Benzothiazazacycloheptatriene compounds and their uses as bile acid regulators

Abstract

This invention relates to 1,2,5-benzothiazazacycloheptatriene derivatives of formula (I). These compounds are bile acid modulators with inhibitory activity against apical sodium-dependent bile acid transporters (ASBT) and / or hepatic bile acid transporters (LBAT). The invention also relates to pharmaceutical compositions comprising these compounds, and the use of these compounds in the treatment of cardiovascular diseases, fatty acid metabolism and glucose utilization disorders, gastrointestinal diseases, and liver diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Indian Application No. 201911049980, filed on December 4, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to 1,2,5-benzothiazazacycloheptatriene derivatives of formula (I). These compounds are bile acid modulators with inhibitory activity against apical sodium-dependent bile acid transporters (ASBT) and / or hepatic bile acid transporters (LBAT). The invention also relates to pharmaceutical compositions comprising these compounds, and the use of these compounds in the treatment of cardiovascular diseases, fatty acid metabolism and glucose utilization disorders, gastrointestinal diseases, and liver diseases. Background Technology

[0004] Bile acids are physiological cleansers, playing a crucial role in intestinal absorption and the transport of lipids, nutrients, and vitamins. They are also signaling molecules, activating nuclear receptors and regulating cellular signaling pathways that regulate lipid, glucose, and energy metabolism. Bile acids are steroid acids, synthesized from cholesterol in the liver and stored in the gallbladder as mixed microparticles. During digestion, the duodenum triggers the release of hormones that cause gallbladder contraction, releasing bile acids in the small intestine, enabling the absorption of fat-soluble vitamins and cholesterol. Upon reaching the ileum, bile acids are reabsorbed from the intestine and secreted into the portal vein bloodstream, returning to the liver via the portal circulation. Thus, over 90% of bile acids are recycled and return to the liver. These bile acids are then transported across the sinusoidal membrane of hepatocytes and through the tubular membrane to be secreted into the bile. In this first pass, 75-90% of bile acids are absorbed by hepatocytes, completing one cycle of enterohepatic circulation. Some bile acids that escape liver clearance enter systemic circulation, where free bile acids are filtered by the glomeruli, efficiently recycled in the proximal tubules, and expelled back into systemic circulation. Interestingly, most bile acids secreted into the bile across the tubular membrane originate from the recirculation pool, while less than 10% are newly synthesized in the liver. A small portion of bile acids that are not reabsorbed in the ileum reaches the colon. In the intestinal lumen, primary bile acids are converted into secondary bile acids by intestinal bacteria, primarily through mono- or di-dehydroxylation reactions of the steroid nucleus. Bile acids that escape intestinal absorption are then excreted in feces.

[0005] In general, an efficient transport system helps maintain a constant bile acid pool, ensuring sufficiently high levels of conjugated bile acids in the gut to promote lipid absorption and reduce small intestinal bacterial load. This system also minimizes fecal and urinary bile acid loss and protects the gut and hepatobiliary compartments by eliminating potentially cytotoxic cleansers (as reviewed by Kosters and Karpen (Xenobiotica 2008, Vol. 38, pp. 1043-1071); Chiang (J. Lipid Res. 2009, Vol. 50, pp. 1955-1966); and Dawson (Handb. Exp. Pharmacol. 2011, Vol. 201, pp. 169-203)).

[0006] The regulation of bile acid pool size has been found to play a crucial role in cholesterol homeostasis via the liver's conversion of cholesterol into bile acids, representing the main pathway for cholesterol elimination by the body. The liver plays a vital role in the body's removal of endogenous and exogenous compounds. Normal hepatobiliary secretion and enterohepatic circulation are essential for the body's elimination of endogenous compounds such as cholesterol and bilirubin and their metabolites, thereby maintaining lipid and bile acid homeostasis. (Kosters and Karpen, Xenobiotica 2008, Vol. 38, pp. 1043-1071).

[0007] The reabsorption of bile acids in the ileum can be inhibited by apical sodium-dependent bile acid transporter (ASBT) inhibitors. Inhibition of bile acid reabsorption has been reported for the treatment of several diseases, including dyslipidemia, diabetes, obesity, constipation, cholestatic liver disease, non-alcoholic steatohepatitis, and other liver diseases. A variety of ASBT inhibitor compounds have been disclosed over the past decades, see for example WO 93 / 16055, WO 94 / 18183, WO 94 / 18184, WO 96 / 05188, WO 96 / 08484, WO 96 / 16051, WO 97 / 33882, WO 98 / 03818, WO 98 / 07449, WO 98 / 40375、WO 99 / 35135、WO 99 / 64409、WO 99 / 64410、WO 00 / 47568、WO 00 / 61568、WO 00 / 38725、WO 00 / 38726、WO 00 / 38727、WO 00 / 38728、WO 00 / 38729、WO 01 / 66533、WO 01 / 68096、WO 02 / 32428、WO 02 / 50051、WO 03 / 020710、WO 03 / 022286、WO 03 / 022825、WO 03 / 022830、WO 03 / 061663、WO 03 / 091232、WO 03 / 106482、WO 2004 / 006899、WO 2004 / 076430、WO 2007 / 009655、WO 2007 / 009656、WO 2011 / 137135、WO 2019 / 234077、WO 2020 / 161216、WO 2020 / 161217、DE 19825804, EP 864582, EP 489423, EP 549967, EP 573848, EP 624593, EP624594, EP 624595, EP 624596, EP 0864582, EP 1173205, EP 1535913 and EP 3210977.

[0008] Although many ASBT inhibitor compounds have been previously reported, additional bile acid modulating compounds with optimized characteristics in terms of potency, selectivity and bioavailability are still needed. Detailed Implementation

[0009] Certain 1,2,5-benzothiazazacycloheptatriene derivatives have been identified as effective inhibitors of apical sodium-dependent bile acid transporter (ASBT) and / or hepatic bile acid transporter (LBAT), and may be used to treat diseases requiring inhibition of bile acid circulation.

[0010] In a first aspect, the present invention relates to a compound of formula (I).

[0011]

[0012] in

[0013] R 1 and R 2 Each independently is C 1-4 alkyl;

[0014] R 3 Independently selected from: hydrogen, halogen, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, cyano, nitro, amino, N-(C 1-4 Alkyl)amino, N,N-di(C) 1-4 alkyl)amino and N-(aryl-C) 1-4 alkyl)amino;

[0015] n is an integer 1, 2, or 3; and

[0016] R 4 Selected from: hydrogen, halogen, hydroxyl, cyano, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyloxy, C 1-4 Alkylthio, C 3-6 Cycloalkylthio, amino, N-(C 1-4 alkyl)amino and N,N-di(C) 1-4 alkyl)amino;

[0017] Or its pharmaceutically acceptable salt.

[0018] The condition is that the compound is not derived from compounds selected from the following:

[0019] 2-((3,3-Dibutyl-7-methyl-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid;

[0020] 2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid; and

[0021] 2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid.

[0022] In some implementation schemes, R 1 It is n-butyl.

[0023] In some implementation schemes, R 2 C 2-4 Alkyl group. In a preferred embodiment, R 2 It is ethyl. In another preferred embodiment, R 2 It is n-propyl. In another preferred embodiment, R... 2 It is n-butyl.

[0024] In some implementation schemes, R 3 Independently selected from: hydrogen, halogen, hydroxyl, cyano, C 1-4 Haloalkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkoxy group. In a preferred embodiment, R 3 Independently selected from: hydrogen, fluorine, chlorine, bromine, hydroxyl, cyano, trifluoromethyl, methoxy, and trifluoromethoxy.

[0025] In a preferred embodiment, n is 1, that is, the phenyl ring is only substituent R. 3 Replacement. In another preferred embodiment, R 3 In alignment.

[0026] In some implementation schemes, R 4 Selected from: halogen, hydroxyl, cyano, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Alkylthio, amino, N-(C 1-4 alkyl)amino and N,N-di(C) 1-4 Alkyl)amino. In a preferred embodiment, R 4 Selected from: fluorine, chlorine, bromine, hydroxyl, cyano, methyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino, and dimethylamino. In another preferred embodiment, R 4 Selected from: fluorine, chlorine, bromine, hydroxyl, cyano, methoxy, ethoxy, methylthio, ethylthio, and dimethylamino.

[0027] In a preferred embodiment, the compound of formula (I) is the compound of formula (Ia).

[0028]

[0029] in

[0030] R 2 C 2-4 alkyl;

[0031] R 3 Independently selected from: hydrogen, halogen, hydroxyl, cyano, C 1-4 Haloalkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkoxy groups;

[0032] n is an integer, either 1 or 2;

[0033] R 4 Selected from: halogen, hydroxyl, cyano, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Alkylthio, amino, N-(C 1-4 alkyl)amino and N,N-di(C) 1-4 alkyl)amino;

[0034] Or its pharmaceutically acceptable salt.

[0035] The condition is that the compound is not derived from compounds selected from the following:

[0036] 2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid; and

[0037] 2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid.

[0038] In another preferred embodiment, the compound of formula (I) is the compound of formula (Ib).

[0039]

[0040] in

[0041] R 2 It is ethyl, n-propyl, or n-butyl;

[0042] R 3 Selected from: hydrogen, fluorine, chlorine, bromine, hydroxyl, cyano, trifluoromethyl, methoxy, and trifluoromethoxy;

[0043] R 4 Selected from: fluorine, chlorine, bromine, hydroxyl, cyano, methoxy, ethoxy, methylthio, ethylthio, and dimethylamino;

[0044] Or its pharmaceutically acceptable salt.

[0045] The condition is that the compound is not derived from compounds selected from the following:

[0046] 2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid; and

[0047] 2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid.

[0048] The preferred compounds of the present invention are compounds of formula (Ib) as defined above, wherein M and R 1 To R 4 As indicated in Table 1 below, or their pharmaceutically acceptable salts:

[0049] Table 1

[0050]

[0051]

[0052]

[0053] In one specific embodiment, the compound of formula (I) is selected from:

[0054] 2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiazazacycloheptatrien-8-yl)oxy)acetic acid;

[0055] (S)-2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid; and

[0056] (R)-2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid;

[0057] Or its pharmaceutically acceptable salt.

[0058] As used in this article, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0059] As used in this article, the term "C" 1-6 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and the term "C" is used to indicate that the alkyl group has a chain of 1 to 6 carbon atoms. 1-4"Alkyl" refers to a straight-chain or branched alkyl group having 1 to 4 carbon atoms. C 1-4 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0060] As used in this article, the term "C" 1-4 "Halogenated alkyl" refers to a straight-chain or branched C-chain as defined herein. 1-4 Alkyl group, wherein one or more hydrogen atoms have been halogenated. C 1-4 Examples of alkyl halogens include chloromethyl, fluoroethyl, and trifluoromethyl.

[0061] As used in this article, the term "C" 1-4 "alkoxy" and "C" 1-4 "Alkylthio" refers to a straight or branched carbon atom connected to the rest of the molecule via an oxygen or sulfur atom. 1-4 alkyl.

[0062] As used in this article, the term "C" 3-6 "Cycloalkyl" refers to a monocyclic saturated hydrocarbon ring with 3 to 6 carbon atoms. C 3-6 Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0063] The term "aryl" refers to an aromatic monocyclic ring consisting of 6 carbon atoms or an aromatic bicyclic system consisting of 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, and azulel.

[0064] The term "amino" refers to the -NH2 group. As used herein, the term "N-(C)" refers to the -NH2 group. 1-4 "alkyl)amino" and "N,N-di(C 1-4 "alkyl)amino" refers to a chain with one or two hydrogen atoms, either linearly or branched. 1-4 Alkyl-substituted amino group. N-(C 1-4 Examples of alkyl)amino groups include methylamino, ethylamino, and tert-butylamino, and N,N-bis(C 1-4 Examples of alkyl)amino groups include dimethylamino and diethylamino.

[0065] As used in this article, the term "N-(aryl-C)" 1-4 "alkyl)amino" refers to a hydrogen atom via an aryl-C group. 1-4 Alkyl-substituted amino group. N-(aryl-C) 1-4 Examples of alkyl)amino groups include benzylamino and phenylethylamino.

[0066] As used herein, the term “pharmaceutically acceptable” means compounds, substances, compositions and / or dosage forms that are intended for human medicinal use and are generally safe, non-toxic and neither biologically nor otherwise undesirable.

[0067] As used herein, the term "about" refers to a value or parameter, including (and description of) embodiments for that value or parameter itself. For example, a description referring to "about 20" includes the description of "20". A numerical range includes the numerical value defining that range. Generally, the term "about" refers to the specified value of a variable and all values ​​of the variable within the experimental error of the specified value (e.g., within the 95% confidence interval of the mean) or within 10% of the specified value, whichever is greater.

[0068] The 1,2,5-benzothiazazacycloheptatriene compound of formula (I), or a pharmaceutically acceptable salt thereof, is an inhibitor of apical sodium-dependent bile acid transporter (ASBT inhibitor), an inhibitor of hepatic bile acid transporter (LBAT inhibitor), or an inhibitor of both apical sodium-dependent bile acid and hepatic bile acid transporter (dual ASBT / LBAT inhibitor). Therefore, it is used to treat or prevent conditions, disorders, and diseases requiring inhibition of bile acid cycling, such as cardiovascular diseases, fatty acid metabolism and glucose utilization disorders, gastrointestinal diseases, and liver diseases.

[0069] Cardiovascular diseases and disorders of fatty acid metabolism and glucose utilization, including but not limited to hypercholesterolemia; fatty acid metabolism disorders; type 1 and type 2 diabetes; complications of diabetes, including cataracts, microvascular and macrovascular diseases, retinopathy, neuropathy, nephropathy and delayed wound healing, tissue ischemia, diabetic foot, arteriosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, arrhythmia and restenosis; diabetes-related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, obesity, dyslipidemia, including hyperlipidemia with hypertriglyceridemia, metabolic syndrome (Syndrome X), atherosclerosis and hypertension; and for increasing high-density lipoprotein levels.

[0070] Gastrointestinal disorders and disturbances include constipation (including chronic constipation, functional constipation, chronic idiopathic constipation (CIC), intermittent / occasional constipation, constipation secondary to diabetes, constipation secondary to stroke, constipation secondary to chronic kidney disease, constipation secondary to multiple sclerosis, constipation secondary to Parkinson's disease, constipation secondary to systemic sclerosis, drug-induced constipation, irritable bowel syndrome with constipation (IBS-C), mixed irritable bowel syndrome (IBS-M), functional constipation in children, and opioid-induced constipation); Crohn's disease; primary bile acid malabsorption; irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ileitis; and reflux diseases and their complications, such as Barrett's esophagus. (esophagus), bile reflux esophagitis, and bile reflux gastritis.

[0071] As defined in this article, liver disease refers to any disease of the liver and its connected organs, such as the pancreas, portal vein, liver parenchyma, intrahepatic bile tree, extrahepatic bile tree, and gallbladder. In some cases, liver disease is bile acid-dependent liver disease. Liver diseases and disorders include, but are not limited to, hereditary liver metabolic disorders; congenital errors in bile acid synthesis; congenital bile duct abnormalities; biliary atresia; post-Kasai biliary atresia; post-liver transplant biliary atresia; neonatal hepatitis; neonatal cholestasis; hereditary forms of cholestasis; cerebral tendinous xanthomas; secondary BA synthesis defects; Zellweger's syndrome; cystic fibrosis-related liver disease; α1-antitrypsin deficiency; Alagilles syndrome (ALGS); Byler syndrome. This includes conditions such as: 1. Primary bile acid (BA) synthesis deficiency; 2. Progressive familial intrahepatic cholestasis (PFIC), including PFIC-1, PFIC-2, PFIC-3 and nonspecific PFIC, post-cholecystectomy PFIC, and post-liver transplantation PFIC; 3. Benign recurrent intrahepatic cholestasis (BRIC), including BRIC1, BRIC2 and nonspecific BRIC, post-cholecystectomy BRIC, and post-liver transplantation BRIC; 4. Autoimmune hepatitis; 5. Primary biliary cirrhosis (PBC); 6. Liver fibrosis; 7. Nonalcoholic fatty liver disease (NAFLD); 8. Nonalcoholic steatohepatitis (NASH); 9. Portal hypertension; 10. Cholestasis; 11. Down syndrome. cholestasis; drug-induced cholestasis; intrahepatic cholestasis of pregnancy (jaundice of pregnancy); intrahepatic cholestasis; extrahepatic cholestasis; parenteral nutrition-associated cholestasis (PNAC); hypophospholipid-associated cholestasis; lymphedema cholestasis syndrome 1 (LSC1); primary sclerosing cholangitis (PSC); immunoglobulin G4-associated cholangitis; primary biliary cholangitis; cholelithiasis (gallstones); biliary duct stones; common bile duct stones; cholelithiasis pancreatitis; Carole disease (Carolei disease) Disease); malignant tumors of the bile ducts; malignant tumors leading to obstruction of the bile duct tree; biliary stricture; AIDS cholangitis; ischemic cholangitis; pruritus caused by cholestasis or jaundice; pancreatitis; chronic autoimmune liver disease leading to progressive cholestasis; hepatic steatosis; alcoholic hepatitis; acute fatty liver; fatty liver of pregnancy; drug-induced hepatitis; iron overload; congenital bile acid synthesis deficiency type 1 (BAS type 1); drug-induced liver injury (DILI); liver fibrosis; congenital liver fibrosis; cirrhosis; Langerhans cell histiocytosis (LCH); neonatal ichthyosis sclerosing cholangitis (NISCH); erythropoietic protoporphyria (EPP);Idiopathic adult-onset bile duct absence (IAD); idiopathic neonatal hepatitis (INH); non-symptomatic interlobular bile duct absence (NS PILBD); North American Indian childhood cirrhosis (NAIC); hepatic sarcoidosis; amyloidosis; necrotizing enterocolitis; serum bile acid toxicity, including arrhythmias with abnormal serum bile acid distribution patterns (e.g., atrial fibrillation), cirrhosis-associated cardiomyopathy (“cholecardia”), and skeletal muscle atrophy associated with cholestatic liver disease; polycystic liver disease; viral hepatitis (including hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E); hepatocellular carcinoma (liver cancer); cholangiocarcinoma; bile acid-associated gastrointestinal cancers; and cholestasis caused by tumors and vegetations of the liver, bile ducts, and pancreas. Compounds of formula (I) or pharmaceutically acceptable salts thereof are also used to enhance corticosteroid therapy for liver diseases.

[0072] Other diseases that can be treated or prevented by compounds of formula (I) or their pharmaceutically acceptable salts include hyperabsorption syndromes (including abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicron retention disease (CRD), and sitosterolemia); hypervitaminosis and osteosclerosis; hypertension; glomerular hyperfiltration; polycystic kidney disease (PKD) (including autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD)); and pruritus associated with renal failure. The compounds are also used for the protection against kidney damage associated with liver disease or metabolic disorders.

[0073] The transport of bile acids in the human body is controlled by members of the SLC10 solute carrier protein family, especially Na+. + Taurocholate cotransporter (NTCP, also known as hepatic bile acid transporter (LBAT); gene symbol SLC10A1) is expressed in the sinusoidal membrane of hepatocytes; and apical sodium-dependent bile acid transporter (ASBT, also known as ileal bile acid transporter (IBAT), ISBT, ABAT, or NTCP2; gene symbol SLC10A2) is expressed in the apical membrane of ileal epithelial cells, proximal renal tubular cells, bile duct epithelial cells, gallbladder duct cells, and gallbladder epithelial cells. In the liver, bile acids are efficiently extracted from portal vein blood via hepatic bile acid transporter (LBAT) and resected across the tubular membrane via the bile salt export pump (BSEP; gene symbol ABCB11). Reabsorption of bile acids in the ileum is handled by apical sodium-dependent bile acid transporter (ASBT), commonly referred to as ileal bile acid transporter (IBAT). Both LBAT and ASBT act as electrostatic sodium-solute cotransporters, transporting two or more Na+ molecules per solute molecule. + ion.

[0074] Exogenous and endogenous substances, including bile acids, are absorbed from the portal vein by the liver and secreted into bile via different transport proteins with individualized substrate specificity. Glycine-conjugated and taurine-conjugated bile acids exist in anionic form and cannot diffuse across the membrane; therefore, they depend entirely on membrane transport proteins for entry into or exit from hepatocytes (Kosters and Karpen, Xenobiotica 2008, Vol. 38, pp. 1043-1071). ASBT and LBAT show a preference for glycine-conjugated and taurine-conjugated bile salts over their unconjugated counterparts, and exhibit a higher affinity for dihydroxy bile salts than for trihydroxy bile salts. No non-bile acid substrates for ASBT have been identified; however, LBAT has been found to transport various steroid sulfates, hormones, and exogenous substances.

[0075] Regarding the requirements for drug inhibition, LBAT is not as thoroughly characterized as ASBT. Dong et al. have identified FDA-approved drugs that inhibit human LBAT and compared the inhibition requirements of LBAT and ASBT. Using FDA-approved drugs, a series of LBAT inhibition studies were conducted using iterative computational models. Screening studies identified 27 drugs as novel LBAT inhibitors, including irbesartan (Ki = 11.9 μM) and ezetimibe (Ki = 25.0 μM). Common pharmacophore characteristics indicate that two hydrophobic compounds and one hydrogen-bonded receptor are important for LBAT inhibition. Of the 72 drugs screened in vitro, 31 inhibited LBAT, while 51 (more than half) inhibited ASBT. Therefore, despite inhibitor overlap, ASBT is surprisingly more tolerant of drug inhibition than LBAT, which may be related to the fact that LBAT has fewer pharmacophore features (Dong et al., Mol. Pharm. 2013, Vol. 10, pp. 1008-1019).

[0076] Vaz et al. described the identification of LBAT deficiency as a novel innate metabolic error with relatively mild clinical phenotypes. The identification of LBAT deficiency confirms that this transporter is the main input system for conjugated bile salts into the liver, and also indicates that helper transporters can maintain enterohepatic circulation in its absence (Vaz et al., Hepatology 2015, Vol. 61, pp. 260-267). These findings support the hypothesis that LBAT inhibition is a safe mechanism of action, as hepatocytes can still absorb the necessary amount of bile acids.

[0077] Liu et al. described a novel method for identifying hypercholanemia associated with homozygosity of the p.Ser267Phe mutation in SLC10A1 (LBAT). The allele frequency of this mutation in the SLC10A1 gene varied across different populations. Increased serum BA levels were also observed in both conjugated and unconjugated individuals in homozygous individuals. Liu et al. proposed that this finding is most likely attributable to reduced BA transport from the portal circulation to hepatocytes. This supports the hypothesis that the physiological function of enterohepatic circulation not only recycles bile acids but also clears them from the circulation to achieve homeostasis (Karpen and Dawson, Hepatology 2015, Vol. 61, pp. 24-27). Alternatively, the liver may synthesize increased bile acid levels to compensate for reduced enterohepatic recirculation in homozygous individuals. Since LBAT also transports unconjugated bile acids, the increase in unconjugated bile acids in this study was not unexpected (Liu et al., Scientific Reports 2017, 7:9214, pp. 1-7).

[0078] LBAT has been found to be downregulated in several forms of cholestatic liver injury and cholestasis, while ASBT has been found to be downregulated in various gastrointestinal disorders, such as Crohn's disease, primary bile acid malabsorption, inflammatory bowel disease, and ileitis, but upregulated in cholestasis. LBAT also acts as a cell receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV), which are major causes of liver disease and hepatocellular carcinoma.

[0079] ASBT inhibition has been studied for its effects in lowering plasma cholesterol levels, improving insulin resistance, and reducing hepatic bile acid load in cholestatic liver disease. Furthermore, ASBT inhibition has been found to restore normal insulin and blood glucose levels, thus establishing ASBT inhibition as a promising treatment for type 2 diabetes. ASBT inhibitors are also used to treat functional constipation.

[0080] Since ASBT is primarily expressed in the ileum (where it is commonly referred to as IBAT), ASBT inhibitors do not necessarily need to be systemically available. On the other hand, ASBT is also expressed in the proximal tubular cells of the kidney. Therefore, systemically available ASBT inhibitors can also inhibit the reabsorption of bile acids in the kidney. This is believed to lead to an increase in urinary bile acid levels and increased removal of bile acids from the body via urine. Therefore, systemically available ASBT inhibitors that act not only in the ileum but also in the kidney are expected to result in a greater reduction in bile acid levels than non-systemically available ASBT inhibitors that act only in the ileum.

[0081] Compounds with high ASBT inhibitory potency are particularly used to treat liver diseases that cause cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), Arajary syndrome, biliary atresia, and nonalcoholic steatohepatitis (NASH).

[0082] Biliary atresia is a rare pediatric liver disease involving partial or complete obstruction (or even absence) of the bile ducts. This obstruction or absence causes bile stasis, leading to the accumulation of bile acids, which damage the liver. In some implementations, bile acid accumulation occurs in the extrahepatic bile duct tree. In other implementations, bile acid accumulation occurs in the intrahepatic bile duct tree. The current standard of care is the Kasai procedure, which involves removing the obstructed bile duct and directly connecting a portion of the small intestine to the liver. There is currently no approved drug therapy for this disorder.

[0083] This document provides a method for treating biliary atresia in an individual of need, the method comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject has undergone a Kasai procedure prior to administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is administered the compound of formula (I) or a pharmaceutically acceptable salt thereof prior to the Kasai procedure. In some embodiments, treatment of biliary atresia reduces the subject's serum bile acid levels. In some embodiments, serum bile acid levels are determined by, for example, an ELISA enzyme assay or an assay measuring total bile acids, as described in Danese et al., PLoS One. 2017, Vol. 12(6): e0179200, which is incorporated herein by reference in its entirety. In some embodiments, serum bile acid levels may be reduced by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to 70%, 50% to 80%, or greater than 90% of the serum bile acid levels prior to administration of the compound of formula (I) or its pharmaceutically acceptable salt. In some embodiments, treatment of biliary atresia includes treatment of pruritus.

[0084] PFIC is a rare genetic disorder that is estimated to affect one in 50,000 to 100,000 children born worldwide and causes progressive, life-threatening liver disease.

[0085] One manifestation of PFIC is pruritus, which often leads to a significant decline in quality of life. In some cases, PFIC can lead to cirrhosis and liver failure. Current treatments include partial biliary diversion (PEBD) and liver transplantation; however, these options may carry a considerable risk of postoperative complications, as well as psychological and social problems.

[0086] Three alternative gene defects have been identified, which are associated with three separate PFIC subtypes known as type 1, type 2, and type 3:

[0087] PFIC type 1, sometimes called "Bayer's disease," is caused by impaired bile secretion due to a mutation in the ATP8B1 gene, which encodes a protein that helps maintain the proper balance of lipids called phospholipids in the bile duct cell membranes. Imbalances in these phospholipids are associated with cholestasis and elevated bile acid levels in the liver. Subjects affected by PFIC type 1 typically develop cholestasis in the first few months after birth and, without surgical treatment, progress to cirrhosis and end-stage liver disease before the end of their first decade of life.

[0088] PFIC type 2, sometimes called "Beyer syndrome," is caused by impaired bile salt secretion due to a mutation in the ABCB11 gene, which encodes a protein called the bile salt export pump, which removes bile acids from the liver. Individuals with PFIC type 2 typically develop liver failure within the first few years of life and have an increased risk of developing a type of liver cancer called hepatocellular carcinoma.

[0089] PFIC type 3, which usually occurs in the first few years of childhood with progressive cholestasis, is caused by a mutation in the ABCB4 gene, which encodes a transport protein that moves phospholipids across the cell membrane.

[0090] Furthermore, mutations in the TJP2, NR1H4, or Myo5b genes have been proposed as causes of PFIC. Additionally, some subjects with PFIC do not have mutations in any of the ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b genes. In these cases, the cause of the condition is unknown.

[0091] Exemplary mutations of the ATP8B1 gene or the resulting protein are listed in Tables 2 and 3, where the numbers are based on the human wild-type ATP8B1 protein (e.g., SEQ ID NO:1) or gene (e.g., SEQ ID NO:2). Exemplary mutations of the ABCB11 gene or the resulting protein are listed in Tables 4 and 5, where the numbers are based on the human wild-type ABCB11 protein (e.g., SEQ ID NO:3) or gene (e.g., SEQ ID NO:4).

[0092] As those skilled in the art will understand, the amino acid positions in the reference protein sequence corresponding to specific amino acid positions of SEQ ID NO:1 or 3 can be determined by comparing the reference protein sequence with SEQ ID NO:1 or 3 (e.g., using software programs such as ClustalW2). Changes to these residues (referred to herein as “mutations”) can include single or multiple amino acid substitutions, intra- or flanking insertions, and intra- or flanking deletions. Similarly, as those skilled in the art will understand, the nucleotide positions in the reference gene sequence corresponding to specific nucleotide positions of SEQ ID NO:2 or 4 can be determined by comparing the reference gene sequence with SEQ ID NO:2 or 4 (e.g., using software programs such as ClustalW2). Changes to these residues (referred to herein as “mutations”) can include single or multiple nucleotide substitutions, intra- or flanking insertions, and intra- or flanking deletions. See also Kooistra et al., “KLIFS: A structural kinase-ligand interaction database”, Nucleic Acids Res. 2016, Vol. 44, No. D1, pp. D365-D371, which is incorporated herein by reference in its entirety.

[0093] Typical protein sequence of ATP8B1 (SEQ ID NO:1) - Uniproto ID O43520

[0094]

[0095]

[0096] Typical DNA sequence of ATP8B1 (SEQ ID NO:2)

[0097]

[0098]

[0099] Table 2. Exemplary ATP8B1 mutations

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Table 3. Selected ATP8B1 mutations associated with PFIC-1

[0110]

[0111]

[0112]

[0113]

[0114] A The mutation to "X" indicates an early stop codon.

[0115] References in Tables 2 and 3

[0116] 1 Folmer et al., Hepatology. 2009, Vol. 50(5), pp. 1597-1605

[0117] 2 Hsu et al., Hepatol Res. 2009, Vol. 39(6), pp. 625-631

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[0119] 4 Davit-Spraul et al., Hepatology 2010, Vol. 51(5), pp. 1645-1655

[0120] 5 Vitale et al., J Gastroenterol. 2018, Vol. 53(8), pp. 945-958

[0121] 6 Klomp et al., Hepatology 2004, Vol. 40(1), pp. 27-38

[0122] 7Zarenezhad et al., Hepatitis Monthly: 2017, Vol. 17(2); e43500.

[0123] 8 Dixon et al., Scientific Reports 2017, Vol. 7, 11823.

[0124] 9 Painter et al., Eur J Hum Genet. 2005, Vol. 13(4), pp. 435-439

[0125] 10 Deng et al., World J Gastroenterol. 2012, Vol. 18(44), pp. 6504-6509

[0126] 11 Giovannoni et al., PLoS One. 2015, Vol. 10(12):e0145021.

[0127] 12 Li et al., Hepatology International 2017, Vol. 11, No. 1, Supp. 1, pp. S180. Abstract No. OP284.

[0128] 13 Togawa et al., Journal of Pediatric Gastroenterology and Nutrition, 2018, Vol. 67, Supp. 1, pp. S363. Abstract No. 615.

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[0130] 15 et al., Zeitschrift fur Gastroenterologie 2015, Vol. 53, No. 12. Abstract No. A3-27. Conference Information: 32. Jahrestagung der Deutschen Arbeitsgemeinschaft zum Studium der Leber, Düsseldorf, Germany, January 22-23, 2016.

[0131] 16 Mizuochi et al., Clin Chim Acta. 2012, Vol. 413(15-16), pp. 1301-1304

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[0136] 21 Kang et al., J Pathol Transl Med. May 16, 2019. doi:10.4132 / jptm.2019.05.03. [Earlier than preprinted electronic publication]

[0137] 22 Sharma et al., BMC Gastroenterol. 2018, Vol. 18(1), p. 107

[0138] 23Uegaki et al., Intern Med. 2008, Vol. 47(7), pp. 599-602

[0139] 24 Goldschmidt et al., Hepatol Res. 2016, Vol. 46(4), pp. 306-311

[0140] 25 Liu et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 50(2), pp. 179-183

[0141] 26 Jung et al., J Pediatr Gastroenterol Nutr. 2007, Vol. 44(4), pp. 453-458

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[0145] 30 Blackmore et al., J Clin Exp Hepatol. 2013, Vol. 3(2), pp. 159-161

[0146] 31Matte et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 51(4), pp. 488-493

[0147] 32 Squires et al., J Pediatr Gastroenterol Nutr. 2017, Vol. 64(3), pp. 425-430

[0148] 33 Hayshi et al., EBioMedicine. 2018, Vol. 27, pp. 187-199

[0149] 34 Nagasaka et al., J Pediatr Gastroenterol Nutr. 2007, Vol. 45(1), pp. 96-105

[0150] 35 Wang et al., PLoS One. 2016; Vol. 11(4): e0153114.

[0151] 36 Narchi et al., Saudi J Gastroenterol. 2017, Vol. 23(5), pp. 303-305

[0152] 37 Alashkar et al., Blood 2015, Vol. 126, No. 23. Conference Information: 57th Annual Meeting of the American Society of Hematology. Orlando, Florida, USA, December 5-8, 2015, Amer Soc Hematol.

[0153] 38 Ferreira et al., Pediatric Transplantation 2013, Vol. 17, Supp. SUPPL. 1, pp. 99. Abstract No. 239. Conference Information: IPTA 7th Congress on Pediatric Transplantation. Warsaw, Poland, July 13-16, 2013.

[0154] 39 Pauli-Magnus et al., J Hepatol. 2005, Vol. 43(2), pp. 342-357

[0155] 40 Jericho et al., Journal of Pediatric Gastroenterology and Nutrition, 2015, Vol. 60(3), pp. 368-374

[0156] 41 van der Woerd et al., PLoS One. 2013, Vol. 8(11):e80553.

[0157] 42 Copeland et al., J Gastroenterol Hepatol. 2013, Vol. 28(3), pp. 560-564

[0158] 43 et al., J Hepatol. 2017, Vol. 67(6), pp. 1253-1264

[0159] 44 Chen et al., Journal of Pediatrics 2002, Vol. 140(1), pp. 119-124

[0160] 45 Jirsa et al., Hepatol Res. 2004, Vol. 30(1), pp. 1-3

[0161] 46 van der Woerd et al., Hepatology 2015, Vol. 61(4), pp. 1382-1391

[0162] In some implementations, the mutations in ATP8B1 are selected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, and G1040R.

[0163] Typical protein sequence of ABCB11 (SEQ ID NO:3) - Uniproto ID O95342

[0164]

[0165] Typical DNA sequence of ABCB11 (SEQ ID NO:4)

[0166]

[0167]

[0168]

[0169] Table 4. Exemplary ABCB11 mutations

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] Table 5. Selected ABCB11 mutations associated with PFIC-2

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] A The mutation to "X" indicates an early stop codon.

[0194] References in Tables 4 and 5

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[0241] 47 Shimizu et al., Am J Transplant. 2011, Vol. 11(2), pp. 394-398

[0242] 48 Krawczyk et al., Ann Hepatol. 2012, Vol. 11(5), pp. 710-744

[0243] 49 Sharma et al., BMC Gastroenterol. 2018, Vol. 18(1), p. 107

[0244] 50Sattler et al., Journal of Hepatology 2017, Vol. 66, No. 1, Suppl.S, pp.S177. Conference Information: International Liver Congress / 52nd Annual Meeting of the European Association for the Study of the Liver. Amsterdam, Netherlands, April 19-23, 2017, European Association for Study Liver.

[0245] 51 Jung et al., J Pediatr Gastroenterol Nutr. 2007, Vol. 44(4), pp. 453-458

[0246] 52 Sciveres. Digestive and Liver Disease 2010, Vol. 42, Supp. SUPPL. 5, pp. S329. Abstract No. CO18. Conference Information: 17th National Congress SIGENP. Pescara, Italy, October 7-9, 2010.

[0247] 53 Sohn et al., Pediatr Gastroenterol Hepatol Nutr. 2019, Vol. 22(2), pp. 201-206

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[0249] 55 Wang et al., Hepatol Res. 2018, Vol. 48(7), pp. 574-584

[0250] 56 Shaprio et al., J Hum Genet. 2010, Vol. 55(5), pp. 308-313

[0251] 57Bounford, University of Birmingham, Dissertation Abstracts International, (2016), Vol. 75, No. 1C, Subscription No.: AAI10588329. ProQuest Dissertations & Theses.

[0252] 58 Stolz et al., Aliment Pharmacol Ther. 2019, Vol. 49(9), pp. 1195-1204

[0253] 59 Jankowska et al., J Pediatr Gastroenterol Nutr. 2014, Vol. 58(1), pp. 92-95

[0254] 60 Kim. Journal of Pediatric Gastroenterology and Nutrition 2016, Vol. 62, Supp. SUPPL. 1, pp. 620. Abstract No. HP-045. Conference Information: 49th Annual Meeting of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition, ESPGHAN 2016, Athens, Greece, May 25-28, 2016.

[0255] 61 Pauli-Magnus et al., Hepatology 2003, Vol. 38, No. 4, Suppl. 1, pp. 518A.print. Conference Information: 54th Annual Meeting of the American Association for the Study of Liver Diseases. Boston, Massachusetts, USA, October 24-28, 2003, American Association for the Study of Liver Diseases.

[0256] 62Li et al., Hepatology International 2017, Vol. 11, No. 1, Supp. 1, pp. S362. Abstract No. PP0347. Conference Information: 26th Annual Conference of the Asian Pacific Association for the Study of the Liver, APASL 2017. Shanghai, China, February 15-19, 2017.

[0257] 63 Rumbo et al., Transplantation 2018, Vol. 102, No. 7, Supp. 1, pp. S848. Abstract p. 752. Conference Information: 27th International Congress of the Transplantation Society, TTS 2018. Madrid, Spain, June 30 – July 5, 2018.

[0258] 64 Lee et al., Pediatr Gastroenterol Hepatol Nutr. 2017, Vol. 20(2), pp. 114-123

[0259] 65 Sherrif et al., Liver International: Official Journal of the International Association for the Study of the Liver 2013, Vol. 33, No. 8, pp. 1266-1270

[0260] 66 Blackmore et al., J Clin Exp Hepatol. 2013, Vol. 3(2), pp. 159-161

[0261] 67 Matte et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 51(4), pp. 488-493

[0262] 68 Lin et al., Zhongguo Dang Dai Er Ke Za Zhi.2018, Volume 20(9), Pages 758-764

[0263] 69 Harmanci et al., Experimental and Clinical Transplantation 2015, Vol. 13, Supp. SUPPL. 2, pp. 76. Abstract number: P62. Conference information: 1st Congress of the Turkic World Transplantation Society. Astana, Kazakhstan, May 20-22, 2015.

[0264] 70 Herbst et al., Mol Cell Probes. 2015, Vol. 29(5), pp. 291-298

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[0266] 72 Holz et al., Zeitschrift for Gastroenterologie 2016, Vol. 54, No. 8. Abstract No. KV275. Conference Information: Viszeralmedizin 2016, 71. Jahrestagung der Deutschen Gesellschaft für Gastroenterologie, Verdauungs-und Stoffwechselkrankheiten mit Sektion Endoskopie-10. Herbsttagung der Deutschen Gesellschaft für Allgemein-und Viszeralchirurgie. Hamburg, Germany, 21 September to 24 September 2016.

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[0268] 74 Hao et al., International Journal of Clinical and Experimental Pathology 2017, Vol. 10(3), pp. 3480-3487.

[0269] 75Arnell et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 51(4), pp. 494-499

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[0271] 77 Beauséjour et al., Can J Gastroenterol. 2011, Vol. 25(6), pp. 311-314

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[0273] 79 Peng et al., Zhonghua Erkezazhi (Chinese Journal of Pediatrics), 2018, Vol. 56, No. 6, pp. 440-444.

[0274] 80 Tibesar et al., Case Rep Pediatr. 2014, Vol. 2014: 185923.

[0275] 81Ng et al., Journal of Pediatric Gastroenterology and Nutrition 2018, Vol. 66, Supp. 2, pp. 860. Abstract number HP-127. Conference information: 51st Annual Meeting European Society for Paediatric Gastroenterology, Hepatology and Nutrition, ESPGHAN 2018. Geneva, Switzerland, May 9-12, 2018.

[0276] 82 Wong et al., Clin Chem. 2008, Vol. 54(7), pp. 1141-1148

[0277] 83 Pauli-Magnus et al., J Hepatol. 2005, Vol. 43(2), pp. 342-357

[0278] 84 Jericho et al., Journal of Pediatric Gastroenterology and Nutrition, Vol. 3, No. 60, pp. 368-374.

[0279] 85 Scheimann et al., Gastroenterology 2007, Vol. 132, No. 4, Suppl. 2, pp. A452. Conference Information: Digestive Disease Week Meeting / 108th Annual Meeting of the American-Gastroenterological-Association. Washington, D.C., USA, May 19-24, 2007. Amer Gastroenterol Assoc; Amer Assoc Study Liver Dis; Amer Soc Gastrointestinal Endoscopy; Soc Surg Alimentary Tract.

[0280] 86 Jaquotot-Haerranz et al., Rev Esp Enferm Dig. 2013, Vol. 105(1), pp. 52-54

[0281] 87Khosla et al., American Journal of Gastroenterology 2015, Vol. 110, Suppl. 1, pp. S397. Conference Information: 80th Annual Scientific Meeting of the American-College-of-Gastroenterology. Honolulu, Hawaii, USA, October 16-21, 2015.

[0282] 88 et al., J Hepatol. 2017, Vol. 67(6), pp. 1253-1264

[0283] 89 Liu et al., Liver International 2010, Vol. 30(6), pp. 809-815

[0284] 90 Chen et al., Journal of Pediatrics 2002, Vol. 140(1), pp. 119-124

[0285] 91 U.S. Patent 9,295,677

[0286] In some implementations, the mutations in ABCB11 are selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V, G982R, R1153C, and R1268Q.

[0287] A method for providing a PFIC (e.g., PFIC-1 and PFIC-2) to a subject includes analyzing a sample obtained from the subject to determine whether the subject has a PFIC-related mutation (e.g., ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b mutation), and administering (e.g., specific or selective administration) a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to the subject determined to have a PFIC-related mutation. In some embodiments, the mutation is an ATP8B1 or ABCB11 mutation. For example, mutations provided in any of Tables 2 through 5. In some embodiments, the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, and G1040R. In some implementations, the mutations in ABCB11 are selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V, G982R, R1153C, and R1268Q.

[0288] Methods for treating PFIC (e.g., PFIC-1 and PFIC-2) in subjects in need are also provided, comprising: (a) detecting a PFIC-related mutation (e.g., ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b mutation) in the subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the method for treating PFIC may include administering to a subject having a PFIC-related mutation (e.g., ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b mutation) a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the mutation is an ATP8B1 or ABCB11 mutation. For example, mutations provided by any of Tables 2 through 5. In some embodiments, the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11 is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V, G982R, R1153C, and R1268Q.

[0289] In some embodiments, the presence of a PFIC-associated mutation in the subject or in a biopsy sample from the subject is determined using any test recognized in the art, including next-generation sequencing (NGS). In some embodiments, the presence of a PFIC-associated mutation in the subject is determined using a regulatory-approved (e.g., FDA-approved) test or analysis for identifying PFIC-associated mutations in the subject or in a biopsy sample from the subject, or by any of the non-limiting examples of the analysis described herein. Additional methods for diagnosing PFIC are described in Gunaydin, M. et al., Hepat Med. 2018, Vol. 10, pp. 95–104, which are incorporated herein by reference in their entirety.

[0290] In some embodiments, treatment with a PFIC (e.g., PFIC-1 or PFIC-2) reduces serum bile acid levels in the subject. In some embodiments, serum bile acid levels are determined by, for example, an ELISA enzyme assay or an assay measuring total bile acids, as described in Danese et al., PLoS One. 2017, Vol. 12(6): e0179200, which is incorporated herein by reference in its entirety. In some embodiments, serum bile acid levels may be reduced by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to 70%, 50% to 80%, or greater than 90% of the serum bile acid levels prior to administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, treatment with a PFIC includes treatment of pruritus.

[0291] Because LBAT is expressed on hepatocytes, LBAT and dual ASBT / LBAT inhibitors need to have at least a certain bioavailability and free fraction in the blood. Since LBAT inhibitor compounds only need to survive from the intestine to the liver, relatively low systemic exposure to such compounds is expected to be sufficient to minimize the potential risk of any side effects in the rest of the body. Inhibition of LBAT and ASBT is expected to have an additive effect, at least in reducing intrahepatic bile acid concentrations. Dual ASBT / LBAT inhibitors are also expected to reduce bile acid levels without inducing diarrhea, which has sometimes been observed with ASBT inhibitors.

[0292] Compounds with high LBAT inhibitory potency and sufficient bioavailability are expected to be particularly useful for the treatment of hepatitis. Compounds with dual ASBT / LBAT inhibitory potency and sufficient bioavailability are expected to be particularly useful for the treatment of non-alcoholic steatohepatitis (NASH).

[0293] Nonalcoholic fatty liver disease (NAFLD) is a common and serious chronic liver disease, similar to alcoholic liver disease, but occurring in people who drink little or no alcohol. In NASH patients, fat accumulation in the liver, known as NAFLD or steatosis, and other factors such as high LDL cholesterol and insulin resistance induce chronic inflammation in the liver and can lead to progressive scarring of tissues, called fibrosis and cirrhosis, ultimately resulting in liver failure and death. Total serum bile acid concentrations have been found to be significantly higher in NASH patients than in healthy subjects at both fasting (2.2 to 2.4-fold increase in NASH) and at all postprandial time points (1.7 to 2.2-fold increase in NASH). These are driven by increases in taurine-conjugated and glycine-conjugated primary and secondary bile acids. NASH patients exhibit greater variability in their fasting and postprandial bile acid profiles. These results suggest that NASH patients have higher exposure to bile acids, including more hydrophobic and cytotoxic secondary types, both fasting and postprandial. Increased bile acid exposure may be involved in liver damage and the pathogenesis of NAFLD and NASH (Ferslew et al., Dig Dis Sci. 2015, Vol. 60, pp. 3318-3328). Therefore, ASBT and / or LBAT inhibition may be beneficial for the treatment of NASH.

[0294] The defining characteristic of NAFLD is hepatic steatosis without secondary causes of hepatic steatosis, including excessive alcohol consumption, other known liver diseases, or long-term use of steatogenic drugs (Chalasani et al., Hepatology 2018, Vol. 67(1), pp. 328-357). NAFLD can be classified as non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH). According to Chalasani et al., NAFL is defined as the presence of ≥5% hepatic steatosis without evidence of hepatocellular damage in the form of hepatocellular swelling. NASH is defined as inflammation with ≥5% hepatic steatosis and hepatocellular damage (e.g., swelling), with or without any liver fibrosis. NASH is also commonly associated with liver inflammation and liver fibrosis, which can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Although liver fibrosis is not always present in NASH, the severity of fibrosis (when present) may be related to long-term outcomes.

[0295] Numerous methods exist for assessing and evaluating whether a subject has NAFLD, and if so, for assessing and evaluating the severity of the disease, including distinguishing NAFLD from NAFLD or NASH. In some implementations, NAS can be used to assess the severity of NAFLD. In some implementations, NAS can be used to assess the treatment of NAFLD. In some implementations, NAS can be determined as described in Kleiner et al., Hepatology. 2005, 41(6):1313-1321, which is incorporated herein by reference in its entirety. For a simplified NAS protocol adapted from Kleiner's, see, for example, Table 6.

[0296] Table 6. Examples of NAFLD Activity Scores (NAS) with Fibrosis Stages

[0297]

[0298]

[0299] In some embodiments, NAS is determined non-invasively, for example, as described in U.S. Application Publication No. 2018 / 0140219, which is incorporated herein by reference in its entirety. In some embodiments, the NAS of a sample from a subject is determined prior to administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the NAS is determined during or after administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a lower NAS score during or after administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof indicates treatment for NAFLD (e.g., NASH) compared to prior administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. For example, a NAS reduction of 1, 2, 3, 4, 5, 6, or 7 indicates treatment for NAFLD (e.g., NASH). In some embodiments, the NAS is 7 or less after administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, during the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the NAS is 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, during the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the NAS is 7 or less. In some embodiments, during the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the NAS is 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, after the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the NAS is 7 or less. In some embodiments, after the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the NAS is 5 or less, 4 or less, 3 or less, or 2 or less.

[0300] Other methods for assessing and evaluating NASH in subjects include identifying hepatic steatosis (e.g., fat accumulation in the liver); liver inflammation; and one or more biomarkers (e.g., serum markers and groups) indicating one or more of liver injury, liver inflammation, liver fibrosis, and / or cirrhosis. Other examples of physiological parameters of NASH may include liver morphology, liver stiffness, and the size or weight of the subject's liver.

[0301] In some implementations, NASH in subjects is demonstrated by the presence of fat accumulation in the liver and the detection of biomarkers indicating liver damage. For example, elevated serum ferritin and low titers of serum autoantibodies are common characteristics of NASH.

[0302] In some implementations, methods for assessing NASH include magnetic resonance imaging, quantification of fatty degeneration via spectral analysis or via proton density fat fraction (MRI-PDFF), and transient elastography. Hepatic venous pressure gradient (HPVG), liver stiffness measurement using MRE, diagnosis of significant liver fibrosis and / or cirrhosis, and assessment of histological features of liver biopsy. In some embodiments, magnetic resonance imaging is used to detect one or more of steatotic hepatitis (NASH-MRI), liver fibrosis (Fibro-MRI), and steatosis. See, for example, U.S. Patent Application Publications 2016 / 146715 and 2005 / 0215882, each of which is incorporated herein by reference in its entirety.

[0303] In some implementations, treatment of NASH may include, following administration of one or more doses of a compound of formula (I) or a pharmaceutically acceptable salt thereof, a reduction in one or more NASH-related symptoms in the subject; a reduction in the amount of hepatic steatosis; a decrease in NAS; a reduction in hepatic inflammation; a decrease in the levels of biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis; and a reduction in fibrosis and / or cirrhosis, no further progression of fibrosis and / or cirrhosis, or a slowing of the progression of fibrosis and / or cirrhosis.

[0304] In some implementations, treatment of NASH includes a reduction in one or more NASH-related symptoms in the subject. Exemplary symptoms may include one or more of the following: enlarged liver, fatigue, right upper quadrant pain, abdominal swelling, dilated blood vessels just below the skin surface, gynecomastia, splenomegaly, palmar erythema, jaundice, and pruritus. In some implementations, the subject is asymptomatic. In some implementations, the subject's total weight does not increase. In some implementations, the subject's total weight decreases. In some implementations, the subject's body mass index (BMI) does not increase. In some implementations, the subject's body mass index (BMI) decreases. In some implementations, the subject's waist-to-hip ratio (WTH) does not increase. In some implementations, the subject's WTH ratio decreases.

[0305] In some embodiments, treatment of NASH can be assessed by measuring hepatic steatosis. In some embodiments, treatment of NASH comprises a reduction in hepatic steatosis following administration of a compound of formula (I) as described herein or a pharmaceutically acceptable salt thereof. In some embodiments, hepatic steatosis is determined by one or more methods selected from: ultrasound examination, computed tomography (CT), magnetic resonance imaging, magnetic resonance spectroscopy (MRS), magnetic resonance elastography (MRE), transient elastography (TE) (e.g., (See, for example, Di Lascio et al., Ultrasound Med Biol. 2018, Vol. 44(8), pp. 1585-1596; Lv et al., J Clin Transl Hepatol. 2018, Vol. 6(2), pp. 217-221; Reeder et al., J MagnReson Imaging. 2011, Vol. 34(4), spcone; and de Lédinghen V et al., J Gastroenterol Hepatol. 2016, Vol. 31(4), pp. 848-855, each of which is incorporated herein by reference in its entirety). Subjects diagnosed with NASH may have more than about 5% hepatic steatosis, for example, more than about 5% to about 25%, about 25% to about 45%, about 45% to about 65%, or more than about 65% hepatic steatosis. In some implementations, subjects with more than about 5% to about 33% of hepatic steatosis are classified as stage 1 hepatic steatosis, subjects with about 33% to about 66% of hepatic steatosis are classified as stage 2 hepatic steatosis, and subjects with more than about 66% of hepatic steatosis are classified as stage 3 hepatic steatosis.

[0306] In some embodiments, the amount of hepatic steatosis is measured before administering the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the amount of hepatic steatosis is measured during or after the period of administering the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a reduction in the amount of hepatic steatosis during or after the period of administering the compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to before administering the compound of formula (I) or a pharmaceutically acceptable salt thereof, indicates treatment for NASH. For example, a reduction in the amount of hepatic steatosis of about 1% to about 50%, about 25% to about 75%, or about 50% to about 100% indicates treatment for NASH. In some implementations, a reduction of approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% in the amount of hepatic steatosis indicates treatment for NASH.

[0307] In some embodiments, the presence of liver inflammation is determined by one or more methods selected from: biomarkers indicating liver inflammation and liver biopsy samples from the subject. In some embodiments, the severity of liver inflammation is determined by liver biopsy samples from the subject. For example, liver inflammation in a liver biopsy sample may be assessed as described in Kleiner et al., Hepatology 2005, Vol. 41(6), pp. 1313-1321 and Brunt et al., Am J Gastroenterol 1999, Vol. 94, pp. 2467-2474, each of which is hereby incorporated by full reference. In some embodiments, the severity of liver inflammation is determined before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the severity of liver inflammation is determined during or after the administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a reduction in the severity of liver inflammation during or after the administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to before administration, indicates treatment for NASH. For example, a reduction in the severity of liver inflammation of about 1% to about 50%, about 25% to about 75%, or about 50% to about 100% indicates treatment for NASH. In some embodiments, a reduction in the severity of liver inflammation of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% indicates treatment for NASH.

[0308] In some implementations, treatment of NASH includes treatment of fibrosis and / or cirrhosis, such as a reduction in the severity of fibrosis, no further progression of fibrosis and / or cirrhosis, or a slowing of the progression of fibrosis and / or cirrhosis. In some implementations, the presence of fibrosis and / or cirrhosis is determined by one or more methods selected from: transient elastography (e.g., This includes non-invasive markers of liver fibrosis and histological features from liver biopsy. In some implementations, the severity of fibrosis (e.g., staging) is determined by one or more methods selected from: transient elastography (e.g., ), fibrosis scoring systems, biomarkers of liver fibrosis (e.g., non-invasive biomarkers), and hepatic venous pressure gradient (HVPG). Non-limiting examples of fibrosis scoring systems include the NAFLD fibrosis scoring system (see, for example, Angulo et al., Hepatology 2007, Vol. 45(4), pp. 846-54), the fibrosis scoring system in Brunt et al., Am. J. Gastroenterol. 1999, Vol. 94, pp. 2467-2474, the fibrosis scoring system in Kleiner et al., Hepatology 2005, Vol. 41(6), pp. 1313-1321, and the ISHAK fibrosis scoring system (see Ishak et al., J. Hepatol. 1995, Vol. 22, pp. 696-699), the contents of which are incorporated herein by reference in their entirety.

[0309] In some embodiments, the severity of fibrosis is determined before administering the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the severity of fibrosis is determined during or after the administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a decrease in the severity of fibrosis during or after the administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to before administration, indicates treatment for NASH. In some embodiments, a decrease in the severity of fibrosis, no further progression of fibrosis and / or cirrhosis, or a slowing of the progression of fibrosis and / or cirrhosis indicates treatment for NASH. In some embodiments, a scoring system, such as any of the fibrosis scoring systems described herein, is used to determine the severity of fibrosis; for example, the score may indicate a stage of fibrosis, such as stage 0 (no fibrosis), stage 1, stage 2, stage 3, and stage 4 (cirrhosis) (see, for example, Kleiner et al.). In some embodiments, a decrease in the fibrosis stage is a decrease in the severity of fibrosis. For example, a decrease in stage 1, 2, 3, or 4 is a decrease in the severity of fibrosis. In some implementations, a reduction in stage, such as from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage 1 to stage 0, indicates treatment for NASH. In some implementations, the stage of fibrosis is reduced from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage 1 to stage 0, after administration of compound (I) or its pharmaceutically acceptable salt, compared to before administration of the compound (I) or its pharmaceutically acceptable salt. In some embodiments, the stage of fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage 1 to stage 0, during the period of administration of compound (I) or its pharmaceutically acceptable salt, compared to before administration of compound (I) or its pharmaceutically acceptable salt. In some embodiments, the stage of fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage 1 to stage 0, after the period of administration of compound (I) or its pharmaceutically acceptable salt, compared to before administration of compound (I) or its pharmaceutically acceptable salt.

[0310] In some embodiments, the presence of NASH is determined by one or more biomarkers or their scoring systems that indicate one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis. In some embodiments, the severity of NASH is determined by one or more biomarkers or their scoring systems that indicate one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis. The levels of biomarkers can be determined, for example, by measuring, quantifying, and monitoring the expression levels of genes or mRNAs encoding the biomarkers and / or the peptides or proteins of the biomarkers. Non-limiting examples of biomarkers and / or scoring systems indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis include the aspartate aminotransferase (AST) to platelet ratio index (APRI); the aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR); the FIB-4 score, which is based on APRI, alanine aminotransferase (ALT) levels, and subject age (see, for example, McPherson et al., Gut 2010, Vol. 59(9), pp. 1265-9, which is incorporated herein by reference in its entirety); hyaluronic acid; pro-inflammatory cytokines; and a set of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, and gamma-glutamyl transferase (GGT) combined with the subject's age and sex to generate a measure of fibrotic and necrotic inflammatory activity in the liver (e.g., A group of biomarkers (e.g., bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin) combined with the subject's age and sex. See, for example, Adams et al., Clin. Chem. 2005, Vol. 51(10), pp. 1867-1873), and a group of biomarkers consisting of tissue inhibitors of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g. A set of biomarkers consisting of tissue inhibitors of metalloproteinase 1 (TIMP-1), N-terminal propeptide of type III procollagen (PIIINP), and hyaluronic acid (HA) (e.g., enhanced liver fibrosis (ELF) score, see, for example, Lichtinghagen R et al., J Hepatol. 2013 Aug; 59(2):236-42, which is incorporated herein by reference in its entirety). In some embodiments, the presence of fibrosis is measured by a set of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, and gamma-glutamyl transferase (GGT) in combination with the subject’s age and sex to generate a measure of fibrotic and necrotic inflammatory activity in the liver (e.g., A group of biomarkers (e.g., bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin) combined with the subject's age and sex. See, for example, Adams et al., Clin. Chem. 2005, Vol. 51(10), pp. 1867-1873), and a group of biomarkers consisting of tissue inhibitors of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g. ), and a group of biomarkers consisting of tissue inhibitors of metalloproteinase 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP), and hyaluronic acid (HA) (e.g., enhanced liver fibrosis (ELF) score).

[0311] In some embodiments, the aspartate aminotransferase (AST) level does not increase. In some embodiments, the aspartate aminotransferase (AST) level decreases. In some embodiments, the alanine aminotransferase (ALT) level does not increase. In some embodiments, the alanine aminotransferase (ALT) level decreases. In some embodiments, the "level" of an enzyme refers to its concentration, such as in blood. For example, the level of AST or ALT may be expressed as U / L.

[0312] In some implementations, the severity of fibrosis is measured by a FIB-4 score, combined with the subject's age and sex, using a panel of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, and gamma-glutamyl transferase (GGT) to quantify fibrotic and necrotic inflammatory activity in the liver (e.g., A group of biomarkers (e.g., bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin) combined with the subject's age and sex. See, for example, Adams et al., Clin. Chem. 2005, Vol. 51(10), pp. 1867-1873 (which is incorporated herein by reference in its entirety), and a group of biomarkers consisting of tissue inhibitors of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g. ), and a group of biomarkers consisting of tissue inhibitors of metalloproteinase 1 (TIMP-1), amino-terminal propeptide of type III procollagen (PIIINP), and hyaluronic acid (HA) (e.g., enhanced liver fibrosis (ELF) score).

[0313] In some implementations, liver inflammation is determined by the levels of liver inflammation biomarkers, such as pro-inflammatory cytokines. Non-limiting examples of biomarkers indicating liver inflammation include interleukin-(IL)6, interleukin-(IL)1β, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, monocyte chemoattractant protein (MCP)-1, C-reactive protein (CRP), PAI-1, and collagen subtypes such as Col1a1, Col1a2, and Col4a1 (see, for example, Neuman et al., Can. J. Gastroenterol. Hepatol. 2014, Vol. 28(11), pp. 607-618 and U.S. Patent No. 9,872,844, each of which is incorporated herein by reference in its entirety). Liver inflammation can also be assessed by changes in macrophage infiltration, such as by measuring changes in CD68 expression levels. In some implementations, liver inflammation can be determined by measuring or monitoring the serum or circulating levels of one or more of interleukin-(IL)6, interleukin-(IL)1β, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, monocyte chemoattractant protein (MCP)-1, and C-reactive protein (CRP).

[0314] In some embodiments, prior to administering the compound of formula (I) or a pharmaceutically acceptable salt thereof, the levels of one or more biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis are measured in samples from the subject. In some embodiments, during or after administering the compound of formula (I) or a pharmaceutically acceptable salt thereof, the levels of one or more biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis are measured. In some embodiments, a reduction in the levels of one or more biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis during or after administering the compound of formula (I) or a pharmaceutically acceptable salt thereof indicates treatment of NASH. For example, a reduction in the levels of one or more biomarkers indicating liver injury, inflammation, liver fibrosis, and / or cirrhosis by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% indicates treatment for NASH. In some embodiments, after administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof, the levels of one or more biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, during the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the levels of one or more biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.In some embodiments, after a period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, the levels of one or more biomarkers indicating one or more of liver injury, inflammation, liver fibrosis, and / or cirrhosis are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

[0315] In some embodiments, treatment of NASH reduces serum bile acid levels in the subject. In some embodiments, serum bile acid levels are determined by, for example, an ELISA enzyme assay or an assay measuring total bile acids, as described in Danese et al., PLoS One. 2017, Vol. 12(6): e0179200, which is incorporated herein by reference in its entirety. In some embodiments, serum bile acid levels may be reduced by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to 70%, 50% to 80%, or greater than 90% of the serum bile acid levels prior to administration of the compound of formula (I) or its pharmaceutically acceptable salts. In some embodiments, NASH is NASH accompanied by cholestasis. In cholestasis, the release of bile (including bile acids) from the liver is obstructed. Bile acids can cause hepatocellular damage (see, for example, Perez MJ, Briz O. World J. Gastroenterol. 2009, Vol. 15(14), pp. 1677-1689), which may lead to or increase the progression of fibrosis (e.g., cirrhosis) and increase the risk of hepatocellular carcinoma (see, for example, Sorrentino P et al., Dig. Dis. Sci. 2005, Vol. 50(6), pp. 1130-1135 and Satapathy SK and Sanyal AJ. Semin. Liver Dis. 2015, Vol. 35(3), pp. 221-235, each of which is incorporated herein by reference in its entirety). In some embodiments, treatment of NASH includes treatment of pruritus. In some embodiments, treatment of NASH with cholestasis includes treatment of pruritus. In some embodiments, a subject with NASH with cholestasis suffers from pruritus.

[0316] Exemplary biomarkers for NASH are provided in Table 7.

[0317] Table 7. Exemplary NASH biomarkers

[0318] Biomarkers of liver fibrosis

[0319] Aspartate aminotransferase (AST) and platelet ratio index (APRI)

[0320] The aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR)

[0321] FIB-4 rating 1

[0322] Hyaluronic acid

[0323] Pro-inflammatory cytokines

[0324] A group combined with the subject's age and sex, including α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, and gamma-glutamyl transferase (GGT), to produce a measure of fibrotic and necrotic inflammatory activity in the liver (e.g., )

[0325] A group combined with the subject's age and sex included bilirubin, gamma-glutamyl transferase, hyaluronic acid, and α2-macroglobulin (e.g., ...). )

[0326] Including tissue inhibitors of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g.) A group of )

[0327] This includes tissue inhibitors of metalloproteinase 1 (TIMP-1), N-terminal propeptide of type III procollagen (PIIINP), and hyaluronic acid (HA) (e.g., enhancing liver fibrosis (ELF) score). 3 A group of )

[0328] Liver inflammation biomarkers 4,5

[0329] Interleukin-(IL)6

[0330] Interleukin-(IL)1β

[0331] Tumor necrosis factor (TNF)-α

[0332] Transforming growth factor (TGF)-β

[0333] Monocyte chemotactic protein (MCP)-1

[0334] C-reactive protein (CRP)

[0335] PAI-1

[0336] Collagen subtypes (e.g., Col1a1, Col1a2, and Col4a1)

[0337] Changes in macrophage infiltration (e.g., changes in CD68 expression levels)

[0338] References in Table 7

[0339] 1 McPherson et al., Gut. 2010, Vol. 59(9), pp. 1265-1269.

[0340] 2 Adams et al., Clin Chem. 2005, Vol. 51(10), pp. 1867-1873.

[0341] 3 Lichtinghagen et al., J Hepatol. 2013, Vol. 59(2), pp. 236-242.

[0342] 4 Neuman et al., Can J Gastroenterol Hepatol. 2014, Vol. 28(11), pp. 607-618.

[0343] 5 US Patent No. 9,872,844

[0344] Some compounds of formula (I) or their pharmaceutically acceptable salts may exhibit a high free fraction in plasma. In some embodiments, the free fraction is greater than about 0.2%, such as greater than about 0.4%, such as greater than about 0.6%, such as greater than about 0.8%, such as greater than about 1.0%, such as greater than about 1.25%, such as greater than about 1.5%, such as greater than about 1.75%, such as greater than about 2.0%, such as greater than about 2.5%, such as greater than about 3%, such as greater than about 4%, such as greater than about 5%, such as greater than about 7.5%, such as greater than about 10%, or such as greater than about 20%.

[0345] Some compounds of formula (I) or their pharmaceutically acceptable salts may be excreted in urine. In some embodiments, the fraction of the compound excreted in urine is greater than about 0.2%, such as greater than about 0.4%, such as greater than about 0.6%, such as greater than about 0.8%, such as greater than about 1.0%, such as greater than about 2%, such as greater than about 3%, such as greater than about 5%, such as greater than about 7.5%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, or such as greater than about 50%.

[0346] Following absorption by the intestine, some compounds of formula (I) or their pharmaceutically acceptable salts may circulate via enterohepatic circulation. In some embodiments, the fraction of the compound circulating via enterohepatic circulation is greater than about 0.1%, such as greater than about 0.2%, such as greater than about 0.3%, such as greater than about 0.5%, such as greater than about 1.0%, such as greater than about 1.5%, such as greater than about 2%, such as greater than about 3%, such as greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, or such as greater than about 50%.

[0347] Some compounds of formula (I) or their pharmaceutically acceptable salts can induce the renal secretion of bile salts. In some embodiments, the fraction of circulating bile acids secreted via the renal pathway is greater than about 1%, such as greater than about 2%, such as greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, or such as greater than about 25%.

[0348] Some compounds of formula (I) or their pharmaceutically acceptable salts may exhibit improved or optimal permeability. Permeability can be measured in Caco2 cells and the value is given as an apparent permeability (Papp) value in cm / s. In some embodiments, the permeability is greater than at least about 0.1 × 10⁻⁶. -6 cm / s, such as greater than approximately 0.2 × 10 -6 cm / s, such as greater than approximately 0.4 × 10 - 6 cm / s, such as greater than approximately 0.7 × 10 cm / s -6 cm / s, such as greater than approximately 1.0 × 10 cm / s -6 cm / s, such as greater than approximately 2 × 10 -6 cm / s, such as greater than approximately 3 × 10 -6 cm / s, such as greater than approximately 5 × 10 -6 cm / s, such as greater than approximately 7 × 10 -6 cm / s, such as greater than approximately 10 × 10 -6 cm / s, such as greater than approximately 15 × 10 -6 cm / second.

[0349] Some compounds of formula (I) or their pharmaceutically acceptable salts may exhibit improved or optimal bioavailability. In some embodiments, oral bioavailability is greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, such as greater than about 40%, such as greater than about 50%, such as greater than about 60%, such as greater than about 70%, or such as greater than about 80%. In other embodiments, oral bioavailability is between about 10% and about 90%, such as between about 20% and about 80%, such as between about 30% and about 70%, or such as between about 40% and about 60%.

[0350] Some compounds of formula (I) or their pharmaceutically acceptable salts can serve as substrates for related transport proteins in the kidney.

[0351] Some compounds of formula (I) or their pharmaceutically acceptable salts can produce concentrations of bile acids in the intestine, liver, and serum that do not cause adverse gastrointestinal effects.

[0352] Some compounds of formula (I) or their pharmaceutically acceptable salts can reduce the concentration of bile acids in the liver without causing gastrointestinal disorders such as diarrhea.

[0353] As used herein, the term "treatment / treat / treating" refers to reversing or alleviating a disease or disorder as described herein, or one or more of its symptoms, delaying its onset, or inhibiting its progression. In some embodiments, treatment may be administered after one or more symptoms have appeared. In other embodiments, treatment may be administered even when symptoms are absent. For example, treatment may be administered to susceptible subjects before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0354] Suitable pharmaceutically acceptable salts of the compounds of the present invention are, for example, base addition salts of the compounds of the present invention that have sufficient acidity, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), ammonium salts, or salts with organic bases that provide physiologically acceptable cations, such as salts with methylamine, dimethylamine, trimethylamine, piperidine, morpholine, or tri-(2-hydroxyethyl)amine.

[0355] Some compounds of formula (I) or pharmaceutically acceptable salts thereof may have chiral centers and / or geometric isomer centers (E- and Z-isomers). It should be understood that this invention covers all such optical, diastereomeric, and geometric isomers having ASBT and / or LBAT inhibitory activity. This invention also covers any and all tautomeric forms of compounds of formula (I) or pharmaceutically acceptable salts thereof having ASBT and / or LBAT inhibitory activity. Some compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in both non-solvated and solvated forms, such as hydrated forms. It should be understood that this invention covers all such solvated forms having ASBT and / or LBAT inhibitory activity.

[0356] In another aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients. The excipients may include, for example, fillers, binders, disintegrants, flow aids, and lubricants. Generally, the pharmaceutical composition can be prepared in a conventional manner using conventional excipients.

[0357] Examples of suitable fillers include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose (such as lactose monohydrate), sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, dry starch, hydrolyzed starch, and pregelatinized starch. In some embodiments, the filler is mannitol and / or microcrystalline cellulose.

[0358] Examples of suitable adhesives include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (such as sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums (such as gum arabic and tragacanth), sodium alginate, cellulose derivatives (such as hydroxypropyl methylcellulose (or hydroxypropyl methylcellulose), hydroxypropyl cellulose, and ethyl cellulose), and synthetic polymers (such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid copolymers, and polyvinylpyrrolidone (polyvinylpyrrolidone)). In some embodiments, the adhesive is hydroxypropyl methylcellulose (hydroxypropyl methylcellulose).

[0359] Examples of suitable disintegrants include, but are not limited to, dry starch, modified starches (such as (partially) pregelatinized starch, sodium glycolate starch, and sodium carboxymethyl starch), alginate, cellulose derivatives (such as sodium carboxymethyl cellulose, hydroxypropyl cellulose, and low-substituted hydroxypropyl cellulose (L-HPC)), and cross-linked polymers (such as carboxymethyl cellulose, croscarmellose sodium, calcium carboxymethyl cellulose, and croscarmellose PVP (croscarmellose polyvinylpyrrolidone)). In some embodiments, the disintegrant is croscarmellose sodium.

[0360] Examples of suitable flow aids and lubricants include, but are not limited to, talc, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, colloidal silica, aqueous silica, synthetic magnesium silicate, fine silica, starch, sodium lauryl sulfate, boric acid, magnesium oxide, waxes (such as carnauba wax), hydrogenated oils, polyethylene glycol, sodium benzoate, and mineral oils. In some embodiments, the flow aid or lubricant is magnesium stearate or colloidal silica.

[0361] Pharmaceutical compositions may conventionally be coated with one or more coating layers. This also includes enteric coatings or coating layers for delayed or targeted release compounds of formula (I) or pharmaceutically acceptable salts thereof. The coating layer may contain one or more coating agents and, where appropriate, plasticizers and / or pigments (or colorants).

[0362] Examples of suitable coating agents include, but are not limited to, cellulose-based polymers (such as ethyl cellulose, hydroxypropyl methylcellulose (or hydroxypropyl methylcellulose), hydroxypropyl cellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methylcellulose succinate, and hydroxypropyl methylcellulose phthalate), vinyl-based polymers (such as polyvinyl alcohol), and acrylic acid-based polymers and their derivatives (such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid copolymers). In some embodiments, the coating agent is hydroxypropyl methylcellulose. In other embodiments, the coating agent is polyvinyl alcohol.

[0363] Examples of suitable plasticizers include, but are not limited to, triethyl citrate, glyceryl triacetate, tributyl citrate, diethyl phthalate, acetyl tributyl citrate, dibutyl phthalate, dibutyl sebacate, and polyethylene glycol. In some embodiments, the plasticizer is polyethylene glycol.

[0364] Examples of suitable pigments include, but are not limited to, titanium dioxide, iron oxide (such as yellow, brown, red or black iron oxide), and barium sulfate.

[0365] The pharmaceutical composition may be in the form of oral administration, parenteral injection (including intravenous, subcutaneous, intramuscular, and intravascular injection), topical administration, or rectal administration. In a preferred embodiment, the pharmaceutical composition is in the form of tablets or capsules for oral administration.

[0366] The dosage required for therapeutic or preventative treatment will depend on the route of administration, the severity of the disease, the patient's age and weight, and other factors that the attending physician typically considers when determining the appropriate regimen and dosage level for a particular patient.

[0367] The amount of the compound to be administered varies from patient to patient and can range from about 1 μg to about 50 mg per kilogram of body weight per day. Unit dosage forms such as tablets or capsules will typically contain about 1 to about 250 mg of the active ingredient, such as about 1 to about 100 mg, or such as about 1 to about 50 mg, or such as about 1 to about 20 mg, for example about 2.5 mg, or about 5 mg, or about 10 mg, or about 15 mg. The daily dose can be administered as a single dose or divided into one, two, three, or more unit doses. The daily dose of orally administered bile acid modifiers is preferably in the range of about 0.1 to about 250 mg, more preferably in the range of about 1 to about 100 mg, such as in the range of about 1 to about 5 mg, such as in the range of about 1 to about 10 mg, such as in the range of about 1 to about 15 mg, or such as in the range of about 1 to about 20 mg.

[0368] In another aspect, the present invention relates to a compound of formula (I).

[0369]

[0370] in

[0371] R 1 and R 2 Each independently is C 1-4 alkyl;

[0372] R 3 Independently selected from: hydrogen, halogen, hydroxyl, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, cyano, nitro, amino, N-(C 1-4 Alkyl)amino, N,N-di(C) 1-4 alkyl)amino and N-(aryl-C) 1-4 alkyl)amino;

[0373] n is an integer, 1, 2, or 3;

[0374] R 4 Selected from: hydrogen, halogen, hydroxyl, cyano, C 1-4 Alkyl, C 3-6 cycloalkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyloxy, C 1-4 Alkylthio, C 3-6 Cycloalkylthio, amino, N-(C 1-4 alkyl)amino and N,N-di(C) 1-4 alkyl)amino;

[0375] Or its pharmaceutically acceptable salt.

[0376] It is used as a medicine.

[0377] In another aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment or prevention of any of the diseases listed herein. The invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of any of the diseases listed herein. The invention further relates to a method of treating or preventing any of the diseases listed herein in a subject (such as a human) comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject requiring such treatment or prevention.

[0378] Combination therapy

[0379] In one aspect of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with at least one other therapeutically active agent, such as in combination with one, two, three or more other therapeutically active agents. The compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one other therapeutically active agent may be administered simultaneously, sequentially or separately. Suitable therapeutically active agents for combination with a compound of formula (I) include, but are not limited to, known active agents for treating any of the aforementioned conditions, disorders, and diseases.

[0380] In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with another ASBT inhibitor. Suitable ASBT inhibitors are disclosed in WO 93 / 16055, WO 94 / 18183, WO 94 / 18184, WO 96 / 05188, WO96 / 08484, WO 96 / 16051, WO 97 / 33882, WO 98 / 03818, WO 98 / 07449, WO 98 / 40375, WO 99 / 35135、WO 99 / 64409、WO 99 / 64410、WO 00 / 47568、WO 00 / 61568、WO 00 / 38725、WO 00 / 38726、WO 00 / 38727、WO 00 / 38728、WO 00 / 38729、WO 01 / 66533、WO 01 / 68096、WO 02 / 32428、WO 02 / 50051、WO 03 / 020710、WO 03 / 022286、WO 03 / 022825、WO 03 / 022830、WO 03 / 061663、WO 03 / 091232、WO 03 / 106482、WO 2004 / 006899、WO 2004 / 076430、WO 2007 / 009655、WO 2007 / 009656、WO 2011 / 137135、WO 2019 / 234077、WO 2020 / 161216、WO 2020 / 161217、DE 19825804,EP EP 864582, EP 489423, EP 549967, EP 573848, EP 624593, EP624594, EP 624595, EP 624596, EP 0864582, EP 1173205, EP 1535913 and EP 3210977 are all incorporated herein by reference in their entirety.

[0381] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a bile acid conjugate (also known as a bile acid chelator or resin), such as colesevelam, cholestyramine, or cholestipol. In a preferred embodiment of such a combination, the bile acid conjugate is formulated for colonic release. Examples of such formulations are disclosed, for example, in WO 2017 / 138877, WO 2017 / 138878, WO 2019 / 032026 and WO 2019 / 032027, all of which are incorporated herein by reference in their entirety.

[0382] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a DPP-IV inhibitor, including gliptins such as sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin, and dutogliptin or a pharmaceutically acceptable salt thereof.

[0383] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an HMG CoA reductase inhibitor, such as fluvastatin, lovastatin, pravastatin, simvastatin, atorvastatin, pitavastatin, cerivastatin, mevastatin, rosuvastatin, bevastatin, or dalvastatin or a pharmaceutically acceptable salt thereof.

[0384] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a cholesterol absorption inhibitor, such as ezetimibe or a pharmaceutically acceptable salt thereof.

[0385] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a PPARα agonist, including fibrates such as clofibrate, bezafibrate, ciprofibrate, clinofribrate, clofibride, fenofibrate, gemfibrozil, ronifibrate, and simfribrate or a pharmaceutically acceptable salt thereof.

[0386] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a PPARγ agonist, including thiazolidinediones such as pioglitazone, rosiglitazone, and lobeglitazone or a pharmaceutically acceptable salt thereof.

[0387] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a dual PPARα / γ agonist, including glitazars such as saroglitazar, alglitazar, muraglitazar, or tesaglitazar or a pharmaceutically acceptable salt thereof.

[0388] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a dual PPARα / δ agonist such as elafibranor.

[0389] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a pan-PPAR agonist (i.e., a PPAR agonist active against all of the following subtypes: α, γ, and δ), such as IVA337.

[0390] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a farnesoid X receptor (FXR) modulator, including FXR agonists such as caffeol, chenodeoxycholic acid, 6α-ethyl-chenodeoxycholic acid (obeticholic acid; INT-747), fexaramine, tropifexor, cilofexor, and MET409.

[0391] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a TGR5 receptor modulator, including a TGR5 agonist, such as 6α-ethyl-23(S)-methylcholic acid (INT-777).

[0392] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a dual FXR / TGR5 agonist such as INT-767.

[0393] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with ursodeoxycholic acid (UDCA). In yet another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with norursodeoxycholic acid (nor-UDCA).

[0394] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an FGF19 modulator, such as NGM282.

[0395] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an FGF21 agonist, such as BMS-986036.

[0396] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an integrin inhibitor, such as PLN-74809 and PLN-1474.

[0397] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a CCR2 / CCR5 inhibitor, such as cenicriviroc.

[0398] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a caspase inhibitor, such as emricasan.

[0399] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a galactolectin-3 inhibitor, such as GR-MD-02.

[0400] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a stearoyl-CoA desaturase (SCD) inhibitor, such as aramchol (eicosylaminocholic acid).

[0401] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an inhibitor of apoptosis signal-regulated kinase 1 (ASK1), such as selonsertib.

[0402] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a LOXL2 inhibitor, such as simtuzumab.

[0403] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an ACC inhibitor, such as GS-0976.

[0404] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a thyroid hormone receptor-β agonist, such as MGL3196.

[0405] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a GLP-1 agonist such as liraglutide.

[0406] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a dual glucagon-like peptide and a glucagon receptor agonist, such as SAR425899.

[0407] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a mitochondrial pyruvate carrier inhibitor, such as MSDC-0602K.

[0408] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an antioxidant, such as vitamin E.

[0409] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an SGLT1 inhibitor, an SGLT2 inhibitor, or a combination of SGLT1 and SGLT2 inhibitors. Examples of such compounds are dapagliflozin, sotagliflozin, canagliflozin, empagliflozin, LIK066, and SGL5213.

[0410] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a diacylglycerol O-acyltransferase 2 (DGAT2) inhibitor, such as DGAT2RX and PF-06865571.

[0411] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a fatty acid synthase (FASN) inhibitor, such as TVB-2640.

[0412] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an AMP-activated protein kinase (AMPK) activator, such as PXL-770.

[0413] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a glucocorticoid receptor antagonist (GR), a mineralocorticoid receptor antagonist (MR), or a dual GR / MR antagonist. Examples of such compounds are MT-3995 and CORT-118335.

[0414] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a cannabinoid receptor 1 (CB1) antagonist, such as IM102.

[0415] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with Klothoβ (KLB) and fibroblast growth factor receptor (FGFR) activators, such as MK-3655 (formerly known as NGM-313).

[0416] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a chemokine (cc motif) ligand 24 (CCL24) inhibitor, such as CM101.

[0417] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an A3 antagonist, such as PBF-1650.

[0418] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a P2x7 receptor antagonist, such as SGM 1019.

[0419] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a P2Y13 receptor agonist, such as CER-209.

[0420] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a sulfated oxysterol, such as Dur-928.

[0421] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a leukotriene D4 (LTD4) receptor antagonist, such as MN-001.

[0422] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a type 1 natural killer T cell (NKT1) inhibitor, such as GRI-0621.

[0423] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an anti-lipopolysaccharide (LPS) compound, such as IMM-124E.

[0424] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a VAP1 inhibitor, such as BI1467335.

[0425] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an A3 adenosine receptor agonist, such as CF-102.

[0426] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a SIRT-1 activator, such as NS-20.

[0427] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a nicotinic acid receptor 1 agonist, such as ARI-3037MO.

[0428] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a TLR4 antagonist, such as JKB-121.

[0429] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a hexylose kinase inhibitor, such as PF-06835919.

[0430] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an adiponectin receptor agonist, such as ADP-335.

[0431] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an inhibitor of autocrine motor factors, such as PAT-505 and PF8380.

[0432] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a chemokine (cc motif) receptor 3 (CCR3) antagonist, such as bertilimumab.

[0433] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt and chloride channel stimulant thereof, such as cobiprostone and lubiprostone, is administered in combination.

[0434] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a heat shock protein 47 (HSP47) inhibitor, such as ND-L02-s0201.

[0435] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a sterol regulatory element binding protein (SREBP) transcription factor inhibitor, such as CAT-2003 and MDV-4463.

[0436] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with biguanides, such as metformin.

[0437] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with insulin.

[0438] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a glycogen phosphorylase inhibitor and / or a glucose-6-phosphatase inhibitor.

[0439] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with sulfonylureas, such as glipizide, glibenklamid, and glimepirid.

[0440] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with meglitinide, such as repaglinide, nateglinide and ormiglitinide.

[0441] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a glucosidase inhibitor, such as acarbose or miglitol.

[0442] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a squalene synthase inhibitor, such as TAK-475.

[0443] In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with a PTPB1 inhibitor, such as trodusquemine, ertiprotafib, JTT-551, and claramine.

[0444] Preparation of compounds

[0445] The compounds of the present invention can be prepared as free acids or pharmaceutically acceptable salts thereof by the methods described below. Throughout the following description of such methods, it should be understood that, where appropriate, suitable protecting groups will be added to and subsequently removed from the reactants and intermediates in a manner readily understood by one skilled in the art of organic synthesis. Conventional procedures using such protecting groups and examples of suitable protecting groups are described, for example, in Greene's Protective Groups in Organic Synthesis, 4th Edition, John Wiley & Sons, Hoboken, 2006, by PGM Wutz and TW Greene.

[0446] General Method

[0447] All solvents used were analytical grade. Commercially available anhydrous solvents were routinely used for the reactions. Starting materials were either commercially available or prepared according to literature procedures. Room temperature refers to 20–25°C. Solvent mixture compositions are given as volume percentages or volume ratios.

[0448] LCMS:

[0449] Instrument Name: Agilent 1290 Infinity II.

[0450] Method A: Mobile phase: A: 0.1% HCOOH / H2O:ACN (95:5), B: ACN; Flow rate: 1.5 mL / min; Column: ZORBAX XDB C-18 (50×4.6 mm) 3.5 μM.

[0451] Method B: Mobile phase: A: 10 mM NH4HCO3 aqueous solution, B: ACN; Flow rate: 1.2 mL / min; Column: XBridge C8 (50 × 4.6 mm), 3.5 μM.

[0452] Method C: Mobile phase: A: 0.1% HCOOH aqueous solution: ACN (95:5), B: ACN; Flow rate: 1.5 mL / min; Column: ATLANTIS dC18 (50 × 4.6 mm), 5 μM.

[0453] Method D: Mobile phase: A: 10 mM NH4OAc aqueous solution, B: ACN; Flow rate: 1.2 mL / min; Column: Zorbax Extend C18 (50 × 4.6 mm) 5 μM.

[0454] Method E: Mobile phase: A: 0.1% TFA aqueous solution: ACN (95:5), B: 0.1% TFA in ACN solution; Flow rate: 1.5 mL / min; Column: XBridge C8 (50 × 4.6 mm), 3.5 μM.

[0455] UPLC:

[0456] Instrument Name: Waters Acquity I Class

[0457] Method A: Mobile phase: A: 0.1% HCOOH aqueous solution, B: 0.1% HCOOH in ACN solution; Flow rate: 0.8 mL / min; Column: Acquity UPLC HSS T3 (2.1 × 50) mm; 1.8 μm.

[0458] HPLC:

[0459] Instrument name: Agilent 1260 Infinity II series instrument, as shown below, using % and UV detection (maxplot).

[0460] Method A: Mobile phase: A: 10 mM NH4HCO3 aqueous solution, B: ACN; Flow rate: 1.0 mL / min; Column: XBridge C8 (50 × 4.6 mm, 3.5 μm).

[0461] Method B: Mobile phase: A: 0.1% TFA aqueous solution, B: 0.1% TFA in ACN solution; Flow rate: 2.0 mL / min; Column: XBridge C8 (50 × 4.6 mm, 3.5 μm).

[0462] Method C: Mobile phase: A: 10 mM NH4OAc in a milli-q aqueous solution, B: ACN; Flow rate: 1.0 ml / min; Column: Phenomenex Gemini C18 (150 × 4.6 mm, 3.0 μm).

[0463] Chiral SFC:

[0464] Instrument Name: PIC SFC 10 (Analytical)

[0465] The ratio of CO2 to cosolvent is between 60:40 and 80:20.

[0466] Method A: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: YMC Amylose-SA (250 × 4.6 mm, 5 μm).

[0467] Method B: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Chiralpak AD-H (250 × 4.6 mm, 5 μm).

[0468] Method C: Mobile phase: 20 mM ammonia / methanol; Flow rate: 3 mL / min; Column: YMC cellulose-SC (250 × 4.6 mm, 5 μm).

[0469] Method D: Mobile phase: methanol; Flow rate: 3 mL / min; Column: Lux A1 (250 × 4.6 mm, 5 μm).

[0470] Method E: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 5 mL / min; Column: Lux C4.

[0471] Method F: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: YMC cellulose-SC.

[0472] Method G: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: Lux A1.

[0473] Method H: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Lux A1 (250 × 4.6 mm, 5 μm).

[0474] Method I: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: chiral CCS (250 × 4.6 mm, 5 μm).

[0475] Method J: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 5 mL / min; Column: YMC cellulose-SC AD-H (250 × 4.6 mm, 5 μm).

[0476] Preparative HPLC:

[0477] Instrument Name: Agilent 1290 Infinity II

[0478] Method A: Mobile phase: A: 0.1% TFA aqueous solution; B: 0.1% TFA in ACN solution; Flow rate: 2.0 mL / min; Column: X-Bridge C8 (50 × 4.6 mm, 3.5 μM).

[0479] Method B: Mobile phase: A: 10 mM NH4OAc aqueous solution; B: ACN; Flow rate: 35 mL / min; Column: X select C18 (30 × 150 mm, 5 μm).

[0480] Method C: Mobile phase: A: 10 mM NH4HCO3 aqueous solution; B: ACN; Flow rate: 1.0 mL / min; Column: XBridge C8 (50 × 4.6 mm, 3.5 μm).

[0481] Method D: Mobile phase: A: 0.1% HCOOH aqueous solution; B: ACN; Flow rate: 1.0 mL / min; Column: X-select C18 (30×150 mm, 5 μm).

[0482] Chiral preparative SFC:

[0483] Instrument Names: PIC SFC 100 and PSC SFC 400

[0484] The ratio of CO2 to cosolvent is between 60:40 and 80:20.

[0485] Method A: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: YMC Amylose-SA (250×30 mm, 5 μm).

[0486] Method B: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Chiralpak AD-H (250 × 30 mm, 5 μm).

[0487] Method C: Mobile phase: 20 mM ammonia / methanol; Flow rate: 3 mL / min; Column: YMC cellulose-SC (250 × 30 mm, 5 μm).

[0488] Method D: Mobile phase: methanol; Flow rate: 3 mL / min; Column: chiral CCS (250 × 30 mm, 5 μm).

[0489] Method E: Mobile phase: methanol; Flow rate: 3 mL / min; Column: Lux A1 (250 × 30 mm, 5 μm).

[0490] Method F: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Lux A1 (250 × 30 mm, 5 μm).

[0491] Method G: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: chiral CCS (250 × 30 mm, 5 μm).

[0492] abbreviation

[0493] ACN Acetonitrile

[0494] DCM dichloromethane

[0495] DMF (dimethylformamide)

[0496] IPA isopropanol

[0497] LCMS (Liquid Chromatography-Mass Spectrometry)

[0498] HPLC (High Performance Liquid Chromatography)

[0499] PE petroleum ether

[0500] SFC Supercritical Fluid Chromatography

[0501] TFA (trifluoroacetic acid)

[0502] THF Tetrahydrofuran

[0503] TLC (Thin Layer Chromatography)

[0504] UPLC (Ultra-High Performance Liquid Chromatography)

[0505] The invention will now be described by way of the following examples, which are not intended to limit the invention in any way. All listed documents and references are incorporated herein by reference.

[0506] Example

[0507] Intermediate 1

[0508] 2-Aminobutyric acid ethyl ester hydrochloride

[0509]

[0510] At 0 °C, thionyl chloride (78 mL, 1.07 mol) was added to a stirred solution of 2-aminobutyric acid (100 g, 0.97 mol) in ethanol (750 mL). The reaction mixture was then heated at 80 °C for 16 hours. After the reaction was complete, the reaction mixture was concentrated under vacuum to give the crude title compound, which was used as is in the next step without any further purification. Yield: 93% (152 g, white solid).

[0511] 1 H NMR (400MHz, DMSO-d6): δ8.66 (bs, 3H), 4.25-4.16 (m, 2H), 3.98-3.85 (m, 1H), 1.84 (t, J = 7.2Hz, 2H), 1.23 (t, J = 6.8Hz, 3H), 0.92 (t, J = 7.6Hz, 3H).

[0512] Intermediate 2

[0513] Ethyl (E)-2-(benzylamino)butyrate

[0514]

[0515] Triethylamine (152 mL, 1.09 mol) was added over a 30-minute period to a stirred solution of ethyl 2-aminobutyrate hydrochloride (intermediate 1; 152 g, 0.91 mol) in DCM (900 mL) at 0 °C. Magnesium sulfate (98 g, 0.82 mol) was added in portions to the reaction mixture at 0 °C. Benzaldehyde (84 mL, 0.82 mol) was then added over a 20-minute period at 0 °C, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum. The resulting crude product was dissolved in petroleum ether (1000 mL) and filtered again through diatomaceous earth, followed by concentration under vacuum to give the title compound. This crude product was transferred unchanged to the next step without any further purification. Yield: 90% (180 g, pale brown liquid).

[0516] 1 H NMR (400MHz, DMSO-d6): δ8.40(s,1H),7.79-7.76(m,2H),7.49-7.47(m,3H),4.16-4.10(m,2H),3.9 8-3.95(m,1H),1.92-1.89(m,1H),1.79-1.74(m,1H),1.19(t,J=7.2Hz,3H),0.85(t,J=7.2Hz,3H).

[0517] Intermediate 3

[0518] (E)-2-(benzylamino)-2-ethylhexanoate ethyl ester

[0519]

[0520] At 0°C, DMF (800 mL) containing ethyl (E)-2-(phenylmethyleneamino)butyrate (intermediate 2; 180 g, 0.82 mol) was slowly added over a 30-minute period to a stirred solution of NaH (60%; 32.8 g, 0.82 mol) in DMF (100 mL). The reaction mixture was then stirred at room temperature for 1.5 h. At 0°C, n-butyl iodide (93 mL, 0.82 mol) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with 2-propanol (100 mL) at 0°C and then diluted with water (1000 mL). The aqueous layer was extracted with petroleum ether (1000 mL). The organic layer was washed with brine (200 mL) and dried over anhydrous Na₂SO₄. The organic fraction was concentrated under vacuum, and the resulting crude product was transferred unchanged to the next step without any further purification. Yield: 88% (200g, yellow liquid).

[0521] 1 H NMR (400MHz, DMSO-d6): δ8.34(s,1H),7.80-7.77(m,2H),7.47-7.44(m,3H),4 .16(q,J=7.0Hz,2H),2.51-1.79(m,4H),1.31-1.18(m,7H),0.88-0.84(m,6H).

[0522] Intermediate 4

[0523] Ethyl 2-amino-2-ethylhexanoate

[0524]

[0525] At 0 °C, dilute HCl (1000 mL, 1.5 N) was added to a stirred solution of ethyl (E)-2-(benzylamino)-2-ethylhexanoate (intermediate 3; 200 g, 0.73 mol) in petroleum ether (500 mL), and the reaction mixture was vigorously stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL), followed by alkalization of the aqueous layer with solid sodium bicarbonate (200 g) (pH ~ 8.5) and extraction with EtOAc (2 × 200 mL). The organic layer was washed with water (2 × 15 mL). The combined organic fractions were dried over anhydrous Na₂SO₄ and concentrated under vacuum to give the title compound. The crude material was transferred to the next step as is without any further purification. Yield: 80% (110 g, pale yellow liquid).

[0526] 1 H NMR (400MHz, DMSO-d6): δ4.08 (q, J=7.1Hz, 2H), 1.68-1.00 (m, 13H), 0.85 (t, J=7.2Hz, 3H), 0.77 (t, J=7.4Hz, 3H).

[0527] Intermediate 5

[0528] 2-Amino-2-ethyl-N-phenylhexanamide

[0529]

[0530] At -78°C, n-BuLi (2.6 M in hexane; 205 mL, 534 mmol) was added dropwise over a 30-minute period to a stirred solution of aniline (48.3 mL, 534 mmol) in THF (250 mL), and the reaction mixture was stirred at -25°C to -30°C for 45 minutes. Subsequently, THF (250 mL) containing ethyl 2-amino-2-ethylhexanoate (intermediate 4; 50 g, 267 mmol) was added to the reaction mixture at -78°C, and the reaction mixture was stirred at -78°C for 2 hours. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water (500 mL) at -78°C. The reaction mixture was extracted with EtOAc (2 × 250 mL), and the organic layer was washed with water (2 × 15 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum to give the title compound as a crude product. The crude product was dissolved in petroleum ether (1000 mL). The organic fraction was washed with 30% methanol aqueous solution (2 × 250 mL) and dried over anhydrous Na₂SO₄. The organic fraction was concentrated under vacuum, and the resulting crude product was transferred to the next step as is without any further purification. Yield: 66 g (crude product, brown liquid).

[0531] 1 H NMR (400MHz, DMSO-d6): δ7.64(d,J=8.4Hz,2H),7.30(t,J=7.4Hz,2H),7.05(t ,J=7.4Hz,1H),6.55(d,J=8.5Hz,1H),1.76-1.07(m,10H),0.86-0.77(m,6H).

[0532] Intermediate 6

[0533] 2-Ethyl-N1-phenylhexane-1,2-diamine

[0534]

[0535] At 0 °C, borane dimethyl sulfide (2 M in THF, 253 mL, 0.51 mol) was added to a stirred solution of 2-amino-2-ethyl-N-phenylhexanamide (intermediate 5; 66 g, 0.28 mol) in 600 mL of THF, and the reaction mixture was heated at 70 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched at 0 °C with 300 mL of methanol. The reaction mixture was then heated at 70 °C for 2 h. The reaction mixture was concentrated under vacuum, and the resulting residue was dissolved in EtOAc (1000 mL). The organic layer was washed with water (2 × 150 mL), dried over anhydrous Na2SO4, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 40% EtOAc / hexane; silica gel: 230-400 mesh) to give the title compound. Yield: 82% (50 g, brown liquid).

[0536] 1 H NMR (400MHz, DMSO-d6): δ7.04(t,J=7.2Hz,2H), 6.61(d,J=8.4Hz,2H), 6.49(t,J=7.2Hz, 1H), 5.15 (t, J = 4.8Hz, 1H), 2.79 (d, J = 5.6Hz, 2H), 1.39-1.17 (m, 10H), 0.88-0.79 (m, 6H).

[0537] Intermediate 7

[0538] 1,2-bis(2,4-dibromo-5-methoxyphenyl)dithion

[0539]

[0540] Bromine (73 mL, 1.4 mol) was added dropwise to a stirred solution of 100 g (0.7 mol) of 3-methoxythiophenol in 1000 mL of methanol at 0 °C, and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated under vacuum, and the crude product was diluted with EtOAc (2000 mL) and washed with water (2 × 500 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was dissolved in glacial acetic acid (600 mL), and bromine (20 mL) was added dropwise at room temperature, and the reaction mixture was stirred at room temperature for 2 h. The resulting solid was filtered off, ground with DCM, and dried under vacuum to give the pure title compound. Yield: 37% (78 g, white solid).

[0541] 1 H NMR (400MHz, DMSO-d6): δ7.69(s,2H),7.17(s,2H),3.84(s,6H).

[0542] Intermediate 8

[0543] 2,4-Dibromo-5-methoxybenzenesulfonyl chloride

[0544]

[0545] Thionyl chloride (13.6 mL, 168.35 mmol) was added dropwise to a stirred suspension of 1,2-bis(2,4-dibromo-5-methoxyphenyl)dithione (intermediate 7; 20.0 g, 33.67 mmol) and potassium nitrate (17.02 g, 168.35 mmol) in acetonitrile (200 mL) at 0 °C. The reaction mixture was stirred at room temperature for 24 hours. After the reaction was complete (monitored by TLC), the reaction mixture was poured into crushed ice and the resulting solid was filtered off. The solid was washed with water and dried under vacuum to give the pure title compound. Yield: 91% (22.5 g, white solid).

[0546] 1 H NMR (400MHz, DMSO-d6): δ8.05(s,1H),7.66(s,1H),4.01(s,3H).

[0547] Intermediate 9

[0548] 2,4-Dibromo-5-methoxy-N-(3-((phenylamino)methyl)hept-3-yl)benzenesulfonamide

[0549]

[0550] At 0 °C, 2,4-dibromo-5-methoxybenzenesulfonyl chloride (intermediate 8; 10.5 g, 28.91 mmol) and triethylamine (9.3 mL, 67.02 mmol) were added to a stirred solution of 2-ethyl-N1-phenylhexane-1,2-diamine (intermediate 6; 4.9 g, 22.34 mmol) in THF (10 mL), and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with EtOAc (50 mL). The organic layer was washed with water (2 × 15 mL) and dried over anhydrous Na2SO4. The organic fraction was concentrated under vacuum and the crude product was purified by Isolera column chromatography (eluent: 10% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 59% (7.2 g, white solid).

[0551] 1 H NMR (400MHz, DMSO-d6): δ8.01(s,1H),7.60(s,1H),7.50(s,1H),7.03(t,J=8.1Hz,2H),6.54-6.46(m,3H),4. 80(t,J=5.1Hz,1H),3.86(s,3H),3.07-2.96(m,2H),1.66-1.41(m,4H),1.15-0.95(m,4H),0.78-0.69(m,6H).

[0552] Intermediate 10

[0553] 7-Bromo-3-butyl-3-ethyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatriene 1,1-dioxide

[0554]

[0555] Potassium carbonate (3.62 g, 26.2 mmol) and copper powder (834 mg, 13.1 mmol) were added to a stirred solution of 2,4-dibromo-5-methoxy-N-(3-((phenylamino)methyl)hept-3-yl)benzenesulfonamide (intermediate 9; 7.2 g, 13.1 mmol) in DMF (50 mL), and the reaction mixture was heated at 150 °C for 24 h. After the reaction was complete (monitored by TLC), the reaction mixture was filtered through diatomaceous earth and washed with EtOAc (25 mL). The filtrate was concentrated under vacuum and purified by Isolera column chromatography (eluent: 20% EtOAc / PE; silica gel: 230-400 mesh) to obtain the title compound. Yield: 83% (5.1 g, white solid).

[0556] 1¹H NMR (400MHz, DMSO-d⁶): δ 7.43–7.30 (m, 4H), 7.15–7.13 (m, 2H), 7.03–7.01 (m, 2H), 4.00–3.60 (m, 5H), 1.62–1.34 (m, 4H), 1.08–0.95 (m, 4H), 0.74–0.71 (m, 6H). LCMS: (Method A) 467.0 (M + ), Rt. 3.06 min, 95.31% (max).

[0557] Intermediate 11

[0558] 7-Bromo-3-Butyl-3-ethyl-8-methoxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatriene 1,1-dioxide

[0559]

[0560] At 0 °C, Cs₂CO₃ (27.8 g, 85.5 mmol) and p-methoxybenzyl bromide (7.98 mL, 39.5 mmol) were added to a stirred solution of 7-bromo-3-butyl-3-ethyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 10; 20.0 g, 42.7 mmol) in N-methyl-2-pyrrolidone (100 mL), and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with EtOAc (200 mL), and the organic layer was washed with water (2 × 50 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 10% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 64% (16g, white solid).

[0561] LCMS: (Method A) 587.2 (M + ), Rt. 3.51 min, 92.94% (max).

[0562] Intermediate 12

[0563] 3-Butyl-3-ethyl-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatriene 1,1-dioxide

[0564]

[0565] Sodium methanethiol (9.5 g, 136.1 mmol) was added to a stirred solution of 7-bromo-3-butyl-3-ethyl-8-methoxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 11; 16.0 g, 27.2 mmol) in DMF (120 mL), and the reaction mixture was heated at 60 °C for 16 h. After the reaction was complete (monitored by LCMS), the reaction mixture was diluted with EtOAc (200 mL), and the organic layer was washed with water (2 × 50 mL). The organic fraction was dried over anhydrous Na₂SO₄, concentrated under vacuum, and purified by Isolera column chromatography (eluent: 10% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 65% (9.2 g, white solid).

[0566] 1 ¹H NMR (400MHz, DMSO-d6): δ 10.37 (bs, 1H), 7.31–7.22 (m, 5H), 7.01–6.65 (m, 6H), 4.32–4.13 (m, 2H), 4.10–3.90 (m, 2H), 3.74 (s, 3H), 2.15 (s, 3H), 1.62–1.34 (m, 4H), 1.08–0.98 (m, 4H), 0.74–0.65 (m, 6H). LCMS: (Method E) 541.2 (M + +H), Rt.2.86min, 93.67% (max).

[0567] Intermediate 13

[0568] 2-((3-Butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)tert-butyl acetate

[0569]

[0570] Anhydrous K₂CO₃ (0.51 g, 3.70 mmol) and tert-butyl bromoacetate (0.25 mL, 2.77 mmol) were added to a solution of 3-butyl-3-ethyl-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 12; 1 g, 1.85 mmol) in DMF (5 mL), and the reaction mixture was heated at 100 °C for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with EtOAc (20 mL). The organic layer was washed with water (15 mL) and brine (15 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (eluent: 15% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 66% (0.8 g, white solid).

[0571] 1 H NMR (400MHz, DMSO-d6): δ7.29-7.27(m,4H),7.11-6.96(m,4H),6.88(d,J=7.8Hz,2H),6.81(s,1H),3.31(s,2 H),4.81(s,2H),4.32(s,2H),3.75(s,3H),2.15(s,3H),1.40(s,13H),1.11-0.81(m,4H),0.78-0.55(m,6H). LCMS:(Method A)655.3(M + +H), Rt.3.51min, 90.95% (max).

[0572] Intermediate 14

[0573] (S)-2-((3-Butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazazepine-8-yl)oxy)tert-butyl acetate and (R)-2-((3-Butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazepine-8-yl)oxy)tert-butyl acetate

[0574]

[0575] Two enantiomers of racemic 2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)tert-butyl acetate (0.52 g, 0.79 mmol) were separated by chiral SFC (Method H). The material was concentrated under vacuum at 50 °C. The first eluent corresponds to enantiomer 1 and the second eluent corresponds to enantiomer 2. The absolute configurations of the two enantiomers are unknown.

[0576] Enantiomer 1: Yield: 38% (200 mg, white solid) Chiral SFC: (Method J) Rt. 3.13 min, 99.90% (Max).

[0577] Enantiomer 2: Yield: 42% (220 mg, white solid). LCMS: (Method E) 654.8 (M + +H), Rt. 3.55 min, 99.21% (Max) HPLC: (Method B) Rt. 7.12 min, 99.02% (Max) Chiral SFC: (Method J) Rt. 3.84 min, 98.09% (Max).

[0578] Example 1

[0579] 2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiazazacycloheptatrien-8-yl)oxy)acetic acid

[0580]

[0581] At 0 °C, TFA (4 mL) and triethylsilane (4 mL) were added to a stirred solution of 2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetate (intermediate 13; 0.8 g, 1.22 mmol) in DCM (10 mL), and the reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete (monitored by TLC), the reaction mixture was poured into ice-cold water and the aqueous layer was extracted with DCM (2 × 10 mL). The combined organic layers were washed with ice-cold water (20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude product was purified by preparative HPLC (Method D) to obtain the title compound. Yield: 20% (0.12 g, grayish-white solid).

[0582] 1¹H NMR (400MHz, DMSO-d⁶): δ 13.18 (s, 1H), 7.27 (t, J = 8.0Hz, 3H), 7.18–7.04 (m, 3H), 6.95–6.93 (m, 1H), 6.60 (s, 1H), 4.77 (s, 2H), 3.82 (s, 2H), 2.12 (s, 3H), 1.62–1.52 (m, 2H), 1.49–1.43 (m, 2H), 1.40–1.35 (m, 1H), 1.24–0.97 (m, 3H), 0.76–0.72 (m, 6H). LCMS: (Method A) 479.1 (M + +H), Rt. 2.65 min, 99.44% (max) HPLC: (Method B) Rt. 5.58 min, 97.46% (max).

[0583] Examples 2 and 3

[0584] (S)-2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazazepine-8-yl)oxy)acetic acid and (R)-2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazepine-8-yl)oxy)acetic acid

[0585]

[0586] Triphenylamine (0.16 g, 0.67 mmol) was added to a solution of enantiomer 1 (intermediate 14; 0.20 g, 0.33 mmol) of tert-butyl 2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiazazacycloheptatrien-8-yl)oxy)acetate in toluene (6 mL), and the reaction mixture was cooled to 0 °C. Subsequently, TFA (0.77 g, 6.72 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with EtOAc (15 mL), and the organic layer was washed with water (2 × 10 mL) and brine (10 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude material was purified by Isolera column chromatography (eluent: 30% EtOAc / PE; silica gel: 230-400 mesh) to obtain enantiomer 1 of the title compound.

[0587] Following the same procedure, enantiomer 2 of the title compound was obtained starting from 0.22 g of enantiomer 2 of intermediate 14. The absolute configurations of the two enantiomers are unknown.

[0588] Enantiomer 1: Yield: 43% (70 mg, white solid) 1 ¹H NMR (400MHz, DMSO-d⁶): δ 13.14 (bs, 1H), 7.29–7.25 (m, 3H), 7.11–7.06 (m, 3H), 6.94 (t, J = 7.2Hz, 1H), 6.60 (s, 1H), 4.78 (s, 2H), 3.85 (bs, 2H), 2.12 (s, 3H), 1.72–1.58 (m, 1H), 1.57–1.45 (m, 1H), 1.43–1.35 (m, 2H), 1.29–1.14 (m, 1H), 1.13–0.88 (m, 3H), 0.74 (t, J = 7.2Hz, 6H). LCMS: (Method E) 478.8 (M + +H), Rt. 2.92 min, 98.18% (max) HPLC: (Method B) Rt. 5.54 min, 97.87% (max) Chiral SFC: (Method G) Rt. 2.0 min, 99.30% (max).

[0589] Enantiomer 2: Yield: 37% (60 mg, white solid) 1 ¹H NMR (400MHz, DMSO-d⁶): δ 13.10 (bs, 1H), 7.29–7.25 (m, 3H), 7.11–7.06 (m, 3H), 6.96–6.94 (m, 1H), 6.60 (s, 1H), 4.78 (s, 2H), 3.80 (bs, 2H), 2.12 (s, 3H), 1.72–1.58 (m, 1H), 1.57–1.45 (m, 1H), 1.43–1.35 (m, 2H), 1.29–1.13 (m, 1H), 1.13–0.88 (m, 3H), 0.74 (t, J = 7.2Hz, 6H). LCMS: (Method E) 478.9 (M + +H), Rt. 2.92 min, 98.99% (max), HPLC: (Method B) Rt. 5.53 min, 97.81% (Max) Chiral SFC: (Method G) Rt. 1.89 min, 100% (Max).

[0590] Bioanalysis

[0591] IBAT (h / m) analysis scheme

[0592] 10,000 cells (human or mouse IBAT-overexpressing cells) were seeded in 200 μL of MEM-α medium (Gibco 12571-063) supplemented with 10% FBS (Gibco 10438026) and containing puromycin (Gibco A1113803) (10 μg / mL) in 96-well plates (Corning CLS3809) and incubated at 37°C in 5% CO2 for 48 hours. After incubation, the medium was decanted from the wells and the cells were washed twice with 300 μL of basal MEM-α medium (without FBS). After each decanting of the basal MEM-α medium, the plate was gently tapped against a paper towel to ensure maximum removal of residual medium.

[0593] The test inhibitor dilution (maximum test concentration 10 μM, 3-fold serial dilution, 10 spots) prepared in DMSO (Sigma D2650) was added to an incubation mixture (maintaining a final DMSO concentration of 0.2%) containing 0.25 μM 3H-taurocholic acid (ARC ART-1368) and 5 μM cold taurocholic acid (Sigma T4009). Then, 50 μL of the incubation mixture containing the test inhibitor was added to each well (in duplicate), and the plate was incubated in a CO2 incubator at 37°C for 20 minutes. After incubation, the reaction was stopped by holding the plate on an ice-water mixture for 2–3 minutes, and the incubation mixture was then completely aspirated from the wells. Wash the wells twice with 250 μL of cooled, unlabeled 1 mM taurine dissolved in (10 mM) HBSS (Gibco 14175079) (pH 7.4) buffered with HEPES (Gibco 15630080). After each wash, gently pat the plate against a paper towel to ensure maximum removal of the blocking buffer.

[0594] 100 μL of MicroScint-20 (PerkinElmer 6013621) was added to the wells and left to incubate overnight at room temperature, followed by analysis using a TopCount NXT from PerkinElmer. TM The micro-board blinking and light emission counter reads the board according to the 3H test scheme (set to 120 seconds reading time per well).

[0595] LBAT (h / m) analysis scheme

[0596] 20,000 cells (human or mouse LBAT-overexpressing cells) were seeded in 100 μL of MEM-α medium (Gibco 12571-063) supplemented with 10% FBS (Gibco 10438026) and containing genistein (Gibco 10131-027) (1 mg / mL) in 96-well plates (Corning CLS3809) and incubated at 37°C in 5% CO2 for 24 hours. After incubation, the medium was decanted from the wells and the cells were washed twice with 300 μL of basal MEM-α medium (FBS-free). After each decanting of the basal MEM-α medium, the plate was gently tapped against a paper towel to ensure maximum removal of residual medium.

[0597] For human LBAT, the incubation mixture was prepared by adding the test inhibitor dilution (3-fold serial dilution in DMSO (Sigma D2650), 10 spots) to MEM-α (FBS-free) (maintaining a final DMSO concentration of 0.2%) containing 0.3 μM 3H-taurocholic acid (ARC ART-1368) and 7.5 μM cold taurocholic acid (Sigma T4009). For mouse LBAT, the incubation mixture was prepared by adding the test inhibitor dilution (3-fold serial dilution in DMSO, 10 spots) to MEM-α (FBS-free) (maintaining a final DMSO concentration of 0.2%) containing 0.3 μM 3H-taurocholic acid and 25 μM cold taurocholic acid.

[0598] Next, add 50 μL of the incubation mixture containing the test inhibitor to each well (in duplicate), and incubate the plate in a CO2 incubator at 37°C for 20 minutes. After incubation, stop the reaction by holding the plate on an ice-water mixture for 2–3 minutes, and then completely aspirate the incubation mixture from the wells. Wash the wells twice with 250 μL of cooled, unlabeled 1 mM taurine dissolved in (10 mM) HBSS (Gibco 14175079) (pH 7.4) in HEPES (Gibco 15630080) buffer. After each wash, gently pat the plate against a paper towel to ensure maximum removal of the blocking buffer.

[0599] 100 μL of MicroScint-20 (PerkinElmer 6013621) was added to the wells and left to incubate overnight at room temperature, followed by analysis using a TopCount NXT from PerkinElmer. TM The micro-board blinking and light emission counter reads the board according to the 3H test scheme (set to 120 seconds reading time per hole, normal board orientation).

[0600] Two-way permeability analysis (Caco-2 cells)

[0601] Caco-2 cells (Evotec) were seeded at a density of 70,000 cells / well. Cells were placed in 24-well insert cell culture plates and kept in an incubator (37°C, 5% CO2, 95% RH) for 21 days, with the culture medium changed every other day.

[0602] Stock solutions (10 mM) of the test compounds atenolol (a low-permeability marker), propranolol (a high-permeability marker), and digoxin (a substrate for the P-gp transport pathway) were prepared in dimethyl sulfoxide (DMSO). Intermediate stock solutions (1 mM) were prepared by diluting 10 μL of the 10 mM mother stock solution with 90 μL of pure DMSO. Working stock solutions (10 μM) were prepared by diluting 50 μL of the 1 mM intermediate stock solution with 4950 μL of FaSSIF buffer. The compounds were added after FaSSIF, and the samples were subjected to acoustic treatment for 2 hours followed by centrifugation at 4000 RPM for 30 minutes at 37 °C. 4 mL of the supernatant was used directly for analysis. The final DMSO concentration in the transport experiments was 1%.

[0603] On the day of analysis, the Caco-2 monolayer was washed twice with transport buffer (HBSS, pH 7.4) and pre-incubated in an incubator for 30 minutes (37°C, 5% CO2, 95% RH). The -ERS system measures the electrical resistance of a single cell layer. Transepithelial resistance (TEER) values ​​exceeding 350 ohms / cm are considered optimal. 2 Single-cell layers were selected for analysis.

[0604] Analysis was performed along the absorption (A2B) and secretion (B2A) directions. The transport assay was initiated by adding a transport analysis buffer (FaSSIF buffer prepared in HBSS) consisting of the compound to the donor chamber (top chamber AB; basal-side chamber BA) in two wells (n=2). Drug-free HBSS buffer (pH 7.4) containing 1% bovine serum albumin (BSA) was introduced into the recipient chamber (AB-basal-side chamber; BA-top chamber). The volumes of the top and basal-side chambers were 0.4 mL and 0.8 mL, respectively. After adding the preparation solution, the plate was incubated at 37°C for 120 minutes. After 120 minutes, donor and recipient samples were collected and matrix-matched with the opposite buffer (1:1, 30 μL study sample + 30 μL blank buffer). The sample matrix was then prepared with the opposite buffer (1:1, 30 μL study sample + 30 μL blank buffer). Samples were treated with acetonitrile containing the internal standard (60 μL study sample + 200 μL acetonitrile containing tolbutamide, 500 ng / mL). The samples were vortexed and centrifuged at 4000 rpm for 10 min. The resulting supernatant (100 μL) was diluted with 100 μL of water and transferred to freshly prepared 96-well plates. If applicable, the concentration of compounds in the samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using discovery-grade bioanalytical methods.

[0605] The average apparent permeability (P) of the compounds atenolol, propranolol and digoxin was tested. app , ×10 -6 The speed (cm / s) is calculated as follows:

[0606]

[0607] Where dq / dt = transport rate (the rate at which the compound is transported in the acceptor chamber), C0 = initial concentration in the donor chamber, and A = surface area of ​​the effective filter membrane.

[0608] HepaRG-based analysis scheme

[0609] Following the protocol provided by Biopredic International, cryopreserved vials (Biopredic International HPR116080) of HepaRG cells were thawed in HepaRG thaw / inoculation / universal medium (Biopredic International ADD670C) supplemented with 200 mM glutamine (Gibco 35050061) for differentiation. 70,000 cells per well were seeded into 100 μL of HepaRG thaw / inoculation / universal medium supplemented with 200 mM Glutamax in a 96-well plate (Corning CLS3809) and incubated at 37°C in 5% CO2 for 24 hours. After incubation, the inoculation medium was replaced with HepaRG maintenance / metabolism medium (Biopredic International ADD620C) and incubated for 6 days, with fresh HepaRG maintenance / metabolism medium added every 48 hours. Seven days after incubation, the incubation medium was decanted from the wells, and the cells were washed twice with 250 μL of Williams's E Basal Media (Gibco 1255 1032). After each decanting of Williams's E Basal Media, the plate was gently patted against a paper towel to ensure maximum removal of residual medium.

[0610] The incubation mixture was prepared by adding a 3-fold serial dilution of the test inhibitor (in DMSO (Sigma D2650)) to Williams E medium (basal) containing 0.3 μM 3H-taurocholic acid (ARC ART-1368) and 7.5 μM cold taurocholic acid (Sigma T4009) (maintaining a final DMSO concentration of 0.2%). Then, 50 μL of the incubation mixture containing the test inhibitor was added to each well (in duplicate), and the plate was incubated in a 5% CO2 incubator at 37°C for 30 minutes. After incubation, the reaction was stopped by holding the plate on an ice-water mixture for 2–3 minutes, and the incubation mixture was then completely aspirated from the wells. Wash the wells twice with 250 μL of cooled, unlabeled 1 mM taurine dissolved in (10 mM) HBSS (Gibco 14175079) (pH 7.4) buffered with HEPES (Gibco 15630080). After each wash, gently pat the plate against a paper towel to ensure maximum removal of the blocking buffer.

[0611] 100 μL of MicroScint-20 (PerkinElmer 6013621) was added to the wells and left to incubate overnight at room temperature, followed by analysis using a TopCount NXT from PerkinElmer. TMThe micro-board blinking and light emission counter reads the board according to the 3H test scheme (set to 120 seconds reading time per hole, normal board orientation).

[0612] Preparation of diluent for test compounds

[0613] All test compounds were provided in powder form at room temperature. A 10 mM DMSO stock solution of each test compound was prepared, aliquoted, and stored at -20°C. Three-fold serial dilutions in DMSO were prepared from the 10 mM DMSO stock solutions of the compounds, resulting in a total of 10 test compound dilutions. 0.5 μL of this DMSO dilution was added to 250 μL of FBS-free basal medium containing 3H-taurocholic acid and cold taurocholic acid to prepare an incubation mixture.

[0614] Bioavailability study

[0615] Male mice (C57BL / 6 or CD1) aged 8 to 9 weeks or Wistar rats were used. For each test compound, two groups of three animals were used. One group received a single intravenous dose of 1 mg / kg (mediator 100% DMSO) via tail vein, and the other group received a single oral dose of 10 mg / kg via feeding tube. The group receiving the oral dose fasted overnight. Blood samples were collected 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intravenous administration, and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral administration. Blood samples were obtained from the saphenous vein. 0.2% EDTA was used as the anticoagulant. Samples were analyzed using an LC-MS / MS system, following a discovery-level bioanalytical method developed for estimating the presence of the test compound in plasma.

[0616] result

[0617] Biological data of the compounds in the examples are shown in Table 8 below.

[0618] Table 8

[0619]

[0620]

[0621] PD model: Evaluation of the total bile acid content in male C57BL6 mice by testing compounds.

[0622] The effects of bile acid modulators on bile acid levels were investigated using 8- to 9-week-old C57BL / 6N Tac mice. After quarantine and acclimatization, animals were randomly assigned to x experimental groups based on body weight: (i) a control group and (ii) a group receiving oral administration of the test compound y mg / kg once daily. Animals were treated with the test compound for 7 days. On day 5, animals were isolated in new cages. On day 7, feces were collected from each cage, and blood was subsequently drawn from each animal via the retroorbital route. Animals were euthanized to collect liver and terminal ileum for further analysis. Body weight and food consumption were measured twice weekly. Serum lipid profiles were analyzed from serum samples taken on day 7. Total bile acids in serum were measured from serum samples taken on day 7. Fecal bile excretion was measured from fecal samples taken on day 7. Hepatic expression of CYP7A1 and SHP was quantified from liver samples taken on day 7. Hepatic triglycerides and total cholesterol were analyzed from liver samples taken on day 7.

[0623] Urinary bile acid model: Evaluation of the effect of test compounds on urinary bile acid levels in male C57BL / 6N mice.

[0624] The effects of bile acid modulators on bile acid levels were investigated using 8- to 9-week-old C57BL / 6N Tac mice. After quarantine and acclimatization, animals were randomly assigned to x experimental groups based on body weight: (i) a control group and (ii) a group receiving oral administration of the test compound y mg / kg once daily. Animals were treated with the test compound for 7 days. On day 6, animals were transferred to metabolic cages. On day 7, feces and urine were collected from each metabolic cage, followed by blood collection via the retroorbital route. Animals were euthanized to collect kidneys for further analysis. Body weight was measured twice weekly. Total bile acids in serum were measured in day 7 serum samples. Fecal bile acid excretion was measured in day 7 fecal samples. Urinary bile acid excretion was measured in day 7 samples. Kidney expression of ASBT, OTa, OSTAb, and MRP2 was quantified in day 7 samples. sequence list <110> Albilio Company <120> Benzothiazazacycloheptatriene compounds and their uses as bile acid regulators <130> NP0503WO <150> IN 201911049980 <151> 2019-12-04 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1251 <212> PRT <213> Homo sapiens <400> 1 Met Ser Thr Glu Arg Asp Ser Glu Thr Thr Phe Asp Glu Asp Ser Gln 1 5 10 15 Pro Asn Asp Glu Val Val Pro Tyr Ser Asp Asp Glu Thr Glu Asp Glu 20 25 30 Leu Asp Asp Gln Gly Ser Ala Val Glu Pro Glu Gln Asn Arg Val Asn 35 40 45 Arg Glu Ala Glu Glu Asn Arg Glu Pro Phe Arg Lys Glu Cys Thr Trp 50 55 60 Gln Val Lys Ala Asn Asp Arg Lys Tyr His Glu Gln Pro His Phe Met 65 70 75 80 Asn Thr Lys Phe Leu Cys Ile Lys Glu Ser Lys Tyr Ala Asn Asn Ala 85 90 95 Ile Lys Thr Tyr Lys Tyr Asn Ala Phe Thr Phe Ile Pro Met Asn Leu 100 105 110 Phe Glu Gln Phe Lys Arg Ala Ala Asn Leu Tyr Phe Leu Ala Leu Leu 115 120 125 Ile Leu Gln Ala Val Pro Gln Ile Ser Thr Leu Ala Trp Tyr Thr Thr 130 135 140 Leu Val Pro Leu Leu Val Val Leu Gly Val Thr Ala Ile Lys Asp Leu 145 150 155 160 Val Asp Asp Val Ala Arg His Lys Met Asp Lys Glu Ile Asn Asn Arg 165 170 175 Thr Cys Glu Val Ile Lys Asp Gly Arg Phe Lys Val Ala Lys Trp Lys 180 185 190 Glu Ile Gln Val Gly Asp Val Ile Arg Leu Lys Lys Asn Asp Phe Val 195 200 205 Pro Ala Asp Ile Leu Leu Leu Ser Ser Ser Glu Pro Asn Ser Leu Cys 210 215 220 Tyr Val Glu Thr Ala Glu Leu Asp Gly Glu Thr Asn Leu Lys Phe Lys 225 230 235 240 Met Ser Leu Glu Ile Thr Asp Gln Tyr Leu Gln Arg Glu Asp Thr Leu 245 250 255 Ala Thr Phe Asp Gly Phe Ile Glu Cys Glu Glu Pro Asn Asn Arg Leu 260 265 270 Asp Lys Phe Thr Gly Thr Leu Phe Trp Arg Asn Thr Ser Phe Pro Leu 275 280 285 Asp Ala Asp Lys Ile Leu Leu Arg Gly Cys Val Ile Arg Asn Thr Asp 290 295 300 Phe Cys His Gly Leu Val Ile Phe Ala Gly Ala Asp Thr Lys Ile Met 305 310 315 320 Lys Asn Ser Gly Lys Thr Arg Phe Lys Arg Thr Lys Ile Asp Tyr Leu 325 330 335 Met Asn Tyr Met Val Tyr Thr Ile Phe Val Val Leu Ile Leu Leu Ser 340 345 350 Ala Gly Leu Ala Ile Gly His Ala Tyr Trp Glu Ala Gln Val Gly Asn 355 360 365 Ser Ser Trp Tyr Leu Tyr Asp Gly Glu Asp Asp Thr Pro Ser Tyr Arg 370 375 380 Gly Phe Leu Ile Phe Trp Gly Tyr Ile Ile Val Leu Asn Thr Met Val 385 390 395 400 Pro Ile Ser Leu Tyr Val Ser Val Glu Val Ile Arg Leu Gly Gln Ser 405 410 415 His Phe Ile Asn Trp Asp Leu Gln Met Tyr Tyr Ala Glu Lys Asp Thr 420 425 430 Pro Ala Lys Ala Arg Thr Thr Thr Leu Asn Glu Gln Leu Gly Gln Ile 435 440 445 His Tyr Ile Phe Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Ile Met 450 455 460 Thr Phe Lys Lys Cys Cys Ile Asn Gly Gln Ile Tyr Gly Asp His Arg 465 470 475 480 Asp Ala Ser Gln His Asn His Asn Lys Ile Glu Gln Val Asp Phe Ser 485 490 495 Trp Asn Thr Tyr Ala Asp Gly Lys Leu Ala Phe Tyr Asp His Tyr Leu 500 505 510 Ile Glu Gln Ile Gln Ser Gly Lys Glu Pro Glu Val Arg Gln Phe Phe 515 520 525 Phe Leu Leu Ala Val Cys His Thr Val Met Val Asp Arg Thr Asp Gly 530 535 540 Gln Leu Asn Tyr Gln Ala Ala Ser Pro Asp Glu Gly Ala Leu Val Asn 545 550 555 560 Ala Ala Arg Asn Phe Gly Phe Ala Phe Leu Ala Arg Thr Gln Asn Thr 565 570 575 Ile Thr Ile Ser Glu Leu Gly Thr Glu Arg Thr Tyr Asn Val Leu Ala 580 585 590 Ile Leu Asp Phe Asn Ser Asp Arg Lys Arg Met Ser Ile Ile Val Arg 595 600 605 Thr Pro Glu Gly Asn Ile Lys Leu Tyr Cys Lys Gly Ala Asp Thr Val 610 615 620 Ile Tyr Glu Arg Leu His Arg Met Asn Pro Thr Lys Gln Glu Thr Gln 625 630 635 640 Asp Ala Leu Asp Ile Phe Ala Asn Glu Thr Leu Arg Thr Leu Cys Leu 645 650 655 Cys Tyr Lys Glu Ile Glu Glu Lys Glu Phe Thr Glu Trp Asn Lys Lys 660 665 670 Phe Met Ala Ala Ser Val Ala Ser Thr Asn Arg Asp Glu Ala Leu Asp 675 680 685 Lys Val Tyr Glu Glu Ile Glu Lys Asp Leu Ile Leu Leu Gly Ala Thr 690 695 700 Ala Ile Glu Asp Lys Leu Gln Asp Gly Val Pro Glu Thr Ile Ser Lys 705 710 715 720 Leu Ala Lys Ala Asp Ile Lys Ile Trp Val Leu Thr Gly Asp Lys Lys 725 730 735 Glu Thr Ala Glu Asn Ile Gly Phe Ala Cys Glu Leu Leu Thr Glu Asp 740 745 750 Thr Thr Ile Cys Tyr Gly Glu Asp Ile Asn Ser Leu Leu His Ala Arg 755 760 765 Met Glu Asn Gln Arg Asn Arg Gly Gly Val Tyr Ala Lys Phe Ala Pro 770 775 780 Pro Val Gln Glu Ser Phe Phe Pro Pro Gly Gly Asn Arg Ala Leu Ile 785 790 795 800 Ile Thr Gly Ser Trp Leu Asn Glu Ile Leu Leu Glu Lys Lys Thr Lys 805 810 815 Arg Asn Lys Ile Leu Lys Leu Lys Phe Pro Arg Thr Glu Glu Glu Arg 820 825 830 Arg Met Arg Thr Gln Ser Lys Arg Arg Leu Glu Ala Lys Lys Glu Gln 835 840 845 Arg Gln Lys Asn Phe Val Asp Leu Ala Cys Glu Cys Ser Ala Val Ile 850 855 860 Cys Cys Arg Val Thr Pro Lys Gln Lys Ala Met Val Val Asp Leu Val 865 870 875 880 Lys Arg Tyr Lys Lys Ala Ile Thr Leu Ala Ile Gly Asp Gly Ala Asn 885 890 895 Asp Val Asn Met Ile Lys Thr Ala His Ile Gly Val Gly Ile Ser Gly 900 905 910 Gln Glu Gly Met Gln Ala Val Met Ser Ser Asp Tyr Ser Phe Ala Gln 915 920 925 Phe Arg Tyr Leu Gln Arg Leu Leu Leu Val His Gly Arg Trp Ser Tyr 930 935 940 Ile Arg Met Cys Lys Phe Leu Arg Tyr Phe Phe Tyr Lys Asn Phe Ala 945 950 955 960 Phe Thr Leu Val His Phe Trp Tyr Ser Phe Phe Asn Gly Tyr Ser Ala 965 970 975 Gln Thr Ala Tyr Glu Asp Trp Phe Ile Thr Leu Tyr Asn Val Leu Tyr 980 985 990 Thr Ser Leu Pro Val Leu Leu Met Gly Leu Leu Asp Gln Asp Val Ser 995 1000 1005 Asp Lys Leu Ser Leu Arg Phe Pro Gly Leu Tyr Ile Val Gly Gln 1010 1015 1020 Arg Asp Leu Leu Phe Asn Tyr Lys Arg Phe Phe Val Ser Leu Leu 1025 1030 1035 His Gly Val Leu Thr Ser Met Ile Leu Phe Phe Ile Pro Leu Gly 1040 1045 1050 Ala Tyr Leu Gln Thr Val Gly Gln Asp Gly Glu Ala Pro Ser Asp 1055 1060 1065 Tyr Gln Ser Phe Ala Val Thr Ile Ala Ser Ala Leu Val Ile Thr 1070 1075 1080 Val Asn Phe Gln Ile Gly Leu Asp Thr Ser Tyr Trp Thr Phe Val 1085 1090 1095 Asn Ala Phe Ser Ile Phe Gly Ser Ile Ala Leu Tyr Phe Gly Ile 1100 1105 1110 Met Phe Asp Phe His Ser Ala Gly Ile His Val Leu Phe Pro Ser 1115 1120 1125 Ala Phe Gln Phe Thr Gly Thr Ala Ser Asn Ala Leu Arg Gln Pro 1130 1135 1140 Tyr Ile Trp Leu Thr Ile Ile Leu Ala Val Ala Val Cys Leu Leu 1145 1150 1155 Pro Val Val Ala Ile Arg Phe Leu Ser Met Thr Ile Trp Pro Ser 1160 1165 1170 Glu Ser Asp Lys Ile Gln Lys His Arg Lys Arg Leu Lys Ala Glu 1175 1180 1185 Glu Gln Trp Gln Arg Arg Gln Gln Val Phe Arg Arg Gly Val Ser 1190 1195 1200 Thr Arg Arg Ser Ala Tyr Ala Phe Ser His Gln Arg Gly Tyr Ala 1205 1210 1215 Asp Leu Ile Ser Ser Gly Arg Ser Ile Arg Lys Lys Arg Ser Pro 1220 1225 1230 Leu Asp Ala Ile Val Ala Asp Gly Thr Ala Glu Tyr Arg Arg Thr 1235 1240 1245 Gly Asp Ser 1250 <210> 2 <211> 3756 <212> DNA <213> Homo sapiens <400> 2 atgagtacag aaagagactc agaaacgaca tttgacgagg attctcagcc taatgacgaa 60 gtggttccct acagtgatga tgaaacagaa gatgaacttg atgaccaggg gtctgctgtt 120 gaaccagaac aaaaccgagt caacagggaa gcagaggaga accgggagcc attcagaaaa 180 gaatgtacat ggcaagtcaa agcaaacgat cgcaagtacc acgaacaacc tcactttatg 240 aacacaaaat tcttgtgtat taaggagagt aaatatgcga ataatgcaat taaaacatac 300 aagtacaacg catttacctt tataccaatg aatctgtttg agcagtttaa gagagcagcc 360 aatttatatt tcctggctct tcttatctta caggcagttc ctcaaatctc taccctggct 420 tggtacacca cactagtgcc cctgcttgtg gtgctgggcg tcactgcaat caaagacctg 480 gtggacgatg tggctcgcca taaaatggat aaggaaatca acaataggac gtgtgaagtc 540 attaaggatg gcaggttcaa agttgctaag tggaaagaaa ttcaagttgg agacgtcatt 600 cgtctgaaaa aaaatgattt tgttccagct gacattctcc tgctgtctag ctctgagcct 660 aacagcctct gctatgtgga aacagcagaa ctggatggag aaaccaattt aaaatttaag 720 atgtcacttg aaatcacaga ccagtacctc caaagagaag atacattggc tacatttgat 780 ggttttattg aatgtgaaga acccaataac agactagata agtttacagg aacactattt 840 tggagaaaca caagttttcc tttggatgct gataaaattt tgttacgtgg ctgtgtaatt 900 aggaacaccg atttctgcca cggcttagtc atttttgcag gtgctgacac taaataatg 960 aagaatagtg ggaaaaccag atttaaaaaga actaaaattg attacttgat gaactacatg 1020 gtttacacga tctttgttgt tcttattctg ctttctgctg gtcttgccat cggccatgct 1080 tattgggaag cacaggtggg caattcctct tggtacctct atgatggaga agacgataca 1140 ccctcctacc gtggattcct cattttctgg ggctatatca ttgttctcaa caccatggta 1200 cccatctctc tctatgtcag cgtggaagtg attcgtcttg gacagagtca cttcatcaac 1260 tgggacctgc aaatgtacta tgctgagaag gacacacccg caaaagctag aaccaccaca 1320 ctcaatgaac agctcgggca gatccattat atcttctctg ataagacggg gacactcaca 1380 caaaatatca tgacctttaa aaagtgctgt atcaacgggc agatatatgg ggaccatcgg 1440 gatgcctctc aacacaacca caacaaata gagcaagttg attttagctg gaatacatat 1500 gctgatggga agcttgcatt ttatgaccac tatcttattg agcaaatcca gtcagggaaa 1560 gagccagaag tacgacagtt cttcttcttg ctcgcagttt gccacacagt catggtggat 1620 aggactgatg gtcagctcaa ctaccaggca gcctctcccg atgaaggtgc cctggtaaac 1680 gctgccagga actttggctt tgccttctc gccaggaccc agaacaccat caccatcagt 1740 gaactgggca ctgaaaggac ttacaatgtt cttgccattt tggacttcaa cagtgaccgg 1800 aagcgaatgt ctatcattgt aagaacccca gaaggcaata tcaagcttta ctgtaaaggt 1860 gctgacactg ttattatga acggttacat cgaatgaatc ctactaagca agaaacacag 1920 gatgccctgg atatctttgc aaatgaaact cttagaaccc tatgcctttg ctacaaggaa 1980 attgaagaaa aagaatttac agaatggaat aaaaagttta tggctgccag tgtggcctcc 2040 accaaccggg acgaagctct ggataaagta tatgaggaga ttgaaaaaga cttaattctc 2100 ctgggagcta cagctattga agacaagcta caggatggag ttccagaaac catttcaaaa 2160 cttgcaaaag ctgacattaa gatctgggtg cttactggag acaaaaagga aactgctgaa 2220 aatataggat ttgcttgtga acttctgact gaagacacca ccatctgcta tggggaggat 2280 attaattctc ttcttcatgc aaggatggaa aaccagagga atagaggtgg cgtctacgca 2340 aagtttgcac ctcctgtgca ggaatctttt tttccacccg gtggaaaccg tgccttaatc 2400 atcactggtt cttggttgaa tgaaattctt ctcgagaaaa agaccaagag aaataagatt 2460 ctgaagctga agttcccaag aacagaagaa gaaagacgga tgcggaccca aagtaaaagg 2520 aggctagaag ctaagaaaga gcagcggcag aaaaactttg tggacctggc ctgcgagtgc 2580 agcgcagtca tctgctgccg cgtcacccc aagcagaagg ccatggtggt ggacctggtg 2640 aagaggtaca agaaagccat cacgctggcc atcggagatg gggccaatga cgtgaacatg 2700 atcaaaactg cccacattgg cgttggaata agtggacaag aaaggaatgca agctgtcatg 2760 tcgagtgact attcctttgc tcagttccga tatctgcaga ggctactgct ggtgcatggc 2820 cgatggtctt acataaggat gtgcaagttc ctacgatact tcttttacaa aaactttgcc 2880 tttactttgg ttcatttctg gtactcttc ttcaatggct actctgcgca gactgcatac 2940 gaggattggt tcatcaccct ctacaacgtg ctgtacacca gcctgcccgt gctcctcatg 3000 gggctgctcg accaggatgt gagtgaaaa ctgagcctcc gattccctgg gttatacata 3060 gtgggacaaa gagacttact attcaactat aagagattct ttgtaagctt gttgcatggg 3120 gtcctaacat cgatgatcct cttcttcata cctcttggag cttatctgca aaccgtaggg 3180 caggatggag aggcaccttc cgactaccag tcttttgccg tcaccattgc ctctgctctt 3240 gtaataacag tcaatttcca gattggcttg gatacttctt attggacttt tgtgaatgct 3300 ttttcaattt ttggaagcat tgcactttat tttggcatca tgtttgactt tcatagtgct 3360 ggatacatg ttctctttcc atctgcattt caatttacag gcacagcttc aaacgctctg 3420 agacagccat acatttggtt aactatcatc ctggctgttg ctgtgtgctt actacccgtc 3480 gttgccattc gattcctgtc aatgaccatc tggccatcag aaagtgataa gatccagaag 3540 catcgcaagc ggttgaaggc ggaggagcag tggcagcgac ggcagcaggt gttccgccgg 3600 ggcgtgtcaa cgcggcgctc ggcctacgcc ttctcgcacc agcggggcta cgcggacctc 3660 atctcctccg ggcgcagcat ccgcaagaag cgctcgccgc ttgatgccat cgtggcggat 3720 ggcaccgcgg agtacaggcg caccggggac agctga 3756 <210> 3 <211> 1321 <212> PRT <213> Homo sapiens <400> 3 Met Ser Asp Ser Val Ile Leu Arg Ser Ile Lys Lys Phe Gly Glu Glu 1 5 10 15 Asn Asp Gly Phe Glu Ser Asp Lys Ser Tyr Asn Asn Asp Lys Lys Ser 20 25 30 Arg Leu Gln Asp Glu Lys Lys Gly Asp Gly Val Arg Val Gly Phe Phe 35 40 45 Gln Leu Phe Arg Phe Ser Ser Ser Thr Asp Ile Trp Leu Met Phe Val 50 55 60 Gly Ser Leu Cys Ala Phe Leu His Gly Ile Ala Gln Pro Gly Val Leu 65 70 75 80 Leu Ile Phe Gly Thr Met Thr Asp Val Phe Ile Asp Tyr Asp Val Glu 85 90 95 Leu Gln Glu Leu Gln Ile Pro Gly Lys Ala Cys Val Asn Asn Thr Ile 100 105 110 Val Trp Thr Asn Ser Ser Leu Asn Gln Asn Met Thr Asn Gly Thr Arg 115 120 125 Cys Gly Leu Leu Asn Ile Glu Ser Glu Met Ile Lys Phe Ala Ser Tyr 130 135 140 Tyr Ala Gly Ile Ala Val Ala Val Leu Ile Thr Gly Tyr Ile Gln Ile 145 150 155 160 Cys Phe Trp Val Ile Ala Ala Ala Arg Gln Ile Gln Lys Met Arg Lys 165 170 175 Phe Tyr Phe Arg Arg Ile Met Arg Met Glu Ile Gly Trp Phe Asp Cys 180 185 190 Asn Ser Val Gly Glu Leu Asn Thr Arg Phe Ser Asp Asp Ile Asn Lys 195 200 205 Ile Asn Asp Ala Ile Ala Asp Gln Met Ala Leu Phe Ile Gln Arg Met 210 215 220 Thr Ser Thr Ile Cys Gly Phe Leu Leu Gly Phe Phe Arg Gly Trp Lys 225 230 235 240 Leu Thr Leu Val Ile Ile Ser Val Ser Pro Leu Ile Gly Ile Gly Ala 245 250 255 Ala Thr Ile Gly Leu Ser Val Ser Lys Phe Thr Asp Tyr Glu Leu Lys 260 265 270 Ala Tyr Ala Lys Ala Gly Val Val Ala Asp Glu Val Ile Ser Ser Met 275 280 285 Arg Thr Val Ala Ala Phe Gly Gly Glu Lys Arg Glu Val Glu Arg Tyr 290 295 300 Glu Lys Asn Leu Val Phe Ala Gln Arg Trp Gly Ile Arg Lys Gly Ile 305 310 315 320 Val Met Gly Phe Phe Thr Gly Phe Val Trp Cys Leu Ile Phe Leu Cys 325 330 335 Tyr Ala Leu Ala Phe Trp Tyr Gly Ser Thr Leu Val Leu Asp Glu Gly 340 345 350 Glu Tyr Thr Pro Gly Thr Leu Val Gln Ile Phe Leu Ser Val Ile Val 355 360 365 Gly Ala Leu Asn Leu Gly Asn Ala Ser Pro Cys Leu Glu Ala Phe Ala 370 375 380 Thr Gly Arg Ala Ala Ala Thr Ser Ile Phe Glu Thr Ile Asp Arg Lys 385 390 395 400 Pro Ile Ile Asp Cys Met Ser Glu Asp Gly Tyr Lys Leu Asp Arg Ile 405 410 415 Lys Gly Glu Ile Glu Phe His Asn Val Thr Phe His Tyr Pro Ser Arg 420 425 430 Pro Glu Val Lys Ile Leu Asn Asp Leu Asn Met Val Ile Lys Pro Gly 435 440 445 Glu Met Thr Ala Leu Val Gly Pro Ser Gly Ala Gly Lys Ser Thr Ala 450 455 460 Leu Gln Leu Ile Gln Arg Phe Tyr Asp Pro Cys Glu Gly Met Val Thr 465 470 475 480 Val Asp Gly His Asp Ile Arg Ser Leu Asn Ile Gln Trp Leu Arg Asp 485 490 495 Gln Ile Gly Ile Val Glu Gln Glu Pro Val Leu Phe Ser Thr Thr Ile 500 505 510 Ala Glu Asn Ile Arg Tyr Gly Arg Glu Asp Ala Thr Met Glu Asp Ile 515 520 525 Val Gln Ala Ala Lys Glu Ala Asn Ala Tyr Asn Phe Ile Met Asp Leu 530 535 540 Pro Gln Gln Phe Asp Thr Leu Val Gly Glu Gly Gly Gly Gln Met Ser 545 550 555 560 Gly Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Ala Leu Ile Arg Asn 565 570 575 Pro Lys Ile Leu Leu Leu Asp Met Ala Thr Ser Ala Leu Asp Asn Glu 580 585 590 Ser Glu Ala Met Val Gln Glu Val Leu Ser Lys Ile Gln His Gly His 595 600 605 Thr Ile Ile Ser Val Ala His Arg Leu Ser Thr Val Arg Ala Ala Asp 610 615 620 Thr Ile Ile Gly Phe Glu His Gly Thr Ala Val Glu Arg Gly Thr His 625 630 635 640 Glu Glu Leu Leu Glu Arg Lys Gly Val Tyr Phe Thr Leu Val Thr Leu 645 650 655 Gln Ser Gln Gly Asn Gln Ala Leu Asn Glu Glu Asp Ile Lys Asp Ala 660 665 670 Thr Glu Asp Asp Met Leu Ala Arg Thr Phe Ser Arg Gly Ser Tyr Gln 675 680 685 Asp Ser Leu Arg Ala Ser Ile Arg Gln Arg Ser Lys Ser Gln Leu Ser 690 695 700 Tyr Leu Val His Glu Pro Pro Leu Ala Val Val Asp His Lys Ser Thr 705 710 715 720 Tyr Glu Glu Asp Arg Lys Asp Lys Asp Ile Pro Val Gln Glu Glu Val 725 730 735 Glu Pro Ala Pro Val Arg Arg Ile Leu Lys Phe Ser Ala Pro Glu Trp 740 745 750 Pro Tyr Met Leu Val Gly Ser Val Gly Ala Ala Val Asn Gly Thr Val 755 760 765 Thr Pro Leu Tyr Ala Phe Leu Phe Ser Gln Ile Leu Gly Thr Phe Ser 770 775 780 Ile Pro Asp Lys Glu Glu Gln Arg Ser Gln Ile Asn Gly Val Cys Leu 785 790 795 800 Leu Phe Val Ala Met Gly Cys Val Ser Leu Phe Thr Gln Phe Leu Gln 805 810 815 Gly Tyr Ala Phe Ala Lys Ser Gly Glu Leu Leu Thr Lys Arg Leu Arg 820 825 830 Lys Phe Gly Phe Arg Ala Met Leu Gly Gln Asp Ile Ala Trp Phe Asp 835 840 845 Asp Leu Arg Asn Ser Pro Gly Ala Leu Thr Thr Arg Leu Ala Thr Asp 850 855 860 Ala Ser Gln Val Gln Gly Ala Ala Gly Ser Gln Ile Gly Met Ile Val 865 870 875 880 Asn Ser Phe Thr Asn Val Thr Val Ala Met Ile Ile Ala Phe Ser Phe 885 890 895 Ser Trp Lys Leu Ser Leu Val Ile Leu Cys Phe Phe Pro Phe Leu Ala 900 905 910 Leu Ser Gly Ala Thr Gln Thr Arg Met Leu Thr Gly Phe Ala Ser Arg 915 920 925 Asp Lys Gln Ala Leu Glu Met Val Gly Gln Ile Thr Asn Glu Ala Leu 930 935 940 Ser Asn Ile Arg Thr Val Ala Gly Ile Gly Lys Glu Arg Arg Phe Ile 945 950 955 960 Glu Ala Leu Glu Thr Glu Leu Glu Lys Pro Phe Lys Thr Ala Ile Gln 965 970 975 Lys Ala Asn Ile Tyr Gly Phe Cys Phe Ala Phe Ala Gln Cys Ile Met 980 985 990 Phe Ile Ala Asn Ser Ala Ser Tyr Arg Tyr Gly Gly Tyr Leu Ile Ser 995 1000 1005 Asn Glu Gly Leu His Phe Ser Tyr Val Phe Arg Val Ile Ser Ala 1010 1015 1020 Val Val Leu Ser Ala Thr Ala Leu Gly Arg Ala Phe Ser Tyr Thr 1025 1030 1035 Pro Ser Tyr Ala Lys Ala Lys Ile Ser Ala Ala Arg Phe Phe Gln 1040 1045 1050 Leu Leu Asp Arg Gln Pro Pro Ile Ser Val Tyr Asn Thr Ala Gly 1055 1060 1065 Glu Lys Trp Asp Asn Phe Gln Gly Lys Ile Asp Phe Val Asp Cys 1070 1075 1080 Lys Phe Thr Tyr Pro Ser Arg Pro Asp Ser Gln Val Leu Asn Gly 1085 1090 1095 Leu Ser Val Ser Ile Ser Pro Gly Gln Thr Leu Ala Phe Val Gly 1100 1105 1110 Ser Ser Gly Cys Gly Lys Ser Thr Ser Ile Gln Leu Leu Glu Arg 1115 1120 1125 Phe Tyr Asp Pro Asp Gln Gly Lys Val Met Ile Asp Gly His Asp 1130 1135 1140 Ser Lys Lys Val Asn Val Gln Phe Leu Arg Ser Asn Ile Gly Ile 1145 1150 1155 Val Ser Gln Glu Pro Val Leu Phe Ala Cys Ser Ile Met Asp Asn 1160 1165 1170 Ile Lys Tyr Gly Asp Asn Thr Lys Glu Ile Pro Met Glu Arg Val 1175 1180 1185 Ile Ala Ala Ala Lys Gln Ala Gln Leu His Asp Phe Val Met Ser 1190 1195 1200 Leu Pro Glu Lys Tyr Glu Thr Asn Val Gly Ser Gln Gly Ser Gln 1205 1210 1215 Leu Ser Arg Gly Glu Lys Gln Arg Ile Ala Ile Ala Arg Ala Ile 1220 1225 1230 Val Arg Asp Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala 1235 1240 1245 Leu Asp Thr Glu Ser Glu Lys Thr Val Gln Val Ala Leu Asp Lys 1250 1255 1260 Ala Arg Glu Gly Arg Thr Cys Ile Val Ile Ala His Arg Leu Ser 1265 1270 1275 Thr Ile Gln Asn Ala Asp Ile Ile Ala Val Met Ala Gln Gly Val 1280 1285 1290 Val Ile Glu Lys Gly Thr His Glu Glu Leu Met Ala Gln Lys Gly 1295 1300 1305 Ala Tyr Tyr Lys Leu Val Thr Thr Gly Ser Pro Ile Ser 1310 1315 1320 <210> 4 <211> 3966 <212> DNA <213> Homo sapiens <400> 4 atgtctgact cagtaattct tcgaagtata aagaaatttg gagaggagaa tgatggtttt 60 gagtcagata aatcatataa taatgataag aaatcaaggt tacaagatga gaagaaaggt 120 gatggcgtta gagttggctt ctttcaattg tttcggtttt cttcatcaac tgacatttgg 180 ctgatgtttg tgggaagttt gtgtgcattt ctccatggaa tagcccagcc aggcgtgcta 240 ctcatttttg gcacaatgac agatgttttt attgactacg acgttgagtt acaagaactc 300 cagattccag gaaaagcatg tgtgaataac accattgtat ggactaacag ttccctcaac 360 cagaacatga caaatggaac acgttgtggg ttgctgaaca tcgagagcga aatgatcaaa 420 tttgccagtt actatgctgg aattgctgtc gcagtactta tcacaggata tattcaaata 480 tgcttttggg tcattgccgc agctcgtcag atacagaaaa tgagaaaatt ttactttagg 540 agaataatga gaatggaaat agggtggttt gactgcaatt cagtggggga gctgaataca 600 agattctctg atgatattaa taaaatcaat gatgccatag ctgaccaaat ggccctttc 660 attcagcgca tgacctcgac catctgtggt ttcctgttgg gatttttcag gggttggaaa 720 ctgaccttgg ttattattc tgtcagccct ctcattggga ttggagcagc caccattggt 780 ctgagtgtgt ccaagtttac ggactatgag ctgaaggcct atgccaaagc aggggtggtg 840 gctgatgaag tcatttcatc aatgagaaca gtggctgctt ttggtggtga gaaaagagag 900 gttgaaaggt atgagaaaaa tcttgtgttc gcccagcgtt ggggaattag aaaaggaata 960 gtgatgggat tctttactgg attcgtgtgg tgtctcatct ttttgtgtta tgcactggcc 1020 ttctggtacg gctccacact tgtcctggat gaaggagaat atacaccagg aacccttgtc 1080 cagatttcc tcagtgtcat agtaggagct ttaaatcttg gcaatgcctc tccttgtttg 1140 gaagcctttg caactggacg tgcagcagcc accagcattt ttgagacaat agacaggaaa 1200 cccatcattg actgcatgtc agaagatggt tacaagttgg atcgaatcaa gggtgaaatt 1260 gaattccata atgtgacctt ccattatcct tccagaccag aggtgaagat tctaaatgac 1320 ctcaacatgg tcattaaacc aggggaaatg acagctctgg taggacccag tggagctgga 1380 aaaagtacag cactgcaact cattcagcga ttctatgacc cctgtgaagg aatggtgacc 1440 gtggatggcc atgacattcg ctctcttaac attcagtggc ttagagatca gattgggata 1500 gtggagcaag agccagttct gttctctacc accattgcag aaaatattcg ctatggcaga 1560 gaagatgcaa caatggaaga catagtccaa gctgccaagg aggccaatgc ctacaacttc 1620 atcatggacc tgccacagca atttgacacc cttgttggag aaggagggagg ccagatgagt 1680 ggtggccaga aacaaagggt agctatcgcc agagccctca tccgaaatcc caagattctg 1740 cttttggaca tggccacctc agctctggac aatgagagtg aagccatggt gcaagaagtg 1800 ctgagtaaga ttcagcatgg gcacacaatc atttcagttg ctcatcgctt gtctacggtc 1860 agagctgcag ataccatcat tggttttgaa catggcactg cagtggaaag agggacccat 1920 gaagaattac tggaaaggaa aggtgtttac ttcactctag tgactttgca aagccaggga 1980 aatcaagctc ttaatgaaga ggacataaag gatgcaactg aagatgacat gcttgcgagg 2040 acctttagca gagggagcta ccaggatagt ttaagggctt ccatccggca acgctccaag 2100 tctcagcttt cttacctggt gcacgaacct ccattagctg ttgtagatca taagtctacc 2160 tatgaagaag atagaaagga caaggacatt cctgtgcagg aagaagttga acctgcccca 2220 gttaggagga ttctgaaatt cagtgctcca gaatggccct acatgctggt agggtctgtg 2280 ggtgcagctg tgaacgggac agtcacaccc ttgtatgcct ttttattcag ccagattctt 2340 gggacttttt caattcctga taagaggaa caaaggtcac agatcaatgg tgtgtgccta 2400 cttttgtag caatgggctg tgtatctctt ttcacccaat ttctacaggg atatgccttt 2460 gctaaatctg gggagctcct aaaaaagg ctacgtaaat ttggtttcag ggcaatgctg 2520 gggcaagata ttgcctggtt tgatgacctc agaaatagcc ctggagcatt gacaacaaga 2580 cttgctacag atgcttccca agttcaaggg gctgccggct ctcagatcgg gatgatagtc 2640 aattccttca ctaacgtcac tgtggccatg atcattgcct tctcctttag ctggaagctg 2700 agcctggtca tcttgtgctt cttccccttc ttggctttat caggagccac acagaccagg 2760 atgttgacag gatttgcctc tcgagataag caggccctgg agatggtggg acagattaca 2820 aatgaagccc tcagtaacat ccgcactgtt gctggaattg gaaaggagag gcggttcatt 2880 gaagcacttg agactgagct ggagaagcccc ttcaagacag ccattcagaa agccaatatt 2940 tacggattct gctttgcctt tgcccagtgc atcatgttta ttgcgaattc tgcttcctac 3000 agatatggag gttacttaat ctccaatgag gggctccatt tcagctatgt gttcagggtg 3060 atctctgcag ttgtactgag tgcaacagct cttggaagag ccttctctta caccccaagt 3120 tatgcaaaag ctaaaatatc agctgcacgc ttttttcaac tgctggaccg acaaccccca 3180 atcagtgtat acaatactgc aggtgaaaaa tgggacaact tccaggggaa gattgatttt 3240 gttgattgta aatttacata tccttctcga cctgactcgc aagttctgaa tggtctctca 3300 gtgtcgatta gtccagggca gacactggcg tttgttggga gcagtggatg tggcaaaagc 3360 actagcattc agctgttgga acgtttctat gatcctgatc aagggaaggt gatgatagat 3420 ggtcatgaca gcaaaaaagt aaatgtccag ttcctccgct caaacattgg aattgtttcc 3480 caggaaccag tgttgtttgc ctgtagcata atggacaata tcaagtatgg agacaacacc 3540 aaagaaattc ccatggaaag agtcatagca gctgcaaaac aggctcagct gcatgatttt 3600 gtcatgtcac tcccagagaa atatgaaact aacgttgggt cccaggggtc tcaactctct 3660 agagggaga aacaacgcat tgctattgct cgggccattg tacgagatcc taaaatcttg 3720 ctactagatg aagccacttc tgccttagac aagaaagtg aaaagacggt gcaggttgct 3780 ctagacaaag ccagagaggg tcggacctgc attgtcattg cccatcgctt gtccaccatc 3840 cagaacgcgg atatcattgc tgtcatggca cagggggtgg tgattgaaaa ggggacccat 3900 gaaactga tggcccaaaa aggagcctac tacaaactag tcaccactgg atcccccatc 3960 agttga 3966

Claims

1. A compound of formula (I) in R 1 and R 2 Each independently is C 1-4 alkyl; R 3 For hydrogen; and R 4 Selected from: C 1-4 Alkoxy and C 1-4 Alkylthio; Or its pharmaceutically acceptable salt. The condition is that the compound is not selected from the following compounds: 2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxide-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid.

2. The compound according to claim 1, wherein R 1 It is n-butyl.

3. The compound according to claim 1 or 2, wherein R 2 It is n-butyl.

4. The compound according to claim 1 or 2, wherein R 2 It is an ethyl group.

5. The compound according to any one of claims 1 to 4, wherein R 4 Selected from: methoxy, ethoxy, methylthio, and ethylthio.

6. The compound according to claim 1, wherein the compound is selected from: 2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiazazacycloheptatrien-8-yl)oxy)acetic acid; (S)-2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid; and (R)-2-((3-Butyl-3-ethyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiazazacycloheptatrien-8-yl)oxy)acetic acid; Or its pharmaceutically acceptable salt.

7. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 6 and one or more pharmaceutically acceptable excipients.

8. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament. in R 1 and R 2 Each independently is C 1-4 alkyl; R 3 Independently selected from: hydrogen; and R 4 Selected from: C 1-4 Alkoxy and C 1-4 Alkylthio, The drug is used to treat or prevent cardiovascular disease or fatty acid metabolism disorder or glucose utilization disorder, selected from any of the following: hypercholesterolemia; fatty acid metabolism disorder and type 2 diabetes.

9. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament. in R 1 and R 2 Each independently is C 1-4 alkyl; R 3 Independently selected from: hydrogen; and R 4 Selected from: C 1-4 Alkoxy and C 1-4 Alkylthio, The medication described herein is used to treat or prevent gastrointestinal disorders or disturbances, and is selected from any of the following: chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C).

10. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament. in R 1 and R 2 Each independently is C 1-4 alkyl; R 3 Independently selected from: hydrogen; and R 4 Selected from: C 1-4 Alkoxy and C 1-4 Alkylthio, The medication described herein is used to treat or prevent liver disease or disorder, and is selected from any of the following: post-Kasai biliary atresia; Alageri syndrome (ALGS); progressive familial intrahepatic cholestasis (PFIC); benign recurrent intrahepatic cholestasis (BRIC); primary biliary cirrhosis (PBC); and nonalcoholic fatty liver disease (NAFLD).

11. The use according to claim 10, wherein the progressive familial intrahepatic cholestasis (PFIC) is selected from PFIC-1, PFIC-2, PFIC-3 and nonspecific PFIC, post-cholesterol shunt PFIC and post-liver transplant PFIC, and wherein the benign recurrent intrahepatic cholestasis (BRIC) is selected from BRIC1, BRIC2 and nonspecific BRIC, post-cholesterol shunt BRIC and post-liver transplant BRIC.

12. The use according to claim 10, wherein the non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic steatohepatitis (NASH).

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