RU2803242C2 - Pyridine sulfonamide compounds for the treatment of conditions associated with interleukin 1 beta - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to new compounds of pyridinesulfonamide formula I

where R¹ is phenyl or a 9-membered fused heteroaromatic ring system containing either 1 or 2 O heteroatoms optionally substituted with one substituent selected from the group consisting of C_1-4alkyl, C_1-4alkyl-Y¹-, C_1-4alkyl-Y¹ substituted with halogen, HO-C_1-4 alkanediyl; Y¹ is O; X¹ represents either NH or O; n1 is either 0 or 1; X³ is either absent or is NR^y; R^x and R^y are independently C_1-4alkyl; L is either O or CH₂; Z¹ and Z³ are selected from N and CH, Z² is CH; R³ is selected from the group consisting of halogen, C(O)NR^2aR^2b where R^2a and R^2b are independently selected from the group consisting of H and C_1-4alkyl; compositions comprising these compounds and use in the treatment of diseases mediated by IL-1β.

EFFECT: obtaining new compounds that can be used as drugs for the treatment of diseases sensitive to IL-1 inhibition, βsuch as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF).

11 cl, 16 ex, 8 dwg

Description

Field of technology to which the invention relates

The present invention relates to a new class of pyridine sulfonamide compounds and to compositions comprising these compounds. The compounds and compositions (such as pharmaceutical compositions) of the present invention can be used as drugs in the treatment of diseases associated with interleukin 1 beta (IL-1β), such as inflammation and fibrosing diseases.

BACKGROUND OF THE INVENTION

IL-1 has been shown to be involved in a wide range of human pathologies from autoinflammatory diseases to rheumatoid arthritis; and IL-1 blocking agents (IL-1Ra, anakira; anti-IL-1b monoclonal antibody [mAb], scanakinumab; and anti-IL-1a monoclonal antibody, MABp1) have been approved for clinical use or are being tested for use in some of the of these disorders (Dinarello, 2009; Gabay et al., 2010; Garlanda et al., 2013; Udalova et al., 2016).

IL-1 has long been associated with inflammation and innate immunity. This cytokine is now known to play various roles in shaping and targeting innate immunity and inflammation in response to various microbiological pathogens or environmental challenges. Moreover, in the last decade, preclinical research on the role of IL-1 beta (IL-1β) has expanded beyond classical inflammation to identify its role in immunopathology, fibrosing disease, degenerative disease, cardiovascular disease, and cancer. In addition, clinical studies of the effect of blocking IL-1β (Aaron et al., 2018; Trankle C.R. et al., 2018; Ridker P.M., 2018; Ridker P.M., 2017) showed that in more than 10,000 patients, blocking IL-1 did not protect not only from cardiovascular death caused by atherosclerosis, but also from a number of diseases, including lung cancer, osteoarthritis and gout. This discovery, highlighting the diversity and at the same time commonality of disease mechanisms, has suggested that IL-1 represents a paradigm for inflammation and immunity, as well as a noteworthy drug target (Montovani et al., 2019).

The production and subsequent secretion of IL-1β depends primarily on the activation of toll-like receptor 4 (TLR4) and the inflammasome. In the first stage, inflammatory stimuli or infection transmitted through the TLR4 receptor trigger the production of pro-IL-1β; in the second step, caspartase-1, activated by inflammasomes, proteolytically releases IL-1β into the bloodstream. This cytokine, among other important functions, is responsible for the activation of T cells, as well as antigen recognition. Interestingly, IL-1β production is dependent on the action of mitogen-activated protein kinase (MAPK), a group of proteins within the TLR4 receptor signaling pathway. MAPKs such as p38, JNK, and ERK activate nuclear factors that bind to the promoters of genes involved in IL-1β production. Thus, MAPK inhibition is a reasonable approach to preventing IL-1β production in the context of the development of inflammatory diseases and conditions.

Among the inflammatory diseases and conditions caused by IL-1β, the most important are non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF). These diseases have an unmet clinical need, and there are only a few therapeutic approaches that focus primarily on symptomatic treatment.

Non-alcoholic steatohepatitis (NASH)

In particular, non-alcoholic fatty liver disease (NAFLD) is a common liver disease with histological evidence 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 lipids within cells (usually defined as steatosis), which most often occurs in the liver, since this organ is primarily responsible for lipid metabolism. NAFLD manifests itself in various histological forms, including hepatic steatosis and non-alcoholic steatohepatitis (NASH), which is characterized by inflammation, steatosis, necrosis and fibrosis of the liver due to the destruction of liver cells.

Various imaging systems are used to assess liver structure and the presence of steatosis. However, liver biopsy remains the gold standard for detecting fibrosis, although this test cannot be used in every single study due to its invasiveness. Non-invasive assessment of liver biochemistry and metabolism is often used to identify 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). When using blood plasma, in addition to serum glucose parameters and insulin resistance parameters, high levels of enzymes such as Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (AP) and/or gamma glutamyl transpeptidase (GGT), as well as the presence of other proteins of liver origin (including haptoglobin, total bilirubin, alpha-2-microglobulin, resistin, cleaved or intact cytokeratin-18).

Effective treatments for fibrosing liver diseases, in particular NAFLD and NASH, are still insufficient. Treatment for patients with NASH has not been developed, and several therapeutic options are being tested in clinical trials (Vuppalanchi R. and Chalasani N., 2009, Hepatology; 49: 306-317; Dowman J.K. et al., 2009, Q.J. Med.; 103: 71 -83). These trials include the use of many different families of chemical compounds (fibrates, thiazolidinediones, biguanides, statins, cannabinoids) and therapeutic targets (nuclear receptors, angiotensin receptors, cannabinoid receptors, HMG-CoA reductase).

c-Jun expression has been shown to be specifically 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 is involved in the development of liver fibrosis by inducing chronic inflammation (Kodama et al., 2009, Gastroenterology; 137(4); 1467-1477).

Mouse animal models have been developed as preclinical in vivo models of NASH (Hansen H. et al., 2017, Drug Discovery Today, 22: 1707-1718). The most commonly used mice are the C57BL/6 mice because they are sensitive to high-fat diets and show the development of symptoms that are also seen in humans with NASH. Injection of streptozotocin is also known to sensitize mouse models to a high-fat diet during the development of NASH.

Idiopathic pulmonary fibrosis (IPF)

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation and subsequent progressive scarring of the lungs. Interstitial lung diseases (ILDs) constitute a heterogeneous group of parenchymal lung diseases characterized by varying degrees of inflammation and fibrosis. Some may occur secondary to a known exacerbating factor such as drugs, autoimmune connective tissue disease, hypersensitivity to inhaled organic antigens, or sarcoidosis, while the causes of other idiopathic interstitial pneumonias (IIPs) have not been identified. Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of IIP, characterized by chronic progressive fibrosis associated with steady regression of lung function, progressive respiratory failure, and high mortality (Shaney et al., 2018).

In recent years, two new antifibrotic therapeutic agents, pirfenidone and nintedanib, have been developed, providing treatment to a large number of patients with IPF. Unfortunately, their profile as disease modifiers leaves much to be desired, and therefore there remains a need for new therapeutic agents.

Elevated levels of IL-1β are known to promote the formation of a proinflammatory and profibrotic environment in the lungs of patients suffering from idiopathic pulmonary fibrosis (IPF) (Barlo et al., 2011). Preclinical studies in animal models of pulmonary fibrosis have shown that transient expression of IL-1β causes acute lung injury and subsequent chronic healing, which leads to fibrosis (Kolb M., 2001; Gasse P. et al., 2011). In addition, a model of bleomycin-induced lung injury in IPF has convincingly demonstrated the role of IL-1β in pulmonary fibrosis (Hoshino et al., 2009; Burgy et al., 2016).

The role of MAPKs in IPF has also been the focus of animal and human studies, leading to the conclusion that activated MAPKs in lung homogenates from IPF patients are significantly elevated compared to controls (Yoshida et al., 2002). In addition, JNK inhibition reduces lung remodeling and systemic markers of fibrosis in animal models of IPF (Van del Velden J.L. et al., 2016). Results from studies of IPF point to activated MAPK and subsequent production of IL-1β as one of the main factors in the occurrence and development of IPF.

Thus, the need for new therapeutic options for the treatment of liver diseases and other fibrosing disorders, particularly those associated with liver and lung fibrosis, remains clear and urgent.

The essence of the invention

The present invention relates to a new class of compounds of formula I and/or formula Ia, which includes their pharmaceutically acceptable salts. The compounds of the present invention are intended for the treatment of diseases that are sensitive to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF).

The compounds of the present invention are compounds of formula I or pharmaceutically acceptable salts thereof:

formula I

Where

R ¹ is an aromatic or heteroaromatic ring system, 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 _1-4 alkyl-Y ¹ -, substituted with a halogen such as fluorine, C _2-4 alkenyl-Y ¹ -, substituted with a halogen, C _2-4 alkynyl-Y ¹ , substituted with a halogen, HO-C _{1- 4} alkanediyl-Y ¹ -, HO-C _2-4 alkendiyl-Y ¹ -, HO-C _2-4 alkendiyl-Y ¹ -, HO-C _1-4 alkanediyl-, HO-C _2-4 alkendiyl-, HO -C _2-4 alkyndiyl-, C _1-4 alkyl-C _2-4 alkenyl-, C _2-4 alkynyl-, C _1-4 alkyl substituted with halogen, C _2-4 alkenyl- substituted with halogen, C _{2- 4} alkynyl-substituted with halogen and halogen;

Y ¹ is selected from the group consisting of O, S, NH, C(O), C(O)O, C(O)NH, O(CO) and NHC(O);

X ¹ represents NH, O or CH ₂ ;

n1 is 0 or 1;

X ³ is absent or represents NR ^y ;

R ^x and R ^y independently represent C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl or H, in particular CH ₃ or H;

L is O, S, S(O), S(O) ₂ , NH, C(O) or CH ₂ ;

Z ¹ , Z ² and Z ³ are independently selected from N and CH;

R ³ is selected from the group consisting of H, halogen, 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 _1-4 alkyl, optionally substituted with one or more halogen atoms, C _2-4 alkenyl, optionally substituted with one or more halogen atoms, and C _2-4 alkynyl, optionally substituted with one or more halogen atoms, where R ^2a and R ^2b are independently selected from the group consisting of H, C _1-4 alkyl, C _2-4 alkenyl and C _2-4 alkynyl.

In another aspect, the present invention relates to a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient or carrier.

In another aspect, the present invention relates to a compound of the present invention or a composition of the present invention for use as a drug.

In yet another additional aspect, the present invention provides a compound of the present invention or a composition of the present invention for use in the treatment of diseases sensitive to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF).

Brief description of the figures

Figure 1 . Effect of compound 5 and compound 7 on the NAFLD activity score (NAS).

Figure 2 . Serum AST levels (U/L) after 28 days of treatment.

Figure 3 . Effect of compounds 5 and 7 on the ratio of liver weight to body weight.

Figure 4 . Effect of compound 7 on pulmonary hydroxyproline levels during treatment of bleomycin-induced IPF.

Figure 5A . Total number of cells in bronchoalveolar lavage fluid (BALF).

Figure 5B. Leukocyte formula BALF.

Figure 6A . Disease severity scoring using Ashcroft's hematoxylin and eosin stain (H&E Ashcroft).

Figure 6B . Collagen Proportion Area (CPA).

Detailed Description of the Invention

Definitions

In the context of the present invention, the term "C _1-4 alkyl" is intended to denote a linear or branched hydrocarbon group containing from 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

Likewise, the term "C _2-4 alkenyl" is intended to denote linear or branched hydrocarbon groups containing from 2 to 4 carbon atoms and including a double bond. Examples of alkenyl groups are vinyl, allyl and butenyl. Preferred examples of alkenyl are vinyl and allyl, especially allyl.

In the context of the present invention, the term “C _2-4 alkynyl” is intended to mean a straight or branched hydrocarbon group containing from 2 to 4 carbon atoms and including a triple bond. Illustrative examples of C _2-4 alkynyl groups include acetylene, propynyl, butynyl, as well as branched forms thereof. An unsaturation (triple bond) can occur anywhere along the carbon chain. Multiple bonds may be unsaturated, so that "C _2-4 alkynyl" is a diyne, as known to one skilled in the art.

As used herein, the term "halo" or "halogen" includes fluorine, chlorine, bromine and iodine, more specifically fluorine, chlorine and bromine.

In the context of the present invention, the term "aromatic ring or aromatic ring system" is intended to refer to a wholly or partially aromatic carbocyclic ring or aromatic ring system, for example phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl.

The term “heteroaromatic ring or heteroaromatic ring system” is intended to refer to a wholly or partially aromatic carbocyclic ring or aromatic ring system in which one or more carbon atoms are replaced by heteroatoms, for example nitrogen (=N- or -NH-), sulfur and/or oxygen. Examples of such heteroaromatic ring or heteroaromatic ring system groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, coumaryl, furyl, thienyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzooc salted, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl, benzofuranyl, dihydrobenzofuranyl and benzodioxolyl.

In the context of the present invention, the term “heterocyclic ring or heterocyclic ring system” is intended to mean a non-aromatic carbocyclic ring or non-aromatic carbocyclic ring system in which one or more carbon atoms are replaced by heteroatoms, for example nitrogen atoms (=N- or -NH-), sulfur and /or oxygen. Examples of such heterocyclic groups are imidazolidine, piperazine, hexahydropyridazine, hexahydropyrimidine, diazepane, diazocane, pyrrolidine, piperidine, azepane, azocane, aziridine, azirine, azetidine, pyroline, tropane, oxazinan (morpholine), azepine, dihydroazepine, tetrahydroazepine, hexahydroazepine , oxazolane, oxazepane, oxazocane, thiazolane, thiazinan, thiazepane, thiazocane, oxazetane, diazetane, thiazetane, tetrahydrofuran, tetrahydropyrane, oxepane, tetrahydrothiophene, tetrahydrothiopyrane, tiepane, dithiane, dithiepane, dioxane, dioxepane, oxathiane and oxathiepane.

In the context of the present invention, the term "optionally substituted" is intended to mean that the group in question may be substituted one or more times, preferably 1-2 times. In addition, the term "optionally substituted" can also mean that the group in question is unsubstituted.

The compounds of the present invention may be in free form or in pharmaceutically acceptable salt form. As used herein, the term "pharmaceutically acceptable salt" means a salt formed by a base or an acid in which the counterion formed does not add substantially to the toxicity of the compound of the present invention.

Examples of pharmaceutically acceptable salts include salts of inorganic acids such as hydrochloride, sulfate, nitrate, phosphate or hydrobromide, etc., salts of organic acids such as acetate, fumarate, oxalate, citrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate or maleate and etc. In addition, when the compound contains a substituent such as a carboxyl group, a salt with a base (for example, an alkali metal salt such as sodium salt, potassium salt, etc., or an alkaline earth metal salt such as calcium salt) may be specified etc.).

Connections

The compounds of the present invention are compounds of formula I or pharmaceutically acceptable salts thereof:

formula I

Where

R ¹ is an aromatic or heteroaromatic ring system, 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 _1-4 alkyl-Y ¹ -, substituted with a halogen such as fluorine, C _2-4 alkenyl-Y ¹ -, substituted with a halogen, C _2-4 alkynyl-Y ¹ , substituted with a halogen, HO-C _{1- 4} alkanediyl-Y ¹ -, HO-C _2-4 alkendiyl-Y ¹ -, HO-C _{2- 4} alkendiyl-Y ¹ -, HO-C _1-4 alkanediyl-, HO-C _2-4 alkendiyl-, HO -C _2-4 alkyndiyl-, C _1-4 alkyl- C _2-4 alkenyl-, C _2-4 alkynyl-, C _1-4 alkyl substituted with halogen, C _2-4 alkenyl- substituted with halogen, C _{2- 4} alkynyl-substituted with halogen and halogen;

Y ¹ is selected from the group consisting of O, S, NH, C(O), C(O)O, C(O)NH, O(CO) and NHC(O);

X ¹ represents NH, O or CH ₂ ;

n1 is 0 or 1;

X ³ is absent or represents NR ^y ;

R ^x and R ^y independently represent C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl or H, for example CH ₃ or H;

L is O, S, S(O), S(O) ₂ , NH, C(O) or CH ₂ ;

Z ¹ , Z ² and Z ³ are independently selected from N and CH;

R ³ is selected from the group consisting of H, halogen, 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 _1-4 alkyl, optionally substituted with one or more halogen atoms, C _2-4 alkenyl, optionally substituted with one or more halogen atoms, and C _2-4 alkynyl, optionally substituted with one or more halogen atoms, where R ^2a and R ^2b are independently selected from the group consisting of H, C _1-4 alkyl, C _2-4 alkenyl and C _2-4 alkynyl.

In one embodiment, the compound of formula I is a compound of formula Ia:

Where

R ¹ is 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 _1-4 alkyl-Y ¹ -, substituted with halogen , such as fluorine, C _2-4 alkenyl-Y ¹ -, substituted with halogen, C _2-4 alkynyl-Y ¹ , substituted with halogen, HO-C _1-4 alkanediyl-Y ¹ -, HO-C _2-4 alkendiyl- Y ¹ -, HO-C _2-4 alkyndiyl-Y ¹ -, HO-C _1-4 alkanediyl-, HO-C _2-4 alkendiyl-, HO-C _2-4 alkyndiyl-, C _1-4 alkyl-C _2-4 alkenyl-, C _2-4 alkynyl-, C _1-4 alkyl-substituted with halogen, C _2-4 alkenyl-substituted with halogen, C _2-4 alkynyl-substituted with halogen, and halogen;

R ² represents hydrogen; or

R ¹ together with R ² forms an aromatic, heteroaromatic, cyclic or heterocyclic five- or six-membered ring, 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 _1-4 alkyl-Y ¹ -, substituted with a halogen such as fluorine, C _2-4 alkenyl-Y ¹ -, substituted with a halogen, C _2-4 alkynyl -Y ¹ substituted with halogen, HO-C _1-4 alkanediyl-, HO-C _2-4 alkendiyl-, HO-C _2-4 alkyndiyl-, C _1-4 alkyl- C _2-4 alkenyl-, C _{2- 4} alkynyl-, C _1-4 alkyl-substituted with halogen, C _2-4 alkenyl-substituted with halogen, C _2-4 alkynyl-substituted with halogen, and halogen; And

X ¹ , n1, Y ¹ , X ³ , R ^x , R ^y , L, Z ¹ , Z ² , Z ³ , R ³ take the values defined for the compound of formula I.

In a further embodiment, in compounds of formula I and formula Ia, X ³ is NR ^y , resulting in compounds of formula II and IIa:

formula II

,

formula IIa

Where

X ¹ , n1, Y ¹ , R ^x , R ^y , L, Z ¹ , Z ² , Z ³ , R ¹ , R ² , R ³ take the values defined for compounds of formula I and formula Ia.

In yet another further embodiment, in compounds of Formula II and Formula IIa, R ³ is in the para position, resulting in compounds of Formula III and Formula IIIa:

formula III

formula IIIa

Where

X ¹ , n1, Y ¹ , R ^x , R ^y , L, Z ¹ , Z ² , Z ³ , R ¹ , R ² , R ³ take the values defined for compounds of formula I and formula Ia.

In yet another further embodiment, in compounds of Formula III and Formula IIIa, Z ² is CH, resulting in compounds of Formula IV and IVa:

formula IV

formula IVa

where X ¹ , n1, Y ¹ , R ^x , R ^y , L, Z ¹ , Z ³ , R ¹ , R ² , R ³ take the values defined for compounds of formula I and formula Ia.

In one embodiment of the compounds of the present invention, Y ¹ is selected from the group consisting of O, S, NH, C(O) and C(O)NH. In a further embodiment, Y ¹ is selected from the group consisting of O, S and NH. In yet another further embodiment, Y ¹ is O.

In another embodiment of the compounds of the present invention, X ¹ is NH or O. In yet another embodiment, X ¹ is NH or O, and n1 is 1.

In a further embodiment of the compounds of the present invention, R ^x and R ^y are independently C _1-4 alkyl. In yet another further embodiment, R ^x and R ^y are methyl.

In one embodiment of the compounds of the present invention, L is selected from the group consisting of O, S and CH ₂ . In another embodiment, L is selected from the group consisting of O and CH ₂ . In yet another embodiment, L is O.

In a further embodiment of the compounds of the present invention, Z ¹ represents CH. In yet another embodiment, Z ¹ is CH and L is O. In yet another further embodiment, Z ¹ is CH, Z ² is CH, and L is O.

In another embodiment of the compounds of the present invention, R ³ is selected from the group consisting of H, halogen and C(O)NH ₂ . In yet another embodiment, R ³ is selected from the group consisting of halogen and C(O)NH ₂ . In yet another embodiment, R ³ is selected from the group consisting of Cl and C(O)NH ₂ . In a further embodiment, R ³ is Cl.

In one embodiment of the compounds of the present invention in formula Ia, IIa, IIIa or IVa, R ² is H.

In another embodiment, the compounds of formula IVa are compounds of formula V below:

where X ¹ , Y ¹ , R ^x , R ^y , R ¹ and R ³ take the values defined for compounds of formula I and formula Ia. In a further embodiment of compounds of formula VX ^{, 1} is O, R ¹ is C _1-4 alkyl-O-, R ³ is halogen or C(O)NH ₂ , and R ^x and R ^y are independently C _{1- 4} alkyl.

In a presently preferred embodiment, a compound of the present invention is selected from the group consisting of the following compounds:

.

Pharmaceutical drug

The compounds of the present invention are intended for use as a medicine. The compounds of the present invention can in principle be used on their own, but are preferably formulated with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is an inert carrier suitable for each route of administration and can be incorporated into a conventional pharmaceutical preparation (tablets, granules, capsules, powder, solution, suspension, emulsion, injection, infusion, etc.). As such a carrier, there may be mentioned, for example, a binder, excipient, lubricant, disintegrating agent and the like, which are pharmaceutically acceptable. When the compounds of the present invention are used as an injection or infusion solution, these preparations can be prepared using distilled water injection, saline solution or aqueous glucose solution.

The administration method of the compounds of the present invention is not particularly limited, and a conventional oral or parenteral administration method (intravenous, intramuscular, subcutaneous, transdermal, intranasal, transmucosal, enteral, etc.) may be used.

The dosage of the tetrahydroisoquinoline derivatives or a pharmaceutically acceptable salt thereof of the present invention may not necessarily be set within the range of an effective amount sufficient to produce a pharmacological effect in accordance with the potency or characteristics of the compound to be used as the active ingredient. Dosage may vary depending on the route of administration, age, body weight, or medical condition of the patient.

Pharmaceutical Application

The compounds of the present invention are intended for the treatment of diseases that are sensitive to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF). Thus, in one aspect, the present invention provides a compound or composition of the present invention for use as a drug. In an additional aspect, the present invention provides a compound or composition of the present invention for use in the treatment of diseases sensitive to inhibition of IL-1β, such as non-alcoholic steatohepatitis (NASH) and idiopathic pulmonary fibrosis (IPF). In one embodiment, the disease responsive to IL-1β inhibition is selected from the group consisting of nonalcoholic steatohepatitis (NASH), idiopathic pulmonary fibrosis (IPF), autoinflammatory diseases, cardiovascular disease, osteoarthritis, lung cancer, and gout. In yet another embodiment, the disease is NASH. In yet another embodiment, the disease is IPF.

Obtaining compounds in which X ¹ represents NR ^y

The substituted pyridine sulfonamides of formula II and IIa of the present invention are typically synthesized via intermediate C, which is prepared according to Scheme 1:

Scheme 1

In the first step, 5,6-dichloropyridine-3-sulfonamide A undergoes alkylation to yield intermediate B , which is then converted to compound C via a Buchwald reaction with the corresponding bicyclic amine under microwave irradiation.

Depending on the values of the variables R ^x , R ^y and R ³ , additional synthetic transformations such as deprotection/deprotection reactions may be required to obtain intermediate C in addition to the transformations shown in Scheme 1.

Depending on the presence and/or value of X ¹ , the compounds of the present invention can be prepared in accordance with schemes 2b, 2c or 2d.

Scheme 2b - Synthesis of Type IIb Compound

Pd-catalyzed Buchwald reaction of intermediate C with a suitable boronic acid at 150°C under microwave irradiation produces type IIb compounds (such as compound 1; synthesis procedure is presented in Bioorg. Med. Chem. Lett. 2011 , 21(10) , 3152-3158).

Scheme 2c - Synthesis of type IIc compound

The Pd-catalyzed Buchwald reaction of intermediate C with substituted anilines at 40°C in the presence of a strong base produces type IIc compounds (such as compounds 2, 3, 4, 6; the synthesis procedure is presented in Org. Lett. 2011 , 13( 8) , 1984-1987).

Scheme 2d - Synthesis of type IId compound

Type IId compounds (such as compounds 5, 7, 8, 9, 10, 12) are prepared according to the Ullman reaction of intermediate C with the corresponding phenol in the presence of sodium hydride and cesium carbonate at 150ºC using microwave irradiation (synthesis procedure presented in J. Am. Chem. Soc. 2013 , 135(24) , 9213-9219).

Obtaining compounds in which X ³ absent

The substituted pyridine sulfonamides of formula I and Ia of the present invention, where X ³ is absent, are typically synthesized via intermediate E , which is prepared according to Scheme 3:

Scheme 3 - Method of synthesis of compound E

The synthesis of the final compound 11 is carried out in accordance with a procedure similar to that for the preparation of compound C , but using compound D, for example 2,3-dichloro-5-(methylsulfonyl)pyridine, as the starting material.

Examples

Example 1 - Synthesis of intermediate B, where R ^x and R ^y are methyl: N,N-dimethyl-5,6-dichloropyridine-3-sulfonamide

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

Example 2 - Synthesis of intermediate C, where R ^x and R ^y represent methyl, Z ¹ , Z ² , Z ³ represent CH, L represents O, and R ³ represents Cl in pair- position: N,N-dimethyl-5-chloro-6-(4-(4-chlorophenoxy)-N-piperidinyl)pyridine-3-sulfonamide

A mixture of 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) heated first to 80ºC and kept for 5 minutes and then to 120°C and kept for 30 minutes under microwave irradiation (200 W) (HPLC control). The solvent is then removed by evaporation and ethyl acetate is added to the residue; the organic mixture is washed twice with water and the combined organic layers are dried over magnesium sulfate. After removal of the solvent by evaporation, the resulting crude product is sufficiently pure to be used in the following examples without further purification (transparent brown solid, 91% yield).

Example 3 - Synthesis of intermediate C, where R ^x and R ^y represent methyl, Z ¹ , Z ³ represent CH, Z ² represents N, L represents O, and R ³ represents C(O)NH ₂ V pair- position: N,N-dimethyl-5-chloro-6-[4-(5-[2-carboxamidopyridin]yloxy)-N’-piperidinyl]pyridine-3-sulfonamide

A mixture of the compound obtained in Example 1 (1 eq.), commercially available 4-(piperidin-4-yloxy)pyridin-2-carboxamide (1.2 eq.) and TEA (2.2 eq.) in EtOH (4. 8 ml/mmol) is first heated to 80°C and maintained for 5 minutes, then to 120°C and maintained for 30 minutes under microwave irradiation (200 W) (HPLC control). The solvent is then removed by evaporation and ethyl acetate is added to the residue; the organic mixture is washed twice with water and the combined organic layers are dried over magnesium sulfate. After removal of the solvent by evaporation, the resulting crude product is sufficiently pure to be used in the next step without further purification (white transparent solid, 93% yield).

Example 4 - Synthesis of Compound 1

A solution of the intermediate obtained in Example 2 (1 eq.), benzo[b]furan-5-boronic acid (1.1 eq.), potassium carbonate (1.4 eq.) and Pd(PhPh ₃ ) ₄ (0 .1 eq.) in DME (5 ml/mmol) is heated to 150°C under microwave irradiation (200 W) and maintained under these conditions for 1 hour (or overnight at 150ºC). The crude mixture is then diluted with EtOAc, filtered through a pad of Celite, washed with EtOAc and MeOH and the solvent removed by evaporation. The resulting crude product was purified using an Isolera Biotage system (C18, acetonitrile/water) to give pure product (white foam, 45% overall yield, HPLC purity 98%).

1H NMR (CDCl3 _, 400 MHz): 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, 2H), 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 ClN ₃ O ₄ S.

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

Example 5 - Synthesis of Compound 2

A screw cap tube equipped with a magnetic stirrer is charged with BrettPhos precatalyst (4 mol%), 2,3-dihydrobenzo[b]furan-5-amine (1 eq.) and the intermediate obtained in Example 2 (1 eq.) . The tube is hermetically sealed with a Teflon screw cap, a vacuum is created in the tube and then filled again with nitrogen; This procedure is repeated two more times. LiHMDS (1M in THF, 2.5 eq.) is then added. The reaction mixture is stirred at 40°C until the reaction is complete (2.5 hours). The solution is allowed to cool to room temperature, the reaction is quenched by adding saturated aqueous NH ₄ Cl (5 ml) and then diluted with EtOAc. The organic phase is separated and the aqueous phase is extracted once more with EtOAc. The combined organic phases are washed with brine and dried over MgSO ₄ . The solvent is removed under reduced pressure and the crude product is purified using the Isolera Biotage system (C18, acetonitrile/water) to obtain the impurity-containing product (115.7 mg), which is again purified using the said system to obtain the product (brown solid, general yield 6%, HPLC purity 96% (254 nm)).

1H NMR (CDCl3 _, 400 MHz): δ (ppm) = 8.34 (s, 1H), 7.96 (br.s, 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 ClN ₄ O ₄ S.

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

Example 6 - Synthesis of Compound 5

2,3-Dihydro-5-hydroxybenzo[b]furan (1.1 eq.) and cesium carbonate (1.2 eq.) were transferred to anhydrous DMF (1.2 ml/mmol) and treated with pure sodium hydride (1. 1 eq.). After the evolution of hydrogen ceases, add the intermediate compound obtained in example 2 (1 eq.) and stir the reaction mixture at 150°C until further development of the reaction ceases in a sealed thick-walled tube (overnight) or under microwave irradiation (200 W) (2 hours). The solvent is then removed in vacuo and the crude product is diluted with ethyl acetate (6 ml/mmol) and water (6 ml/mmol). The aqueous layer was extracted with ethyl acetate three times. The organic layer was washed with 2N NaOH, dried and concentrated in vacuo. The crude product was purified using the Isolera Biotage system (C18, acetonitrile-water gradient elution) to give the crude product, which was purified by preparative chromatography (silica, hexane/ethyl acetate 4:1) to give the pure product (clear oil, overall yield 12 %, HPLC purity 100% (254 nm)).

1H NMR (CDCl3 _, 400 MHz): 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 ClN ₃ O ₅ S.

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

Example 7 - Synthesis of Compound 7

4-Ethoxyphenol (1.1 eq.) and cesium carbonate (1.2 eq.) were transferred to anhydrous DMF (1.2 ml/mmol) and treated with pure sodium hydride (1.1 eq.). After the end of hydrogen evolution, add the intermediate compound obtained in example 2 (1 eq.) and stir the reaction mixture at 150ºC until further development of the reaction ceases in a sealed thick-walled test tube (overnight) or under microwave irradiation (200 W, 2 hours). The solvent is then removed in vacuo and the crude product is diluted with ethyl acetate (6 ml/mmol) and water (6 ml/mmol). The aqueous layer was extracted with ethyl acetate three times. The organic layer was washed with 2N NaOH, dried and concentrated in vacuo. The crude product was purified using an Isolera Biotage system (C18, acetonitrile-water gradient elution), separating unreacted starting materials and pure product (brown clear solid, 41% yield, HPLC purity 100% (254 nm)).

1H NMR (CDCl3 _, 400 MHz): 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), 1.43 (t, 3H); C ₂₆ H ₃₀ ClO ₅ S.

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

Example 8 - Synthesis of Compound 8

4-Ethoxyphenol (1.1 eq.) and cesium carbonate (1.2 eq.) were transferred to anhydrous DMF (1.2 ml/mmol) and treated with pure sodium hydride (1.1 eq.). After the evolution of hydrogen ceases, add the intermediate compound obtained in example 3 (1 eq.) and stir the reaction mixture at 150°C until further development of the reaction ceases in a sealed thick-walled tube (overnight) or under microwave irradiation (200 W) (3 hours). The solvent is removed in vacuo and the crude product is diluted with ethyl acetate (6 ml/mmol) and water (6 ml/mmol). The aqueous layer was extracted with ethyl acetate three times. The organic layer was washed with 2N NaOH, dried and concentrated in vacuo. The crude product was purified using an Isolera Biotage system (C18, acetonitrile-water gradient elution), separating unreacted starting materials and pure product (brown clear solid, 16% yield, HPLC purity 100% (254 nm)).

1H NMR (CDCl3 _, 400 MHz): δ (ppm) = 8.38 (d, 1H), 8.34 (s, 1H), 6.98-6.97 (m, 1H), 6 .89 (s, 1H), 5.59 (br.s, 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 ); _{C26H31N5O6S . _ _}

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

Example 9 - Synthesis of Compound 12

2,3-Dihydro-5-hydroxybenzo[b]furan (1.1 eq.) and cesium carbonate (1.2 eq.) were transferred to anhydrous DMF (1.2 ml/mmol) and treated with pure sodium hydride (1. 1 eq.). After the evolution of hydrogen ceases, add the intermediate compound obtained in example 3 (1 eq.) and stir the reaction mixture at 150°C until the reaction stops further development in a sealed thick-walled tube (overnight) or under microwave irradiation (200 W) (2 ,5 hours). The solvent is then removed in vacuo and the crude product is diluted with ethyl acetate (6 ml/mmol) and water (6 ml/mmol). The aqueous layer was extracted with ethyl acetate three times. The organic layer was washed with 2N NaOH, dried and concentrated in vacuo. The crude product was purified using an Isolera Biotage system (C18, acetonitrile-water gradient elution), separating unreacted starting materials and pure product (white foam, 13% yield, HPLC purity 98% (254 nm)).

1H NMR (CDCl3 _, 400 MHz): 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 (br.s, 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); _{C26H29N5O6S . _ _}

MS (electrospray): m/z= 540.1944 (M+1).

Example 10 - Synthesis of intermediate E in which R ^x represents methyl, Z ¹ , Z ² , Z ³ represent CH, L represents O, and R ³ represents Cl in pair -position: 3-chloro-2-[4-(4-chlorophenoxy)-N-piperidinyl]-5-methylsulfonylpyridine

The title compound (white solid, 96% overall yield) was prepared following the procedure for the intermediate described in Example 2, but using commercially available 2,3-dichloro-5-(methylsulfonyl)pyridine as the starting material.

Example 11 - Synthesis of Compound 11

The compound is prepared (6 hours; clear oil, 16% overall yield, HPLC purity 95% (254 nm)) according to the procedure for the synthesis of compounds 5, 7, 8 and 12 of Examples 6-9, but using the intermediate obtained in Example 10 as starting material.

1H NMR (CDCl3 _, 400 MHz): 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 ClN ₂ O ₅ S.

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

Example 12 - IL-1β Inhibition in vitro in primary human macrophages stimulated with lipopolysaccharides (LPS)

Materials

No. Materials Catalog number Provider 1 LEGEND MAX Human IL-1β Enzyme-Linked Immunosorbent Assay (ELISA) Kit 437008 Biolegend 2 Cell culture medium RPMI 1640 R6504 Sigma-Aldrich 3 Phosphate Buffer Solution (PBS) P3813 Sigma-Aldrich 4 Dimethyl sulfoxide D2650 Sigma-Aldrich 5 Fetal bovine serum (FBS) 10270-106 Gibco 6 Penicillin-streptomycin 15140-122 Gibco 7 Histopaque-1077 10771 Sigma-Aldrich 8 Tribasic sodium citrate solution 91150 Sigma-Aldrich 9 96-well polypropylene microplate with 'V' bottom 15160 Greener 10 Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) 572902 Biolegend eleven Lipopolysaccharides (LPS) L6529 Sigma-Aldrich 12 CellTiter-Glo® Luminescence Assay Kit G7570 Promega

Preparation of growth medium RPMI 1640

RPMI 1640 Basal Medium is prepared according to the manufacturer's instructions on the data sheet. Sterility testing is carried out using 5 ml of medium for 48 hours at 37°C in an incubator with a constant supply of 5% CO _2. After checking sterility, the basal medium is made complete by adding FBS and the antibiotic penicillin/streptomycin. The medium is stored at 4°C until further use.

Preparation of phosphate buffer solution (PBS)

One sachet of dry PBS is dissolved in a liter of Milli-Q water. PBS is filtered through a 0.22 μm filter membrane and stored at 4°C until further use.

Obtaining peripheral blood mononuclear cells (PBMC)

5 ml of serum-free RPMI medium (1:1) is added to a sample of 5 ml of human whole blood in EDTA or sodium citrate and mixed thoroughly by inversion. Add 3 mL of Histopaque-1077 to a 15 mL conical centrifuge tube and allow to reach room temperature. Using a transfer pipette, 10 ml of the blood-RPMI mixture was carefully layered onto Histopaque-1077 and centrifuged at 400 xg for exactly 30 minutes at room temperature.

After centrifugation, the top layer within 0.5 cm of the opaque interface containing mononuclear cells is aspirated using a Pasteur pipette. The top layer is removed. Using a Pasteur pipette, the opaque interface is carefully transferred to a clean conical centrifuge tube. 5 ml of RPMI is added to the tube and mixed by inversion followed by centrifugation at 250×g strictly for 10 minutes. The supernatant is aspirated and removed.

The leukocyte pellet is resuspended in 5 ml RPMI, mixed carefully using a Pasteur pipette, followed by centrifugation at 250 xg for strictly 10 minutes.

The pellet is washed 3 times with PBS and resuspended in RPMI medium.

The number of viable PBMC/ml was counted.

Counting the number of PBMCs

10 μl of PBMC suspension is diluted with 90 μl of PBS medium (1:10 dilution). 20 µl of cell suspension is added to 20 µl of triptan blue solution (1:1 ratio) and mixed carefully to prevent aerosol formation. The hemocytometer is loaded with the cell culture mixture until the area under the coverslip is sufficiently filled. Before counting, the suspension is allowed to settle in the hemocytometer for at least 10 seconds.

In four 1 mm squares with right angles of one chamber, the number of viable cells as well as the number of dead cells are counted. Viable cells are transparent; non-viable cells are colored blue. When calculating, cells that touch the top line or the vertical line of the perimeter of any rectangular square are taken into account. Cells that touch the bottom or right vertical perimeter line of any rectangular square are not counted.

Calculation of the number of cells

Calculation of the number of viable PBMC/ml:

PBMC/ml=PBMC in all four squares × 10 × 2 × 10 ⁴ /4

10 ⁴⁼ Volume conversion factor up to 1 ml;

10=cell suspension dilution factor;

2=dilution factor of triptan blue.

Total number of cells=PBMC/ml × total volume (ml) of PBMC suspension

% cell viability = (Counted number of viable cells/total number of cells (viable + dead)) × 100.

Cell seeding, differentiation and treatment

2x10 ⁵ PBMC with a total volume of 200 μl/well are placed in a 96-well microplate and incubated in an incubator for 4 hours at 37°C in a CO ₂ atmosphere to sediment monocytes, leaving lymphocytes in suspension. After incubation, 100 μl is aspirated from each well to ensure removal of the lymphocyte population.

Monocytes are differentiated into macrophages by adding 200 ng/ml recombinant human GM-CSF (4 mg/ml stock solution) and incubating the mixture at 37°C/5% CO ₂ for 6 hours. The medium is changed every two days by removing half the volume of medium in the well and replenishing with fresh complete RPMI medium and recombinant human GM-CSF.

After differentiation, cells are treated in duplicate with the compound of the present invention in a total volume of 50 μl, maintaining a final DMSO percentile of 0.5%. The plate is transferred to an incubator at 37°C/5% CO ₂ and incubated for 2 hours. Cells are stimulated with 100 ng/ml LPS (4×) (1 mg/ml stock solution) in a total volume of 50 μl.

The plate is transferred to an incubator at 37°C/5% CO ₂ and incubated for 16 hours.

Cytokine assessment using ELISA

1. Each well of the pre-coated ELISA plate is aspirated and washed with wash buffer (0.05% Tween 20 in PBS; pH 7.2-7.4) by filling each well with wash buffer (300 µl). The process is repeated twice, performing a total of three washes. The liquid is completely removed at each stage. After the last wash, any remaining buffer is removed.

2. Block the plates by adding 300 μl of blocking buffer (1% bovine serum albumin (BSA) in PBS) to each well and incubate at room temperature for 1 hour.

3. The aspiration/washing stage, as in stage 1, is repeated.

4. Add 100 µl of sample or standard prepared in reagent diluent (0.1% BSA, 0.05% Tween 20 in PBS, pH 7.2-7.4). The wells are covered with adhesive tape and incubated for 2 hours at room temperature.

5. Aspiration/washing as in stage 1 is repeated.

6. Add 100 µl of detection antibody diluted with reagent diluent to each well. The wells are covered with new adhesive tape and incubated for 2 hours at room temperature.

7. Aspiration/washing as in stage 1 is repeated.

8. Add 100 µl of working dilution (1:200 from the original solution) and streptavidin-HRP to each well. The plate is covered and incubated for 20 minutes at room temperature.

9. Add 100 µl of substrate solution (1:1 mixture of H ₂ O ₂ and tetramethylbenzidine) to each well and incubate for 20 minutes at room temperature.

10. Add 50 µl of stop reagent (2N H ₂ SO ₄ ) to each well. The plate is gently tapped to ensure thorough mixing.

11. The optical density of each well is immediately determined using a microplate reader (Spectramax Plus) set at 450 nm.

Cytokine viability assessment

CellTiter-Glo® luminescent reagent is added to the assay plates in a volume of 100 µl/well and the plates are incubated at room temperature for 30 minutes on a plate shaker. After incubation, the luminescent signal value is determined in each well using a microplate reader (Perkin Elmer ENVISION 2104).

Analysis data

The %inhibition by test compounds is determined using the following formula:

%Inhibition=100-(100*(Average amount using test compounds - Average amount obtained using negative control)/(Average amount obtained using positive control - Average amount obtained using negative control))

All "calculated values" in this formula are derived from the "optical density values" determined as described above using a microplate reader (step 11).

The cytotoxicity of the test compounds is determined using the following formula:

%Cytotoxicity=100-(100*(Average amount obtained using test compounds - Average amount obtained using negative control)/(Average amount obtained using positive control - Average amount obtained using negative control))

All "amounts" indicated in this formula are derived from the "luminescent signal" determined as described in the cytokine viability assessment section.

results

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

Example 13 - Linking in vitro JNK1 (MAPK8)

Recombinant human MAPK8, dissolved in water at various concentrations, was manually printed onto a bare gold-coated chip (47 nm density) PlexArray Nanocapture Sensor (Plexera Bioscience, Seattle, WA, US) at 40% humidity. Each concentration is printed in duplicate, and each spot contains 0.2 μL of protein solution. The chip is incubated at 80% humidity and 4°C overnight and washed with 10× phosphate-buffered saline with Tween (PBST) for 10 minutes, 1× PBST for 10 minutes, and twice deionized water for 10 minutes. The chip is then blocked with 5% (w/v) skim milk in water overnight, then washed with 10× PBST for 10 minutes, 1× PBST for 10 minutes, and deionized water twice for 10 minutes, then Dry under a stream of nitrogen before use.

Surface Plasmon Resonance Imaging (SPRi) measurements were performed using a PlexAray HT (Plexera Bioscience, Seattle, WA, US). Collimated light (660 nm) passes through the coupling prism, is reflected from the SPR-active gold surface and is detected by the CCD camera. Buffers and samples are injected using a non-pulsing piston pump into a 30 μL flow cell mounted on a coupling prism. Each measurement cycle consists of four steps: wash with PBST running buffer at a constant rate of 2 μL/s to obtain a stable baseline, sample injection at 5 μL/s for binding, surface wash with PBST at 2 μL/s for 300 seconds, and regeneration 0.5% (v/v) H ₃ PO ₄ at 2 µl/s for 300 seconds. All measurements are carried out at 4°C.

Signal changes after binding and washing are recorded in AU as the assay value. Selected protein-grafted areas are analyzed on SPR images, and the average reflectance changes of the selected areas are used to plot as a function of time. Real-time binding signals were recorded and analyzed using a Data Analysis Module (DAM, Plexera Bioscience, Seattle, WA, US). Kinetic analysis was performed using BIAevaluation 4.1 software (Biacore, Inc.).

The equilibrium dissociation constant (KD value) determined for compound 7 is 3.41×10 ^-8 M. (Ka= 2.29×10 ⁴ M ^-1 s ^-1 , Kd=7.82×10 ^-4 s ^-1 ).

Example 14 - p38 MAPK binding under conditions in vitro

p38 MAPK, dissolved in water at various concentrations, was manually printed onto a bare gold-coated (47 nm density) PlexArray Nanocapture Sensor chip (Plexera Bioscience, Seattle, WA, US) at 40% humidity. Each concentration is printed in duplicate, and each spot contains 0.2 μL of protein solution. The chip is incubated at 80% humidity and 4°C overnight and washed with 10× PBST for 10 minutes, 1× PBST for 10 minutes, and twice deionized water for 10 minutes. The chip is then blocked with 5% (w/v) skim milk in water overnight and washed with 10×PBST for 10 minutes, 1×PBST for 10 minutes, and deionized water twice for 10 minutes, then dried in nitrogen flow before use.

Surface plasmon resonance imaging (SPRi) measurements were performed using a PlexAray HT (Plexera Bioscience, Seattle, WA, US). Collimated light (660 nm) passes through the coupling prism, is reflected from the SPR-active gold surface and is detected by the CCD camera. Buffers and samples are injected using a non-pulsing piston pump into a 30 μL flow cell mounted on a coupling prism. Each measurement cycle consists of four steps: wash with PBST running buffer at a constant rate of 2 μL/s to obtain a stable baseline, sample injection at 5 μL/s for binding, surface wash with PBST at 2 μL/s for 300 seconds, and regeneration with using 0.5% (v/v) H ₃ PO ₄ at 2 µl/s for 300 seconds. All measurements are carried out at 4°C.

Signal changes after binding and washing (in AU) are recorded as the assay result. Selected protein-grafted areas are analyzed on SPR images and the average changes in reflectance of the selected areas are plotted as a function of time based on the values obtained. Real-time binding signals were recorded and analyzed using a Data Analysis Module (DAM, Plexera Bioscience, Seattle, WA, US). Kinetic analysis was performed using BIAevaluation 4.1 software (Biacore, Inc.).

The equilibrium dissociation constant (KD value) determined for compound 7 is 1.19×10 ^-8 M. (Ka= 2.08×104 M ^-1 s ^-1 , Kd=2.48×10 ^-4 s ¹ ).

Example 15: Effect of Compounds 5 and 7 in the Treatment of Non-Alcoholic Steatohepatitis (NASH) in Male C57BL/6 Mice

Inducing the development of NASH

Mice with a known gestational age (n=30) were selected for the study. Newborn mice on the 2nd day after birth (post-natal day 2 - PND-2) are subcutaneously injected with 2200 μg of streptozotocin and left with their mother until they reach the age of weaning from mother's milk. After weaning, male mice were selected and fed a 60% kcal fat diet (research diet-D12492) for the next 2 weeks. All animals are examined twice daily for clinical signs.

Research methodology

Mice are administered vehicle, test compounds, and control compound (elafibranor) twice daily in the morning (9:00 AM) and in the evening before the start of the dark cycle (6:00 PM) on days 0 to 28.

The body weight of the animals was measured daily throughout the experiment.

Test and control compounds are administered to animals for 28 days (from 6 to 10 weeks).

Blood glucose levels are assessed before treatment and at the end - on day 28. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are measured in plasma before treatment and on day 28.

Histopathological analysis of liver tissue includes hematoxylin & eosin (H&E) staining, Masson trichome staining, and Oil-Red-O (ORO) staining.

Sample analysis

Non-alcoholic fatty liver disease (NAFLD) severity score: H&E staining

All H&E-stained tissue sections were examined using optical microscopy. According to the scoring system below (Kleiner et al., 2005) using a 200× lens, NAFLD is scored by assessing steatosis, lobular inflammation, and liver ballooning.

Collagen Proportion Area (%CPA) measurement: Trichome Masson staining

All trichomely Meysson-stained tissue sections were examined by optical microscopy using a 100× objective lens. On randomly selected five areas (approximately 684.85 μm × 917.11 μm per area) from each liver, the collagen lobe area was measured using Image Pro Premier 9.1 software. The percentage that constitutes the area of the collagen lobe is calculated by dividing the area of the collagen lobe by the total area of the tissue section.

Measuring the percentage of area stained: oil red O-staining

All tissue sections stained with oil red O dye were examined by optical microscopy using a 100× lens objective. In randomly selected five areas (approximately 688.33 μm × 922.45 μm per area) from each liver, the stained area was measured using Image Pro Premier 9.1 software. The percentage that is stained area is calculated by dividing the area of stained lipid tissue by the total tissue area.

The effect of compound 5 and compound 7 on the NAFLD activity score (NAS) is presented in Figure 1.

The study clearly shows that compound 7 at 3 mg/kg significantly reduces steatosis and lobular inflammation in the liver and shows better NAS compared to elafibranor.

Compound 5 at a dose of 3 mg/kg significantly reduced lobular inflammation and showed a trend towards reduced steatosis and ballooning of the liver, significantly reducing NAS compared to control.

Compounds 5 and 7 exhibited a significant reduction in AST levels after 28 days of treatment, as shown in Figure 2.

In contrast to the reference compound (elafibranor), both test compounds (compounds 5 and 7) did not cause hepatomegaly and an increase in the liver weight to body weight ratio, as shown in Figure 3.

In conclusion, the tested compounds lead to significant improvements in liver chemistry and histological NASH activity. The therapeutic profile of compounds 5 and 7 suggests they have potential for the treatment of NASH in humans. All treated animals experienced an increase in body weight and no clinical signs of toxicity or side effects were observed.

Example 16: Effect of Compound 7 in the Treatment of Idiopathic Pulmonary Fibrosis (IPF) Caused by Bleomycin

Induction of IPF

Mice with a known gestational age (n=30) were selected for the study. Newborn mice on postnatal day 2 (PND-2) are injected subcutaneously with 1.5 U/kg bleomycin (intratracheal) and left with their mother until they reach weaning age. After weaning, male mice were selected and fed a 60% kcal fat diet (Research Diet-D12492) for the following week. All animals are examined twice daily for clinical signs.

Research methodology

Mice are orally administered a dose of vehicle, a dose of test compounds, or a control compound (pirfenidone) twice daily: in the morning (9:00 AM) and in the evening before the start of the dark cycle (6:00 PM) on days 0 to 14.

The body weight of the animals was measured daily throughout the experiment.

Test compounds and control compounds are administered to animals for 14 days.

The level of hydroxyproline in the lungs is assessed before the start of treatment and at the end on the 14th day. Total cell count and white blood cell count in bronchoalveolar lavage fluid (BALF) are measured before treatment and on day 28.

Lung tissues are subjected to histopathological analysis according to Ashcroft's H&E staining score.

Sample analysis

Assessment of IPF severity: H&E staining

All H&E stained tissue sections were examined using optical microscopy. The severity of IPF is assessed according to the scoring system below (Kleiner et al., 2005) using a 200× lens objective.

Collagen Proportion Area (% CPA) measurement: Trichome Masson staining

All tissue sections stained with trichome Meysson were examined by optical microscopy using a 100× objective lens. On randomly selected five areas (approximately 684.85 μm × 917.11 μm per area) from each liver, the stained area was measured using Image Pro Premier 9.1 software. The percentage that constitutes the area of the collagen lobe is calculated by dividing the area of the collagen lobe by the total area of the tissue section.

The effect of compound 7 on lung hydroxyproline levels is presented in Figure 4.

Lung hydroxyproline levels are significantly increased in the bliomycin control compared to the no-drug control. The study clearly shows that compound 7 at a dose of 15 mg/kg significantly reduces lung hydroxyproline levels and that this reduction is superior to the reduction achieved with 100 mg/kg pirfenidone.

In BALF, the number of total and differential leukocytes (macrophages and lymphocytes) is significantly increased in the bleomycin control group. The number of macrophages in BALF is reduced by treatment with compound 7 (p>0.05) and pirfenidone (p<0.05) compared to bleomycin control, as shown in Figures 5A and 5B.

The fibrosis severity score and %CPA increased significantly in the bleomycin control group compared to the no-drug control group (Naïve control). In lungs treated with Compound 7 (p<0.05) and pirfenidone (p<0.05), samples showed a significant reduction in fibrosis severity score and %CPA compared to bleomycin control, as shown in Figures 6A and 6B. These data are consistent with the results for hydroxyproline.

In conclusion, the test compound leads to significant improvement in lung chemistry and histological activity of IPF. The therapeutic profile of compound 7 suggests that it has potential for the treatment of human IPF. All treated animals showed an increase in body weight compared to the bleomycin control group and showed no clinical manifestations of toxicity of the specified compound or side effects.

Claims (32)

1. Compound of formula I and its pharmaceutically acceptable salts: where R ¹ represents phenyl or a 9-membered fused heteroaromatic ring system containing 1 or 2 O heteroatoms, optionally substituted with one substituent selected from the group consisting of C _1-4 alkyl, C _1-4 alkyl-Y ¹ -, C _1-4 alkyl-Y ¹ -, substituted with halogen, HO-C _1-4 alkanediyl; Y ¹ represents O; X ¹ represents NH or O; n1 is 0 or 1; X ³ is absent or represents NR ^y ; R ^x and R ^y independently represent C _1-4 alkyl; L represents O or CH ₂ ; Z ¹ and Z ³ are selected from N and CH, Z ² represents CH; R ³ is selected from the group consisting of halogen, C(O)NR ^2a R ^2b , where R ^2a and R ^2b are independently selected from the group consisting of H and C _1-4 alkyl. 2. Compound according to claim 1 of formula Ia where R ¹ is selected from the group consisting of C _1-4 alkyl-Y ¹ -, C _1-4 alkyl-Y ¹ - substituted with halogen; R ² represents hydrogen; or R ¹ together with R ² forms a heterocyclic five-membered ring, optionally substituted with one substituent selected from HO-C _1-4 alkanediyl-; And X ¹ , n1, Y ¹ , X ³ , R ^x , R ^y , L, Z ¹ , Z ² , Z ³ , R ³ take the values defined in paragraph 1. 3. A compound according to any one of claims 1 or 2, where X ¹ represents NH or O and n1 represents 1. 4. A compound according to any one of claims 1 to 3, where X ³ represents NR ^y . 5. A compound according to any one of claims 1 to 4, where R ^x and R ^y represent methyl. 6. A compound according to any one of claims 1 to 5, where L represents O. 7. A compound according to any one of claims 1 to 6, wherein R ³ is selected from the group consisting of halogen and C(O)NH ₂ . 8. The compound according to claim 7, where R ³ is selected from the group consisting of Cl and C(O)NH ₂ . 9. A compound according to claim 1, which is selected from the group consisting of the following compounds: 10. A pharmaceutical composition having inhibitory activity against IL-1β, comprising an effective amount of 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 treatment of diseases mediated by IL-1β selected from non-alcoholic steatohepatitis (NASH), idiopathic pulmonary fibrosis (IPF), autoinflammatory diseases, cardiovascular diseases, osteoarthritis, lung cancer or gout, preferably for use in the treatment of NASH or IPF.