Medicine for treating steatotic liver diseases

Abstract

Provided is a pharmaceutical composition for treating and / or preventing steatotic liver diseases, particularly non-alcoholic fatty liver diseases, which has an excellent MGAT2 inhibitory effect and does not involve adverse side effects such as hemostatic abnormality, hepatic and renal disorders or embryonic lethality. The pharmaceutical composition contains a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.　Formula (I): (wherein: R2a and R2b together, in conjunction with the adjacent carbon atom, form a ring B; the ring B is represented by any one of the formulae shown in the middle part of FIG. 1 (wherein R6's each independently represent a halogen atom or the like); R4a is represented by the formula shown in the lower part of FIG. 1 (wherein L3 represents an alkylene group, and R7 represents an alkylsulfonyl group); R4b represents an alkyl group which may be substituted by a group α substituent, or the like; the group α substituent is a halogen atom, or the like; and a group β substituent is a halogen atom, or the like.)

Description

Medicine for treating fatty liver disease

[0001] The present invention relates to a new pharmaceutical composition for treating and / or preventing fatty liver disease, particularly non-alcoholic fatty liver disease.

[0002] Liver disease is a leading cause of death worldwide. Liver disease can be caused by infection, injury, exposure to drugs or toxic compounds, alcohol, or dietary impurities, as well as abnormal accumulation of normal substances in the blood, autoimmune processes, genetic defects (e.g., hemochromatosis), or unknown causes. Liver disease is generally classified as acute or chronic based on the duration of the disease. Fatty liver disease (SLD) is a general term for diseases in which triglycerides accumulate in the liver, causing liver damage. Fatty liver is defined as a condition in which 30% or more hepatocytes contain lipid droplets. Fatty liver is a condition in which triglycerides accumulate in the liver. Because liver fat accumulation is a non-progressive (reversible) change, removal of the cause will result in normal liver fat accumulation. However, if fat accumulation progresses and liver dysfunction occurs, it may develop into cirrhosis or liver cancer. Alcoholic steatohepatitis (ASH), caused by alcohol consumption, is a typical example of fatty liver. However, fatty liver can also occur in people who consume little alcohol, a condition known as nonalcoholic fatty liver disease (NAFLD). A more severe form of NAFLD is called nonalcoholic steatohepatitis (NASH). NASH is considered an important cause of cirrhosis and liver cancer. The histological features of alcoholic and nonalcoholic hepatitis are similar, and common pathogenic mechanisms, such as oxidative stress in the liver, are being studied. According to the American Liver Association, more than 20% of the population has NAFLD. If left untreated, NAFLD can progress to NASH, which can cause serious adverse effects. Furthermore, if untreated, NASH can lead to liver fibrosis, cirrhosis, liver failure, or hepatocellular carcinoma. An estimated 16 million adults in the United States have NASH, and roughly 50% have advanced liver fibrosis (bridging fibrosis or cirrhosis) associated with NASH. NASH is currently the leading cause of hepatocellular carcinoma in the United States and the second most common indication for liver transplantation after hepatitis C.NASH is characterized by the presence of steatosis and other characteristics, including hepatocellular degeneration (balloon, Mallory hyaline), inflammatory cell infiltration, and the development of progressive fibrosis. There are currently no approved therapies for the treatment of NASH, nor therapies that reduce fibrosis and / or steatosis in NASH patients. Therefore, there remains a need to provide new, effective drugs for treating liver disease or symptoms of liver disease.

[0003] Patent Document 1 describes a method for treating and / or preventing NASH and / or primary biliary cirrhosis, which comprises administering a pharmaceutical composition containing eicosapentaenoic acid or a derivative thereof to a subject in need thereof. It has been reported that obeticholic acid, a semisynthetic bile acid and agonist of the nuclear receptor FXR (farnesoid X receptor), has been approved in Europe and the United States for the treatment of fibrosis caused by NASH.

[0004] In monoacylglycerol acyltransferase 2 (MGAT2) knockout mice, suppression of high-fat diet-induced weight gain, suppression of insulin resistance, suppression of elevated blood cholesterol levels, suppression of fatty liver formation, and enhanced energy expenditure has been confirmed (Non-Patent Document 1). Furthermore, compounds with MGAT2 inhibitory activity (JTP-103237, BMS-963272) have been reported to improve fatty liver in high-sucrose-fed model mice and liver fibrosis in NASH model mice (Non-Patent Documents 2 and 3). Furthermore, increased expression of MGAT2 has been reported in the livers of NAFLD patients (Non-Patent Document 4). Based on these findings, MGAT2 inhibitors are expected to be promising therapeutic agents for fatty liver disease, including NASH; however, no drugs demonstrating efficacy in humans have yet been developed (Non-Patent Document 5).

[0005] Patent documents 2 to 4 disclose MGAT2 inhibitors of the formula: Patent documents 5 and 6 disclose compounds of the formula: However, Patent Documents 2 to 6 do not state that the compounds are effective in treating and / or preventing non-alcoholic fatty liver disease.

[0006] U.S. Patent Application Publication No. 2014 / 187633, International Publication No. 2019013311, International Publication No. 2019013312, Japanese Patent Application No. 2020-02515, International Publication No. 2020145369, Japanese Patent Application No. 2021-113901

[0007] Nature Medicine volume 15, pages442-446 (2009)Journal of Pharmacological Sciences 128 (2015) 150e157Cell Metabolism 34, 1732-1748, 2022Journal of Lipid Research Volume 53, 2012, p990-999J. Med. Chem. 2018, 61, 9879-9888

[0008] An object of the present invention is to provide a pharmaceutical composition for the treatment and / or prevention of fatty liver disease, particularly non-alcoholic fatty liver disease, which has excellent MGAT2 inhibitory activity and is not accompanied by side effects such as hemostatic abnormalities, liver damage, nephropathy, or embryonic lethality.

[0009] As a result of extensive investigations to solve the above problems, the present inventors have found that, among the compounds having MGAT2 inhibitory activity described in Patent Documents 2 to 4, certain compounds are effective in treating and / or preventing non-alcoholic fatty liver disease without causing side effects such as hemostatic abnormalities, liver damage, nephropathy, or embryonic lethality, and have thus completed the present invention.

[0010] [1] Formula (I): (In the formula, R ２ａ and R ２ｂ together with the adjacent carbon atom to form ring B; ring B is a ring of the formula: (In the formula, R ６ are each independently halogen, haloalkyl, alkyloxy, haloalkyloxy, or cyclopropanyl; R ４ａ is the formula: (In the formula, L ３ is alkylene, and R ７ is alkylsulfonyl); R ４ｂ [2] A pharmaceutical composition for treating and / or preventing fatty liver disease, comprising a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, wherein R is an alkyl optionally substituted with substituent group α, a phenyl group optionally substituted with substituent group β, or a 5- to 6-membered aromatic heterocyclic group optionally substituted with substituent group β; substituent group α is halogen, haloalkyloxy, and cyclopropanyl; and substituent group β is halogen, cyano, alkyl, haloalkyl, and cyclopropanyl. ６ are each independently halogen, haloalkyl, or haloalkyloxy; R ４ａ But the formula: and R ４ｂ is haloalkyloxyalkyl, a phenyl group optionally substituted with halogen, or a 5- to 6-membered aromatic heterocyclic group optionally substituted with substituent group β, and substituent group β is halogen and alkyl. [3] The pharmaceutical composition according to [1], wherein ring B is a group represented by the formula: (wherein each symbol is as defined in [1] or [2]). [4] The pharmaceutical composition according to [1] or [2], wherein the compound is selected from the group consisting of compounds I-8, I-23, I-34, I-190, I-212, I-236, I-253, I-275, I-276, II-93, II-103, II-121, II-151, II-168, II-174, II-203, II-225, II-233, and II-295, or a pharmaceutically acceptable salt thereof. [5] The pharmaceutical composition according to [1], wherein the compound is selected from the group consisting of compounds I-236, I-253, I-275, II-103, II-121, II-174, II-203, II-225, and II-233, or a pharmaceutically acceptable salt thereof. [6] The pharmaceutical composition of [1], wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD). [7] The pharmaceutical composition of [1], wherein the fatty liver disease is metabolic dysfunction-associated fatty liver disease (MASLD), cryptogenic fatty liver disease (cryptogenic SLD), or specific aetiology fatty liver disease (specific aetiology SLD). [8] The pharmaceutical composition of [1], wherein the fatty liver disease is non-alcoholic steatohepatitis (NASH). [9] The pharmaceutical composition of [1], wherein the fatty liver disease is metabolic dysfunction-associated steatohepatitis (MASH).

[10] The pharmaceutical composition of [1], wherein the fatty liver disease is NASH or hepatic fibrosis caused by MASH.

[11] The pharmaceutical composition of [1], wherein the fatty liver disease is hepatic cirrhosis caused by NASH or MASH.

[12] The pharmaceutical composition of [1], wherein the fatty liver disease is hepatocellular carcinoma (HCC) caused by NASH or MASH.

[13] The pharmaceutical composition of [1], wherein administration of the pharmaceutical composition does not involve at least one side effect of hemostatic abnormalities, liver damage, kidney damage, and embryonic lethality.

[14] The pharmaceutical composition of [1], wherein the body mass index (BMI) is 25 kg / m or less. ２

[15] The pharmaceutical composition according to any one of [1] to

[14] , for treating and / or preventing fatty liver disease in a subject having a blood cholesterol level of less than 1000 mg / kg.

[16] The pharmaceutical composition according to

[15] , for treating and / or preventing fatty liver disease in a subject having a blood cholesterol level of less than 1000 mg / kg.

[17] The pharmaceutical composition according to

[16] , wherein an effective amount of the compound is in the range of 0.5 mg to 100 mg per day.

[18] The pharmaceutical composition according to

[17] , wherein an effective amount of the compound is in the range of 3 mg to 30 mg per day.

[19] A method for treating and / or preventing fatty liver disease, comprising administering to a subject a compound of the formula: (In the formula, R ２ａ and R ２ｂ together with the adjacent carbon atom to form ring B; ring B is a ring of the formula: (In the formula, R ６ are each independently halogen, haloalkyl, alkyloxy, haloalkyloxy, or cyclopropanyl; R ４ａ is the formula: (In the formula, L ３ is alkylene, and R ７ is alkylsulfonyl); R ４ｂ

[20] A method for treating and / or preventing fatty liver disease, comprising the step of administering an effective amount of a compound represented by the formula: (In the formula, R ２ａ and R ２ｂ together with the adjacent carbon atom to form ring B; ring B is a ring of the formula: (In the formula, R ６are each independently halogen, haloalkyl, alkyloxy, haloalkyloxy, or cyclopropanyl; R ４ａ is the formula: (In the formula, L ３ is alkylene, and R ７ is alkylsulfonyl); R ４ｂ is an alkyl optionally substituted with substituent group α, a phenyl group optionally substituted with substituent group β, or a 5- to 6-membered aromatic heterocyclic group optionally substituted with substituent group β; substituent group α is halogen, haloalkyloxy, and cyclopropanyl; and substituent group β is halogen, cyano, alkyl, haloalkyl, and cyclopropanyl) or a pharmaceutically acceptable salt thereof.

[21] Use of a compound represented by the formula: (In the formula, R ２ａ and R ２ｂ together with the adjacent carbon atom to form ring B; ring B is a ring of the formula: (In the formula, R ６ are each independently halogen, haloalkyl, alkyloxy, haloalkyloxy, or cyclopropanyl; R ４ａ is the formula: (In the formula, L ３ is alkylene, and R ７ is alkylsulfonyl); R ４ｂ is an alkyl optionally substituted with substituent group α, a phenyl group optionally substituted with substituent group β, or a 5- to 6-membered aromatic heterocyclic group optionally substituted with substituent group β; substituent group α is halogen, haloalkyloxy, and cyclopropanyl, and substituent group β is halogen, cyano, alkyl, haloalkyl, and cyclopropanyl), or a pharmaceutically acceptable salt thereof.

[0011] The pharmaceutical composition of the present invention is effective in treating and / or preventing fatty liver disease, particularly non-alcoholic fatty liver disease, and is highly safe, being free from at least one side effect of hemostatic abnormalities, liver damage, nephropathy, and embryonic lethality.

[0012] indicates the intracellular fat accumulation content when HepG2 cells were treated overnight with compounds II-203, II-121, and BMS-963272. indicates the intracellular ATP content when HepG2 cells were treated for 48 hours with compounds II-203, II-121, and BMS-963272. indicates the LDH activity in the culture supernatant when HepG2 cells were treated for 24 hours with compounds II-203, II-121, and BMS-963272.

[0013] The meaning of each term used in this specification is explained below. Unless otherwise specified, each term has the same meaning whether used alone or in combination with other terms. The term "consisting of" means having only the constituent elements. The term "comprise" means not being limited to the constituent elements and does not exclude unrecited elements. The present invention will be explained below with reference to embodiments. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0014] The term "halogen" includes fluorine, chlorine, bromine, and iodine atoms. Particularly preferred are fluorine and chlorine atoms.

[0015] The term "alkyl" encompasses straight-chain or branched hydrocarbon groups having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, and n-decyl. Preferred embodiments of "alkyl" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and n-pentyl. More preferred embodiments include methyl, ethyl, n-propyl, isopropyl, and tert-butyl.

[0016] The term "alkylene" includes a straight-chain or branched divalent hydrocarbon group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms. Examples include methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, and hexamethylene.

[0017] The term "aromatic heterocyclic group" refers to a monocyclic or bicyclic or more aromatic cyclic group having one or more identical or different heteroatoms selected from O, S, and N in the ring. Bicyclic or more aromatic heterocyclic groups also include those in which the rings in the above-mentioned "aromatic carbocyclic groups" are fused to a monocyclic or bicyclic or more aromatic heterocyclic group, and the bond may be on any of the rings. Monocyclic aromatic heterocyclic groups are preferably 5- to 8-membered, and more preferably 5- or 6-membered. Examples of 5-membered aromatic heterocyclic groups include pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl, thiadiazolyl, etc. Examples of 6-membered aromatic heterocyclic groups include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc. The bicyclic aromatic heterocyclic group is preferably 8- to 10-membered, and more preferably 9- or 10-membered. Examples thereof include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyridazinyl, oxazolopyridyl, and thiazolopyridyl. The tricyclic or more aromatic heterocyclic group is preferably 13- to 15-membered. Examples include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, dibenzofuryl, and the like.

[0018] In this specification, the phrase "may be substituted with substituent group α" means "may be substituted with one or more groups selected from substituent group α." The same applies to substituent group β.

[0019] Fatty liver disease (SLD) is a general term for diseases caused by the accumulation of triglycerides in the liver, resulting in liver damage. While previously thought to be primarily caused by alcohol, SLD has recently become more prevalent in obese individuals who do not drink alcohol. Liver damage characterized by hepatic fat deposition in liver tissue findings similar to alcoholic liver disease, despite the absence of a clear drinking history, is called nonalcoholic fatty liver disease (NAFLD).

[0020] NAFLD is a pathological condition in which fatty liver is confirmed by histological or imaging diagnosis and other liver diseases such as alcoholic liver disease have been excluded. NAFLD is characterized by the accumulation of fat in hepatocytes and is often associated with some aspects of metabolic syndrome (e.g., type 2 diabetes mellitus, insulin resistance, hyperlipidemia, and hypertension). The frequency of this disease is becoming increasingly common due to the consumption of carbohydrate-rich and high-fat diets. A subset of NAFLD patients develops nonalcoholic steatohepatitis (NASH).

[0021] NASH, a subtype of fatty liver disease, is a more severe form of NAFLD. It is characterized by inflammation that ultimately leads to macrovesicular steatosis, balloon degeneration of hepatocytes, and / or scarring of the liver (i.e., fibrosis). Patients diagnosed with NASH progress to advanced stages of liver fibrosis and ultimately to cirrhosis. The current treatment for end-stage cirrhotic NASH patients is liver transplantation.

[0022] NAFLD as defined above includes metabolic dysfunction-associated fatty liver disease (MASLD), cryptogenic fatty liver disease (cryptogenic SLD), and specific aetiology fatty liver disease (specific aetiology SLD).

[0023] Metabolic dysfunction-associated fatty liver disease (MASLD) refers to a disease that, in addition to fatty liver, meets one or more of the criteria for obesity, impaired glucose tolerance, hypertension, hypertriglyceridemia, and hypo-HDL cholesterol, such as those described in J Hepatol, 2023; 79 (6): 1542-56.

[0024] Cryptogenic fatty liver disease (Cryptogenic SLD) refers to a disease among the NAFLD / NASH defined above that does not meet any of the criteria for obesity, impaired glucose tolerance, hypertension, hypertriglyceridemia, or hypo-HDL cholesterol. Examples of the criteria include those described in J Hepatol, 2023; 79 (6): 1542-56.

[0025] Specific aetiology fatty liver disease (SLD) refers to fatty liver disease without accompanying metabolic dysfunction, which is caused by a specific drug or other factor.

[0026] NASH as defined above is intended to include metabolic dysfunction-associated steatohepatitis (MASH). Thus, a subset of patients with NAFLD or MASLD may develop MASH.

[0027] Metabolic dysfunction-associated steatohepatitis (MASH) refers to the MASLD defined above in which ballooning, Mallory-Denk bodies, etc. are histologically observed.

[0028] Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, that occurs in most types of chronic liver disease. Advanced liver fibrosis leads to cirrhosis, liver failure, and portal hypertension, often requiring liver transplantation.

[0029] As liver fibrosis progresses, the liver cells become surrounded by fibrosis, leading to cirrhosis. As cirrhosis progresses, symptoms such as edema, ascites, and jaundice appear, and if gastrointestinal lesions such as esophageal and gastric varices occur concomitantly, vomiting blood may occur. Furthermore, as liver fibrosis progresses, the patient becomes more susceptible to developing liver cancer.

[0030] Hepatocellular carcinoma (HCC) is the most common cancer originating in the liver and usually occurs in patients with severe scarring of the liver (cirrhosis). Patients with advanced liver fibrosis and cirrhosis are reported to develop liver cancer within five years at a rate of 5-30%.

[0031] Alcoholic liver disease (ALD) encompasses a wide range of diseases, including alcoholic fatty liver (AFL), alcoholic steatosis (ASH), severe alcoholic hepatitis (SAH), alcoholic liver fibrosis, and liver cirrhosis. The compounds according to the present invention have excellent MGAT2 inhibitory activity and are therefore useful as therapeutic and / or preventive agents for ALD.

[0032] In this specification, Body Mass Index (BMI) means a simple index of weight-to-height ratio commonly used to classify adult (age 15 and over) populations or individuals as overweight or obese, and is defined as weight in kilograms divided by the square of height in meters (kg / m ２ ) BMI is 25 kg / m ２ It is effective for the above subjects, but especially for those with a BMI of 25 kg / m ２ It is effective for subjects with less than

[0033] As the compound represented by formula (I), the following compounds or pharmaceutically acceptable salts thereof are particularly preferred.

[0034] The compound used in the present invention is not limited to a specific isomer, and includes all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereoisomers, optical isomers, rotational isomers, etc.), racemates, or mixtures thereof.

[0035] One or more hydrogen, carbon and / or other atoms of the compounds used in the present invention may be replaced with isotopes of the hydrogen, carbon and / or other atoms, respectively. Examples of such isotopes include, for example, ２ H. ３ H.１１ C. １３ C. １４ C. １５ N. １８ O. １７ O. ３１ P. ３２ P. ３５ S. １８ F. 123 I and ３６ The isotopes of the compounds used in the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as Cl. The compounds used in the present invention also include compounds substituted with such isotopes. The isotope-substituted compounds are also useful as pharmaceuticals and include all isotopes and radiolabeled compounds of the compounds used in the present invention. The present invention also includes a "radiolabeling method" for producing the "radiolabeled compounds," and the "radiolabeled compounds" are useful as research and / or diagnostic tools in metabolism pharmacokinetic studies and binding assays.

[0036] Radiolabeled compounds used in the present invention can be prepared by methods well known in the art. For example, tritium-labeled compounds of formula (I) can be prepared by introducing tritium into a specific compound of formula (I) via catalytic dehalogenation using tritium. This method involves reacting a precursor of a compound of formula (I) in which the appropriate halogen is substituted with tritium gas in the presence of a suitable catalyst, such as Pd / C, with or without a base. Other suitable methods for preparing tritium-labeled compounds can be found in "Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987)." １４ C-labeled compounds are １４ It can be prepared by using a raw material having C carbon.

[0037] Pharmaceutically acceptable salts of the compounds used in the present invention include, for example, salts of the compounds used in the present invention with alkali metals (e.g., lithium, sodium, potassium, etc.), alkaline earth metals (e.g., calcium, barium, etc.), magnesium, transition metals (e.g., zinc, iron, etc.), ammonia, organic bases (e.g., trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, pico- Examples of suitable salts include salts with inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.) and amino acids, and salts with inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid, etc.) and organic acids (e.g., formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, succinic acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, trifluoroacetic acid, etc.). These salts can be formed by a commonly used method.

[0038] The compound used in the present invention or a pharmaceutically acceptable salt thereof may form a solvate (e.g., hydrate, etc.), co-crystal, and / or crystalline polymorph, and the present invention also encompasses such various solvates, co-crystals, and crystalline polymorphs. A "solvate" may be coordinated with the compound used in the present invention with any number of solvent molecules (e.g., water molecules, etc.). When the compound used in the present invention or a pharmaceutically acceptable salt thereof is left in the atmosphere, it may absorb moisture, and adsorbed water may adhere, or a hydrate may be formed. Furthermore, a crystalline polymorph may be formed by recrystallization of the compound used in the present invention or a pharmaceutically acceptable salt thereof. A "co-crystal" means that the compound or salt used in the present invention and a counter molecule exist in the same crystal lattice, and may contain any number of counter molecules.

[0039] The compounds used in the present invention or pharmaceutically acceptable salts thereof may form prodrugs, and the present invention also encompasses such various prodrugs. Prodrugs are derivatives of the compounds of the present invention having chemically or metabolically decomposable groups, and are compounds that become pharmaceutically active compounds of the present invention in vivo by solvolysis or under physiological conditions. Prodrugs include compounds that are converted to the compounds of formula (I) by enzymatic oxidation, reduction, hydrolysis, etc. under physiological conditions in vivo, and compounds that are converted to the compounds of formula (I) by hydrolysis with gastric acid, etc. Methods for selecting and preparing appropriate prodrug derivatives are described, for example, in "Design of Prodrugs, Elsevier, Amsterdam, 1985." Prodrugs may themselves be active.

[0040] The compounds according to the present invention have excellent MGAT2 inhibitory activity and are therefore useful as therapeutic and / or preventive agents for fatty liver disease, particularly non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease is not limited to non-alcoholic steatohepatitis (NASH), and they are also useful as therapeutic and / or preventive agents for liver fibrosis, cirrhosis, or hepatocellular carcinoma (HCC) caused by NASH. Furthermore, the compounds according to the present invention are useful as pharmaceuticals and preferably have one or more of the following excellent characteristics: a) weak inhibitory activity against CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.); b) good pharmacokinetics, such as high bioavailability and moderate clearance; c) high metabolic stability; d) no irreversible inhibitory activity against CYP enzymes (e.g., CYP3A4) within the concentration range of the measurement conditions described herein; and e) no mutagenicity. f) Low cardiovascular risk, g) High solubility, and h) Free from at least one of the side effects of hemostatic abnormalities, liver damage, kidney damage, and embryonic lethality.

[0041] The pharmaceutical composition of the present invention can be administered orally or parenterally, including transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, transmucosal, inhalation, nasal, ophthalmic, otic, and vaginal administration.

[0042] For oral administration, the composition may be prepared and administered in any of the commonly used dosage forms, such as solid preparations for internal use (e.g., tablets, powders, granules, capsules, pills, films, etc.) and liquid preparations for internal use (e.g., suspensions, emulsions, elixirs, syrups, lemonades, spirits, perfumes, extracts, decoctions, tinctures, etc.), according to conventional methods. Tablets may be sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, troches, sublingual tablets, buccal tablets, chewable tablets, or orally disintegrating tablets; powders and granules may be dry syrups; and capsules may be soft capsules, microcapsules, or sustained-release capsules.

[0043] In the case of parenteral administration, the compound can be suitably administered in any of the commonly used dosage forms, such as injections, infusions, and topical preparations (e.g., eye drops, nasal drops, ear drops, aerosols, inhalants, lotions, infusions, liniments, mouthwashes, enemas, ointments, plasters, jellies, creams, patches, poultices, powders for topical use, suppositories, etc.). Injections may be emulsions such as O / W, W / O, O / W / O, and W / O / W types.

[0044] Pharmaceutical compositions can be prepared by mixing an effective amount of the compound of the present invention with various pharmaceutical additives, such as excipients, binders, disintegrants, and lubricants, appropriate for the dosage form, as needed. Furthermore, by appropriately modifying the effective amount of the compound of the present invention, the dosage form, and / or the various pharmaceutical additives, the pharmaceutical composition can also be prepared as a pharmaceutical composition for pediatrics, the elderly, critically ill patients, or surgical patients. For example, pediatric pharmaceutical compositions can be administered to newborns (less than 4 weeks old), infants (4 weeks old to less than 1 year old), toddlers (1 year old to less than 7 years old), children (7 years old to less than 15 years old), or patients aged 15 to 18 years. For example, pharmaceutical compositions for the elderly can be administered to patients aged 65 years or older.

[0045] The dosage of the compound according to the present invention is desirably determined taking into consideration the patient's age, body weight, type and severity of the disease, route of administration, etc., but typically, in the case of oral administration, the dosage for an adult is about 0.5 mg to 100 mg, preferably about 1 mg to 50 mg, and more preferably about 3 mg to 30 mg per day, which may be administered in divided doses if necessary. Furthermore, in the case of children, the dosage is about 0.5 to 100 mg per day. Furthermore, in the case of parenteral administration, the dosage for an adult is about 0.1 mg to 100 mg, preferably about 0.5 mg to 50 mg, or about 1 mg to 30 mg per day. This dosage may be administered once or in divided doses several times a day.

[0046] The formula of the present invention: (wherein each symbol has the same meaning as defined above) or a pharmaceutically acceptable salt thereof can be used in combination with at least one drug selected from the group consisting of drugs having anti-obesity effects, drugs for controlling blood glucose levels, drugs for controlling cholesterol and / or triglycerides in the blood, and drugs for controlling blood pressure, for the purpose of enhancing the effect of the compound or reducing the dose of the compound, etc.

[0047] The "at least one drug selected from the group consisting of drugs having anti-obesity effects, drugs for controlling blood glucose levels, drugs for controlling blood cholesterol and / or triglycerides, and drugs for controlling blood pressure" is not limited to drugs on the market or under development, but examples of drugs on the market or under development include orlistat, cetilistat, phentermine, mazindol, benzphetamine, amfepramone, methamphetamine, phentermine hydrochloride / topiramate, naltrexone hydrochloride / bupropion hydrochloride, liraglutide, semaglutide, setomelanotide (s etmelanotide, RM-493), metreleptin, topiramate, naltrexone, bupropion, acarbose, voglibose, miglitol, ipragliflozin, dapagliflozin, remogliflozin, KGT-1075, luseogliflozin, tofogliflozin, canagliflozin, empagliflozin, ertugliflozin, bexagliflozin, enavogliflozin, henagliflozin, janagliflozin, sotagliflozin, insulin Aspart, insulin lispro, insulin glulisine, biosynthetic human neutral insulin, human insulin, biosynthetic human isophane insulin, human isophane insulin, intermediate-acting insulin lispro, insulin detemir, insulin glargine, insulin degludec, glibenclamide, gliclazide, glimepiride, glipizide, gliquidone, nateglinide, mitiglinide calcium hydrate, repaglinide, metformin hydrochloride, buformin hydrochloride, pioglitazone hydrochloride, rosiglitazone, lobeglitazone ne), sitagliptin phosphate, vildagliptin, alogliptin benzoate, linagliptin, teneligliptin hydrobromide, anagliptin, saxagliptin, trelagliptin succinate, omarigliptin, gemigliptin, evogliptin, lixisenatide, exenatide, dulaglutide, tirzepatide, imeglimin,Sitagliptin phosphate / ipragliflozin, pioglitazone hydrochloride / metformin, pioglitazone hydrochloride / glimepiride, teneligliptin hydrobromide / canagliflozin, alogliptin benzoate / pioglitazone hydrochloride, alogliptin benzoate / metformin hydrochloride, vildagliptin / metformin hydrochloride, mitiglinide calcium / voglibose, pravastatin, simvastatin, fluvastatin, atorvastatin, pitavastatin, rosuvastatin, lovastatin, clinofibrate, clofibrate fibrate, bezafibrate, fenofibrate, ciprofibrate, pemafibrate, gemfibrozil, colestimide, cholestyramine, colesevelam hydrochloride, colestipol, ezetimibe, brobucol, nicomol, tocopherol nicotinate, niceritrol, ethyl icosapentate, omega-3 fatty acid ethyl ester, evolocumab, alirocumab, nifedipine, amlodipine, efonidipine, cilnidipine, nicardipine, nisoldipine, nitrendipine, nilvadipine, Barnidipine, felodipine, benzipine, manidipine, azelnidipine, aranidipine, diltiazem, tolchlormethiazide, benzylhydrochlorothiazide, hydrochlorothiazide, meicran, invadamide, tripamide, mefruside, furosemide, triamterene, spinololactone, eplerenone, tolvaptan, torasemide, hydrochlorothiazide, bumetanide, chlorthalidone, isosorbide, metolazone, losartan, cande Sartan, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, captopril, enalapril, alacepril, delapril, cilazapril, lisinopril, benazepril, imidapril, temocapril, quinapril, trandolapril, perindopril erbumine, urapidil, terazosin, brazosin, doxazosin, bunazosin, atenolol, bisobrolol, metoprolol, acebutolol, celiprolol, propranolol, nadolol, nipradilol, carteolol, pindolol, nebivolol,Carvedilol, labetalol, sotalol, landiolol, arotinolol, amosulalol, arotinolol, carvedilol, labetalol, bevantolol, clonidine, guanabenz, methyldopa, reserpine, hydralazine, nitroprusside, aliskiren, kallidinogenase, alprostadil alfadex, dihydroergotoxine, doxazosin, urapidil, hydralazine, prazosin, moxonidine, guanfacine, rilmenidine, amlo Dipine / atorvastatin, losartan / hydrochlorothiazide, valsartan / hydrochlorothiazide, candesartan / hydrochlorothiazide, telmisartan / hydrochlorothiazide, irbesartan / trichlorthiazide, valsartan / amlodipine, olmesartan / azelnidipine, candesartan / amlodipine, telmisartan / amlodipine, irbesartan / amlodipine, valsartan / cilnidipine, azilsartan / amlodipine, tirzepatide, SCO-094, efinopegdutide (HM12525A), BI -456906, DD01, NN-9423, LY-3437943, danuglipron, PF07081532, LY-3502970, RGT-075, efpeglenatide, exenatide, noiglutide, GMA105, HM-15136, noliglycopeptide (SHR-20004), carbetocin (LV-101), PYY-1562 (NNC0165-1562), PYY-1875 (NNC0165-187 5), cotadutide / AM833 (NN9838, NNC-01740833), cotadutide (cotadutide, MEDI0382), LY3305677, pegapamodutide (pegapamodutide, LY2944876, OPK88003), NGM395, YH34160, CT-388, CT-868, SCO-267, SCO-792, diazoxide, tesofensine, namodenoson, ERX1000, AMG133, ASC41, Xla1,HDV Biotin, EMP16, metoprolol / tesofensine, RZL-012, CB4211, BI1356225, AMG171, NO-13065, bardoxolone methyl, HSG4112, YHC2129, YHC2134, KTX-0200, obeticholic acid, cilofexor (GS-9674), tropifexor (LJN452) , EDP-305, EYP-001, resmetirom (resmetirom), VK-2809, cenicriviroc (cenicriviroc), saroglitazar (saroglitazar), lanifibranor (lanifibranor), selonsertib, PF-06835919, pegbelfermin (pegbelfermin), efrugxifermin (efrugxifermin), Aldafermin, aramcol, MK-3655, MSDC-0602K, belapectin, firsocostat (GS-0976), PF-05221304, ervogastat, ION-224, AXA-1125, HU-6, MET-409, MET-642, TERN-101, TERN-501, LPC Examples of such drugs include N-1144, denifanstate, fluxifermin, leronlimab, pegozafermin, rencofilstat, retatrutide, tipelukast, S-237648, and S-723595. Furthermore, when these drugs do not form a salt, pharmaceutically acceptable salts thereof are also included, and when they form a salt, other pharmaceutically acceptable salts may be formed. Preferably, orlistat, cetilistat, phentermine, mazindol, benzphetamine, amfepramone, methamphetamine, phentermine hydrochloride / topiramate, naltrexone hydrochloride / bupropion hydrochloride, liraglutide, semaglutide, setmelanotide (RM-493), metreleptin, topiramate, naltrexone, bupropion,Tirzepatide, SCO-094, efinopegdutide (HM12525A), BI-456906, DD01, NN-9423, LY-3437943, danuglipron, PF07081532, LY-3502970, RGT-075, efpeglenatide, exenatide, noiglutide, GMA105, HM-15136, noliglycopeptide (SHR-20004), carbetocin (LV-101), PYY-1562 (NNC0165-1562), PYY-1875 (NNC0165-1875), cotadutide / AM833 (NN9838, NNC-01740833), cotadutide (MEDI0382), LY3305677, pegapamodutide (LY294 4876, OPK88003), NGM395, YH34160, CT-388, CT-868, SCO-267, SCO-792, diazoxide, tesofensine, namodenoson, ERX1000, AMG133, ASC41, Xla1, HDV Biotin, EMP16, metoprolol / tesofensine, RZL-012, CB4211, BI1356225, AMG171, NO-13065, bardoxolone methyl, HSG4112, YHC2129, YHC2134, KTX-0200, S-237648, S-723595, etc. More preferred examples include liraglutide, semaglutide, tirzepatide, BI-456906, danuglipron, PF07081532, LY-3502970, RGT-075, S-237648, S-723595, etc.

[0048] The administration timing of the compound of the present invention and the concomitant drug used in combination as described above is not limited, and they may be administered to a subject simultaneously or sequentially, or may be administered at staggered times. Furthermore, the compound of the present invention and the concomitant drug may be administered as two or more types of preparations containing the respective active ingredients, or as a single preparation containing those active ingredients.

[0049] The dose of the concomitant drug can be appropriately selected based on the clinically used dose. The compounding ratio of the compound of the present invention to the concomitant drug can be appropriately selected depending on the administration subject, administration route, target disease, symptoms, combination, etc. For example, when the administration subject is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 1 part by weight of the compound of the present invention.

[0050] The present invention will be explained in more detail below with reference to examples and test examples of the present invention, but the present invention is not limited thereto. Furthermore, changes may be made within the scope of the present invention. The names of compounds shown in the following examples and comparative examples do not necessarily conform to the IUPAC nomenclature.

[0051] (Synthesis Example 1) Compound II-121 Compound II-121 was synthesized from a commercially available compound according to the method described in Patent Document 2. 1 H-NMR(CDCl3) δ: 2.09-2.18(m, 2H), 2.29-2.36(m, 2H), 3.03(d, J=16.3 Hz, 1H), 3.21(s, 3H), 3.31(d, J=16.3 Hz, 1H), 3.99-4.35(m, 10H), 5.92(s, 1H), 6.40(d, J=2.4 Hz, 1H), 6.60(dd, J=8.8, 2.4 Hz, 1H), 7.41(d, J=8.8 Hz, 1H), 9.56(s, 1H).

[0052] The following compounds were synthesized from commercially available compounds according to the methods described in Patent Documents 2 to 4.

[0053]

[0054] The physical data for each compound is shown below. In the table, "MS" indicates the mass (M+H) measured by LC / MS. [Measurement Condition A] Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm id 2.1 x 50 mm) (Waters) Flow rate: 0.8 mL / min UV detection wavelength: 254 nm Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid A linear gradient of 5% to 100% solvent [B] was run over 3.5 minutes, followed by maintaining 100% solvent [B] for 0.5 minutes. [Measurement Condition B] Column: Shim-pack XR-ODS (2.2 μm id50×3.0 mm) (Shimadzu) Flow rate: 1.6 mL / min UV detection wavelength: 254 nm; Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid Gradient: A linear gradient of 10%-100% solvent [B] was performed over 3 minutes, and 100% solvent [B] was maintained for 0.5 minutes. [Measurement Condition C] Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm id 2.1 × 50 mm) (Waters) Flow rate: 0.55 mL / min UV detection wavelength: 254 nm Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [B] is an acetonitrile solution containing 0.1% formic acid Gradient: A linear gradient of 5% to 100% solvent [B] was performed over 3 minutes, and 100% solvent [B] was maintained for 0.5 minutes.

[0055] The NMR analysis obtained in each example was performed at 300 MHz, and DMSO-d ６ , CDCl ３ was measured using

[0056] Test Example 1: Measurement of human MGAT2 inhibitory activity To a 384-well polystyrene microplate (manufactured by Corning) into which 0.2 μL of a DMSO solution of each compound according to the present invention had been dispensed, 5 μL of an enzyme solution prepared with an assay buffer (100 mmol / L phosphate buffer (pH 7.4) containing 2 mmol / L DTT) and 5 μL of a substrate solution (100 mmol / L phosphate buffer (pH 7.4), 30 μmol / L 2-oleoylglycerol, 10 μmol / L oleoyl-CoA) were added, followed by stirring and centrifugation, and then incubation in a humidified box at room temperature for 1 hour. After the enzymatic reaction, 50 μL of stop solution (containing 0.2 μmol / L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing an internal standard (IS) was added to terminate the reaction. The mixture was sealed on a Shimadzu GLC plate, stirred, and centrifuged. The mass was analyzed by electrospray ionization using a RapidFire 360 ​​and an Agilent 6550 Q-TOF mass spectrometer. The ammonium adduct ion (P) of the reaction product (P) Diolein and IS of the substrate 2-oleoylglycerol was detected, and the peak height was used to calculate the peak intensity ratio (P / IS) to evaluate inhibitory activity. The inhibitory activity was calculated using TIBCO Spotfire (TIBCO Software) using the following formula: Control (+) / Control (-) with and without enzyme addition, with the percentage inhibition defined as 0% and 100%, respectively. The peak intensity ratio (P / IS) with the addition of the compound of the present invention was defined as Sample. Inhibitory activity (%) = [1 - {Sample - Control (-)} / {Control (+) - Control (-)}] * 100 The inhibitory activity results for each compound according to the present invention are shown in the following table. ５０ (nM) indicates the concentration exhibiting 50% enzyme inhibition.

[0057]

[0058] Test Example 2: Verification of the effect on hepatic fat accumulation in high-fat diet-fed obese mice. Five-week-old male C57BL / 6J mice were fed a high-fat diet (TestDiet; 58Y1) for four weeks to create high-fat diet-fed obese mice. Starting three weeks prior to compound administration, vehicle (0.5% HPMC) was administered twice daily. Randomization was performed based on changes in body weight and food intake during this acclimation period, and mice were assigned to groups. From Day 0 to Day 35 or Day 88, 0.5% HPMC was administered by gavage twice daily (hereinafter referred to as the control group) or the compounds of the present invention (hereinafter referred to as the II-121 and II-203 groups). After the completion of repeated oral administration, plasma and liver samples were collected, and liver weight and hepatic triglyceride content were measured. Furthermore, plasma liver damage markers and liver fibrosis-related gene expression were analyzed. (Results) At the time of autopsy, the reduction rates of liver weight, hepatic triglyceride, plasma liver damage markers, and hepatic fibrosis-related gene expression compared to the control group were as follows: From these results, the level of hepatic fat accumulation and NASH pathology markers in the liver was evaluated, and it was confirmed that the compound according to the present invention suppressed hepatic fat accumulation and NASH pathology marker levels.

[0059] Test Example 3: Verification of Effects on Liver Fat Accumulation and Hepatotoxicity Using an In Vitro Hepatic Cell Line The in vitro hepatic fat accumulation inhibitory and cytotoxic effects of the compounds and / or drugs according to the present invention were verified using the human hepatic cell line, HepG2 cells. PBS, the compounds according to the present invention, or BMS-963272 were added to HepG2 cells at 10 or 30 μM, and intracellular lipid droplet accumulation after overnight incubation was evaluated. (Results) The results are shown in Figure 1. Compared to the PBS-treated group, the compound II-121 and II-203-treated groups inhibited lipid droplet accumulation in a concentration-dependent manner. On the other hand, the BMS-963272-treated group did not exhibit significant lipid droplet accumulation inhibitory effects. Next, the cytotoxicity of the compounds according to the present invention was evaluated using HepG2 cells. Cytotoxicity was assessed by adding each compound (12.5-100 μM) and culturing for 48 hours, followed by measuring the intracellular ATP content as an indicator of cell viability and LDH activity in the culture supernatant as an indicator of cell death. (Results) The results are shown in Figures 2 and 3. Compound II-203 (100 µM) reduced the intracellular ATP content by approximately 5%, but no change was observed in LDH activity in the culture supernatant. Similarly, compound II-121 (100 µM) reduced the intracellular ATP content by approximately 7%, but no change was observed in LDH activity in the culture supernatant. On the other hand, BMS-963272 (100 µM) reduced the intracellular ATP content by approximately 67%, and increased LDH activity in the culture supernatant by approximately 9-fold. These results suggest that BMS-963272 has stronger cytotoxicity than compounds II-203 and II-121.

[0060] Test Example 4: Verification of the effect on the progression of liver fibrosis in mice fed a high-fat, choline-deficient, methionine-reduced diet. Seven-week-old male C57BL / 6J mice were fed a high-fat, choline-deficient, methionine-reduced diet (RESEARCH DIETES, A06071302). Starting 4 weeks after feeding, mice received repeated oral administration of a vehicle (0.5% methylcellulose solution) once daily (hereinafter referred to as the control group) or a compound of the present invention suspended in 0.5% methylcellulose solution at a dose of 3-30 mg / kg / 10 ml for 8 weeks. After the completion of the repeated oral administration, livers were harvested, and the hepatic hydroxyproline content was measured. (Results) The percentage reduction in hepatic hydroxyproline content compared to the control group at the time of autopsy was as follows. Based on these results, the degree of progression of liver fibrosis was evaluated, and the inhibition of fibrosis progression by the compound of the present invention was confirmed.

[0061] Test Example 5: Evaluation of Toxicity in Rats by Repeated Administration for 4 Days The toxicity of the compounds and / or drugs according to the present invention was evaluated in Crl:CD (SD) rats. Six-week-old male Crl:CD (SD) rats were administered the compounds according to the present invention or BMS-963272 by gavage once daily for 4 days, followed by observation of general condition, measurement of body weight and food intake, hematology and blood chemistry tests, and pathology tests. (Results) No toxicity was observed in the groups administered with compounds II-121 and II-203 up to the maximum dose of 500 mg / kg / day. On the other hand, the BMS-963272 group showed prolongation of blood clotting time and elevation of hepatobiliary damage markers at doses of 500 and 1000 mg / kg / day, while liver damage, kidney damage, deterioration of general condition, and reductions in body weight and food intake were observed at the dose of 1000 mg / kg / day.

[0062] Test Example 6: Metabolic stability test Commercially available pooled human liver microsomes were reacted with the compound of the present invention for a certain period of time, and the residual rate was calculated by comparing the reacted sample with the unreacted sample, thereby evaluating the extent to which the compound of the present invention was metabolized in the liver.

[0063] Human liver microsomes (0.5 mg protein / mL) were incubated in 0.2 mL of buffer (50 mmol / L Tris-HCl pH 7.4, 150 mmol / L potassium chloride, 10 mmol / L magnesium chloride) in the presence of 1 mmol / L NADPH at 37°C for 0 or 30 minutes (oxidative reaction). After the reaction, 50 μL of the reaction mixture was added to 100 μL of a 1 / 1 (v / v) methanol / acetonitrile solution, mixed, and centrifuged at 3,000 rpm for 15 minutes. The compound of the present invention in the supernatant was quantified by LC / MS / MS, and the remaining amount of the compound of the present invention after the reaction was calculated, with the amount of compound at 0 minutes of reaction being 100%. (Results) The remaining percentage at a compound concentration of 0.5 μmol / L is shown.

[0064] Test Example 7: Solubility Test The solubility of the compound according to the present invention was determined under conditions where 1% DMSO was added. A 10 mmol / L compound solution was prepared in DMSO, and 6 μL of the compound solution according to the present invention was added to 594 μL of pH 6.8 artificial intestinal fluid (250 mL of 0.2 mol / L potassium dihydrogen phosphate test solution, 118 mL of 0.2 mol / L NaOH test solution, and water added to make 1000 mL). After standing at 25°C for 16 hours, the mixture was filtered under suction. The filtrate was diluted 2-fold with methanol / water = 1 / 1 (V / V), and the concentration in the filtrate was measured using HPLC or LC / MS / MS according to the absolute calibration curve method. (Results)

[0065] Test Example 8: Phototoxicity Test As an in vitro phototoxicity test, an erythrocyte photohemolysis test (Wolfgang JW Pepe et al., ATLA29, 145-162, 2001), which is an evaluation method using the effect on biological membranes and photoperoxidation as indicators, was performed. In this method, a mixed solution (concentration: 0.1 to 0.0008%) was used, in which 2.5% (v / v) sheep erythrocyte solution was added to a preparation solution of the compound of the present invention in dimethyl sulfoxide as a medium. Two microplates containing this mixed solution were prepared, and one microplate was irradiated with light (10 J / cm) in the UVA and UVB regions using an ultraviolet fluorescent lamp (GL20SE lamp, Sankyo Electric and FL20S-BLB lamp, Panasonic). ２, 290-400 nm), and then centrifuged together with a microplate that had not been irradiated with light. The absorbance (540 nm or 630 nm) of the supernatant was measured. To determine the two indices used to assess phototoxicity (action on biomembranes and photoperoxidation), the absorbance obtained from the compound of the present invention was subtracted from the absorbance in the medium for both the irradiated and unirradiated cases, and the resulting values ​​were used in the subsequent calculations. Regarding the action on biomembranes, the photohemolysis rate was calculated from the difference in absorbance (540 nm) between the irradiated and unirradiated cases. Regarding photoperoxidation, the change in absorbance (630 nm) between the irradiated and unirradiated cases was calculated. In calculating the photohemolysis rate, the absorbance (540 nm) obtained from a 2.5% (v / v) sheep red blood cell solution subjected to forced hemolysis using distilled water was used as the standard for a 100% photohemolysis rate. The case where the photohemolysis rate was less than 10% and the change in absorbance at 630 nm was less than 0.05 was evaluated as (-), and the case where the photohemolysis rate was 10% or more and the change in absorbance at 630 nm was 0.05 or more was evaluated as (+). (Results) Compound I-34: (-)

[0066] Test Example 9: Cytotoxicity Test Using a cell image analyzer, Toxinsight (Thermofisher Scientific), cell numbers after compound exposure were automatically counted to evaluate the cytotoxicity of the compounds of the present invention. HepG2 cells (derived from human hepatoma cells) were seeded into a 384-well plate at 60,000 cells / mL. After 24 hours, a compound solution was added to each well. The compound solutions used were a DMSO solution containing the compound of the present invention (maximum concentration set at 50 μmol / L, diluted 2-fold in five steps, with the lowest concentration being approximately 3.1 μmol / L), a DMSO-only solution as a negative control, and a camptothecin solution as a positive control. A DMSO solution of the compound of the present invention, a negative control solution, or a positive control solution was added to each well. After 71 hours, a Hoechst 33342 solution diluted with Dulbecco's phosphate buffer solution (D-PBS) to a final concentration of 1 μg / mL was added to each well, and the wells were incubated at 37°C and 5% CO ２ The nuclei were stained for 1 hour in an incubator. After staining, the cells were incubated with 4% paraformaldehyde for 20 minutes at 37°C in CO ２The cells were fixed in an incubator. Finally, after washing three times with D-PBS, the number of fluorescently colored nuclei per well was counted using Toxinsight (Thermofisher Scientific). Four wells were set up per concentration, and the mean and variance (SD) of the number of nuclei (number of cells in which no damage was observed) in the four wells were calculated. Compared to the negative control group, the compound exposure concentration (IC) at which the mean value was reduced by 50% or more from the mean value of the negative control was defined as ５０ ) is calculated. ５０ The smaller the value, the higher the risk of cytotoxicity.

[0067] Test Example 10: CYP inhibition test Using commercially available pooled human liver microsomes, typical substrate metabolic reactions of major human CYP5 molecular species (CYP1A2, 2C9, 2C19, 2D6, 3A4) were used as indicators, and the extent to which the production of each metabolite was inhibited by the compound of the present invention was evaluated. The reaction conditions were as follows: substrates, 0.5 μmol / L ethoxyresorufin (CYP1A2), 100 μmol / L tolbutamide (CYP2C9), 50 μmol / L S-mephenytoin (CYP2C19), 5 μmol / L dextromethorphan (CYP2D6), and 1 μmol / L terfenadine (CYP3A4); reaction time, 15 minutes; reaction temperature, 37°C; enzyme, pooled human liver microsomes, 0.2 mg protein / mL; and concentrations of the compound of the present invention, 1, 5, 10, and 20 μmol / L (4 points). A 96-well plate was prepared as a reaction solution, and five types of substrates, human liver microsomes, and the compound of the present invention were added in the above-mentioned composition to a 50 mmol / L Hepes buffer solution. The coenzyme NADPH was then added to initiate the metabolic reaction used as an indicator. After 15 minutes of reaction at 37°C, the reaction was stopped by adding a 1 / 1 (V / V) methanol / acetonitrile solution. After 15 minutes of centrifugation at 3000 rpm, resorufin (a CYP1A2 metabolite) in the supernatant was quantified using a fluorescence multilabel counter or LC / MS / MS. Hydroxylated tolbutamide (a CYP2C9 metabolite), 4'-hydroxylated mephenytoin (a CYP2C19 metabolite), dextrorphan (a CYP2D6 metabolite), and alcoholic terfenadine (a CYP3A4 metabolite) were quantified using LC / MS / MS. A control (100%) was prepared by adding only DMSO, the solvent used to dissolve the drug, to the reaction system. Residual activity (%) was calculated, and the IC was calculated by inverse estimation using a logistic model based on the concentration and inhibition rate. ５０ (Results) Compound II-103: 5 types >20 μmol / L

[0068] Test Example 11: BA Test Materials and Methods for Oral Absorption Studies (1) Animals Used: Mice or SD rats were used. (2) Breeding Conditions: Mice or SD rats were allowed free access to solid feed and sterilized tap water. (3) Dosage and Grouping: Oral and intravenous administration was performed at the specified doses. Groups were set up as follows. (Dosages varied for each compound) Oral Administration: 1-30 mg / kg (n=2-3) Intravenous Administration: 0.5-10 mg / kg (n=2-3) (4) Preparation of Dosage Solution: Oral administration was performed as a solution or suspension. Intravenous administration was performed after solubilization. (5) Administration Method: Oral administration was performed by forced intragastric administration using an oral probe. Intravenous administration was performed via the tail or femoral vein using a syringe with an injection needle. (6) Evaluation Items: Blood samples were collected over time, and plasma concentrations of the compound of the present invention were measured using LC / MS / MS. (7) Statistical analysis: The area under the plasma concentration-time curve (AUC) of the compound according to the present invention was calculated using the nonlinear least squares program WinNonlin (registered trademark) for the plasma concentration change of the compound according to the present invention, and the bioavailability (BA) of the compound according to the present invention was calculated from the AUC of the oral administration group and the intravenous administration group.

[0069] Test Example 12: CYP3A4 (MDZ) MBI Test This test evaluated the mechanism-based inhibition (MBI) ability of the compound of the present invention in terms of metabolic potentiation of CYP3A4 inhibition. Using pooled human liver microsomes, CYP3A4 inhibition was evaluated using the 1-hydroxylation of midazolam (MDZ) as an index. The reaction conditions were as follows: substrate, 10 μmol / L MDZ; pre-reaction time, 0 or 30 minutes; reaction time, 2 minutes; reaction temperature, 37°C; pooled human liver microsomes, 0.5 mg / mL for pre-reaction and 0.05 mg / mL (at 10-fold dilution) for reaction; and compound of the present invention, 1, 5, 10, and 20 μmol / L (4 concentrations) for pre-reaction. Pooled human liver microsomes and a solution of the compound according to the present invention were added to a 96-well plate as a pre-reaction solution in K-Pi buffer (pH 7.4) in the above pre-reaction composition. A portion of this mixture was transferred to another 96-well plate so that it was diluted 1 / 10 with substrate and K-Pi buffer, and the coenzyme NADPH was added to initiate the reaction (no pre-reaction). After the specified reaction time, the reaction was stopped by adding a 1 / 1 (V / V) methanol / acetonitrile solution. NADPH was also added to the remaining pre-reaction solution to initiate the pre-reaction (pre-reaction). After the specified reaction time, a portion of this mixture was transferred to another plate so that it was diluted 1 / 10 with substrate and K-Pi buffer, and the reaction was started. After the specified reaction time, the reaction was stopped by adding a 1 / 1 (V / V) methanol / acetonitrile solution. Each plate in which the indicator reaction was performed was centrifuged at 3000 rpm for 15 minutes, and 1-hydroxymidazolam in the supernatant was quantified by LC / MS / MS. A control (100%) was prepared by adding only DMSO, the solvent in which the compound according to the present invention was dissolved, to the reaction system. The residual activity (%) was calculated when the compound according to the present invention was added at each concentration, and the IC was calculated by inverse estimation using a logistic model using the concentration and inhibition rate. The IC at 0 min preincubation / IC at 30 min preincubation was defined as the Shifted IC value, and a Shifted IC of 1.5 or more was considered positive, and a Shifted IC of 1.0 or less was considered negative. (Results) Compound II-103: Negative

[0070] Test Example 13: Powder Solubility Test An appropriate amount of the compound according to the present invention was placed in an appropriate container, and 200 μL of JP-1 solution (2.0 g of sodium chloride, 7.0 mL of hydrochloric acid, and water added to make 1000 mL), JP-2 solution (500 mL of pH 6.8 phosphate buffer solution added to 500 mL of water), or 20 mmol / L sodium taurocholate (TCA) / JP-2 solution (1.08 g of TCA and JP-2 solution added to make 100 mL) was added to each container. If the entire amount was dissolved after adding the test solution, additional compound according to the present invention was added as appropriate. The container was sealed and shaken at 37°C for 1 hour, then filtered. 100 μL of each filtrate was diluted 2-fold by adding 100 μL of methanol. The dilution ratio was changed as necessary. The container was checked for the presence of bubbles and precipitates, sealed, and shaken. The compound according to the present invention was quantified using HPLC using the absolute calibration curve method.

[0071] Test Example 14: Fluctuation Ames Test The mutagenicity of the compound according to the present invention was evaluated. 20 μL of frozen Salmonella typhimurium (Salmonella typhimurium TA98 strain, TA100 strain) was inoculated into 10 mL of liquid nutrient medium (2.5% Oxoid nutrient broth No. 2) and pre-cultured with shaking at 37°C for 10 hours. For the TA98 strain, 8.0 mL of the bacterial solution was centrifuged (2000 × g, 10 minutes) to remove the culture medium. 8.0 mL of Micro F buffer (K ２ HPO ４ :3.5g / L, KH ２ P.O. ４ : 1 g / L, (NH ４ ) ２ SO ４ : 1 g / L, trisodium citrate dihydrate: 0.25 g / L, MgSO ４ ・7H ２The bacteria were suspended in a 0.0:0.1 g / L solution and added to 120 mL of Exposure medium (MicroF buffer containing biotin: 8 μg / mL, histidine: 0.2 μg / mL, glucose: 8 mg / mL). 3.1 mL of the TA100 strain bacterial solution was added to 120 mL of Exposure medium to prepare a test bacterial solution. The compound according to the present invention was used in a DMSO solution (diluted in several steps at a 2- to 3-fold common ratio from the maximum dose of 50 mg / mL), DMSO as a negative control, and as a positive control, under non-metabolic activation conditions, 50 μg / mL 4-nitroquinoline-1-oxide DMSO solution for the TA98 strain, 0.25 μg / mL 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide DMSO solution for the TA100 strain, and under metabolic activation conditions, 40 μg / mL 2-aminoanthracene DMSO solution for the TA98 strain, and 20 μg / mL 2-aminoanthracene DMSO solution for the TA100 strain, 12 μL each, and 588 μL of test bacterial solution (under metabolic activation conditions, a mixture of 498 μL of test bacterial solution and 90 μL of S9 mix) were mixed and cultured with shaking at 37 ° C. for 90 minutes. 230 μL of the bacterial solution exposed to the compound of the present invention was mixed with 1150 μL of indicator medium (MicroF buffer containing biotin: 8 μg / mL, histidine: 0.2 μg / mL, glucose: 8 mg / mL, and bromocresol purple: 37.5 μg / mL), and 50 μL of the mixture was dispensed into 48 wells of a microplate per dose and incubated statically at 37°C for 3 days. Wells containing bacteria that had acquired the ability to grow due to a mutation in the amino acid (histidine) synthase gene changed color from purple to yellow due to a pH change. The number of wells with bacterial growth that had turned yellow was counted per 48 wells per dose and evaluated by comparison with the negative control group. Negative mutagenicity is indicated by (-), and positive mutagenicity is indicated by (+).

[0072] Test Example 15: hERG Test For the purpose of evaluating the risk of electrocardiogram QT interval prolongation of the compound according to the present invention, a delayed rectifier K channel, which plays an important role in the ventricular repolarization process, was measured using CHO cells expressing the human ether-a-go-go related gene (hERG) channel. ＋ Current (I ＫｒThe effect of the compound of the present invention on the I-cell response was investigated using a fully automated patch clamp system (QPatch; Sophion Bioscience A / S). The cells were held at a membrane potential of −80 mV by the whole-cell patch clamp method, and a leak potential of −50 mV was applied. After that, a depolarizing stimulus of +20 mV was applied for 2 seconds, followed by a repolarizing stimulus of −50 mV for 2 seconds. Ｋｒ After the generated current became stable, the cells were placed in an extracellular solution (NaCl: 145 mmol / L, KCl: 4 mmol / L, CaCl ２ :2 mmol / L, MgCl ２ : 1 mmol / L, glucose: 10 mmol / L, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid): 10 mmol / L, pH = 7.4) was applied to the cells at room temperature for 10 minutes. Ｋｒ The absolute value of the maximum tail current was measured using analysis software (Falster Patch; Sophion Bioscience A / S) based on the current value at the resting membrane potential. Furthermore, the inhibition rate relative to the maximum tail current before application of the compound of the present invention was calculated, and the I Ｋｒ The effect on the growth of the serotonin-producing cells was evaluated. (Results) The inhibition rate at a compound concentration of 10 μmol / L is shown. Compound I-253: 6.9%

[0073] Test Example 16: Reproductive and Developmental Toxicity Test The embryo-fetal developmental toxicity of the compound of the present invention was evaluated in Crl:CD (SD) rats. Female Crl:CD (SD) rats aged 10 to 14 weeks were gavaged with the compound of the present invention once daily for 12 days, from days 6 to 17 of gestation, and then underwent cesarean section on day 21 of gestation. The maternal general condition was observed, and weight and food intake were measured, followed by necropsy and corpus luteum count. In addition, the viability and sex of the embryos and fetuses were confirmed, and weight and placental weight were measured. External and placental macroscopic examinations, and internal organ and skeletal examinations were also performed. (Results) No toxicity was observed in the II-203-treated group up to a dose of 1,000 mg / kg / day.

[0074] The following formulation examples are illustrative only and are not intended to limit the scope of the invention. The compounds of the present invention can be administered as pharmaceutical compositions by any conventional route, particularly enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injection solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments, or creams, or intranasally or in the form of suppositories. Pharmaceutical compositions containing the compounds of the present invention in free form or in the form of a pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier or diluent can be prepared by conventional mixing, granulation, or coating methods. For example, oral compositions can be in the form of tablets, granules, or capsules containing excipients, disintegrants, binders, lubricants, etc., and active ingredients, etc. Injectable compositions can be in the form of solutions or suspensions, which may be sterilized and may contain preservatives, stabilizers, buffers, etc.

[0075] The method for treating and / or preventing non-alcoholic fatty liver disease of the present invention, and the therapeutic pharmaceutical composition used therein, are believed to exhibit excellent therapeutic effects when a predetermined amount of the active ingredient, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, is administered to a patient with non-alcoholic fatty liver disease. Furthermore, the administration of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof does not cause side effects such as hemostatic abnormalities, liver damage, kidney damage, or embryonic lethality, and can be applied extremely safely and is suitable for long-term administration, making the therapeutic and / or preventive method and therapeutic pharmaceutical composition of the present invention extremely excellent.

Claims

1. Formula: (In the formula, R ２ａ and R ２ｂ together with the adjacent carbon atom to form ring B; Ring B is of the formula: (In the formula, R ６ are each independently halogen, haloalkyl, alkyloxy, haloalkyloxy, or cyclopropanyl; R ４ａ is the formula: (In the formula, L ３ is alkylene; R ７ is alkylsulfonyl; R ４ｂ is alkyl optionally substituted with substituent group α, phenyl group optionally substituted with substituent group β, or 5- to 6-membered aromatic heterocyclic group optionally substituted with substituent group β; substituent group α is halogen, haloalkyloxy, and cyclopropanyl, and substituent group β is halogen, cyano, alkyl, haloalkyl, and cyclopropanyl, or a pharma- ceutical composition for treating and / or preventing fatty liver disease, comprising a compound represented by the following formula (1):

2. R ６ are each independently halogen, haloalkyl, or haloalkyloxy; R ４ａ But the formula: And R ４ｂ The pharmaceutical composition according to claim 1, wherein: is haloalkyloxyalkyl, a phenyl group optionally substituted with halogen, or a 5- to 6-membered aromatic heterocyclic group optionally substituted with substituent group β, and substituent group β is halogen and alkyl.

3. Ring B is of the formula: The pharmaceutical composition according to claim 1 or 2, wherein each symbol is as defined in claim 1 or 2.

4. The pharmaceutical composition according to claim 1, comprising a compound selected from the group consisting of compounds I-8, I-23, I-34, I-190, I-212, I-236, I-253, I-275, I-276, II-93, II-103, II-121, II-151, II-168, II-174, II-203, II-225, II-233 and II-295, or a pharma- ceutical acceptable salt thereof.

5. The pharmaceutical composition according to claim 1, comprising a compound selected from the group consisting of compounds I-236, I-253, I-275, II-103, II-121, II-174, II-203, II-225 and II-233, or a pharma- ceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is nonalcoholic fatty liver disease (NAFLD).

7. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is metabolic dysfunction-associated fatty liver disease (MASLD), cryptogenic SLD or specific aetiology SLD.

8. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is nonalcoholic steatohepatitis (NASH).

9. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is metabolic dysfunction-associated steatohepatitis (MASH).

10. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is hepatic fibrosis caused by NASH or MASH.

11. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is cirrhosis caused by NASH or MASH.

12. The pharmaceutical composition according to claim 1, wherein the fatty liver disease is hepatocellular carcinoma (HCC) caused by NASH or MASH.

13. The pharmaceutical composition according to claim 1, wherein administration of said pharmaceutical composition is not associated with at least one of hemostatic abnormalities, liver damage, kidney damage and embryonic lethality.

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