halogens (optionally) 2-4 Alkenyl groups and C groups optionally substituted with one or more halogens 2-4 alkynyl group, wherein R 2a and R 2b Independently select H and C 1-4 Alkyl, C 2-4 alkenyl and C 2-4 A group composed of alkynyl groups.

[0068] In one embodiment, the compound of formula I is a compound having formula Ia:

[0069]

[0070] in

[0071] R 1 Selected from the group consisting of: C 1-4 Alkyl-Y 1 -、C 2-4 alkenyl-Y 1 -、C 2-4 alkynyl-Y 1 - C substituted with halogens such as fluorine 1-4 Alkyl-Y 1 - C replaced by halogens 2-4 alkenyl-Y 1 - C replaced by halogens 2-4 alkynyl-Y 1 HO-C 1-4 Alkyl-Y 1 -、HO-C 2-4 alkendiyl-Y 1 -、HO-C 2-4 ynedi-Y 1-、HO-C 1-4 Alkyl-, HO-C 2-4 alkenadiyl-, HO-C 2-4 yndiyl-, C 1-4 Alkyl-C 2-4 alkenyl-, C 2-4 Alkyne-, halogenated C 1-4 Alkyl groups, halogenated C 2-4 Alkenyl-, halogenated C 2-4 Alkynes and halogens;

[0072] R 2 It is hydrogen; or

[0073] R 1 With R 2 Together they form an aromatic, heteroaromatic, cyclic, or heterocyclic five- or six-membered ring, wherein the five- or six-membered ring is optionally substituted with one or two substituents selected from the group consisting of: C 1-4 Alkyl-Y 1 -、C 2-4 alkenyl-Y 1 -、C 2-4 alkynyl-Y 1 - C substituted with halogens such as fluorine 1-4 Alkyl-Y 1 - C replaced by halogens 2-4 alkenyl-Y 1 - C replaced by halogens 2-4 alkynyl-Y 1 HO-C 1-4 Alkyl-, HO-C 2-4 alkenadiyl-, HO-C 2-4 yndiyl-, C 1-4 Alkyl-C 2-4 alkenyl-, C 2-4 Alkyne-, halogenated C 1-4 Alkyl groups, halogenated C 2-4 Alkenyl-, halogenated C 2-4 Alkynes and halogens; and

[0074] X 1 n1, Y 1 、X 3 、R x 、R y L, Z 1 , Z 2 , Z 3 、R 3 As defined for compounds of formula I.

[0075] In another embodiment, in compounds of formula I and formula Ia, X3 It is NR y Compounds of formula II and IIa were obtained:

[0076] Formula II

[0077]

[0078] Formula IIa

[0079]

[0080] X 1 n1, Y 1 、R x 、R y L, Z 1 , Z 2 , Z 3 、R 1 、R 2 、R 3 As defined for compounds of formula I and Ia. In yet another embodiment, in compounds of formula II and IIa, R 3 At the para position, compounds of formula III and IIIa are obtained: Formula III

[0081]

[0082] Formula IIIa

[0083]

[0084] X 1 n1, Y 1 、R x 、R y L, Z 1 , Z 2 , Z 3 、R 1 、R 2 、R 3 As defined for compounds of formula I and Ia. In yet another embodiment, in compounds of formula III and IIIa, Z 2 CH, yielding compounds of formula IV and IVa: Formula IV

[0085]

[0086] Formula IVa

[0087]

[0088] X 1 n1, Y 1 、R x 、R y L, Z1 , Z 3 、R 1 、R 2 、R 3 As defined for compounds of formula I and Ia.

[0089] In one embodiment of the compound according to the invention, Y 1 The group consisting of O, S, NH, C(O), and C(O)NH is selected. In another embodiment, Y 1 The group consisting of O, S, and NH is selected. In another embodiment, Y... 1 It is O.

[0090] In another embodiment of the compound according to the invention, X 1 It is NH or O. In yet another embodiment, X 1 It is NH or O, and n1 is 1.

[0091] In another embodiment of the compound according to the invention, R x and R y Independently C 1-4 Alkyl group. In yet another embodiment, R x and R y It is a methyl group.

[0092] In one embodiment of the compound according to the invention, L is selected from the group consisting of O, S, and CH2. In another embodiment, L is selected from the group consisting of O and CH2. In yet another embodiment, L is O.

[0093] In another embodiment of the compound according to the invention, Z 1 It is CH. In another implementation, Z 1 It is CH, and L is O. In another embodiment, Z 1 It is CH, Z 2 It is CH, and L is O.

[0094] In another embodiment of the compound according to the invention, R 3 The group consisting of H, halogens, and C(O)NH2 is selected. In another embodiment, R 3 The group consisting of free halogens and C(O)NH2 is selected. In another embodiment, R 3 The group consisting of Cl and C(O)NH2 is selected. In a further embodiment, R 3 It is Cl.

[0095] In one embodiment of the compound according to the invention having formula Ia, IIa, IIIa or IVa, R 2 It’s H.

[0096] In another embodiment, the compound of formula IVa has the following formula V:

[0097]

[0098] X 1 、Y 1 、R x 、R y 、R 1 and R 3 As defined for compounds of formula I and Ia. In another embodiment of compound V, X 1 It is O, R 1 It is C 1-4 Alkyl-O-,R 3 It is a halogen or C(O)NH2, and R x and R y Independently C 1-4 alkyl.

[0099] In the presently preferred embodiments, the compounds according to the invention are selected from the group consisting of:

[0100]

[0101] pharmaceutical preparations

[0102] The compounds of this invention are intended for use as pharmaceuticals. While the compounds can be used alone in principle, they are preferably formulated with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are inert carriers suitable for each method of administration and can be formulated into conventional pharmaceutical dosage forms (tablets, granules, capsules, powders, solutions, suspensions, emulsions, injections, infusions, etc.). Examples of such carriers include, for instance, pharmaceutically acceptable binders, excipients, lubricants, disintegrants, etc. When used as injection or infusion solutions, they can be formulated using distilled water for injection, physiological saline, or glucose aqueous solution.

[0103] There are no particular limitations on the administration method of the compounds of the present invention, and conventional oral or parenteral administration methods (intravenous, intramuscular, subcutaneous, transdermal, intranasal, transmucosal, enteric, etc.) can be used.

[0104] The dosage of the tetrahydroisoquinoline derivative of the present invention or its pharmaceutically acceptable salt thereof may be optionally set within an effective range sufficient to exhibit pharmacological effects, depending on the potency or characteristics of the compound used as the active ingredient. The dosage may vary depending on the method of administration, the patient's age, weight, or condition.

[0105] Drug efficacy

[0106] The compounds of the present invention are intended to treat diseases that respond to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF). Therefore, in one aspect, the present invention relates to compounds or compositions according to the present invention for use as medicaments. In another aspect, the present invention relates to compounds or compositions according to the present invention for treating diseases that respond to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF). In one embodiment, the disease responding to inhibition of IL-1β is selected from the group consisting of non-alcoholic steatohepatitis (NASH), idiopathic pulmonary fibrosis (IPF), autoinflammatory diseases, cardiovascular diseases, osteoarthritis, lung cancer, and gout. In a further embodiment, the disease is NASH. In yet another embodiment, the disease is IPF.

[0107] Preparation of compounds – X 1 NR y

[0108] The substituted pyridine-sulfonamides of formulas II and IIa of the present invention are typically prepared via intermediate C, which is prepared as described in scheme 1:

[0109]

[0110] Solution 1

[0111] In the first step, 5,6-dichloropyridine-3-sulfonamide A was alkylated to give intermediate compound B, which was then converted to compound C by the Buchwald reaction under microwave irradiation.

[0112] According to R x 、R y and R 3 Due to the properties of the intermediate C, the preparation of the intermediate may require additional synthetic transformations, such as the protection / deprotection reaction described in Scheme 1.

[0113] Depends on X 1 The compounds of the present invention can be prepared according to schemes 2b, 2c or 2d, depending on their presence and / or identity.

[0114]

[0115] Synthesis of compounds of type 2b–IIb

[0116] Under microwave irradiation, intermediate compound C reacts with the corresponding boric acid at 150 °C via a Pd-catalyzed Suzuki reaction to generate type IIb compounds (e.g., compound 1; synthetic procedure as shown in Bioorg. Med. Chem. Lett. 2011, 21(10), 3152-3158).

[0117]

[0118] Synthesis of compounds of type 2c–IIc

[0119] In the presence of a strong base, intermediate compound C reacts with substituted aniline at 40 °C via a Pd-catalyzed Buchwald reaction to generate type IIc compounds (such as compounds 2, 3, 4, and 6; the synthetic procedure can be found in Org. Lett. 2011, 13(8), 1984-1987).

[0120]

[0121] Synthesis of Scheme 2d–IId type compounds

[0122] Under microwave irradiation, in the presence of sodium hydride and cesium carbonate, intermediate C was reacted with the corresponding phenols via an Ullman-type reaction to obtain IId-type compounds (such as compounds 5, 7, 8, 9, 10, and 12) (the synthetic procedure can be found in J. Am. Chem. Soc. 2013, 135(24), 9213-9219).

[0123] Preparation of compounds – X 3 does not exist

[0124] The substituted pyridine-sulfonamides of formulas I and Ia of the present invention (wherein X) 3 (Not present) is typically prepared via intermediate E, which is prepared as described in Scheme 3:

[0125]

[0126] Scheme 3 – Synthesis procedure for compound E

[0127] The preparation procedure is similar to that of intermediate compound C, except that a D-type compound is used as the starting material, such as 2,3-dichloro-5-(methylsulfonyl)pyridine in the case of synthesizing the final compound 11.

[0128] Example

[0129] Example 1 – Synthesis of intermediate B, wherein R x and R y Methyl: N,N-dimethyl-5,6-dichloropyridine-3-sulfonamide

[0130] A solution of iodomethane (2 eq) in DMF (5 ml / mmol) was added to a suspension of commercially available 5,6-dichloropyridine-3-sulfonamide A (1 eq) and K₂CO₃ (2 eq) in anhydrous DMF (1 ml / eq), and the resulting mixture was stirred at room temperature until the reaction was complete (overnight, TLC control). The solvent was then evaporated and ethyl acetate was added. The organic mixture was washed twice with water, and the organic layer was dried over magnesium sulfate. After solvent evaporation, the resulting crude product was sufficiently pure to be used in Example 2 without further purification (yellow solid, 95% yield).

[0131] Example 2 – Synthesis of intermediate C, wherein R x and R y For methyl, Z 1 , Z 2 , Z 3 CH is given, L is given, and R is given. 3 For the opposite position Cl: N,N-Dimethyl-5-chloro-6-(4-(4-chlorophenoxy)-N-piperidinyl)-pyridine-3-sulfonamide

[0132] The compound obtained in Example 1 (1 eq), commercially available 4-(4-chlorophenoxy)piperidine hydrochloride (1.2 eq), and TEA (2.2 eq) in EtOH (4.8 mL / mmol) were first heated at 80 °C for 5 min, followed by microwave irradiation (200 W) at 120 °C for 30 min (HPLC control). The solvent was then evaporated and ethyl acetate was added; the organic mixture was washed twice with water, and the combined organic layers were dried with magnesium sulfate. After solvent evaporation, the resulting crude product was sufficiently pure to be used in subsequent examples without further purification (clear brown solid, 91% yield).

[0133] Example 3 – Synthesis of intermediate C, wherein R x and R y For methyl, Z 1 , Z 3 For CH, Z 2 Let N be an integer, L be an integer, and R be an integer. 3 For located on C(O)NH2 at position: N,N-dimethyl-5-chloro-6-[4-(5-[2-carboxamidopyridinyl]oxy)-N'-piperidinyl]-pyridine-3- sulfonamide

[0134] The compound obtained in Example 1 (1 eq), commercially available 4-(piperidin-4-yloxy)pyridine-2-carboxamide (1.2 eq), and TEA (2.2 eq) in EtOH (4.8 mL / mmol) were first heated at 80 °C for 5 min, followed by microwave irradiation (200 W) at 120 °C for 30 min (HPLC control). The solvent was then evaporated and ethyl acetate was added; the organic mixture was washed twice with water, and the combined organic layers were dried with magnesium sulfate. After solvent evaporation, the resulting crude product was sufficiently pure to be used in the next step without further purification (clear white solid, 93% yield).

[0135] Example 4 – Synthesis of Compound 1

[0136] The intermediate obtained in Example 2 (1 eq.), 2,3-dihydrobenzo[b]furan-5-boric acid (1.1 eq), potassium carbonate (1.4 eq), and Pd(PhPh3)4 (0.1 eq) in DME (5 mL / mmol) were heated at 150 °C for 1 hour (or overnight) under microwave irradiation (200 W). The crude mixture was then diluted with EtOAc, filtered through a diatomaceous earth stopper, washed with EtOAc and MeOH, and the solvent was evaporated. The resulting crude product was purified using an Isolera Biotage system (C18, acetonitrile / water) to obtain a pure product. The product was white foam, with an overall yield of 45% and an HPLC purity of 98%.

[0137] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.51 (d, 1H), 7.68 (d, 1H), 7.33 (s, 1H), 7.27 (s, 1H), 7.22 (d, 2H), 6.86-6.81 (m, 3H), 4.64 (t, 2 H),4.42-4.38(m,2H),3.61-3.55(m,2H),3.27(t,2H),3.19-3.18(m,2H),2.76(s,6H),1.94-1.89(m,2H),1.76-1.70(m,2H).C 26 H 29 ClN3O4S

[0138] MS (electrospray): m / z = 514.1583 (M+1).

[0139] Example 5 – Synthesis of Compound 2

[0140] In a screw-capped test tube equipped with a magnetic stir bar, BrettPhos precatalyst (4 mol%), 2,3-dihydrobenzo[b]furan-5-amine (1 eq.), and the intermediate compound obtained in Example 2 (1 eq.) were loaded. The vial was sealed with a PTFE screw cap, evacuated, and backfilled with nitrogen; this process was repeated twice. Then, LiHMDS (1 M, in THF, 2.5 eq.) was added. The reaction mixture was stirred at 40 °C until the reaction was complete (2.5 h). The solution was cooled to room temperature, quenched by adding saturated NH4Cl aqueous solution (5 mL), and then diluted with EtOAc. The organic phase was separated, and the aqueous phase was extracted again with EtOAc. The combined organic phases were washed with brine and dried over MgSO4. The solvent was removed under reduced pressure, and the crude product was purified by an Isolera Biotage System (C18, acetonitrile / water) to give an impure product (115.7 mg), which was purified again by the same system. Brown solid, with an overall yield of 6% and an HPLC purity of 96% (254 nm).

[0141] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.34 (s, 1H), 7, 96 (bs, 1H), 7.24 (dd, 2H), 7.03 (s, 1H), 6.96 (dd, 1H), 6.85-6.79 (m, 3H), 5.88 (s,1H),4.63(t,2H),4.48(m,1H),3.76-3.73(m,2H),3.45(m,2H),3.23(t,2H),2.80(s,6H),1.99-1.94(m,2H),1.82(m,2H).C 26 H 30 ClN4O4S

[0142] MS (electrospray): m / z = 529.1677 (M+1).

[0143] Example 6 – Synthesis of Compound 5

[0144] 2,3-Dihydro-5-hydroxybenzo[b]furan (1.1 eq) and cesium carbonate (1.2 eq) were added to anhydrous DMF (1.2 mL / mmol) and treated with pure sodium hydride (1.1 eq). After hydrogen evolution stopped, the intermediate compound obtained in Example 2 (1 eq) was added and the reaction was stirred at 150 °C under microwave irradiation (200 W) for 2 hours in a sealed pressure tube until no further reaction occurred. The solvent was then removed under vacuum, and the crude product was diluted with ethyl acetate (6 mL / mmol) and water (6 mL / mmol). The aqueous layer was extracted three times with ethyl acetate. The organic layer was washed with 2N NaOH, dried, and concentrated under vacuum. The crude product was purified by an Isolera Biotage system (C18, acetonitrile-water gradient) to obtain an impurity, which was purified by preparative chromatography (silica, hexane / ethyl acetate 4:1). The clarified oil had an overall yield of 12% and an HPLC purity of 100% (254 nm).

[0145] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.30 (dd, 1H), 7.247-7.22 (m, 3H), 6.86-6.81 (m, 3H), 6.75-6.70 (m, 2H), 4.61 (t, 2H), 4. 50-4.47(m,1H),4.00-3.97(m,2H),3.66-3.63(m,2H),3.21(t,2H),2.67(s,6H),2-06-1.99(m,2H),1.90-1.84(m,2H).C 26 H 28 ClN3O5S

[0146] MS (electrospray): m / z = 530.1550 (M+1).

[0147] Example 7 – Synthesis of Compound 7

[0148] 4-Ethoxyphenol (1.1 eq) and cesium carbonate (1.2 eq) were dissolved in anhydrous DMF (1.2 mL / mmol) and treated with pure sodium hydride (1.1 eq). After hydrogen evolution stopped, the intermediate obtained in Example 2 (1 eq) was added, and the reaction was stirred at 150 °C under microwave irradiation (200 W) for 2 hours in a sealed pressure tube until no further reaction occurred. The solvent was then removed under vacuum, and the crude product was diluted with ethyl acetate (6 mL / mmol) and water (6 mL / mmol). The aqueous layer was extracted three times with ethyl acetate. The organic layer was washed with 2N NaOH, dried, and concentrated under vacuum. The crude product was purified using an Isolera Biotage system (C18, acetonitrile-water gradient) to recover unreacted starting material and pure product. A clear brown solid was obtained in 41% yield with HPLC purity of 100% (254 nm).

[0149] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.31 (d, 1H), 7.26-7.23 (m, 4H), 6.88-6.84 (m, 5H), 4.51-4.46 (1H, m), 4.05-4.00(m,4H),3.66-3.59(m,2H),2.67(s,6H),2.00-1.86(m,2H),1.86-1.83(m,2H),.43(t,3H).C 26 H 30 ClO5S

[0150] MS (electrospray): m / z = 532.1680 (M+1).

[0151] Example 8 – Synthesis of Compound 8

[0152] 4-Ethoxyphenol (1.1 eq) and cesium carbonate (1.2 eq) were dissolved in anhydrous DMF (1.2 mL / mmol) and treated with pure sodium hydride (1.1 eq). After hydrogen evolution stopped, the intermediate obtained in Example 3 (1 eq) was added, and the reaction was carried out in a sealed pressure tube (overnight) or under microwave irradiation (200 W) at 150 °C with stirring until no further reaction occurred. The solvent was then removed under vacuum, and the crude product was diluted with ethyl acetate (6 mL / mmol) and water (6 mL / mmol). The aqueous layer was extracted three times with ethyl acetate. The organic layer was washed with 2N NaOH, dried, and concentrated under vacuum. The crude product was purified using an Isolera Biotage system (C18, acetonitrile-water gradient) to recover unreacted starting material and pure product. A clear brown solid was obtained in 16% yield with HPLC purity of 100% (254 nm).

[0153] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.38 (d, 1H), 8.34 (s, 1H), 6.98-6.97 (m, 1H), 6.89 (s, 1H), 5.59 (bs, 1H), 4.79-4. 75(m,1H),4.06-3.63(m,4H),3.69-3.63(m,2H),2.68(s,6H),2-10-2.06(m,2H),1.92-1.88(m,2H),1.44(t,3H).C 26 H 31 N5O6S

[0154] MS (electrospray): m / z = 542.2084 (M+1).

[0155] Example 9 – Synthesis of Compound 12

[0156] 2,3-Dihydro-5-hydroxybenzo[b]furan (1.1 eq) and cesium carbonate (1.2 eq) were added to anhydrous DMF (1.2 mL / mmol) and treated with pure sodium hydride (1.1 eq). After hydrogen evolution stopped, the intermediate obtained in Example 3 (1 eq) was added and the reaction was carried out in a sealed pressure tube (overnight) or under microwave irradiation (200 W) (2.5 h) at 150 °C with stirring until no further reaction occurred. The solvent was then removed under vacuum, and the crude product was diluted with ethyl acetate (6 mL / mmol) and water (6 mL / mmol). The aqueous layer was extracted three times with ethyl acetate. The organic layer was washed with 2N NaOH, dried, and concentrated under vacuum. The crude product was purified by an Isolera Biotage system (C18, acetonitrile-water gradient) to recover unreacted starting material and pure product. White foam, overall yield 13%, HPLC purity 98% (254 nm).

[0157] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.38 (d, 1H), 8.31 (d, 1H), 7.24 (d, 1H), 6.97-6.96 (m, 1H), 6.82 (d, 1H), 6.76-6.69 (m, 2H), 5.63 (bs, 1H) ,4.79-4.76(m,2H),4.62(t,2H),4.02-4.00(m,2H),3.68-3.63(m,2H),3.22(t,2H),2.68(s,6H),2.12-2.08(m,2H),1.93-1.89(m,2H).C 26 H 29 N5O6SMS (electrospray): m / z = 540.1944 (M+1).

[0158] Example 10 – Synthesis of intermediate E, wherein R x For methyl, Z 1 , Z 2 , Z 3 CH is given, L is given, and R is given. 3 For Cl located in the para position: 3-Chloro-2-[4-(4-chlorophenoxy)-N-piperidinyl]-5-methylsulfonyl-pyridine

[0159] The synthesis procedure was similar to that of the intermediate compound according to Example 2, except that commercially available 2,3-dichloro-5-(methylsulfonyl)pyridine was used as the starting material. White solid, overall yield 96%.

[0160] Example 11 – Synthesis of Compound 11

[0161] The synthetic procedure was similar to that of compounds 5, 7, 8, and 12 in Examples 6 through 9, except that the intermediate compound obtained in Example 10 was used as the starting material. 6 hours. Clarified oil, with an overall yield of 16% and an HPLC purity of 95% (254 nm).

[0162] 1HNMR (CDCl3, 400MHz): δ (ppm) = 8.45 (s, 1H), 7.35 (d, 1H), 7.26 (dd, 2H), 6.85 (d, 2H), 4.52-4.48 (m, 1H) ,4.04-4.00(m,4H),3.72-3.66(m,2H),3.01(s,3H),2.05-1.99(m,2H),1.90-1.84(m,2H),1.43(t,3H).C 25 H 28 ClN2O5S

[0163] MS (electrospray): m / z = 503.1398 (M+1).

[0164] Example 12 – In vitro inhibition of IL-1β in LPS-attacked human primary macrophages

[0165] Material

[0166]

[0167] Preparation of RPMI 1640 growth medium

[0168] Prepare RPMI 1640 basal medium according to the manufacturer's instructions in the datasheet. Perform a sterility check at 48 hours using 5 mL of the medium in a 37°C incubator with a continuous 5% CO2 supply. After the sterility check, complete the basal medium by adding FBS and Pen / Strep antibiotics. Store the medium at 4°C for later use.

[0169] Preparation of phosphate-buffered saline (PBS)

[0170] Dissolve one packet of PBS in 1 liter of Milli-Q water. Filter the PBS through a 0.22 μm filter membrane and store at 4°C for later use.

[0171] Preparation of peripheral blood mononuclear cells (PBMCs)

[0172] Add 5 mL of serum-free RPMI medium (1:1) to 5 mL of human whole blood sample in EDTA or sodium citrate and mix by inversion. Add 3 mL of Histopaque-1077 to a 15 mL conical centrifuge tube and allow it to reach room temperature. Using a pipette, carefully spread 10 mL of the blood-RPMI mixture onto the Histopaque-1077 and centrifuge at 400 x g for 30 minutes at room temperature.

[0173] After centrifugation, aspirate the supernatant using a Pasteur pipette until it reaches within 0.5 cm of the opaque interface containing mononuclear cells. Discard the supernatant. Using a Pasteur pipette, carefully transfer the opaque interface to a clean conical centrifuge tube. Add 5 mL of RPMI to the tube and mix by inverting, then centrifuge at 250 x g for exactly 10 minutes. Aspirate and discard the supernatant.

[0174] Resuspend the leukocyte clumps in 5 mL of RPMI and gently mix with a Pasteur pipette, then centrifuge at 250 x g for exactly 10 minutes.

[0175] The pellets were washed three times with PBS and resuspended in RPMI medium.

[0176] The number of live PBMCs per mL was counted.

[0177] PBMC count

[0178] Dilute 10 μL of PBMC suspension with 90 μL of PBS medium (1:10 dilution). Add 20 μL of cell suspension to 20 μL of trypan blue solution (1:1 ratio), mixing carefully to avoid aerosol formation. Load the cell culture mixture into a hemocytometer until the area under the coverslip is fully filled. Allow the suspension to settle in the hemocytometer for at least 10 seconds before counting.

[0179] Live cells and dead cells are counted at the four 1mm square corners of a small chamber. Live PBMCs are transparent; non-viable PBMCs are blue. Cells touching the top line or vertical circumference of any corner square are also included. Cells touching the bottom line or right vertical circumference of any corner square are not counted.

[0180] Cell count calculation

[0181] Calculation of the number of live PBMCs per mL

[0182] PBMC / mL = PBMC in all four squares × 10 × 2 × 10 4 / 4

[0183] 10 4 =Conversion factor for 1 mL volume

[0184] 10 = Dilution factor of cell suspension;

[0185] 2 = the dilution factor of trypan blue

[0186] Total cell count = PBMCs / mL × Total volume of PBMC suspension (mL)

[0187] % cell viability = (Number of live cells / Total number of cells (live cells + dead cells)) × 100

[0188] Cell seeding, differentiation and treatment

[0189] 2×10 5 One PBMC was seeded into a 96-well plate at a total volume of 200 μL / well and incubated at 37°C in a CO2 incubator for 4 hours to allow monocytes to settle, leaving lymphocytes in suspension. After incubation, 100 μL was aspirated from each well to ensure removal of the lymphocyte population.

[0190] Monocytes were differentiated into macrophages by adding 200 ng / mL recombinant human GM-CSF (4 mg / mL stock solution) and incubated at 37°C / 5% CO2 for 6 days. The culture medium was changed every two days by removing half the volume of medium from the wells and replenishing with fresh RPMI complete medium and recombinant human GM-CSF.

[0191] After differentiation, cells were treated with two portions of the compound according to the invention, each containing 50 μL, maintaining a final DMSO percentile of 0.5%. The plates were then transferred to an incubator maintained at 37°C / 5% CO2 for 2 hours. Cells were stimulated with 100 ng / mL LPS (4X) (1 mg / mL stock solution) (total volume 50 μL).

[0192] Transfer the plate to an incubator maintained at 37°C / 5% CO2 for 16 hours.

[0193] Assessing cytokines using ELISA

[0194] 1. Aspirate each well of the pre-coated ELISA plate and wash with wash buffer (0.05% Tween 20 in PBS; pH 7.2–7.4) by filling each well with wash buffer (300 μL). Repeat this process twice, for a total of three washes. Completely remove liquid at each step. After the final wash, remove any remaining wash buffer.

[0195] 2. Block the plate by adding 300 μL of blocking buffer (1% BSA in PBS) to each well and incubate at room temperature for 1 hour.

[0196] 3. Repeat the vacuuming / washing process in step 1.

[0197] 4. Add 100 μL of the prepared sample or standard to reagent diluent (0.1% BSA, 0.05% Tween 20 in PBS, pH 7.2-7.4). Cover the wells with adhesive tape and incubate at room temperature for 2 hours.

[0198] 5. Repeat the vacuuming / washing process in step 1.

[0199] 6. Add 100 μL of the detection antibody diluted in the reagent diluent to each well. Cover the wells with new adhesive tape and incubate at room temperature for 2 hours.

[0200] 7. Repeat the vacuuming / washing process in step 1.

[0201] 8. Add 100 μL of working dilution (diluted 1:200 from stock solution) and streptavidin-HRP to each well. Cover the plate and incubate at room temperature for 20 minutes.

[0202] 9. Add 100 μL of substrate solution (a 1:1 mixture of H2O2 and tetramethylbenzidine) to each well and incubate at room temperature for 20 minutes.

[0203] 10. Add 50 μL of stop solution (2N H2SO4) to each well. Gently tap the plate to ensure thorough mixing.

[0204] 11. Immediately determine the optical density of each well using a microplate reader (Spectramax Plus) set to 450 nm.

[0205] Cytokine activity assessment

[0206] 100 μl / well volume The luminescent reagent was added to the assay plate and incubated for 30 minutes at room temperature on a plate shaker. After incubation, the luminescence signal data for each well were determined using a microplate reader (Perkin Elmer ENVISION 2104).

[0207] The inhibition percentage of the test compound was determined using the following formula:

[0208] % Inhibition = 100 - (100 * (Average test compound count - Average negative control count) / (Average positive control count - Average negative control count))

[0209] All “counts” mentioned in this formula are derived from the “optical density values” determined as described above using an ELISA reader (step 11).

[0210] The cytotoxicity of the test compound is determined using the following formula:

[0211] %Cytotoxicity = 100 - (100 * (mean test compound count - mean negative control count) / (mean positive control count - mean negative control count))

[0212] All “counts” mentioned in the formula are derived from “luminescent signals” as determined in the cytokine activity assessment.

[0213] result

[0214] IC50 (IL-1β inhibition) Cell viability Compound 1 115nM 100％ Compound 2 29nM 100％ Compound 3 121nM 100％ Compound 4 300nM 100％ Compound 5 28nM 100％ Compound 6 13nM 100％ Compound 7 27nM 100％ Compound 8 337nM 100％ Compound 9 531nM 100％ Compound 10 410nM 100％ Compound 11 753nM 100％ Compound 12 2.3 μM 100％

[0215] Example 13 – In vitro binding of JNK1 (MAPK8)

[0216] At 40% humidity, various concentrations of recombinant human MAPK8 dissolved in water were manually stained onto bare gold-plated (47 nm thick) PlexArray nanocapture sensor chips (Plexera Bioscience, Seattle, WA, USA). Each concentration was stained repeatedly, with each spot containing 0.2 μL of protein solution. The chips were incubated overnight at 4°C with 80% humidity, and then rinsed with 10×PBST for 10 min, 1×PBST for 10 min, and deionized water twice for 10 min. Then, prior to use, the chips were blocked overnight in 5% (w / v) skim milk in water, washed with 10×PBST for 10 min, 1×PBST for 10 min, and deionized water twice for 10 min, and then dried under a nitrogen stream.

[0217] Surface plasmon resonance imaging (SPRi) measurements were performed using a PlexAray HT (Plexera Bioscience, Seattle, WA, USA). Collimated light (660 nm) passed through a coupling prism, was reflected from the SPR active gold surface, and received by a CCD camera. Buffer and sample were injected into a 30 μL flowcell mounted on the coupling prism using a non-pulsating piston pump. Each measurement cycle consisted of four steps: washing with PBST run buffer at a constant rate of 2 μL / sec to obtain a stable baseline, injecting sample at 5 μL / sec for binding, washing the surface with PBST at 2 μL / sec for 300 sec, and regenerating with 0.5% (v / v) H3PO4 at 2 μL / sec for 300 sec. All measurements were performed at 4 °C.

[0218] Signal changes after binding and washing were recorded as measurements in AU. Selected protein transplantation regions in SPR images were analyzed, and the average reflectance changes of selected regions were plotted as a function of time. Real-time binding signals were recorded and analyzed using the data analysis module (DAM, Plexera Bioscience, Seattle, Washington, USA). Kinetic analysis was performed using BIAevaluation 4.1 software (Biacore, Inc.).

[0219] The equilibrium dissociation constant (KD value) determined for compound 7 is 3.41 × 10⁻⁶. -8 M. (Ka = 2.29 × 10⁻⁶) 4 M -1 ·s -1 Kd = 7.82 × 10 -4 s -1 )

[0220] Example 14 – In vitro binding of p38MAPK

[0221] At 40% humidity, various concentrations of p38 MAPK dissolved in water were manually imprinted onto bare gold-plated (47 nm thick) PlexArray nanocapture sensor chips (Plexera Bioscience, Seattle, WA, USA). Each concentration was imprinted repeatedly, with each spot containing 0.2 μL of protein solution. The chips were incubated overnight at 4°C with 80% humidity, and then rinsed with 10×PBST for 10 min, 1×PBST for 10 min, and deionized water twice for 10 min. Then, prior to use, the chips were blocked overnight in 5% (w / v) skim milk in water, washed with 10×PBST for 10 min, 1×PBST for 10 min, and deionized water twice for 10 min, and then dried under a nitrogen stream.

[0222] Surface plasmon resonance imaging (SPRi) measurements were performed using a PlexAray HT (Plexera Bioscience, Seattle, WA, USA). Collimated light (660 nm) passed through a coupling prism, was reflected from the SPR active gold surface, and received by a CCD camera. Buffer and sample were injected into a 30 μL flowcell mounted on the coupling prism using a non-pulsating piston pump. Each measurement cycle consisted of four steps: washing with PBST run buffer at a constant rate of 2 μL / sec to obtain a stable baseline, injecting sample at 5 μL / sec for binding, washing the surface with PBST at 2 μL / sec for 300 sec, and regenerating with 0.5% (v / v) H3PO4 at 2 μL / sec for 300 sec. All measurements were performed at 4 °C.

[0223] Signal changes after binding and washing were recorded as measurements in AU. Selected protein transplantation regions in SPR images were analyzed, and the average reflectance changes of selected regions were plotted as a function of time. Real-time binding signals were recorded and analyzed using the data analysis module (DAM, Plexera Bioscience, Seattle, Washington, USA). Kinetic analysis was performed using BIAevaluation 4.1 software (Biacore, Inc.).

[0224] The equilibrium dissociation constant (KD value) determined for compound 7 is 1.19 × 10⁻⁶. -8 M. (Ka = 2.08 × 10⁻⁶) 4 M -1 ·s -1 Kd = 2.48 × 10 -4 s -1 )

[0225] Example 15: The effects of compounds 5 and 7 in the treatment of non-alcoholic steatohepatitis (NASH) in male C57BL / 6 mice effect

[0226] NASH inducement

[0227] Time-pregnant mice (n=30) were selected for this study. Pups were injected subcutaneously with 200 μg streptozotocin on day 2 postnatal day (PND-2) and remained with their mothers until weaning age. After weaning, male pups were selected and fed a 60% kcal fat diet (Research Diet-D12492) for the next two weeks. Clinical signs in all animals were observed twice daily.

[0228] Research Procedures

[0229] From day 0 to day 28, mice were administered the solvent, test compound, and reference compound (elafibranor) twice daily, once in the morning (9:00 a.m.) and once in the evening before the start of the dark cycle (6:00 p.m.).

[0230] Animal weight was measured daily throughout the experiment.

[0231] The test compound and the reference compound were administered to animals for 28 days (from week 6 to week 10).

[0232] Blood glucose levels were assessed before treatment began and on day 28 after treatment ended. Serum ALT and AST levels were measured from plasma before treatment began and on day 28.

[0233] Histopathological analysis of liver tissue was performed, including H&E staining, Masson trichrome staining, and Oil Red-O (ORO) staining.

[0234] Sample analysis

[0235] NAFLD (Non-Alcoholic Fatty Liver Disease) score: H&E staining

[0236] All H&E-stained tissue sections were examined by optical microscopy. Nafantiosis, lobular inflammation, and hepatic ballooning were scored using a grading system (Kleiner et al., 2005) under a 200X objective.

[0237] Collagen percentage area (%CPA) measurement: Masson trichrome staining

[0238] All Masson trichrome stained tissue sections were examined under a 100X objective using an optical microscope. Collagen proportion area was measured using ImagePro Premier 9.1 software, with five randomly selected fields of view (approximately 684.85 μm × 917.11 μm) from each liver section. The collagen proportion area percentage was calculated by dividing the collagen tissue area by the total tissue area.

[0239] Measurement of staining area percentage: Oil Red O staining

[0240] All Oil Red O stained tissue sections were examined under a 100X objective using an optical microscope. Five fields of view (approximately 688.33 μm × 922.45 μm each) were randomly selected from each liver section using Image ProPremier 9.1 software for staining area measurement. The percentage of stained area was calculated by dividing the lipid-stained tissue area by the total tissue area.

[0241] Figure 1 The effects of compounds 5 and 7 on the NAFLD activity score (NAS) were shown.

[0242] This study clearly demonstrates that a 3 mg / kg dose of compound 7 significantly reduces steatosis and lobular inflammation in the liver and shows a better NAS score than efrenolin.

[0243] Compound 5 at a dose of 3 mg / kg significantly reduced lobular inflammation and showed a trend toward reduced steatosis and hepatic ballooning degeneration, resulting in a significantly lower NAS score compared to disease controls.

[0244] Compounds 5 and 7 showed significantly reduced AST levels after 28 days of treatment, such as Figure 2 As shown.

[0245] Unlike the reference compound (efellano), neither of the two test compounds (compounds 5 and 7) showed any increase in hepatomegaly or liver-to-body weight ratio, such as Figure 3 As shown.

[0246] In summary, the tested compounds significantly improved the hepatic chemical and histological activity of NASH. The therapeutic properties of compounds 5 and 7 suggest their potential for treating human NASH. All treated animals showed weight gain and no clinical signs of toxicity or side effects.

[0247] Example 16: The role of compound 7 in the treatment of bleomycin-induced IPF

[0248] IPF-induced

[0249] Time-pregnant mice (n=30) were selected for this study. Pups were injected subcutaneously with 1.5 U / kg bleomycin on day 2 postnatal day (PND-2) and remained with their mothers until weaning age. After weaning, male pups were selected and fed a 60% kcal fat diet (Research Diet-D12492) for the following week. Clinical signs in all animals were observed twice daily.

[0250] Research Procedures

[0251] From day 0 to day 14, mice were administered the solvent, test compound, and reference compound (pirfenidone) twice daily, once in the morning (9:00 a.m.) and once in the evening before the start of the dark cycle (6:00 p.m.).

[0252] Animal weight was measured daily throughout the experiment.

[0253] The test compound and the reference compound were administered to animals 14 days later.

[0254] Assess pulmonary hydroxyproline levels before treatment initiation and on day 14 after treatment termination. Measure total cell count and white blood cell differential count in BALF before treatment initiation and on day 28.

[0255] Histopathological analysis of lung tissue was performed using the H&E Ashcroft scoring method.

[0256] Sample analysis

[0257] IPF score: H&E staining

[0258] All H&E-stained tissue sections were examined by optical microscopy. IPF scores were performed under a 200X objective according to the following grading system (Kleiner et al., 2005).

[0259] Collagen percentage area (%CPA) measurement: Masson trichrome staining

[0260] All Masson trichrome stained tissue sections were examined under a 100X objective using an optical microscope. Collagen proportion area was measured using ImagePro Premier 9.1 software, with five randomly selected fields of view (approximately 684.85 μm × 917.11 μm) from each lung. The collagen proportion area percentage was calculated by dividing the collagen tissue area by the total tissue area.

[0261] The effect of compound 7 on pulmonary hydroxyproline levels is as follows: Figure 4 As shown.

[0262] Compared to the original control, the pulmonary hydroxyproline level was significantly increased in the bleomycin control. This study clearly demonstrates that compound 7 at a dose of 15 mg / kg significantly reduced pulmonary hydroxyproline levels, and the reduction was greater than that at 100 mg / kg pirfenidone.

[0263] In BALF, the total white blood cell count and white blood cell differential (macrophage and lymphocyte) counts were significantly increased in the bleomycin control group. Figure 5A and 5B As shown, treatment with compound 7 (p>0.05) and pirfenidone (p<0.05) reduced BALF macrophage counts compared to the bleomycin control.

[0264] Compared to the original control, the fibrosis score and %CPA of the lungs in the bleomycin control group were significantly increased. Figure 6A and 6B As shown, compared with the bleomycin control, lung samples treated with compound 7 (p<0.05) and pirfenidone (p<0.05) exhibited significantly lower fibrosis scores and %CPA. The data are consistent with the results for hydroxyproline.

[0265] In summary, the tested compounds significantly improved the pulmonary chemical and histological activity of IPF. The therapeutic properties of compound 7 suggest its potential for treating human IPF. Compared to the bleomycin control, all treated animals showed weight gain and no clinical signs of toxicity or side effects.

Claims

1. A compound of formula V or a pharmaceutically acceptable salt thereof: Formula V in R 1 Selected from the group consisting of: C 1-4 Alkyl-Y 1 -、C substituted by halogen 1-4 Alkyl-Y 1 -、HO-C 1-4 Alkanediyl-Y 1 -、HO-C 1-4 Alkanediyl-, C 1-4 Alkyl, halogen-substituted C 1-4 Alkyl and halogen; Y 1 is O; X 1 is NH, O or CH2; R x and R y Independently for C 1-4 Alkyl or H; R 3 Selected from the group consisting of: H, halogen and C(O)NR 2a R 2b , where R 2a and R 2b Independently selected from H and C 1-4 Alkyl group.

2. The compound according to claim 1, wherein C 1-4 Alkyl-Y 1 -substituted by fluorine.

3. The compound according to claim 1, wherein R x and R y are independently CH3 or H.

4. The compound according to claim 1, wherein X 1 is NH or O.

5. The compound according to claim 1, wherein R x and R y Independently for C 1-4 alkyl.

6. The compound according to claim 5, wherein R x and R y It is methyl.

7. The compound according to claim 1, wherein R 3 Selected from the group consisting of H, halogen and C(O)NH2.

8. The compound according to claim 7, wherein R 3 Selected from the group consisting of Cl and C(O)NH2.

9. A compound selected from the group consisting of:

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient or carrier.

11. Use of a compound according to any one of claims 1 to 9 or a composition according to claim 10 in the preparation of a medicament for treating non-alcoholic steatohepatitis, idiopathic pulmonary fibrosis, cardiovascular disease, osteoarthritis, lung cancer or gout.

12. Use of a compound according to any one of claims 1 to 9 or a composition according to claim 10 in the preparation of a medicament for treating an autoinflammatory disease.

13. The use according to claim 11, wherein The medicament is used to treat non-alcoholic steatohepatitis or idiopathic pulmonary fibrosis.

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