Benzothia(d)azepine compounds and their use as bile acid modulators

Abstract

The present invention relates to certain 1,5-benzothiazepine and 1,2,5-benzothiadiazepine derivatives as defined herein. These compounds are bile acid modulators having apical sodium-dependent bile acid transporter (ASBT) and / or liver bile acid transporter (LBAT) inhibitory activity. The invention also relates to pharmaceutical compositions comprising these compounds, and to the use of these compounds in the treatment of cardiovascular diseases, disorders of fatty acid metabolism and glucose utilization, gastrointestinal diseases and liver diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Indian Application No. 202011033169, filed on August 3, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to certain 1,5-benzothiazazaheptatrienes and 1,2,5-benzothiadiazaheptatriene derivatives as defined herein. These compounds are bile acid modulators with apical sodium-dependent bile acid transporter (ASBT) and / or hepatic bile acid transporter (LBAT) inhibitory activity. 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、WO 2021 / 110883, WO 2021 / 110884, WO 2021 / 110885, WO 2021 / 110886, WO 2021 / 110887, 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 benzothiazazazetazone hepttrienes and benzothiazazazetazone hepttriene derivatives have been identified as effective inhibitors of apical sodium-dependent bile acid transporter (ASBT) and / or hepatic bile acid transporter (LBAT), and can be used to treat diseases requiring inhibition of bile acid circulation.

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

[0011]

[0012] Among them, M and R 1 R 2 R 3 R 4 R 5A and R 5B As indicated in Table 1 below, or their pharmaceutically acceptable salts:

[0013] Table 1

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021] In one specific implementation, the compound of formula (I) is selected from:

[0022] (S)-3-(((S)-3-butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionic acid;

[0023] (S)-3-(((R)-3-butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionic acid;

[0024] (R)-3-(((S)-3-butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionic acid;

[0025] (R)-3-(((R)-3-butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazazetrin-8-yl)oxy)-2-hydroxypropionic acid;

[0026] 1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0027] (S)-1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0028] (R)-1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid;

[0029] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid;

[0030] (S)-3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazazetrin-8-yl)oxy)-2,2-dimethylpropionic acid;

[0031] (R)-3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazazetrin-8-yl)oxy)-2,2-dimethylpropionic acid;

[0032] 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid;

[0033] (S)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid;

[0034] (R)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid;

[0035] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid;

[0036] 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid;

[0037] (S)-3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid;

[0038] (R)-3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid;

[0039] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionic acid;

[0040] 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionic acid;

[0041] 3-((3,3-diethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid; and

[0042] 2-Hydroxy-3-((7-(methylthio)-1,1-dioxo-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)propionic acid;

[0043] Or its pharmaceutically acceptable salt.

[0044] 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.

[0045] 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.

[0046] The 1,5-benzothiazazaheptatriene and 1,2,5-benzothiadiazaheptatriene compounds of formula (I), or pharmaceutically acceptable salts thereof, are inhibitors of apical sodium-dependent bile acid transporters (ASBT inhibitors), inhibitors of hepatic bile acid transporters (LBAT inhibitors), or inhibitors of both apical sodium-dependent bile acids and hepatic bile acid transporters (dual ASBT / LBAT inhibitors). Therefore, they are 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.

[0047] 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.

[0048] 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.

[0049] 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 defects in bile acid synthesis; congenital bile duct abnormalities; biliary atresia; post-Kasai biliary atresia; post-liver transplant biliary atresia; neonatal hepatitis; neonatal cholestasis; hereditary cholestasis; encephalotendinosis xanthoma; 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 (LCS1); 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 deficiency 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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).

[0054] 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.

[0055] Liu et al. described the identification of a novel 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, with the highest incidence observed in southern China (8% in Han Chinese and 12% in Dai Chinese) and Vietnam (11%). This “hidden” hypercholanemia is believed to affect 0.64% of the Han Chinese population in southern China, 1.44% of the Dai population in China, and 1.21% of the Vietnamese population. 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 attributed 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 bile acids from the circulation to achieve homeostasis (Karpen and Dawson, Hepatology 2015, Vol. 61, pp. 24-27). Alternatively, the liver may synthesize increased levels of bile acids to compensate for reduced enterohepatic recirculation in homozygous carriers. Since LBAT also transports unconjugated bile acids, the increase in unconjugated bile acids in this study is not unexpected (Liu et al., Scientific Reports 2017, 7:9214, pp. 1-7).

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Three alternative gene defects have been identified, which are associated with three separate PFIC subtypes known as type 1, type 2, and type 3:

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] Typical protein sequence of ATP8B1 (SEQ ID NO:1) - Uniproto ID O43520

[0072]

[0073] Typical DNA sequence of ATP8B1 (SEQ ID NO:2)

[0074]

[0075]

[0076] Table 2. Exemplary ATP8B1 mutations

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Table 3. Selected ATP8B1 mutations associated with PFIC-1

[0086]

[0087]

[0088]

[0089] A The mutation to "X" indicates an early stop codon.

[0090] References in Tables 2 and 3

[0091] 1 Folmer et al., Hepatology. 2009, Vol. 50(5), pp. 1597-1605

[0092] 2 Hsu et al., Hepatol Res. 2009, Vol. 39(6), pp. 625-631

[0093] 3 Alvarez et al., Hum Mol Genet. 2004, Vol. 13(20), pp. 2451-2460

[0094] 4 Davit-Spraul et al., Hepatology 2010, Vol. 51(5), pp. 1645-1655

[0095] 5 Vitale et al., J Gastroenterol. 2018, Vol. 53(8), pp. 945-958

[0096] 6 Klomp et al., Hepatology 2004, Vol. 40(1), pp. 27-38

[0097] 7 Zarenezhad et al., Hepatitis Monthly: 2017, Vol. 17(2); e43500.

[0098] 8 Dixon et al., Scientific Reports 2017, Vol. 7, 11823.

[0099] 9 Painter et al., Eur J Hum Genet. 2005, Vol. 13(4), pp. 435-439

[0100] 10 Deng et al., World J Gastroenterol. 2012, Vol. 18(44), pp. 6504-6509

[0101] 11 Giovannoni et al., PLoS One. 2015, Vol. 10(12):e0145021.

[0102] 12 Li et al., Hepatology International 2017, Vol. 11, No. 1, Supp. 1, pp. S180. Abstract No. OP284.

[0103] 13 Togawa et al., Journal of Pediatric Gastroenterology and Nutrition, 2018, Vol. 67, Supp. 1, pp. S363. Abstract No. 615.

[0104] 14 Miloh et al., Gastroenterology 2006, Vol. 130, No. 4, Suppl. 2, pp. A759-A760. Conference Information: Digestive Disease Week Meeting / 107th Annual Meeting of the American-Gastroenterological-Association. Los Angeles, California, USA, May 19

[0105] 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.

[0106] 16Mizuochi et al., Clin Chim Acta. 2012, Vol. 413(15-16), pp. 1301-1304

[0107] 17 Liu et al., Hepatology International 2009, Vol. 3, No. 1, pp. 184-185. Abstract No. PE405. Conference Information: 19th Conference of the Asian Pacific Association for the Study of the Liver. Hong Kong, China, February 13-16, 2009.

[0108] 18 McKay et al., Version 2. F1000Res. 2013; 2:32. DOI:10.12688 / f1000research.2-32.v2

[0109] 19 Hasegawa et al., Orphanet J Rare Dis. 2014, Vol. 9:89

[0110] 20 Stone et al., J Biol Chem. 2012, Vol. 287(49), pp. 41139-51

[0111] 21 Kang et al., J Pathol Transl Med. May 16, 2019. doi:10.4132 / jptm.2019.05.03. [Earlier than preprinted electronic publication]

[0112] 22 Sharma et al., BMC Gastroenterol. 2018, Vol. 18(1), p. 107

[0113] 23 Uegaki et al., Intern Med. 2008, Vol. 47(7), pp. 599-602

[0114] 24 Goldschmidt et al., Hepatol Res. 2016, Vol. 46(4), pp. 306-311

[0115] 25Liu et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 50(2), pp. 179-183

[0116] 26 Jung et al., J Pediatr Gastroenterol Nutr. 2007, Vol. 44(4), pp. 453-458

[0117] 27 Bounford, University of Birmingham, Dissertation Abstracts International, (2016), Vol. 75, No. 1C, Subscription No.: AAI10588329. ProQuest Dissertations & Theses.

[0118] 28 Stolz et al., Aliment Pharmacol Ther. 2019, Vol. 49(9), pp. 1195-1204

[0119] 29 Ivashkin et al., Hepatology International 2016, Vol. 10, No. 1, Supp. SUPPL. 1, pp. S461. Abstract No. LBO-38. Conference Information: 25th Annual Conference of the Asian Pacific Association for the Study of the Liver, APASL 2016. Tokyo, Japan, February 20-24, 2016.

[0120] 30 Blackmore et al., J Clin Exp Hepatol. 2013, Vol. 3(2), pp. 159-161

[0121] 31 Matte et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 51(4), pp. 488-493

[0122] 32 Squires et al., J Pediatr Gastroenterol Nutr. 2017, Vol. 64(3), pp. 425-430

[0123] 33Hayshi et al., EBioMedicine. 2018, Vol. 27, pp. 187-199

[0124] 34 Nagasaka et al., J Pediatr Gastroenterol Nutr. 2007, Vol. 45(1), pp. 96-105

[0125] 35 Wang et al., PLoS One. 2016; Vol. 11(4): e0153114.

[0126] 36 Narchi et al., Saudi J Gastroenterol. 2017, Vol. 23(5), pp. 303-305

[0127] 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.

[0128] 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.

[0129] 39 Pauli-Magnus et al., J Hepatol. 2005, Vol. 43(2), pp. 342-357

[0130] 40 Jericho et al., Journal of Pediatric Gastroenterology and Nutrition, 2015, Vol. 60(3), pp. 368-374

[0131] 41 van der Woerd et al., PLoS One. 2013, Vol. 8(11):e80553.

[0132] 42 Copeland et al., J Gastroenterol Hepatol. 2013, Vol. 28(3), pp. 560-564

[0133] et al., J Hepatol. 2017, Vol. 67(6), pp. 1253-1264

[0134] 44 Chen et al., Journal of Pediatrics 2002, Vol. 140(1), pp. 119-124

[0135] 45 Jirsa et al., Hepatol Res. 2004, Vol. 30(1), pp. 1-3

[0136] 46 van der Woerd et al., Hepatology 2015, Vol. 61(4), pp. 1382-1391

[0137] In some implementations, the mutations in ATP8B1 are selected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, and G1040R.

[0138] Typical protein sequence of ABCB11 (SEQ ID NO:3) - Uniproto ID O95342

[0139]

[0140] Typical DNA sequence of ABCB11 (SEQ ID NO:4)

[0141]

[0142]

[0143]

[0144] Table 4. Exemplary ABCB11 mutations

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Table 5. Selected ABCB11 mutations associated with PFIC-2

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] A The mutation to "X" indicates an early stop codon.

[0165] References in Tables 4 and 5

[0166] 1 Noe et al., J Hepatol. 2005, Vol. 43(3), pp. 536-543

[0167] 2 Lam et al., Am J Physiol Cell Physiol. 2007, Vol. 293(5), pp. C1709-16

[0168] 3 Stindt et al., Liver Int. 2013, Vol. 33(10), pp. 1527-1735

[0169] 4Gao et al., Shandong Yiyao 2012, Vol. 52(10), pp. 14-16

[0170] 5 Strautnieks et al., Gastroenterology. 2008, Vol. 134(4), pp. 1203-1214

[0171] 6 Kagawa et al., Am J Physiol Gastrointest Liver Physiol. 2008, Vol. 294(1), pp. G58-67

[0172] 7 Byrne et al., Hepatology. 2009, Vol. 49(2), pp. 553-567

[0173] 8 Chen et al., J Pediatr. 2008, Vol. 153(6), pp. 825-832

[0174] 9 Davit-Spraul et al., Hepatology 2010, Vol. 51(5), pp. 1645-1655

[0175] et al., Sci Rep. 2016, Vol. 6: 24827.

[0176] 11 Lang et al., Pharmacogenet Genomics. 2007, Vol. 17(1), pp. 47-60

[0177] 12 Ellinger et al., World J Gastroenterol. 2017, Vol. 23(29), pp. 5295-5303

[0178] 13 Vitale et al., J Gastroenterol. 2018, Vol. 53(8), pp. 945-958

[0179] 14 Knisely et al., Hepatology. 2006, Vol. 44(2), pp. 478-86

[0180] 15 Ellis et al., Hepatology. 2018, Vol. 67(4), pp. 1531-1545

[0181] 16 Lam et al., J Hepatol. 2006, Vol. 44(1), pp. 240-242

[0182] 17 Varma et al., Hepatology 2015, Vol. 62(1), pp. 198-206

[0183] 18 Treepongkaruna et al., World J Gastroenterol. 2009, Vol. 15(34), pp. 4339-4342

[0184] 19 Zarenezhad et al., Hepatitis Monthly: 2017, Vol. 17(2); e43500.

[0185] 20 Hayashi et al., Hepatol Res. 2016, Vol. 46(2), pp. 192-200

[0186] 21 Guorui et al., Linchuang Erke Zazhi 2013, Vol. 31(10), pp. 905-909

[0187] 22 van Mil et al., Gastroenterology. 2004, Vol. 127(2), pp. 379-384

[0188] 23 Anzivino et al., Dig Liver Dis. 2013, Vol. 45(3), pp. 226-232

[0189] 24 Park et al., World J Gastroenterol. 2016, Vol. 22(20), pp. 4901-4907 25 Imagawa et al., J Hum Genet. 2018, Vol. 63(5), pp. 569-577

[0190] 26 Giovannoni et al., PLoS One. 2015, Vol. 10(12):e0145021.

[0191] 27Hu et al., Mol Med Rep. 2014, Vol. 10(3), pp. 1264-1274

[0192] 28 Lang et al. Drug Metab Dispos. 2006, Vol. 34(9), pp. 1582-1599

[0193] 29 Masahata et al., Transplant Proc. 2016, Vol. 48(9), pp. 3156-3162

[0194] 30 Holz et al., Hepatol Commun. 2018, Vol. 2(2), pp. 152-154

[0195] 31 Li et al., Hepatology International 2017, Vol. 11, No. 1, Supp. 1, pp. S180. Abstract No. OP284.

[0196] 32 Francalanci et al., Laboratory Investigation 2011, Vol. 91, Supp. SUPPL. 1, pp. 360A. Abstract No. 1526.

[0197] 33 Francalanci et al., Digestive and Liver Disease, 2010, Vol. 42, Supp. SUPPL. 1, pp. S16. Abstract No. TN5.

[0198] 34 Shah et al., J Pediatr Genet. 2017, Vol. 6(2), pp. 126-127

[0199] 35 Gao et al., Hepatitis Monthly 2017, Vol. 17(10), e55087 / 1-e55087 / 6.

[0200] 36 Evason et al., Am J Surg Pathol. 2011, Vol. 35(5), pp. 687-696

[0201] 37Davit-Spraul et al., Mol Genet Metab. 2014, Vol. 113(3), pp. 225-229

[0202] 38 Maggiore et al., J Hepatol. 2010, Vol. 53(5), pp. 981-96

[0203] 39 McKay et al., Version 2. F1000Res. 2013; 2:32. DOI:10.12688 / f1000research.2-32.v2

[0204] 40 Liu et al., Pediatr Int. 2013, Vol. 55(2), pp. 138-144

[0205] 41 Waisbourd-Zinman et al., Ann Hepatol. 2017, Vol. 16(3), pp. 465-468

[0206] 42 Griffin et al., Canadian Journal of Gastroenterology and Hepatology 2016, Vol. 2016, Abstract A200. Conference Information: 2016 Canadian Digestive Diseases Week, CDDW2016. Montreal, QC, USA, February 26-29, 2016.

[0207] 43 Qiu et al., Hepatology 2017, Vol. 65(5), pp. 1655-1669

[0208] 44 Imagawa et al., Sci Rep. 2017, 7:41806.

[0209] 45 Kang et al., J Pathol Transl Med. 2019 May 16. doi:10.4132 / jptm.2019.05.03. [Earlier than preprinted electronic publication]

[0210] 46 Takahashi et al., Eur J Gastroenterol Hepatol. 2007, Vol. 19(11), pp. 942-6

[0211] 47 Shimizu et al., Am J Transplant. 2011, Vol. 11(2), pp. 394-398

[0212] 48 Krawczyk et al., Ann Hepatol. 2012, Vol. 11(5), pp. 710-744

[0213] 49 Sharma et al., BMC Gastroenterol. 2018, Vol. 18(1), p. 107

[0214] 50 Sattler 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.

[0215] 51 Jung et al., J Pediatr Gastroenterol Nutr. 2007, Vol. 44(4), pp. 453-458

[0216] 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.

[0217] 53 Sohn et al., Pediatr Gastroenterol Hepatol Nutr. 2019, Vol. 22(2), pp. 201-206

[0218] 54 Ho et al., Pharmacogenet Genomics. 2010, Vol. 20(1), pp. 45-57

[0219] 55 Wang et al., Hepatol Res. 2018, Vol. 48(7), pp. 574-584

[0220] 56 Shaprio et al., J Hum Genet. 2010, Vol. 55(5), pp. 308-313

[0221] 57 Bounford, University of Birmingham, Dissertation Abstracts International, (2016), Vol. 75, No. 1C, Subscription No.: AAI10588329. ProQuest Dissertations & Theses.

[0222] 58 Stolz et al., Aliment Pharmacol Ther. 2019, Vol. 49(9), pp. 1195-1204

[0223] 59 Jankowska et al., J Pediatr Gastroenterol Nutr. 2014, Vol. 58(1), pp. 92-95

[0224] 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.

[0225] 61Pauli-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.

[0226] 62 Li 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.

[0227] 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.

[0228] 64 Lee et al., Pediatr Gastroenterol Hepatol Nutr. 2017, Vol. 20(2), pp. 114-123

[0229] 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

[0230] 66Blackmore et al., J Clin Exp Hepatol. 2013, Vol. 3(2), pp. 159-161

[0231] 67 Matte et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 51(4), pp. 488-493

[0232] 68 Lin et al., Zhongguo Dang Dai Er Ke Za Zhi.2018, Volume 20(9), Pages 758-764

[0233] 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.

[0234] 70 Herbst et al., Mol Cell Probes. 2015, Vol. 29(5), pp. 291-298

[0235] 71 Moghadamrad et al., Hepatology. 2013, Vol. 57(6), pp. 2539-2541.

[0236] 72Holz 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.

[0237] 73 Wang et al., PLoS One. 2016; Vol. 11(4): e0153114.

[0238] 74 Hao et al., International Journal of Clinical and Experimental Pathology 2017, Vol. 10(3), pp. 3480-3487.

[0239] 75 Arnell et al., J Pediatr Gastroenterol Nutr. 2010, Vol. 51(4), pp. 494-499

[0240] 76 Sharma et al., Indian Journal of Gastroenterology 2017, Vol. 36, No. 1, Supp. 1, pp. A99. Abstract No. M-20. Conference Information: 58th Annual Conference of the Indian Society of Gastroenterology, ISGCON 2017. Bhubaneswar, India, December 14-17, 2017.

[0241] 77 Beauséjour et al., Can J Gastroenterol. 2011, Vol. 25(6), pp. 311-314

[0242] 78Imagawa et al., Journal of Pediatric Gastroenterology and Nutrition 2016, Vol. 63, Supp. 2, pp. S51. Abstract No. 166. Conference Information: World Congress of Pediatric Gastroenterology, Hepatology and Nutrition 2016. Montreal, Canada, October 5-8, 2016.

[0243] 79 Peng et al., Zhonghua Erkezazhi (Chinese Journal of Pediatrics), 2018, Vol. 56, No. 6, pp. 440-444.

[0244] 80 Tibesar et al., Case Rep Pediatr. 2014, Vol. 2014: 185923.

[0245] 81 Ng 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.

[0246] 82 Wong et al., Clin Chem. 2008, Vol. 54(7), pp. 1141-1148

[0247] 83 Pauli-Magnus et al., J Hepatol. 2005, Vol. 43(2), pp. 342-357

[0248] 84 Jericho et al., Journal of Pediatric Gastroenterology and Nutrition, Vol. 3, No. 60, pp. 368-374.

[0249] 85Scheimann 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.

[0250] 86 Jaquotot-Haerranz et al., Rev Esp Enferm Dig. 2013, Vol. 105(1), pp. 52-54

[0251] 87 Khosla 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.

[0252] et al., J Hepatol. 2017, Vol. 67(6), pp. 1253-1264

[0253] 89 Liu et al., Liver International 2010, Vol. 30(6), pp. 809-815

[0254] 90 Chen et al., Journal of Pediatrics 2002, Vol. 140(1), pp. 119-124

[0255] 91 U.S. Patent 9,295,677

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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).

[0263] 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.

[0264] 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.

[0265] 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.

[0266] Table 6. Examples of NAFLD Activity Scores (NAS) with Fibrosis Stages

[0267]

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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).

[0280] 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.

[0281] 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).

[0282] 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).

[0283] 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%.

[0284] 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.

[0285] Exemplary biomarkers for NASH are provided in Table 7.

[0286] Table 7. Exemplary NASH Biomarkers

[0287] Biomarkers of liver fibrosis

[0288] Aspartate aminotransferase (AST) and platelet ratio index (APRI)

[0289] The aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR)

[0290] FIB-4 rating 1

[0291] Hyaluronic acid

[0292] Pro-inflammatory cytokines

[0293] 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., )

[0294] A group combined with the subject's age and sex included bilirubin, gamma-glutamyl transferase, hyaluronic acid, and α2-macroglobulin (e.g., ...). )

[0295] Including tissue inhibitors of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g.) A group of )

[0296] 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 )

[0297] Liver inflammation biomarkers 4,5

[0298] Interleukin-(IL)6

[0299] Interleukin-(IL)1β

[0300] Tumor necrosis factor (TNF)-α

[0301] Transforming growth factor (TGF)-β

[0302] Monocyte chemotactic protein (MCP)-1

[0303] C-reactive protein (CRP)

[0304] PAI-1

[0305] Collagen subtypes (e.g., Col1a1, Col1a2, and Col4a1)

[0306] Changes in macrophage infiltration (e.g., changes in CD68 expression levels)

[0307] References in Table 7

[0308] 1 McPherson et al., Gut. 2010, Vol. 59(9), pp. 1265-1269.

[0309] 2 Adams et al., Clin Chem. 2005, Vol. 51(10), pp. 1867-1873.

[0310] 3 Lichtinghagen et al., J Hepatol. 2013, Vol. 59(2), pp. 236-242.

[0311] 4 Neuman et al., Can J Gastroenterol Hepatol. 2014, Vol. 28(11), pp. 607-618.

[0312] 5 US Patent No. 9,872,844

[0313] 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%.

[0314] 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%.

[0315] 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%.

[0316] 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%.

[0317] 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.

[0318] 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%.

[0319] Some compounds of formula (I) or their pharmaceutically acceptable salts can serve as substrates for related transport proteins in the kidney.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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)).

[0328] Examples of suitable disintegrants include, but are not limited to, dry starch, modified starch (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, cross-linked sodium carboxymethyl cellulose, calcium carboxymethyl cellulose and cross-linked PVP (cross-linked polyvinylpyrrolidone)).

[0329] 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, polyethylene glycol, and mineral oils.

[0330] 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).

[0331] Examples of suitable coating agents include, but are not limited to, cellulose-based polymers (such as ethyl cellulose, hydroxypropyl methyl cellulose (or hydroxypropyl methyl cellulose), hydroxypropyl cellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose succinate and hydroxypropyl methyl cellulose phthalate), vinyl-based polymers (such as polyvinyl alcohol), and polymers based on acrylic acid and its derivatives (such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid copolymers).

[0332] Examples of suitable plasticizers include, but are not limited to, triethyl citrate, triacetin, tributyl citrate, diethyl phthalate, acetyl tributyl citrate, dibutyl phthalate, dibutyl sebacate, and polyethylene glycol.

[0333] 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.

[0334] The pharmaceutical composition may be in a form suitable for 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 a form suitable for oral administration, such as tablets or capsules.

[0335] 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.

[0336] 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.

[0337] In another aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof used as a medicine. The invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as a medicine.

[0338] 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.

[0339] Combination therapy

[0340] 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.

[0341] 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、WO 2021 / 110883,WO The following documents are incorporated herein by reference in their entirety: 2021 / 110884, WO 2021 / 110885, WO 2021 / 110886, WO 2021 / 110887, 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.Specific examples of suitable ASBT inhibitors include 1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N-{(R)-1'-phenyl-1'-[N'-(carboxymethyl)carbamoyl]methyl}carbamoylmethoxy)-2,3,4,5-tetrahydro-1,5-benzothiazazaheptatriene (eloxibat) and 1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N-{(R)-α-[N-((S)-1-carboxypropyl)carbamoyl]-4-hydroxybenzyl}carbamoylmethoxy)-2,3,4,5-tetrahydro-1,2,5-benzothiazazazaheptatriene (ovixibat).

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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).

[0353] 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.

[0354] 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).

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 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).

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] 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.

[0371] 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.

[0372] 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.

[0373] 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.

[0374] 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.

[0375] 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.

[0376] 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).

[0377] 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.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] 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.

[0390] 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.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] 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.

[0396] 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.

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

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

[0399] 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.

[0400] 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.

[0401] 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.

[0402] 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.

[0403] 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.

[0404] 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.

[0405] Preparation of compounds

[0406] 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.

[0407] General Method

[0408] All solvents used were analytical grade. Commercially available anhydrous solvents were routinely used for the reactions. Starting materials were available from commercial sources or prepared according to literature procedures. 3,3-Dibutyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptadiene 1,1-dioxide and 8-hydroxy-7-(methylthio)-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptadiene 1,1-dioxide were prepared as described in WO 02 / 50051 (methods 26 and 117, respectively). 7-Bromo-3,3-diethyl-8-methoxy-2,3-dihydro-1,5-benzothiazazonium-heptanetriene-4(5H)-one and 3,3-diethyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazonium-heptanetriene 1,1-dioxide can be prepared as described in WO 2019 / 234077 (intermediates 56 and 60, respectively). Room temperature refers to 20-25°C. The composition of the solvent mixture is given as a volume percentage or volume ratio.

[0409] LCMS:

[0410] Instrument Name: Agilent 1290infinity II.

[0411] Method A: Mobile phase: A: 0.1% HCOOH aqueous solution: ACN (95:5), B: ACN; Flow rate: 1.5 mL / min; Column: ZORBAX XDB C-18 (50 × 4.6 mm, 3.5 μm).

[0412] 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).

[0413] 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).

[0414] 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).

[0415] 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).

[0416] Method F: Mobile phase: A: 0.1% TFA aqueous solution, B: 0.1% TFA in ACN solution; Flow rate: 0.8 mL / min; Column: ZORBAX ECLIPSE PLUS C18 (50 × 2.1 mm), 1.8 μm.

[0417] Method G: Mobile phase: A: 0.1% TFA aqueous solution, B: 0.1% TFA in ACN solution; Flow rate: 0.8 mL / min; Column: Acquity UPLC BEH C18 (2.1 × 50 mm), 1.7 μm.

[0418] Method H: Mobile phase: A: 10 mM NH4OAc, B: 100% ACN; Flow rate: 0.8 mL / min; Column: AcquityUPLC BEH C18 (2.1 × 50) mm; 1.7 μm.

[0419] Method I: Mobile phase: A: 0.1% HCOOH aqueous solution: ACN (95:5), B: ACN; Flow rate: 0.8 mL / min; Column: ZORBAX ECLIPSE PLUS C18 (2.1×50) mm, 1.8 μm.

[0420] Method J: Mobile phase: A: 0.1% TFA aqueous solution, B: ACN; Flow rate: 1.0 mL / min; Column: Zorbax Extend C18 (50 x 4.6 mm), 5 μM.

[0421] Method K: Mobile phase: A: 0.1% TFA aqueous solution, B: 0.1% TFA in ACN; Flow rate: 1.5 mL / min; Column: XBridge C8 (50 x 4.6 mm), 3.5 μM.

[0422] Method L: Mobile phase: A: 0.1% TFA aqueous solution, B: 100% ACN; Flow rate: 1.5 mL / min; Column: XBridge C8 (50 x 4.6 mm), 3.5 μM.

[0423] UPLC:

[0424] Instrument Name: Waters Acquity I Class

[0425] 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.

[0426] Instrument Name: Shimadzu Nexera X2 LC / 2020MSD

[0427] Method B: Mobile phase: A: 0.1% HCOOH aqueous solution, B: ACN; Flow rate: 0.8 mL / min; Column: Acquity UPLCBEH C18 (2.1 x 50) mm; 1.7 μm.

[0428] HPLC:

[0429] Instrument name: Agilent 1260 Infinity II series instrument, as shown below, using % and UV detection (maxplot).

[0430] 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).

[0431] 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).

[0432] 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).

[0433] Method D: Mobile phase: A: 0.1% TFA aqueous solution, B: ACN; Flow rate: 1.0 mL / min; Column: ATLANTIS dC18 (250 × 4.6 mm, 5.0 μm).

[0434] Method E: Mobile phase: A: 0.1% TFA aqueous solution, B: CAN, flow rate: 2.0 mL / min; Column: X-Bridge C8 (50 × 4.6 mm, 3.5 μm).

[0435] Chiral SFC:

[0436] Instrument Name: PIC SFC 10 (Analytical)

[0437] The ratio of CO2 to cosolvent is between 60:40 and 80:20.

[0438] Method A: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: YMC amylose-SA (250 × 4.6 mm, 5 μm).

[0439] Method B: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Chiralpak AD-H (250 × 4.6 mm, 5 μm).

[0440] Method C: Mobile phase: 20 mM ammonia / methanol; Flow rate: 3 mL / min; Column: YMC cellulose-SC (250 × 4.6 mm, 5 μm).

[0441] Method D: Mobile phase: methanol; Flow rate: 3 mL / min; Column: Lux A1 (250 × 4.6 mm, 5 μm).

[0442] Method E: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 5 mL / min; Column: Lux C4.

[0443] Method F: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: YMC cellulose-SC.

[0444] Method G: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: Lux A1

[0445] Method H: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Lux A1 (250 × 4.6 mm, 5 μm).

[0446] Method I: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: chiral CCS (250 × 4.6 mm, 5 μm).

[0447] 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).

[0448] Method K: Mobile phase: 0.5% isopropylamine in methanol; Flow rate: 4 mL / min; Column: (R,R)-Whelk-01 (250 x 4.6 mm, 5 μm).

[0449] Method L: Mobile phase: 0.5% isopropylamine in IPA solution; Flow rate: 3 mL / min; Column: Chiralcel OX-H (250 x 4.6 mm, 5 μm).

[0450] Method M: Mobile phase: 0.5% isopropylamine in IPA solution; Flow rate: 5 mL / min; Column: YMC cellulose-SC (250 x 4.6 mm, 5 μm).

[0451] Method N: Mobile phase: methanol, flow rate: 5 mL / min; Column: Chiralcel OX-H (250 × 4.6 mm, 5 μm).

[0452] Method O: Mobile phase: 0.1% isopropylamine / IPA:methanol (1:1), flow rate: 3 mL / min; column: Chiralpak AS-H (250 × 4.6 mm, 5 μm).

[0453] Method P: Mobile phase: 0.5% isopropylamine / methanol, flow rate: 3 mL / min; Column: Chiralpak AS-H (250 × 4.6 mm, 5 μm).

[0454] Method Q: Mobile phase: IPA, flow rate: 3 mL / min; column: Lux A1 (250 × 4.6 mm, 5 μm).

[0455] Method R: Mobile phase: 0.1% isopropylamine / IPA:methanol (1:1), flow rate: 3 mL / min; column: Lux A1 (250 × 4.6 mm, 5 μm).

[0456] Method S: Mobile phase: 0.5% isopropylamine / methanol, flow rate: 3 mL / min; Column: Chiralpak OX-H (250 x 4.6 mm, 5 μm).

[0457] Method T: Mobile phase: 0.5% isopropylamine / IPA, flow rate: 4 mL / min; Column: YMC cellulose-SB (250 x 4.6 mm, 5 μm).

[0458] Method U: Mobile phase: 0.5% isopropylamine / IPA, flow rate: 3 mL / min; Column: Chiralpak AS-H (250 x 4.6 mm, 5 μm).

[0459] Preparative HPLC:

[0460] Instrument Name: Agilent 1290 Infinity II

[0461] 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).

[0462] 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).

[0463] 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).

[0464] 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).

[0465] Chiral preparative SFC:

[0466] Instrument Names: PIC SFC 100 and PSC SFC 400

[0467] The ratio of CO2 to cosolvent is between 60:40 and 80:20.

[0468] Method A: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: YMC amylose-SA (250 × 30 mm, 5 μm).

[0469] Method B: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Chiralpak AD-H (250 × 30 mm, 5 μm).

[0470] Method C: Mobile phase: 20 mM ammonia / methanol; Flow rate: 3 mL / min; Column: YMC cellulose-SC (250 × 30 mm, 5 μm).

[0471] Method D: Mobile phase: methanol; Flow rate: 3 mL / min; Column: chiral CCS (250 × 30 mm, 5 μm).

[0472] Method E: Mobile phase: methanol; Flow rate: 3 mL / min; Column: Lux A1 (250 × 30 mm, 5 μm).

[0473] Method F: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Lux A1 (250 × 30 mm, 5 μm).

[0474] Method G: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 3 mL / min; Column: chiral CCS (250 × 30 mm, 5 μm).

[0475] Method H: Mobile phase: 0.5% isopropylamine / IPA, flow rate: 5 mL / min; Column: YMC amylose-SC (250×30 mm, 5 μm).

[0476] Method J: Mobile phase: 0.5% isopropylamine / IPA; Flow rate: 3 mL / min; Column: Chiralcel OX-H (250 × 30 mm, 5 μm).

[0477] Method K: Mobile phase: 0.5% isopropylamine / methanol; Flow rate: 5 mL / min; Column: YMC cellulose-SC (250 × 30 mm, 5 μm).

[0478] Method L: Mobile phase: methanol; Flow rate: 5 mL / min; Column: Chiralcel OX-H (250 × 30 mm, 5 μm).

[0479] Method M: Mobile phase: 0.5% isopropylamine / methanol, flow rate: 3 mL / min; Column: Lux A1 (250 x 30 mm, 5 μm).

[0480] abbreviation

[0481] ACN Acetonitrile

[0482] DCM dichloromethane

[0483] DBAD di-tert-butyl azodicarbonate

[0484] DMF (dimethylformamide)

[0485] IPA isopropanol

[0486] LCMS (Liquid Chromatography-Mass Spectrometry)

[0487] HPLC (High Performance Liquid Chromatography)

[0488] PE petroleum ether

[0489] SFC Supercritical Fluid Chromatography

[0490] TFA (trifluoroacetic acid)

[0491] THF Tetrahydrofuran

[0492] TLC (Thin Layer Chromatography)

[0493] UPLC (Ultra-High Performance Liquid Chromatography)

[0494] The present invention will now be described by way of the following embodiments, which do not limit the invention in any way. All listed documents and references are incorporated herein by reference.

[0495] Example

[0496] Intermediate 1

[0497] 2-(((2-amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylhexanoic acid

[0498]

[0499] KOH (218.2 g, 3.89 mol) was added to a stirred solution of 5-bromo-6-methoxybenzo[d]thiazol-2-amine (63 g, 0.243 mol) in water (630 mL), and the reaction mixture was stirred at 120 °C for 16 h. After the reaction was complete (monitored by LCMS), the reaction mixture was cooled to room temperature. Then, a solution of 2-(bromomethyl)-2-methylhexanoic acid (70.5 g, 6.31 mol) in THF (210 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 16 h. After the reaction was complete (monitored by LCMS), the reaction mixture was cooled to 0 °C and acidified with concentrated HCl (pH ~2). The reaction mixture was extracted with EtOAc (2 x 350 mL), and the combined organic layers were washed with water (150 mL) and brine (150 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was transported as is to the next step without any further purification. Yield: 75g (crude matter, brown gelatinous substance).

[0500] LCMS: (Method A) 376.1 (M + ), 378.0 (M) + +2), Rt.2.44 minutes, 92.97% (maximum).

[0501] Intermediate 2

[0502] 7-Bromo-3-Butyl-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazazetacycloheptatrien-4(5H)-one

[0503]

[0504] Triethylamine (60.4 g, 0.59 mol) and a solution of 1-propanephosphonic anhydride (50% in EtOAc, 95.1 g, 0.29 mol) were added dropwise to a stirred solution of 2-(((2-amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylhexanoic acid (intermediate 1; 75.0 g, 0.199 mol) in EtOAc (750 mL) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. After the reaction was complete (monitored by UPLC), the reaction mixture was quenched with water (150 mL) and the aqueous layer was extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with brine (150 mL) and dried over anhydrous Na2SO4. The organic fraction was concentrated under vacuum and the resulting crude product was purified by Isolera column chromatography (eluent: 10–12% EtOAc / PE; silica gel: 230–400 mesh) to give the title compound. Yield: 63% (45g, grayish-white solid).

[0505] 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 9.62 (s, 1H), 7.33 (s, 1H), 7.13 (s, 1H), 3.83 (s, 3H), 3.17 (s, 2H), 1.46–1.44 (m, 2H), 1.22 (s, 3H), 1.17–1.14 (m, 4H), 0.79 (t, J = 6.8 Hz, 3H). LCMS: (Method A) 360.0 (M + +2), Rt.2.64 minutes, 97.14% (maximum).

[0506] Intermediate 3

[0507] 7-Bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazazetacycloheptatrien-4(5H)-one

[0508]

[0509] Anhydrous K₂CO₃ (3.9 g, 28 mmol), CuI (0.26 g, 1.4 mmol), and tris[2-(2-methoxyethoxy)ethyl]amine (0.9 g, 2.8 mmol) were added to a solution of 7-bromo-3-butyl-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one (intermediate 2.5 g, 14 mmol) in 1-bromo-4-fluorobenzene (50 mL), and the reaction mixture was heated at 135 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was concentrated under vacuum. The resulting residue was then partitioned between water (25 mL) and EtOAc (25 mL). The aqueous fraction was extracted with EtOAc (2 x 100 mL), and the combined organic layers were washed with ice-cold water (100 mL) and brine (100 mL). The organic fraction was dried with anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The resulting residue was ground with petroleum ether. The resulting solid was then filtered off and dried to give the title compound. Yield: 85% (5.5 g, light brown solid).

[0510] LCMS: (Method E) 451.9 (M + +H), Rt. 3.26 minutes, 81.86% (maximum).

[0511] Intermediate 4

[0512] 7-Bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-3-methyl-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptadiene

[0513]

[0514] Borane dimethyl sulfide (1 M in THF, 58 mL, 58 mmol) was added to a solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one (intermediate 3; 5.2 g, 11.5 mmol) in THF (50 mL), and the reaction mixture was heated at 75 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to 0 °C, methanol (60 mL) was added, and the reaction mixture was heated at 60 °C for 2 h. The reaction mixture was then cooled to room temperature, concentrated under vacuum, and the resulting residue was partitioned between water (50 mL) and EtOAc (50 mL). The aqueous layer was extracted with EtOAc (2 x 100 mL), and the combined organic layers were washed with ice-cold water (100 mL) and brine (100 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to obtain the title compound. Yield: 5.3 g (crude matter, colorless gel).

[0515] LCMS: (Method E) 439.9 (M) + +H), Rt. 3.55 minutes, 87.61% (maximum).

[0516] Intermediate 5

[0517] 7-Bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-3-methyl-2,3,4,5-tetrahydro-1,5-benzothiazazepine-heptane-1,1-dioxide

[0518]

[0519] A solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-3-methyl-2,3,4,5-tetrahydro-1,5-benzothiazazetane heptatriene (intermediate 4; 5.3 g, 12.1 mmol) in a mixture of THF and water (8:2, 55 mL) was mixed with potassium peroxymonosulfonate (37.16 g, 120.8 mmol), and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the aqueous layer was extracted with EtOAc (2 x 100 mL), and the combined organic layers were washed with ice-cold water (100 mL) and brine (100 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 15% EtOAc / PE; silica gel: 230–400 mesh) to give the title compound. Yield: 75% (4.3 g, white solid).

[0520] 1 ¹H NMR (400 MHz, DMSO-d6): δ 7.45 (s, 1H), 7.21 (s, 1H), 7.12–7.05 (m, 4H), 3.93 (s, 3H), 3.85–3.61 (m, 2H), 3.29 (s, 2H), 1.52–1.39 (m, 1H), 1.37–1.26 (m, 1H), 1.25–1.03 (m, 4H), 0.98 (s, 3H), 0.81–0.74 (m, 3H). LCMS: (Method E) 470.1 (M + Rt. 3.21 minutes, 98.04% (maximum).

[0521] Intermediate 6

[0522] 3-Butyl-5-(4-fluorophenyl)-8-hydroxy-3-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide

[0523]

[0524] Sodium methanethiol (3.2 g, 45.7 mmol) was added to a solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-3-methyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 5; 4.3 g, 9.14 mmol) in DMF (43 mL), and the reaction mixture was stirred at 80 °C for 12 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with ice-cold water (25 mL), and the aqueous layer was extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with ice-cold water (50 mL) and brine (50 mL) and dried over anhydrous Na₂SO₄. The organic fraction was concentrated under vacuum, and the resulting crude product was purified by Isolera column chromatography (eluent: 30% EtOAc / PE; silica gel: 230–400 mesh) to give the title compound. Yield: 90% (3.5g, grayish-white solid).

[0525] 1 ¹H NMR (400MHz, DMSO-d6): δ 10.57 (s, 1H), 7.31 (s, 1H), 7.04–6.98 (m, 2H), 6.92–6.88 (m, 2H), 6.71 (s, 1H), 3.81–3.65 (m, 2H), 3.34–3.20 (m, 2H), 2.20 (s, 3H), 1.55–1.39 (m, 1H), 1.38–1.05 (m, 5H), 0.99 (s, 3H), 0.81–0.77 (m, 3H). LCMS: (Method E) 424.2 (M+ +H), Rt. 2.78 minutes, 98.08% (maximum).

[0526] Separate enantiomers:

[0527] (S)-3-Butyl-5-(4-fluorophenyl)-8-hydroxy-3-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazonium-heptatriene 1,1-dioxide and (R)-3-Butyl-5-(4-fluorophenyl)-8-hydroxy-3-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazonium-heptatriene 1,1-dioxide

[0528]

[0529] Two enantiomers of racemic 3-butyl-5-(4-fluorophenyl)-8-hydroxy-3-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (2.8 g, 6.61 mmol) were separated by chiral SFC (Method M). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponds to enantiomer 1 and the second elution fraction corresponds to enantiomer 2. The absolute configurations of the two enantiomers are unknown.

[0530] Enantiomer 1: Yield: 42% (1.2 g, white solid). 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 10.56 (s, 1H), 7.30 (s, 1H), 7.04–6.98 (m, 2H), 6.92–6.88 (m, 2H), 6.71 (s, 1H), 3.80–3.50 (m, 2H), 3.20–3.15 (m, 2H), 2.21 (s, 3H), 1.58–1.36 (m, 1H), 1.35–1.03 (m, 5H), 0.99 (s, 3H), 0.80 (t, J = 9.20 Hz, 3H). LCMS: (Method E) 424.2 (M + +H), Rt. 2.79 min, 93.0% (maximum). HPLC: (Method B) Rt. 5.56 min, 93.77% (maximum). Chiral SFC: (Method M) Rt. 1.70 min, 99.78% (maximum).

[0531] Enantiomer 2: Yield: 44% (1.25 g, white solid). 1¹H NMR (400 MHz, DMSO-d⁶): δ 10.57 (s, 1H), 7.30 (s, 1H), 7.03–6.99 (m, 2H), 6.92–6.91 (m, 2H), 6.71 (s, 1H), 4.00–3.40 (m, 2H), 3.24–3.16 (m, 2H), 2.21 (s, 3H), 1.62–1.38 (m, 1H), 1.38–1.10 (m, 5H), 0.99 (s, 3H), 0.80 (t, J = 8.40 Hz, 3H). LCMS: (Method E) 424.2 (M + +H), Rt. 2.79 min, 98.86% (maximum). HPLC: (Method B) Rt. 5.56 min, 97.15% (maximum). Chiral SFC: (Method M) Rt. 2.51 min, 99.72% (maximum).

[0532] Intermediate 7

[0533] 3-((3-Butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate (single diastereomer)

[0534]

[0535] diastereomers 1 and 2

[0536] To an enantiomer 1 (intermediate 6; 400 mg, 0.94 mmol) of 3-butyl-5-(4-fluorophenyl)-8-hydroxy-3-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptatriene 1,1-dioxide in a stirred solution of DMF (4 mL), Cs₂CO₃ (615 mg, 1.83 mol) and methyl oxadiazine-2-carboxylate (867 mg, 8.49 mmol) were added in portions, and the reaction mixture was stirred at room temperature for 72 hours. The reaction mixture was quenched with dilute HCl (1.5 N, 5 mL) and diluted with water (5 mL). The aqueous layer was extracted with EtOAc (2 x 50 mL), and the combined organic layers were washed with water (10 mL) and brine (10 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 35% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 50% (250 mg, white gel).

[0537] 1H NMR (400MHz, DMSO-d6): δ7.34 (s, 1H), 7.07-6.99 (m, 4H), 6.71 (s, 1H), 5.90 (d, J = 6.0Hz, 1H), 4.48 (d, J = 4.8Hz, 1H), 4.27 (s, 2H), 3.69 (s, 5H), 3.40-3.20 (m, 2H), 2.19 (s, 3H), 1.52-1.38 (m, 1H), 1.38-1.02 (m, 5H), 0.99 (s, 3H), 0.79 (t, J=7.20Hz, 3H). LCMS: (Method E)526.0(M + +H), Rt. 2.95 min, 97.92% (maximum). HPLC: (Method B) Rt. 5.50 min, 96.34% (maximum).

[0538] Two diastereomers (210 mg, 0.39 mmol) were separated by chiral SFC (Method G). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponded to diastereomer 1 and the second elution fraction corresponded to diastereomer 2.

[0539] diastereomers 3 and 4

[0540] A mixture of diastereomers 3 and 4 of the title compound was obtained according to the same procedure, starting with 400 mg of enantiomer 2 of intermediate 6. Yield: 50% (250 mg, yellow gel).

[0541] 1 H NMR (400MHz, DMSO-d6): δ7.34 (s, 1H), 7.08-6.99 (m, 4H), 6.72 (s, 1H), 5.89 (d, J=7.6Hz, 1H), 4.49-4.47 (m, 1H), 4.27-4.26 (m, 2H) ), 3.69-3.67 (m, 5H), 3.23-3.20 (m, 2H), 2.19 (s, 3H), 1.55-1.40 (m, 1H), 1.40-1.05 (m, 5H), 0.99 (s, 3H), 0.79 (t, J=9.60Hz, 3H). LCMS: (Method E)526.0(M + +H), Rt. 2.95 min, 97.93% (maximum). HPLC: (Method B) Rt. 5.50 min, 96.46% (maximum).

[0542] Two diastereomers (210 mg, 0.39 mmol) were separated by chiral SFC (Method G). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponded to diastereomer 3 and the second elution fraction corresponded to diastereomer 4.

[0543] Diastereomer 1: Yield: 38% (80 mg, pale yellow solid). 1 H NMR (400MHz, DMSO-d6): δ7.34 (s, 1H), 7.08-6.99 (m, 4H), 6.71 (s, 1H), 5.89 (d, J=8.0Hz, 1H), 4.48 (d, J=6.0Hz, 1H), 4.27 (t, J=4. 0Hz, 2H), 3.69 (s, 5H), 3.31-3.22 (m, 2H), 2.19 (s, 3H), 1.52-1.38 (m, 1H), 1.30-1.10 (m, 5H), 0.99 (s, 3H), 0.79 (t, J=9.20Hz, 3H). LCMS: (Method G)526.2(M + +H), Rt. 2.32 min, 99.80% (maximum). HPLC: (Method B) Rt. 5.58 min, 99.40% (maximum). Chiral SFC: (Method G) Rt. 2.68 min, 99.38% (maximum).

[0544] Diastereomer 2: Yield: 38% (80 mg, pale yellow solid). 1 H NMR (400MHz, DMSO-d6): δ7.34 (s, 1H), 7.08-6.96 (m, 4H), 6.72 (s, 1H), 5.89 (d, J=7.6Hz, 1H), 4.48 (d, J=6.8Hz, 1H), 4.27 (d, J=5. 6Hz, 2H), 3.69 (s, 5H), 3.31-3.22 (m, 2H), 2.19 (s, 3H), 1.55-1.40 (m, 1H), 1.24-1.11 (m, 5H), 0.99 (s, 3H), 0.79 (t, J=9.20Hz, 3H). LCMS: (Method G)526.2(M + +H), Rt. 2.32 min, 98.65% (maximum). HPLC: (Method B) Rt. 5.50 min, 99.46% (maximum). Chiral SFC: (Method G) Rt. 4.02 min, 99.75% (maximum).

[0545] Diastereomer 3: Yield: 40% (85 mg, pale yellow solid). 1¹H NMR (400MHz, DMSO-d⁶): δ 7.35 (s, 1H), 7.08–6.99 (m, 4H), 6.72 (s, 1H), 5.89 (d, J = 7.6Hz, 1H), 4.49–4.48 (m, 1H), 4.28–4.26 (m, 2H), 3.75–3.50 (m, 5H), 3.23–3.17 (m, 2H), 2.19 (s, 3H), 1.55–1.35 (m, 1H), 1.32–1.06 (m, 5H), 1.00 (s, 3H), 0.90–0.70 (m, 3H). LCMS: (Method E) 526.0 (M + +H), Rt. 2.98 min, 97.37% (maximum). HPLC: (Method B) Rt. 5.58 min, 96.35% (maximum). Chiral SFC: (Method G) Rt. 2.36 min, 99.90% (maximum).

[0546] Diastereomer 4: Yield: 38% (80 mg, pale yellow solid). 1 H NMR (400MHz, DMSO-d6): δ7.35 (s, 1H), 7.07-6.99 (m, 4H), 6.72 (s, 1H), 5.89 (d, J=6.0Hz, 1H), 4.50-4.47 (m, 1H), 4.30-4 .23 (m, 2H), 3.95-3.45 (m, 5H), 3.27-3.23 (m, 2H), 2.19 (s, 3H), 1.27-1.08 (m, 6H), 0.99 (s, 3H), 0.79 (t, J=7.20Hz, 3H). LCMS: (Method E)526.1(M + +H), Rt. 2.96 min, 97.20% (maximum). HPLC: (Method B) Rt. 5.51 min, 99.09% (maximum). Chiral SFC: (Method G) Rt. 3.31 min, 99.48% (maximum).

[0547] The absolute configurations of the four diastereomers are unknown.

[0548] Intermediate 8

[0549] 2-(((2-amino-5-methoxyphenyl)thio)methyl)-2-ethylhexanoic acid

[0550]

[0551] KOH (1345 g, 23.96 mol) was added to a stirred solution of 6-methoxybenzo[d]thiazol-2-amine (270 g, 1.498 mol) in water (2700 mL), and the reaction mixture was stirred at 120 °C for 16 h. After the reaction was complete (monitored by LCMS), the reaction mixture was cooled to room temperature. Subsequently, a solution of 2-(bromomethyl)-2-ethylhexanoic acid (533 g, 2.25 mol) in THF (1000 mL) was added dropwise, and the resulting reaction mixture was stirred at room temperature for 16 h. After the reaction was complete (monitored by LCMS), the reaction mixture was cooled to 0 °C and acidified with concentrated HCl (pH ~2). The reaction mixture was extracted with EtOAc (2 × 4000 mL), and the combined organic layers were washed with water (1000 mL) and brine (1000 mL). The organic fraction was then dried with anhydrous Na₂SO₄ and concentrated under vacuum to obtain crude material, which was transferred to the next step as is without any further purification. Yield: 590 g (crude material, brown gum).

[0552] LCMS: (Method A) 312.1 (M + +H), Rt.2.24min, 97.34% (maximum).

[0553] Intermediate 9

[0554] 3-Butyl-3-ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazazepine-4(5H)-one

[0555]

[0556] At 0 °C, triethylamine (530 mL, 3.78 mol) and a solution of 1-propanephosphonic anhydride (50% in EtOAc; 785 g, 2.46 mol) were added dropwise to a stirred solution of 2-(((2-amino-5-methoxyphenyl)thio)methyl)-2-ethylhexanoic acid (intermediate 8; 590 g, 1.89 mol) in 2500 mL of EtOAc, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by LCMS), water (2000 mL) was added to the reaction mixture, and the aqueous layer was extracted with EtOAc (2 × 2000 mL). The combined organic layers were washed with brine (800 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude substance was purified by washing with methanol to give the title compound. Yield: 48% (265 g, off-white solid).

[0557] 1¹H NMR (300MHz, DMSO-d6): δ 9.53 (s, 1H), 7.04–7.01 (m, 2H), 6.87–6.86 (m, 1H), 3.72 (s, 3H), 2.50 (s, 2H), 1.68–1.66 (m, 4H), 1.50–1.48 (m, 4H), 0.79–0.72 (m, 6H). LCMS: (Method A) 294.3 (M + +H), Rt. 2.68 min, 99.47% (maximum).

[0558] Intermediate 10

[0559] 7-Bromo-3-Butyl-3-ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazazepine-4(5H)-one

[0560]

[0561] N-bromosuccinimide (209 g, 1.17 mol) was added fractionally to a stirred solution of 3-butyl-3-ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one (intermediate 9; 265 g, 0.903 mol) in a 1:1 mixture of DCM and acetonitrile (2650 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 concentrated. The obtained crude product was treated with cold acetonitrile and stirred for 30 minutes. The obtained precipitate was filtered off, washed with cold acetonitrile (2 × 100 mL), and dried under vacuum to give the title compound. Yield: 179 g (79%, crude product, brown solid).

[0562] 1 ¹H NMR (300MHz, DMSO-d⁶): δ 9.61 (s, 1H), 7.33 (s, 1H), 7.10 (s, 1H), 3.82 (s, 3H), 2.98 (s, 2H), 1.70–1.68 (m, 4H), 1.48–1.45 (m, 4H), 0.84–0.82 (m, 6H). LCMS: (Method A) 372.0 (M + +H), Rt. 2.83 min, 99.20% (maximum).

[0563] Intermediate 11

[0564] 7-Bromo-3-butyl-3-ethyl-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one and 3-butyl-3-ethyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one

[0565]

[0566] A stirred solution of 7-bromo-3-butyl-3-ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one (intermediate 10; 15 g, 40.2 mmol) in 1-fluoro-4-iodobenzene (50 mL) was added with cuprous iodide (I) (1.58 g, 0.8 mmol) and K₂CO₃ (11 g, 80.5 mmol), and the reaction mixture was purged with nitrogen for 20 min to degas. Tris[2-(2-methoxyethoxy)ethyl]amine (1.3 mL, 4.0 mmol) was then added under a nitrogen atmosphere, and the resulting reaction mixture was heated at 135 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was filtered through diatomaceous earth, and the diatomaceous earth pad was washed with EtOAc (200 mL). The filtrate was washed with water (100 mL) and brine (75 mL) and dried over anhydrous Na₂SO₄. The crude product was purified by Isolera column chromatography (eluent: 5% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 64% (12.2 g, off-white solid).

[0567] LCMS: (Method E) 467.1 (M + +2), for 7-bromosubstituted compounds, and 514.1 (M + +H), for 7-iodine-substituted compounds), Rt. 3.33 min, 92.83% (maximum).

[0568] Intermediate 12

[0569] 7-Bromo-3-butyl-3-ethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonylheptanetriene and 3-butyl-3-ethyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonylheptanetriene

[0570]

[0571] At 0 °C, a mixture of 7-bromo-3-butyl-3-ethyl-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazazazetacycloheptatrien-4(5H)-one and 3-butyl-3-ethyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3-dihydro-1,5-benzothiazazetacycloheptatrien-4(5H)-one (intermediate 11; 12 g, 25.7 mmol) in a stirred solution of THF (100 mL) was added dropwise with borane dimethyl sulfide (2 M in THF; 38 mL, 77 mmol), and the reaction mixture was refluxed at 65 °C for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with methanol (20 mL), and heated at 65 °C for 2 hours. The resulting reaction mixture was then cooled to room temperature and concentrated under vacuum. The residue was diluted with water (100 mL) and the aqueous layer was extracted with DCM (2 × 100 mL). The combined organic layers were then washed with water (50 mL) and brine (50 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: 10 g (crude product, black gel).

[0572] LCMS: (Method E) 451.8 (M) + +H), for 7-bromosubstituted compounds, and 499.7 (M + +H), for 7-iodine-substituted compounds, Rt. 3.78 min, 75.13% (maximum).

[0573] Intermediate 13

[0574] 7-Bromo-3-butyl-3-ethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonylheptanetriene 1,1-dioxide and 3-butyl-3-ethyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonylheptanetriene 1,1-dioxide

[0575]

[0576] At 0 °C, potassium persulfate (oxone) (81 g, 26.6 mmol) was added to a stirred solution of 7-bromo-3-butyl-3-ethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazazetane and 3-butyl-3-ethyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazetane (intermediate 12; 10 g, 26.6 mmol) in THF (100 mL) and water (60 mL). The resulting reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was filtered through a Buchner funnel, and the filtrate was extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with water (100 mL) and brine (100 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 15% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 54% (7 g, white solid).

[0577] LCMS: (Method E) 486.0 (M) + +2), for 7-bromosubstituted compounds, and 532.0 (M + +H), for 7-iodine-substituted compounds, Rt. 2.87 min, 91.53% (maximum).

[0578] Intermediate 14

[0579] 3-Butyl-3-ethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide

[0580]

[0581] Sodium methanethiol (2.1 g, 31 mmol) was added to a stirred solution of a mixture of 7-bromo-3-butyl-3-ethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazazetane 1,1-dioxide and 3-butyl-3-ethyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazetane 1,1-dioxide (intermediate 13; 3 g, 6.2 mmol) in DMF (16 mL), and the reaction mixture was stirred at 65 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to room temperature and quenched with water (25 mL). The aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude material was purified by Isolera column chromatography (eluent: 10% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 77% (2.13 g, brown solid).

[0582] 1 H NMR (400MHz, DMSO-d6): δ10.49(s,1H),7.28(s,1H),7.06-6.96(m,4H),6.61(s,1H),3.62( bs,2H),3.21(s,2H),2.17(s,3H),1.61-1.25(m,4H),1.20-1.01(m,4H),0.81-0.74(m,6H). LCMS:(Method A)438.1(M + +H), Rt. 2.78 min, 87.79% (maximum).

[0583] Intermediate 15

[0584] 1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid ethyl ester

[0585]

[0586] Triphenylphosphine (180 mg, 0.68 mmol), ethyl (1-hydroxymethyl)cyclopropanecarboxylate (99 mg, 0.68 mmol), and DBAD (210 mg, 0.91 mmol) were added to a stirred solution of 3-butyl-3-ethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 14; 200 mg, 0.45 mmol) in DMF (5 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 diluted with water (10 mL), and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (15 mL) and brine (15 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 11% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 93% (240 mg, colorless gel).

[0587] LCMS: (Method E) 564.1 (M + +H), Rt. 3.33 minutes, 95.41% (maximum).

[0588] Intermediate 16

[0589] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionate methyl ester

[0590]

[0591] Methyl 3-hydroxy-2,2-dimethylpropionate (0.15 g, 1.10 mmol) and triphenylphosphine (0.43 g, 1.60 mmol) were added to a stirred solution of 3-butyl-3-ethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 14; 0.5 g, 1.10 mmol) in THF (10 mL), and the reaction mixture was stirred for 5 min. DBAD (0.50 g, 2.20 mmol) was then added, and the reaction mixture was stirred at room temperature for 24 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water (10 mL), and the aqueous layer was extracted with EtOAc (2 x 15 mL). The combined organic layers were washed with water (2 x 15 mL) and brine (15 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude product was purified by Isolera column chromatography (elution: 10% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 47% (0.30 g, white solid).

[0592] LCMS: (Method E) 552.1 (M + +H), Rt. 3.32 minutes, 85.43% (maximum).

[0593] Intermediate 17

[0594] 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxy-2-methylpropionate

[0595]

[0596] Cs₂CO₃ (6.11 g, 18.8 mmol) was added to a stirred solution of 3,3-diethyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (3.0 g, 9.4 mmol) in DMF (10 mL), and the reaction mixture was stirred at room temperature for 15 min. Then, methyl 2-methylglycidyl ester (3.27 g, 28.2 mmol) was added, and the reaction mixture was stirred at room temperature for 72 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (15 mL) and diluted with water (10 mL). The aqueous layer was extracted with EtOAc (2 x 15 mL), and the combined organic layers were washed with water (15 mL) and brine (15 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 10% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 29.35% (1.5 g, colorless solid).

[0597] LCMS: (Method E) 508.2 (M + +H), Rt. 2.72 minutes, 99.88% (maximum).

[0598] Intermediate 18

[0599] 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionate methyl ester

[0600]

[0601] Diethylaminosulfur trifluoride (0.09 g, 0.70 mmol) was added to a stirred solution of methyl 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxy-2-methylpropionate (intermediate 17; 300 mg, 0.59 mmol) in DCM (10 mL) at -10 °C, and the reaction mixture was stirred for 2 hours. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water (5 mL), and the aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 20% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 43% (130 mg, off-white solid).

[0602] LCMS: (Method E) 510.3 (M + +H), Rt. 2.89 minutes, 98.90% (maximum).

[0603] Separate enantiomers:

[0604] (R)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiaza ...

[0605]

[0606] Two enantiomers of racemic methyl 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazatrien-8-yl)oxy)-2-fluoro-2-methylpropionate (100 mg, 0.19 mmol) were separated by chiral SFC (Method B). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponds to enantiomer 1 and the second elution fraction corresponds to enantiomer 2. The absolute configurations of the two enantiomers are unknown.

[0607] Enantiomer 1: Yield: 30% (30 mg, white solid). 1H NMR (400MHz, DMSO-d6): δ7.36 (s, 1H), 7.22 (t, J=8.4Hz, 2H), 6.99 (d, J=7.2Hz, 2H), 6.86 (t, J=7.6Hz, 1H), 6.71 (s, 1H), 4.50-4 .39 (m, 2H), 3.76 (s, 3H), 3.69 (bs, 2H), 3.26 (s, 2H), 2.16 (s, 3H), 1.60-1.52 (m, 5H), 1.38-1.32 (m, 2H), 0.75 (t, J=7.2Hz, 6H). LCMS: (Method E)510.0(M + +H), Rt. 3.04 min, 96.42% (maximum). HPLC: (Method B) Rt. 5.90 min, 97.43% (maximum).

[0608] Enantiomer 1: Yield: 30% (30 mg, white solid). 1 H NMR (400MHz, DMSO-d6): 7.35 (s, 1H), 7.22 (t, J=8.4Hz, 2H), 6.99 (d, J=7.2Hz, 2H), 6.86 (t, J=7.6Hz, 1H), 6.70 (s, 1H), 4.50-4 .42 (m, 2H), 3.70 (s, 3H), 3.60 (m, 2H), 3.26 (s, 2H), 2.16 (s, 3H), 1.65-1.52 (m, 5H), 1.37-1.32 (m, 2H), 0.75 (t, J=7.6Hz, 6H). LCMS: (Method E)510.0(M + +H), Rt. 3.04 min, 98.16% (maximum). HPLC: (Method B) Rt. 5.90 min, 99.29% (maximum).

[0609] Intermediate 19

[0610] Methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate

[0611]

[0612] Cs₂CO₃ (1.11 g, 3.42 mmol) was added to a stirred solution of 3-butyl-3-ethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptatriene 1,1-dioxide (intermediate 14; 750 mg, 1.71 mmol) in DMF (6 mL), and the reaction mixture was stirred at room temperature for 15 min. Then, methyl oxacyclopropane-2-carboxylate (0.42 mL, 5.14 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 1 mL) and diluted with water (1 mL). The aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 20-30% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 50% (300 mg, colorless gel).

[0613] 1 HNMR: (400MHz, DMSO-d6): δ7.34 (s, 1H), 7.17-7.07 (m, 4H), 6.61 (s, 1H), 5.89 (d, J=6.0Hz, 1H), 4.49-4.46 (m, 1H), 4.25 (t, J =2.8Hz, 2H), 3.68 (s, 3H), 3.62 (bs, 2H), 3.29 (s, 2H), 2.15 (s, 3H), 1.40-1.33 (m, 4H), 1.20-1.10 (m, 4H), 0.78-0.72 (m, 6H). LCMS: (Method E)540.2(M + +H), Rt.2.90 minutes, 94.13% (maximum).

[0614] Intermediate 20

[0615] Methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionate

[0616]

[0617] Sodium hydride (60%, 3.71 mg, 0.09 mmol) was added to a stirred solution of methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate (intermediate 19; 100 mg, 0.18 mmol) in 2 mL of DMF and the reaction mixture was stirred at room temperature for 15 min. Then, a solution of 0.04 mL, 0.55 mmol of iodomethane in 1 mL of DMF was added dropwise and the reaction mixture was stirred at room temperature for 1 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 1 mL) and diluted with water (1 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (10 mL) and brine (10 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The resulting crude product was purified by Isolera column chromatography (eluent: 20-30% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 57% (60 mg, white solid).

[0618] 1 H NMR (400MHz, DMSO-d6): δ7.32 (s, 1H), 7.07 (d, J=6.80Hz, 4H), 6.60 (s, 1H), 4.38-4.27 (m, 3H), 3.70 (s, 3H), 3 .68(s, 2H), 3.41(s, 3H), 3.28(s, 2H), 2.15(s, 3H), 1.37-1.32(m, 4H), 1.16-1.09(m, 4H), 0.77-0.72(m, 6H). LCMS: (Method E)554.3(M + +H), Rt. 3.01 minutes, 98.12% (maximum).

[0619] Intermediate 21

[0620] 7-Bromo-3,3-dibutyl-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one and 3,3-dibutyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one

[0621]

[0622] A mixture of 7-bromo-3,3-dibutyl-8-methoxy-2,3-dihydro-1,5-benzothiazazepine-4(5H)-one (3 g, 7.49 mmol) in 4-fluoroiodobenzene (30 mL) was stirred and copper iodide (I) (0.14 g, 0.74 mmol) and K₂CO₃ (2.07 g, 14.9 mmol) were added, and the reaction mixture was degassed by purging with nitrogen for 20 min. Tris[2-(2-methoxyethoxy)ethyl]amine (0.49 g, 1.49 mmol) was then added under nitrogen atmosphere, and the resulting reaction mixture was heated at 135 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was filtered through diatomaceous earth, and the diatomaceous earth pad was washed with EtOAc (25 mL). The filtrate was washed with water (15 mL) and brine (15 mL) and dried over anhydrous Na₂SO₄. The crude substance was purified by Isolera column chromatography (elution buffer: 20% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 95% (3.5 g, pale yellow solid).

[0623] LCMS: (Method E) 7-bromosubstituted compounds yielded 494.0 (M) + And the 7-iodine-substituted compound is 541.9 (M) + +H), Rt. 3.50 minutes, 96.61% (maximum).

[0624] Intermediate 22

[0625] 7-Bromo-3,3-dibutyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonylheptanetriene and 3,3-dibutyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonylheptanetriene

[0626]

[0627] To a stirred solution of a mixture of 7-bromo-3,3-dibutyl-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazazazetacycloheptatrien-4(5H)-one and 3,3-dibutyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3-dihydro-1,5-benzothiazazetacycloheptatrien-4(5H)-one (intermediate 21; 3.5 g, 7.07 mmol) in THF (35 mL), borane dimethyl sulfide (2 M in THF; 5.3 mL, 10.61 mmol) was added dropwise, and the reaction mixture was refluxed at 65 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with methanol (10 mL), and heated at 65 °C for 2 h. The resulting reaction mixture was then cooled to room temperature, concentrated under vacuum, and the residue was partitioned between water (50 mL) and EtOAc (50 mL). The aqueous layer was extracted with DCM (2 x 50 mL), and the combined organic layers were washed with water (25 mL) and brine (25 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum to give crude material. The crude material was carried to the next step as is without any further purification. Yield: 3.6 g (crude material, pale yellow gel).

[0628] LCMS: (Method E) 7-bromosubstituted compounds were 482.0 (M + Compounds with +2H) and 7-iodine substitution have a molecular weight of 527.9 (M). + +H), Rt. 3.86 min, 81.04% (combination of bromine and iodine substituted compounds) (maximum).

[0629] Intermediate 23

[0630] 7-Bromo-3,3-dibutyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonium-heptanetriene 1,1-dioxide and 3,3-dibutyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonium-heptanetriene 1,1-dioxide

[0631]

[0632] Sodium tungstate (360 mg, 0.01 mmol) and hydrogen peroxide (30% in H₂O; 2.6 mL, 22.47 mmol) were added to a stirred solution of a mixture of 7-bromo-3,3-dibutyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene (intermediate 22; 3.6 g, 7.49 mmol) in acetic acid (36 mL), and the resulting reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete (monitored by TLC), the reaction mixture was filtered through a Büchner funnel, and the filtrate was extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with water (25 mL) and brine (25 mL) and dried over anhydrous Na₂SO₄. The organic fraction was concentrated under vacuum, and the resulting crude product was purified by Isolera column chromatography (eluent: 12% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 79% (3.4 g, off-white solid).

[0633] LCMS: (Method A) 7-bromosubstituted compounds are 512.2 (M) + Compounds with +H) and 7-iodine substitution have a molecular weight of 560.2 (M). + +H); Rt. 3.40 minutes, 70.63% (maximum).

[0634] Intermediate 24

[0635] 3,3-Dibutyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide

[0636]

[0637] Sodium methanethiol (1.09 g, 15.6 mmol) was added to a stirred solution of a mixture of 7-bromo-3,3-dibutyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazaheptatriene 1,1-dioxide and 3,3-dibutyl-5-(4-fluorophenyl)-7-iodo-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazaheptatriene 1,1-dioxide (intermediate 23; 1.6 g, 3.12 mmol) in DMF (16 mL), and the reaction mixture was stirred at 65 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to room temperature and quenched with water (15 mL). The aqueous layer was extracted with EtOAc (2 x 25 mL), and the combined organic layers were washed with brine (10 mL). The organic fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 30% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 90% (1.3 g, off-white solid).

[0638] 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 10.48 (s, 1H), 7.28 (d, J = 4.4 Hz, 1H), 7.08–7.01 (m, 4H), 6.59 (s, 1H), 3.80–3.67 (m, 2H), 3.22 (s, 2H), 2.16 (s, 3H), 1.36–1.33 (m, 4H), 1.12–1.03 (m, 8H), 0.79–0.77 (m, 6H). LCMS: (Method E) 466.0 (M + +H), Rt. 3.23 minutes, 88.86% (maximum).

[0639] Intermediate 25

[0640] Methyl 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate

[0641]

[0642] Cs₂CO₃ (560 mg, 1.72 mmol) was added to a stirred solution of 3,3-dibutyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptatriene 1,1-dioxide (intermediate 24; 400 mg, 0.8602 mmol) in DMF (4 mL), and the reaction mixture was stirred at room temperature for 15 min. Then, methyl oxacyclopropane-2-carboxylate (0.2 mL, 2.58 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 5 mL), and the aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 20-30% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 44% (215 mg, colorless gel).

[0643] LCMS: (Method E) 568.1 (M + +H), Rt. 3.18 minutes, 93.04% (maximum).

[0644] Intermediate 26

[0645] Methyl 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionate

[0646]

[0647] Sodium hydride (60%, 8.80 mg, 0.22 mmol) was added to a stirred solution of methyl 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate (intermediate 25; 125 mg, 0.22 mmol) in DMF (2.5 mL) at 0 °C, and the reaction mixture was stirred for 5 min. Iodomethane (0.08 mL, 1.32 mmol) was then added, and the reaction mixture was stirred at room temperature for 1 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 5 mL), and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (10 mL) and brine (10 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 20-30% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 38% (50 mg, white solid).

[0648] LCMS: (Method E) 582.2 (M + +H), Rt. 3.29 minutes, 97.76% (maximum).

[0649] Intermediate 27

[0650] Methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate

[0651]

[0652] To a solution of 3-butyl-3-ethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazetane-heptanetriene 1,1-dioxide (intermediate 14; 750 mg, 1.71 mmol) in DMF (10 mL), Cs₂CO₃ (1.11 g, 3.42 mmol) and methyl oxadiazine-2-carboxylate (0.52 g, 5.14 mmol) were added, and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water (10 mL), and the aqueous layer was extracted with EtOAc (2 x 15 mL). The combined organic layers were washed with water (15 mL) and brine (15 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 45% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 50% (300 mg, colorless gel).

[0653] LCMS: (Method E) 540.2 (M + +H), Rt.2.90 minutes, 94.11% (maximum).

[0654] Intermediate 28

[0655] Methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionate

[0656]

[0657] A solution of methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazaheptatrien-8-yl)oxy)-2-hydroxypropionate (intermediate 27; 300 mg, 0.55 mmol) in DMF (5 mL) was added to a suspension of NaH (60%, 21 mg, 0.55 mmol) in anhydrous DMF (2 mL) at 0 °C, and the reaction mixture was stirred for 5 min. Then, ethyl iodine (260 mg, 1.67 mmol) was added, and the reaction mixture was stirred at room temperature for 3 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 5 mL) at 0 °C, and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (15 mL) and brine (15 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude product was purified by Isolera column chromatography (elution: 25% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 31% (98 mg, colorless gel).

[0658] LCMS: (Method E) 568.3 (M) + +H), Rt. 3.09 minutes, 65.63% (maximum).

[0659] Intermediate 29

[0660] Methyl 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionate

[0661]

[0662] A solution of 1,1-dioxide (1.5 g, 3.35 mmol) of 3,3-dibutyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptatriene 1,1-dioxide in DMF (10 mL) was added to Cs₂CO₃ (2.18 g, 6.70 mmol) and methyl oxadiazine-2-carboxylate (1.02 g, 10.05 mmol), and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water (50 mL), and the aqueous layer was extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 45% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 45% (600 mg, colorless gel).

[0663] LCMS: (Method E) 550.1 (M + +H), Rt.3.20 minutes, 98.81% (maximum).

[0664] Intermediate 30

[0665] Methyl 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionate

[0666]

[0667] A solution of methyl 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazaheptatrien-8-yl)oxy)-2-hydroxypropionate (intermediate 29; 500 mg, 0.91 mmol) in DMF (7 mL) was added to a suspension of NaH (60%, 35 mg, 0.55 mmol) in anhydrous DMF (3 mL) at 0 °C, and the reaction mixture was stirred for 10 min. Then, ethyl iodine (426 mg, 2.73 mmol) was added, and the reaction mixture was stirred at room temperature for 3 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 10 mL) at 0 °C, and the aqueous layer was extracted with EtOAc (2 x 15 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude product was purified by Isolera column chromatography (elution: 25% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 30% (300 mg, colorless gel).

[0668] LCMS: (Method E) 578.3 (M) + +H), Rt. 3.28 minutes, 71.90% (maximum).

[0669] Intermediate 31

[0670] 7-Bromo-3,3-diethyl-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazazetacycloheptatrien-4(5H)-one

[0671]

[0672] Copper iodide (I) (1.10 g, 0.006 mol) and K₂CO₃ (16.05 g, 0.12 mol) were added to a stirred solution of 7-bromo-3,3-diethyl-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one (20 g, 0.06 mol) in 4-bromofluorobenzene (200 mL), and the reaction mixture was degassed by purging with nitrogen for 20 min. Tris[2-(2-methoxyethoxy)ethyl]amine (3.75 g, 0.01 mol) was then added under nitrogen atmosphere, and the resulting reaction mixture was heated at 135 °C for 3 days. After the reaction was complete (monitored by TLC and UPLC), the reaction mixture was filtered through diatomaceous earth, and the diatomaceous earth pad was washed with EtOAc (100 mL). The filtrate was concentrated under vacuum, and the resulting residue was partitioned between water (100 mL) and EtOAc (50 mL). The aqueous layer was extracted with EtOAc (2 x 100 mL), and the combined organic layers were washed with water (70 mL) and brine (70 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 2-4% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 70% (18.0 g, white solid).

[0673] 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 7.36 (s, 1H), 7.22–7.14 (m, 5H), 3.90 (s, 3H), 3.15 (s, 2H), 1.50–1.45 (m, 4H), 0.78 (t, J = 9.6 Hz, 6H). LCMS: (Method E) 440.1 (M + +2), Rt. 2.97 min, 96.42% (max). HPLC: (Method E) Rt. 5.97 min, 94.16% (max).

[0674] Intermediate 32

[0675] 7-Bromo-3,3-diethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazetane heptadiene

[0676]

[0677] To a stirred solution of 7-bromo-3,3-diethyl-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazazacycloheptatrien-4(5H)-one (intermediate 31; 18.0 g, 0.04 mol) in THF (90 mL), borane dimethyl sulfide (2 M in THF, 102 mL, 0.21 mol) was added dropwise, and the reaction mixture was refluxed at 70 °C for 48 h. After the reaction was complete (monitored by UPLC), the reaction mixture was cooled to 0 °C and quenched with methanol (50 mL). The resulting solution was heated at 70 °C for 2 h, then cooled to room temperature and concentrated under vacuum. The resulting residue was separated by water (100 mL) and EtOAc (50 mL), and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with water (70 mL) and brine (70 mL) and dried over anhydrous Na₂SO₄. The organic fraction was concentrated under vacuum, and the resulting crude material was transported to the next step without any further purification. Yield: 21 g (crude material, white solid).

[0678] 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 7.12–6.95 (m, 5H), 6.88 (s, 1H), 3.81 (s, 3H), 3.70–3.45 (m, 2H), 2.78 (s, 2H), 1.45–1.18 (m, 4H), 0.70 (t, J = 7.6 Hz, 6H). LCMS: (Method E) 424.9 (M + +2), Rt. 3.44 min, 86.38% (max). HPLC: (Method E) Rt. 6.97 min, 94.07% (max).

[0679] Intermediate 33

[0680] 7-Bromo-3,3-diethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazonium heptadiene 1,1-dioxide

[0681]

[0682] Potassium peroxide (152.1 g, 0.49 mol) was added to a stirred solution of 7-bromo-3,3-diethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazetane (intermediate 32; 21.0 g, 0.05 mol) in THF (147 mL) and water (63 mL), and the reaction mixture was stirred at room temperature for 48 hours. After the reaction was complete (monitored by TLC), the reaction mixture was filtered. The filtrate was diluted with water (100 mL), and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with water (70 mL) and brine (70 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 10% MeOH / DCM; silica gel: 230-400 mesh), and the product was ground with petroleum ether (2 x 100 mL) to give the title compound. Yield: 93% (21.0 g, brown solid).

[0683] 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 7.44 (s, 1H), 7.25–7.02 (m, 5H), 3.92 (s, 3H), 3.66 (s, 2H), 3.33 (s, 2H), 1.60–1.42 (m, 2H), 1.42–1.28 (m, 2H), 0.71 (t, J = 9.6 Hz, 6H). LCMS: (Method E) 458.1 (M + +2), Rt.2.94 minutes, 95.31% (maximum).

[0684] Intermediate 34

[0685] 3,3-Diethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazetane-1,1-dioxide

[0686]

[0687] Sodium methanethiol (7.67 g, 0.11 mol) was added to a stirred solution of 7-bromo-3,3-diethyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 33; 10.0 g, 0.02 mol) in DMF (100 mL), and the resulting mixture was stirred at 70 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was quenched with water (100 mL), and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 36–50% EtOAc / PE; silica gel: 230–400 mesh) to give the title compound. Yield: 88% (7.90 g, grayish-white solid).

[0688] 1 ¹H NMR (400MHz, DMSO-d6): δ 10.54 (s, 1H), 7.30 (s, 1H), 7.09–6.99 (m, 2H), 6.99–6.91 (m, 2H), 6.64 (s, 1H), 3.72–3.50 (m, 2H), 3.25–3.18 (m, 2H), 2.18 (s, 3H), 1.65–1.46 (m, 2H), 1.43–1.25 (m, 2H), 0.74 (t, J = 7.20Hz, 6H). LCMS: (Method I) 410.0 (M + +H), Rt. 2.46 min, 92.37% (max). HPLC: (Method E) Rt. 5.33 min, 94.90% (max).

[0689] Intermediate 35

[0690] 3-((3,3-diethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionate methyl ester

[0691]

[0692] To a stirred solution of 3,3-diethyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 35; 300 mg, 0.73 mmol) in THF (10 mL), methyl 3-hydroxy-2,2-dimethylpropionate (96 mg, 0.73 mmol), triphenylphosphine (288 mg, 1.09 mmol), and DBAD (336 mg, 1.46 mmol) were 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 water (10 mL), and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with water (25 mL) and brine (25 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 10-20% EtOAc / PE; silica gel: 230-400 mesh) to give the title compound. Yield: 78% (300 mg, white gel).

[0693] LCMS: (Method E) 524.0 (M) + +H), Rt. 3.17 minutes, 93.5% (maximum).

[0694] Intermediate 36

[0695] 2-Hydroxy-3-((7-(methylthio)-1,1-dioxo-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazazetrin-8-yl)oxy)methyl propionate

[0696]

[0697] Cs₂CO₃ (777 mg, 2.38 mmol) and methyl oxacyclopropane-2-carboxylate (365 mg, 3.57 mmol) were added to a stirred solution of 8-hydroxy-7-(methylthio)-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazepine heptatriene 1,1-dioxide (500 mg, 1.19 mmol) in DMF (10 mL), and the reaction mixture was stirred at room temperature for 4 days. After the reaction was complete (monitored by TLC; ~50% conversion), the reaction mixture was quenched with dilute HCl (1.5 N, 10 mL), and the aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 30–40% EtOAc / PE; silica gel: 230–400 mesh) to give the title compound. Yield: 24% (150 mg, yellow gel).

[0698] LCMS: (Method K) 521.9 (M + +H), Rt. 3.03 minutes, 95.10% (maximum).

[0699] Example 1

[0700] 3-((3-Butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-hydroxypropionic acid (single diastereomer)

[0701]

[0702] diastereomer 1

[0703] To a stirred solution of methyl 3-((3-butyl-5-(4-fluorophenyl)-3-methyl-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazatrien-8-yl)oxy)-2-hydroxypropionate diastereomer 1 (intermediate 7; 80 mg, 0.15 mmol) in 1,4-dioxane (1 mL), an aqueous solution of HCl (6 N, 3 mL) was added, and the reaction mixture was heated at 80 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with water (5 mL), and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 10-20% MeOH / DCM; silica gel: 230-400 mesh) to obtain the title compound.

[0704] The diastereomers 2, 3 and 4 of the title compound were obtained according to the same procedure, starting from 80 to 85 mg of diastereomers 2, 3 and 4 of intermediate 7, respectively.

[0705] Diastereomer 1: Yield: 32% (25 mg, white solid). 1¹H NMR (400MHz, DMSO-d⁶): δ 7.36 (s, 1H), 7.07–6.99 (m, 4H), 6.73 (s, 1H), 4.33 (d, J = 6.8Hz, 1H), 4.12 (s, 2H), 3.69–3.52 (m, 2H), 3.31–3.22 (m, 2H), 2.20 (s, 3H), 1.55–1.38 (m, 1H), 1.30–1.10 (m, 5H), 0.99 (s, 3H), 0.76 (t, J = 8.4Hz, 3H). LCMS: (Method E) 512.0 (M + +H), Rt. 2.83 min, 98.75% (maximum). HPLC: (Method B) Rt. 5.10 min, 98.25% (maximum). Chiral SFC: (Method G) Rt. 2.59 min, 99.42% (maximum).

[0706] Diastereomer 2: Yield: 32% (25 mg, white solid). 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 7.35 (s, 1H), 7.06–6.98 (m, 4H), 6.72 (s, 1H), 4.35 (d, J = 9.2 Hz, 1H), 4.07–4.04 (m, 2H), 3.62 (m, 2H), 3.21 (m, 2H), 2.20 (s, 3H), 1.55–1.39 (m, 1H), 1.35–1.05 (m, 5H), 1.03 (s, 3H), 0.79 (t, J = 8.4 Hz, 3H). LCMS: (Method E) 512.0 (M + +H), Rt. 2.82 min, 99.25% (maximum). HPLC: (Method B) Rt. 5.11 min, 98.71% (maximum). Chiral SFC: (Method G) Rt. 4.19 min, 99.14% (maximum).

[0707] Diastereomer 3: Yield: 49% (40 mg, grayish-white solid). 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 7.36 (s, 1H), 7.07–6.98 (m, 4H), 6.72 (s, 1H), 4.33–4.30 (m, 1H), 4.21–4.15 (m, 2H), 3.88–3.44 (m, 2H), 3.22 (s, 2H), 2.20 (s, 3H), 1.46–1.33 (m, 1H), 1.32–1.10 (m, 5H), 1.04 (s, 3H), 0.80 (t, J = 7.2 Hz, 3H). LCMS: (Method E) 512.0 (M ++H), Rt. 2.83 min, 98.66% (maximum). HPLC: (Method B) Rt. 5.10 min, 98.19% (maximum). Chiral SFC: (Method G) Rt. 2.50 min, 99.54% (maximum).

[0708] Diastereomer 4: Yield: 52% (40 mg, grayish-white solid). 1 ¹H NMR (400MHz, DMSO-d⁶): δ 7.36 (s, 1H), 7.07–6.98 (m, 4H), 6.73 (s, 1H), 4.33 (d, J = 8.4Hz, 1H), 4.17–4.11 (m, 2H), 3.90–3.45 (m, 2H), 3.33–3.30 (m, 2H), 2.20 (s, 3H), 1.58–1.39 (m, 1H), 1.33–1.08 (m, 5H), 1.00 (s, 3H), 0.80 (t, J = 7.2Hz, 3H). LCMS: (Method H) 512.2 (M + +H), Rt. 2.06 min, 96.13% (maximum). HPLC: (Method B) Rt. 5.10 min, 96.16% (maximum). Chiral SFC: (Method G) Rt. 3.88 min, 99.19% (maximum).

[0709] The absolute configurations of the four diastereomers are unknown.

[0710] Example 2

[0711] 1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid

[0712]

[0713] Lithium hydroxide (36 mg, 0.85 mmol) was added to a stirred solution of ethyl 1-(((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylate (intermediate 15; 240 mg, 0.42 mmol) in a mixture of 1,4-dioxane and water (7:3, 10 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 acidified with dilute HCl (1.5 N, pH ~4) and diluted with ice-cold water (2 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL), and the combined organic layers were washed with water (10 mL) and brine (10 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude material was ground with hexane and then filtered to give the title compound. Yield: 22% (50 mg, white solid).

[0714] 1 ¹H NMR (400 MHz, DMSO-d6): δ 12.42 (s, 1H), 7.27 (s, 1H), 7.05–7.07 (m, 4H), 6.63 (s, 1H), 4.19 (s, 2H), 3.84–3.54 (bs, 2H), 3.27 (s, 2H), 2.16 (s, 3H), 1.59–1.36 (m, 4H), 1.35–0.98 (m, 8H), 0.79–0.69 (m, 6H). LCMS: (Method E) 536.2 (M + +H), Rt. 2.92 min, 98.33% (maximum). HPLC: (Method B) Rt. 5.91 min, 96.58% (maximum).

[0715] Example 3

[0716] (S)-1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazon-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid and (R)-1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazon-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid)

[0717]

[0718] Two enantiomers of racemic 1-(((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid (Example 2; 40 mg, 0.074 mmol) were separated by chiral SFC (Method O). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponds to enantiomer 1 and the second elution fraction corresponds to enantiomer 2. The absolute configurations of the two enantiomers are unknown.

[0719] Enantiomer 1: Yield: 32% (13 mg, grayish-white solid). 1 H NMR (400MHz, DMSO-d6): δ12.40 (s, 1H), 7.28 (s, 1H), 7.06 (d, J=8.0Hz, 4H), 6.63 (s, 1H), 4.19 (s, 2H), 3.65 (b s, 2H), 3.27 (s, 2H), 2.15 (s, 3H), 1.45-1.35 (m, 4H), 1.24-1.19 (m, 3H), 1.11-1.07 (m, 5H), 0.82-0.68 (m, 6H). LCMS: (Method E)536.2(M + +H), Rt. 2.91 min, 97.46% (maximum). HPLC: (Method E) Rt. 5.99 min, 96.04% (maximum). Chiral SFC: (Method O) Rt. 2.74 min, 97.81% (maximum).

[0720] Enantiomer 2: Yield: 10% (4 mg, grayish-white solid). 1 H NMR (400MHz, DMSO-d6): δ12.41 (s, 1H), 7.28 (s, 1H), 7.06 (d, J=8.4Hz, 4H), 6.63 (s, 1H), 4.19 (s, 2H), 3.66 (b s, 2H), 3.32 (s, 2H), 2.16 (s, 3H), 1.51-1.36 (m, 4H), 1.31-1.24 (m, 3H), 1.11-1.07 (m, 6H), 0.80-0.70 (m, 6H). LCMS: (Method E)536.1(M + +H), Rt. 3.07 min, 95.83% (maximum). HPLC: (Method E) Rt. 5.995 min, 95.29% (maximum). Chiral SFC: (Method O) Rt. 3.70 min, 99.39% (maximum).

[0721] Example 4

[0722] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid

[0723]

[0724] Lithium hydroxide (45 mg, 1.0 mmol) was added to a stirred solution of methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionate (intermediate 16; 300 mg, 0.54 mmol) in a mixture of 1,4-dioxane and water (7:3, 10 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 acidified with dilute HCl (1.5 N, pH ~4) and diluted with ice-cold water (2 mL). The aqueous layer was extracted with EtOAc (2 x 5 mL), and the combined organic layers were washed with water (5 mL) and brine (5 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (eluent: 9% MeOH / DCM; silica gel: 230-400 mesh), and the residue was further purified by prep HPLC (method A) to give the title compound. Yield: 41% (130 mg, white solid).

[0725] 1 H NMR (400MHz, DMSO-d6): δ12.43 (s, 1H), 7.27 (s, 1H), 7.07-7.05 (m, 4H), 6.26 (s, 1H), 4.05 (s, 2H), 3.67 (bs, 2H), 3.34-3.28(m, 2H), 2.15(s, 3H), 1.50-1.34(m, 4H), 1.25(s, 6H), 1.22-1.04(m, 4H), 0.78-0.72(m, 6H). LCMS: (Method E)538.1(M + +H), Rt. 3.13 min, 97.56% (maximum). HPLC: (Method B) Rt. 6.04 min, 96.19% (maximum).

[0726] Example 5

[0727] (S)-3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazon-8-yl)oxy)-2,2-dimethylpropionic acid and (R)-3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazon-8-yl)oxy)-2,2-dimethylpropionic acid

[0728]

[0729] Two enantiomers of racemic 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid (Example 4; 100 mg, 0.18 mmol) were separated by SFC (Method H). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponded to enantiomer 1 and the second elution fraction corresponded to enantiomer 2. The absolute configurations of the two enantiomers are unknown.

[0730] Enantiomer 1: Yield: 33% (33 mg, white solid). 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 12.43 (s, 1H), 7.27 (s, 1H), 7.07 (t, J = 8.00 Hz, 4H), 6.63 (s, 1H), 4.06 (s, 2H), 3.66 (s, 2H), 3.28 (s, 2H), 2.16 (s, 3H), 1.31–1.33 (m, 10H), 1.02–1.04 (m, 4H), 0.73–0.75 (m, 6H). LCMS: (Method H) 538.5 (M + +H), Rt. 2.33 min, 96.81% (maximum). HPLC: (Method B) Rt. 6.05 min, 95.30% (maximum). Chiral SFC: (Method H) Rt. 3.35 min, 99.93% (maximum).

[0731] Enantiomer 2: Yield: 26% (26 mg, white solid). 1¹H NMR (400 MHz, DMSO-d6): δ 12.40 (s, 1H), 7.27 (s, 1H), 7.05–7.07 (m, 4H), 6.63 (s, 1H), 4.06 (s, 1H), 3.65 (s, 2H), 3.28 (s, 2H), 2.16 (s, 3H), 1.31–1.33 (m, 11H), 1.02–1.04 (m, 4H), 0.73–0.75 (m, 6H). LCMS: (Method H) 538.5 (M + +H), Rt. 2.33 min, 97.41% (maximum). HPLC: (Method B) Rt. 6.02 min, 95.58% (maximum). Chiral SFC: (Method H) Rt. 2.74 min, 99.96% (maximum).

[0732] Example 6

[0733] 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid

[0734]

[0735] Lithium hydroxide (4.9 mg, 0.11 mmol) was added to a stirred solution of 3,3-diethyl-8-(2-hydroxy-2-methoxypropoxy)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatriene 1,1-dioxide (intermediate 18; 30 mg, 0.05 mmol) in a mixture of 1,4-dioxane and water (2:3, 5 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 acidified with dilute HCl (1.5 N, pH ~4) and diluted with ice-cold water (2 mL). The aqueous layer was extracted with EtOAc (2 x 5 mL), and the combined organic layers were washed with water (5 mL) and brine (5 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude compound was purified by Isolera column chromatography (eluent: 9% MeOH / DCM; silica gel: 230-400 mesh), and the residue was further purified by prep HPLC (method A) to give the title compound. Yield: 68% (20 mg, white solid).

[0736] 1H NMR (400MHz, DMSO-d6): 13.53 (bs, 1H), 7.35 (s, 1H), 7.22 (t, J = 8.4Hz, 2H), 6.99 (d, J = 7.2Hz, 2H), 6.84 (t, J = 7.6Hz, 1H), 6.72 ( s, 1H), 4.49-4.38 (m, 2H), 3.70 (s, 2H), 3.26 (s, 2H), 2.17 (s, 3H), 1.62-1.57 (m, 5H), 1.37-1.32 (m, 2H), 0.75 (t, J=7.2Hz, 6H). LCMS: (Method E)496.0(M + +H), Rt. 2.84 min, 99.03% (maximum). HPLC: (Method B) Rt. 5.25 min, 97.49% (maximum).

[0737] Example 7

[0738] (S)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazon-8-yl)oxy)-2-fluoro-2-methylpropionic acid and (R)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazon-8-yl)oxy)-2-fluoro-2-methylpropionic acid

[0739]

[0740] Enantiomer 1

[0741] Lithium hydroxide (5 mg, 0.11 mmol) was added to a stirred solution of methyl 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionate enantiomer 1 (intermediate 18; 30 mg, 0.07 mmol) in a mixture of 1,4-dioxane and water (2:3, 5 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 acidified with dilute HCl (1.5 N, pH ~4) and diluted with ice-cold water (2 mL). The aqueous layer was extracted with EtOAc (2 x 5 mL), and the combined organic layers were washed with water (5 mL) and brine (5 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude substance was purified by Isolera column chromatography (elution buffer: 9% MeOH DCM; silica gel: 230-400 mesh) to obtain the title compound.

[0742] Enantiomer 2 of the title compound was obtained according to the same procedure, starting with 30 mg of enantiomer 2 of intermediate 18.

[0743] Enantiomer 1: Yield: 77% (22 mg, white solid). 1 H NMR (400MHz, DMSO-d6): 13.54 (bs, 1H), 7.36 (s, 1H), 7.22 (dd, J=7.2, 8.8Hz, 2H), 6.96 (d, J=7.2Hz, 2H), 6.84 (t, J=7.2Hz, 1H), 6. 72 (s, 1H), 4.47-4.38 (m, 2H), 3.67 (s, 2H), 3.26 (s, 2H), 2.17 (s, 3H), 1.62-1.57 (m, 5H), 1.37-1.32 (m, 2H), 0.75 (t, J=7.2Hz, 6H). LCMS: (Method E)496.2(M + +H), Rt. 2.64 min, 98.33% (max). HPLC: (Method B) Rt. 5.35 min, 98.75% (max). Chiral SFC (Method G): Rt. 1.80 min, 98.82% (max).

[0744] Enantiomer 2: Yield: 77.54% (22 mg, white solid). 1 H NMR (400MHz, DMSO-d6): 13.52 (bs, 1H), 7.36 (s, 1H), 7.22 (t, J = 8.4Hz, 2H), 6.96 (d, J = 7.6Hz, 2H), 6.84 (t, J = 7.6Hz, 1H), 6.72 ( s, 1H), 4.47-4.38 (m, 2H), 3.68 (s, 2H), 3.27 (s, 2H), 2.17 (s, 3H), 1.62-1.57 (m, 5H), 1.37-1.32 (m, 2H), 0.75 (t, J=7.2Hz, 6H). LCMS: (Method E)496.2(M + +H), Rt. 2.64 min, 98.23% (maximum). HPLC: (Method B) Rt. 5.35 min, 98.42% (maximum). Chiral SFC (Method G): Rt. 2.58 min, 98.47% (maximum).

[0745] The absolute configurations of the two enantiomers are unknown.

[0746] Example 8

[0747] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid

[0748]

[0749] Lithium hydroxide (5.19 mg, 0.217 mmol) was added to a stirred solution of methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionate (intermediate 20; 60 mg, 0.10 mmol) in 1,4-dioxane (1.5 mL), and the reaction mixture was stirred at room temperature for 2 h. After the reaction was complete (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, 2 mL), and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The resulting crude product was purified by preparative HPLC (Method A) to give the title compound. Yield: 27% (16 mg, white solid).

[0750] 1 H NMR (400MHz, DMSO-d6): δ13.20 (s, 1H), 7.33 (s, 1H), 7.07 (d, J=7.20Hz, 4H), 6.62 (s, 1H), 4.36-4.09 (m, 3H), 3 .65(bs, 2H), 3.34(s, 2H), 3.29(s, 3H), 2.16(s, 3H), 1.52-1.37(m, 4H), 1.11-0.99(m, 4H), 0.77-0.72(m, 6H). LCMS: (Method E)540.0(M + +H), Rt. 2.97 min, 98.12% (maximum). HPLC: (Method B) Rt. 5.59 min, 99.49% (maximum).

[0751] Example 9

[0752] 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid

[0753]

[0754] Lithium hydroxide (7.22 mg, 0.17 mmol) was added to a stirred solution of methyl 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionate (intermediate 26; 50 mg, 0.08 mmol) in a mixture of 1,4-dioxane and water (3 mL), and the reaction mixture was stirred at room temperature for 2 h. After the reaction was complete (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, 2 mL), and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The resulting crude product was purified by preparative HPLC (Method A) to give the title compound. Yield: 24% (12 mg, white solid).

[0755] 1 H NMR (400MHz, DMSO-d6): δ13.06 (s, 1H), 7.32 (s, 1H), 7.09 (d, J=6.8Hz, 4H), 6.59 (s, 1H), 4.36-4.33 (m, 1H), 4.27-4.23 (m, 1H), 4 .12 (bs, 1H), 3.64 (bs, 2H), 3.40 (s, 3H), 3.29 (s, 2H), 2.15 (s, 3H), 1.37-1.34 (m, 4H), 1.24-1.02 (m, 8H), 0.76 (t, J=6.80Hz, 6H). LCMS: (Method E)568.1(M + +H), Rt. 3.15 min, 98.26% (maximum). HPLC: (Method B) Rt. 6.07 min, 97.56% (maximum).

[0756] Example 10

[0757] (S)-3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazettacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid and (R)-3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazettacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid

[0758]

[0759] Two enantiomers of racemic 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid (Example 9; 30 mg, 5.29 mmol) were separated by chiral SFC (Method H). The material was concentrated under vacuum at 40 °C. The first elution fraction corresponds to enantiomer 1 and the second elution fraction corresponds to enantiomer 2. The absolute configurations of the two enantiomers are unknown.

[0760] Enantiomer 1: Yield: 16% (5.0 mg, white solid). 1 H NMR (400MHz, DMSO-d6): δ13.12 (s, 1H), 7.33 (s, 1H), 7.09 (d, J=6.8Hz, 4H), 6.59 (s, 1H), 4.35-4.33 (m, 1H), 4.27-4.23 (m, 1H), 4.1 5-4.13 (m, 1H), 3.62 (bs, 2H), 3.40 (s, 3H), 3.38 (s, 2H), 2.15 (s, 3H), 1.41-1.37 (m, 6H), 1.35-1.03 (m, 6H), 0.76 (t, J=6.40Hz, 6H). LCMS: (Method E)568.2(M + +H), Rt. 2.78 min, 94.49% (maximum). HPLC: (Method B) Rt. 6.09 min, 97.7% (maximum). Chiral SFC: (Method H) Rt. 3.55 min, 99.58% (maximum).

[0761] Enantiomer 2: Yield: 26% (8 mg, white solid). 1 H NMR (400MHz, DMSO-d6): δ13.05 (s, 1H), 7.33 (s, 1H), 7.09 (d, J=6.8Hz, 4H), 6.59 (s, 1H), 4.36-4.33 (m, 1H), 4.27-4.23 (m, 1H), 4.1 5-4.13 (m, 1H), 3.62 (bs, 2H), 3.40 (s, 3H), 3.29 (s, 2H), 2.14 (s, 3H), 1.41-1.30 (m, 6H), 1.30-1.02 (m, 6H), 0.76 (t, J=6.80Hz, 6H). LCMS: (Method E)568.2(M + +H), Rt. 2.78 min, 87.94% (maximum). HPLC: (Method B) Rt. 6.09 min, 90.72% (maximum). Chiral SFC: (Method H) Rt. 4.32 min, 95.74% (maximum).

[0762] Example 11

[0763] 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionic acid

[0764]

[0765] Lithium hydroxide (14 mg, 0.34 mmol) was added to a stirred solution of methyl 3-((3-butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionate (intermediate 28; 97 mg, 0.17 mmol) in a mixture of 1,4-dioxane and water (7:3, 4 mL), and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, pH ~3) and diluted with ice-cold water (2 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL), and the combined organic layers were washed with water (10 mL) and brine (10 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude material was ground with hexane, filtered, and dried under vacuum to give the title compound. Yield: 40% (20 mg, white solid).

[0766] 1 ¹H NMR (400MHz, DMSO-d6): δ 12.91 (bs, 1H), 7.37 (s, 1H), 7.08–7.06 (m, 4H), 6.62 (s, 1H), 4.35–4.33 (m, 1H), 4.28–4.24 (m, 2H), 3.72–3.68 (m, 2H), 3.57–3.52 (m, 2H), 3.29–3.21 (m, 2H), 2.16 (s, 3H), 1.59–1.33 (m, 4H), 1.22–0.95 (m, 7H), 0.79–0.68 (m, 6H). LCMS: (Method E) 554.3 (M + +H), Rt. 2.87 min, 99.83% (maximum). HPLC: (Method B) Rt. 5.89 min, 97.41% (maximum).

[0767] Example 12

[0768] 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionic acid

[0769]

[0770] Lithium hydroxide (7.2 mg, 0.17 mmol) was added to a stirred solution of methyl 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionate (intermediate 30; 50 mg, 0.08 mmol) in a mixture of 1,4-dioxane and water (5:1, 3 mL), and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, 2 mL), and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic fraction was filtered and concentrated under vacuum. The resulting crude product was purified by preparative HPLC (Method A) to give the title compound. Yield: 51% (25 mg, white solid).

[0771] 1 H NMR (400MHz, DMSO-d6): δ7.38 (s, 1H), 7.23 (t, J=8.4Hz, 2H), 7.01 (d, J=8. 0Hz, 2H), 6.88 (t, J=6.8Hz, 1H), 6.67 (s, 1H), 4.36-4.34 (m, 1H), 4.27-4.23 (m, 2H), 3.70-3.67 (m, 2H), 3.56 (s, 2H), 3.28 (s, 2H), 2.15 (s, 3H), 1.43-1 .36 (m, 2H), 1.33-1.24 (m, 3H), 1.17-1.03 (m, 10H), 0.77 (t, J=6.80Hz, 6H). LCMS: (Method E) 564.3 (M + +H), Rt. 3.08 min, 99.82% (maximum). HPLC: (Method B) Rt. 6.39 min, 97.45% (maximum).

[0772] Example 13

[0773] 3-((3,3-diethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid

[0774]

[0775] Lithium hydroxide (48 mg, 1.14 mmol) was added to a solution of methyl 3-((3,3-diethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionate (intermediate 35; 300 mg, 0.57 mmol) in a mixture of 1,4-dioxane and water (7:3, 10 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 acidified with dilute HCl (1.5 N, pH ~3) and diluted with ice-cold water (2 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL), and the combined organic layers were washed with water (20 mL) and brine (20 mL). The organic fraction was dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude material was ground with hexane and then filtered to give the title compound. Yield: 15% (45 mg, white solid).

[0776] 1 H NMR (400MHz, DMSO-d6): δ12.43(s, 1H), 7.28(s, 1H), 7.09-7.02(m, 4H), 6.66(s, 1H), 4.07(s, 2H), 3.69(s , 2H), 3.27 (s, 2H), 2.17 (s, 3H), 1.55-1.49 (m, 2H), 1.38-1.31 (m, 2H), 1.25 (s, 6H), 0.74 (t, J=7.6Hz, 6H). LCMS: (Method E)510.2(M + +H), Rt. 2.78 min, 98.4% (maximum). HPLC: (Method B) Rt. 5.55 min, 96.41% (maximum).

[0777] Example 14

[0778] 2-Hydroxy-3-((7-(methylthio)-1,1-dioxo-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)propionic acid

[0779]

[0780] Methyl 2-hydroxy-3-((7-(methylthio)-1,1-dioxo-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)propionate (intermediate 36; 150 mg, 0.29 mmol) in 1,4-dioxane (5 mL) was added to a stirred solution at room temperature, and the reaction mixture was stirred at 100 °C for 16 h. After the reaction was complete (monitored by TLC), the reaction mixture was diluted with water (10 mL), and the aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by Isolera column chromatography (elution: 40–50% EtOAc / PE; silica gel: 230–400 mesh) to give the title compound. Yield: 46% (70 mg, white solid).

[0781] 1 H NMR (400MHz, DMSO-d6): δ12.81 (s, 1H), 7.35 (s, 1H), 7.23 (t, J=8.4Hz, 2H) , 7.00 (d, J=7.6Hz, 2H), 6.87 (t, J=7.2Hz, 1H), 6.68 (s, 1H), 5.80-5.40 (m, 1 H), 4.42-4.32(m, 1H), 4.32-4.15(m, 2H), 3.88-3.58(m, 2H), 3.28(s, 2H), 2.15 (s, 3H), 1.52-1.35 (m, 2H), 1.32-1.12 (m, 6H), 0.70 (t, J=6.80Hz, 6H). LCMS: (Method K) 507.9 (M + +H), Rt. 2.69 min, 97.77% (max). HPLC: (Method E) Rt. 5.32 min, 95.61% (max).

[0782] Bioanalysis

[0783] IBAT (h / m) analysis scheme

[0784] 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 (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.

[0785] 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.

[0786] 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).

[0787] LBAT (h / m) analysis scheme

[0788] 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 h. 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.

[0789] 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.

[0790] 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.

[0791] 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).

[0792] Two-way permeability analysis (Caco-2 cells)

[0793] 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.

[0794] 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%.

[0795] 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.

[0796] 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.

[0797] 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:

[0798]

[0799] 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.

[0800] HepaRG-based analysis scheme

[0801] Following the protocol provided by Biopredic International, cryopreserved vials (Biopredic International HPR116080) of differentiated HepaRG cells were thawed in HepaRG thaw / inoculation / universal medium (Biopredic International ADD670C) supplemented with 200 mM glutamine (Gibco 35050061). 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.

[0802] 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. The wells were washed twice with 250 μL of cooled, unlabeled 1 mM taurocholic acid 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.

[0803] 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).

[0804] Preparation of diluent for test compounds

[0805] 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.

[0806] Bioavailability study

[0807] 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 an 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.

[0808] result

[0809] Biological data of the compounds in the examples are shown in Table 8 below.

[0810] Table 8

[0811]

[0812]

[0813] PD model: Evaluation of the effect of test compounds on total bile acid content in male C57BL / 6 mice.

[0814] 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.

[0815] Urinary bile acid model: Evaluation of the effect of test compounds on urinary bile acid levels in male C57BL / 6N mice.

[0816] 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.

Claims

1. A compound selected from: 1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid; (S)-1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid; (R)-1-(((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)methyl)cyclopropane-1-carboxylic acid; 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid; (S)-3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazazetrin-8-yl)oxy)-2,2-dimethylpropionic acid; (R)-3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazazetrin-8-yl)oxy)-2,2-dimethylpropionic acid; 3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid; (S)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid; (R)-3-((3,3-diethyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-fluoro-2-methylpropionic acid; 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid; 3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid; (S)-3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid; (R)-3-((3,3-dibutyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-methoxypropionic acid; 3-((3-Butyl-3-ethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionic acid; 3-((3,3-dibutyl-7-(methylthio)-1,1-dioxo-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2-ethoxypropionic acid; 3-((3,3-diethyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxo-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)-2,2-dimethylpropionic acid; and 2-Hydroxy-3-((7-(methylthio)-1,1-dioxo-5-phenyl-3,3-dipropyl-2,3,4,5-tetrahydro-1,5-benzothiazazacycloheptatrien-8-yl)oxy)propionic acid; Or its pharmaceutically acceptable salt.

2. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 and one or more pharmaceutically acceptable excipients.

3. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of cardiovascular disease or fatty acid metabolism disorders or glucose utilization disorders; type 1 and type 2 diabetes; complications of diabetes; diabetes-related diseases; and for increasing high-density lipoprotein levels.

4. Use of the compound of claim 1 in the preparation of medicaments for the treatment or prevention of hypercholesterolemia; 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; restenosis; insulin resistance; hyperglycemia; hyperinsulinemia; elevated blood levels of fatty acids or glycerol; obesity; dyslipidemia; and metabolic syndrome.

5. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of syndrome X; atherosclerosis; hyperlipidemia; hypertension; and impaired glucose homeostasis.

6. Use of the compound of claim 1 in the preparation of a medicament for treating or preventing gastrointestinal diseases or disorders.

7. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of Crohn's disease; primary bile acid malabsorption; irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ileitis; or reflux disease and its complications.

8. Use of the compound of claim 1 in the preparation of a medicament for treating or preventing constipation; Barrett's esophagus; bile reflux esophagitis or bile reflux gastritis.

9. Use of the compound of claim 1 in the preparation of a medicament for treating or preventing chronic constipation; functional constipation; chronic idiopathic constipation (CIC); intermittent / occasional constipation; constipation secondary to diabetes mellitus; 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.

10. Use of the compound of claim 1 in the preparation of a medicament for treating or preventing liver disease or disorder.

11. The compound of claim 1 is used in the preparation of a treatment for or prevention of congenital defects in bile acid synthesis; congenital bile duct abnormalities; biliary atresia; neonatal hepatitis; encephalotendinosis xanthomatosis; secondary defects in bile acid (BA) synthesis; Zieweger syndrome; cystic fibrosis-associated liver disease; α1-antitrypsin deficiency; Alageri syndrome (ALGS); Bayer syndrome; primary defects in bile acid (BA) synthesis; autoimmune hepatitis; primary biliary cirrhosis (PBC); liver fibrosis; non-alcoholic fatty liver disease (NAFLD); non- Alcoholic steatohepatitis (NASH); portal hypertension; cholestasis; lymphedema-cholestasis syndrome 1 (LCS1); primary sclerosing cholangitis (PSC); immunoglobulin G4-associated cholangitis; primary biliary cholangitis; cholelithiasis; cholelithic pancreatitis; Carole disease; malignant tumors of the bile ducts; malignant tumors leading to obstruction of the bile duct tree; biliary stricture; AIDS-related cholangopathy; ischemic cholangopathy; pruritus due to 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 deficiency type 1); drug-induced liver injury (DILI); 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 (NSPILBD); North American Indian childhood cirrhosis (NAIC); hepatic sarcoidosis; amyloidosis; necrotizing enterocolitis; serum bile acid toxicity; polycystic liver disease; viral hepatitis; hepatocellular carcinoma; bile acid-related gastrointestinal cancers; or use in medicines that enhance corticosteroid therapy for liver diseases.

12. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of hereditary liver metabolic disorders.

13. The use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of progressive familial intrahepatic cholestasis (PFIC); benign recurrent intrahepatic cholestasis (BRIC); gallstones; arrhythmias with abnormal serum bile acid distribution patterns; cirrhosis-related cardiomyopathy; skeletal muscle atrophy associated with cholestatic liver disease; Down syndrome cholestasis; drug-induced cholestasis; intrahepatic cholestasis of pregnancy; intrahepatic cholestasis; extrahepatic cholestasis; parenteral nutrition-associated cholestasis (PNAC); hypophospholipid-associated cholestasis; neonatal cholestasis; hereditary cholestasis; cholestasis caused by tumors and vegetations of the liver, bile ducts, and pancreas; biliary atresia after Kasai surgery; biliary atresia after liver transplantation; bile duct stones; common bile duct stones; or congenital liver fibrosis.

14. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of PFIC-1; PFIC-2; PFIC-3; nonspecific PFIC; PFIC after bile shunt surgery; PFIC after liver transplantation; BRIC1; BRIC2; nonspecific BRIC; BRIC after bile shunt surgery; BRIC after liver transplantation; jaundice during pregnancy; atrial fibrillation with abnormal serum bile acid distribution pattern; hepatitis A; hepatitis B; hepatitis C; hepatitis D; hepatitis E; or liver cancer.

15. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of hyperabsorption syndrome; hypervitaminosis and osteosclerosis; hypertension; glomerular hyperfiltration; polycystic kidney disease (PKD); or pruritus of renal failure; or for the protection against kidney damage associated with liver disease or metabolic disease.

16. Use of the compound of claim 1 in the preparation of a medicament for the treatment or prevention of abetalipoproteinemia; familial hypobetalipoproteinemia (FHBL); chylomicron retention disease (CRD); sitosterolemia; autosomal dominant polycystic kidney disease (ADPKD); or autosomal recessive polycystic kidney disease (ARPKD).

Images