RU2837840C1 - Benzothia(di)azepine compounds and use thereof as bile acid modulators - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to organic chemistry and includes compounds: (Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid, (R)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid, (S)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid, (E)-3-((3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-sludge)oxy)acrylic acid, (S)-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid, (R)-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid, (Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid, (R)-(Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid, (S)-(Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid, (Z)-3-((3-ethyl-3-methyl-7-(methylthio)-1,1-dioxide-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine-8-yl)oxy)-2-fluoroacrylic acid, pharmaceutically acceptable salts thereof, a pharmaceutical composition based thereon and use.

EFFECT: technical result is 1,5-benzothiazepine derivatives possessing activity of inhibition of apical sodium-dependent bile acid transporter (ASBT) and/or liver bile acid transporter (LBAT).

7 cl, 1 dwg, 8 tbl, 10 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Indian Patent Application No. 201911049983, filed on December 4, 2019, the disclosure of which is incorporated herein by reference in its entirety.

AREA OF TECHNOLOGY

The invention relates to derivatives of 1,5-benzothiazepine and 1,2,5-benzothiadiazepine of formula (I). These compounds are bile acid modulators that have activity of inhibiting the apical sodium-dependent bile acid transporter (ASBT) and/or the liver bile acid transporter (LBAT). The invention also relates to pharmaceutical compositions containing 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.

PRIOR ART

Bile acids are physiological detergents that play an important role in the intestinal absorption and transport of lipids, nutrients, and vitamins. They are also signaling molecules that activate nuclear receptors and cell signaling pathways that regulate lipid, glucose, and energy metabolism. Bile acids are steroid acids that are synthesized from cholesterol in the liver and stored in the gallbladder as mixed micelles. During digestion, the duodenum initiates a release of hormones that cause contraction of the gallbladder, thereby releasing bile acids into the small intestine, where they mediate the absorption of fat-soluble vitamins and cholesterol. When bile acids reach the ileum, they are reabsorbed from the intestine and secreted into the portal venous blood for return to the liver via the portal venous circulation. More than 90% of bile acids are processed and returned to the liver in this way. These bile acids are then transported across the sinusoidal membrane of hepatocytes and resecreted across the canalicular membrane into bile. During the first pass, 75–90% of the bile acids are taken up by hepatocytes, completing one cycle of enterohepatic circulation. The fraction of bile acids that is not excreted by the liver enters the systemic circulation, where the free bile acids are filtered by the renal glomerulus, efficiently regenerated in the proximal tubule, and secreted back into the systemic circulation. Interestingly, the majority of the bile acids secreted across the canalicular membrane into bile come from the recirculating pool, with less than 10% coming from new de novo synthesis in the liver. The small fraction of bile acids that are not reabsorbed in the ileum reaches the colon. Within the intestinal lumen, primary bile acids are converted into secondary bile acids by intestinal bacteria, mainly through single or double dehydroxylation reactions of the steroid nucleus. Bile acids that are not absorbed in the intestine are then excreted in the feces.

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

Regulation of the bile acid pool size has been found to play a key role in cholesterol homeostasis through the conversion of cholesterol to bile acid in the liver, which is the major route of cholesterol clearance from the body. The liver plays an important role in the removal of endogenous and xenobiotic compounds from the body. Normal hepatobiliary secretion and enterohepatic circulation are required to clear endogenous compounds such as cholesterol, bilirubin, and their metabolites from the body, thereby maintaining lipid and bile acid homeostasis (Kosters and Karpen, Xenobiotica 2008, vol. 38, p.1043-1071).

Bile acid reabsorption in the ileum can be inhibited by compounds that inhibit the apical sodium-dependent bile acid transporter (ASBT). Inhibition of bile acid reabsorption has been reported to be useful in the treatment of several diseases, including dyslipidemia, diabetes, obesity, constipation, cholestatic liver disease, nonalcoholic steatohepatitis, and other liver diseases. A number of ASBT inhibitor compounds have been disclosed over the past decades, see, for example, WO 93/16055, WO 94/18183, WO 94/18184, WO 96/05188, WO 96/08484, WO 96/16051, WO 97/33882, WO 98/03818, WO 98/07449, WO 98/40375, WO 99/35135, WO 99/64409, WO 99/64410, WO 00/47568, WO 00/61568, WO 00/38725, WO 00/38726, WO 00/38727, WO 00/38728, WO 00/38729, WO 01/66533, WO 01/68096, WO 02/32428, WO 02/50051, WO 03/020710, WO 03/022286, WO 03/022825, WO 03/022830, WO 03/061663, WO 03/091232, WO 03/106482, WO 2004/006899, WO 2004/076430, WO 2007/009655, WO 2007/009656, WO 2011/137135, WO 2019/234077, WO 2020/161216, WO 2020/161217, DE 19825804, EP 864582, EP 489423, EP 549967, EP 573848, EP 624593, EP 624594, EP 624595, EP 624596, EP 0864582, EP 1173205, EP 1535913 and EP 3210977.

Despite a number of previously reported ASBT inhibitor compounds, there is a need for additional bile acid modulating compounds that have an optimized profile with respect to potency, selectivity, and bioavailability.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows the excretion of bile acids in urine in dogs after treatment with the compound of Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Some 1,5-benzothiazepine and 1,2,5-benzothiadiazepine derivatives have been found to be potent inhibitors of the apical sodium-dependent bile acid transporter (ASBT) and/or the hepatic bile acid transporter (LBAT) and may be useful for the treatment of diseases in which inhibition of bile acid circulation is desirable.

In a first aspect, the invention relates to a compound of formula (I)

Where

M is selected from -CH ₂ - and -NR ⁷ -;

R ¹ is C _1-4 alkyl;

R ² is independently selected from the group consisting of hydrogen, halogen, hydroxy, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, cyano, nitro, amino, N-(C _1-4 alkyl)amino, N,N-di(C _1-4 alkyl)amino, N-(aryl-C _1-4 alkyl)amino, C _1-4 alkylcarbonylamino, C _3-6 cycloalkylcarbonylamino, N-(C _1-4 alkyl)aminocarbonyl, N,N-di(C _1-4 alkyl)aminocarbonyl, C _1-4 alkyloxycarbonylamino, C _3-6 cycloalkyloxy-carbonylamino, C _1-4 alkylsulfonamido and C _3-6 cycloalkylsulfonamido;

n is an integer 1, 2, or 3;

R ³ is selected from the group consisting of hydrogen, halogen, cyano, C _1-4 alkyl, C _3-6 cycloalkyl, C _1-4 alkoxy, C _3-6 cycloalkyloxy, C _1-4 alkylthio, C _3-6 cycloalkylthio, amino, N-(C _1-4 alkyl)amino and N,N-di(C _1-4 alkyl)amino;

one of R ⁴ and R ⁵ is carboxyl and the other of R ⁴ and R ⁵ is selected from the group consisting of hydrogen, fluoro, C _1-4 alkyl and C _1-4 haloalkyl;

R ⁶ is selected from the group consisting of hydrogen and C _1-4 alkyl; and

R ⁷ is selected from the group consisting of hydrogen and C _1-4 alkyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, R ¹ is C _2-4 alkyl. In a preferred embodiment, R ¹ is n-propyl. In another preferred embodiment, R ¹ is n-butyl.

In some embodiments, R ² is independently selected from the group consisting of hydrogen, halogen, hydroxy, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, amino, N-(C _1-4 alkyl)amino, N,N-di(C _1-4 alkyl)amino, C _1-6 alkylcarbonylamino, C _3-6 cycloalkylcarbonylamino, N-(C _1-4 alkyl)aminocarbonyl, N,N-di(C _1-4 alkyl)aminocarbonyl, C _1-4 alkyloxycarbonylamino, C _1-4 alkylsulfonamido, and C _3-6 cycloalkylsulfonamido. In a preferred embodiment, R ² is independently selected from the group consisting of hydrogen, fluoro, chloro, bromine, hydroxy, methoxy, amino, methylamino, dimethylamino, isopropylcarbonylamino, tert-butylcarbonylamino, tert-butylaminocarbonyl, tert-butoxycarbonylamino, methylsulfonamido, and cyclopropylsulfonamido. In another preferred embodiment, R ² is independently selected from the group consisting of hydrogen, fluoro, chloro, bromine, hydroxy, and methoxy.

In a preferred embodiment, n is 1, i.e. the phenyl ring is substituted with only one substituent R ² . In another preferred embodiment, R ² is in the para position.

In some embodiments, R ³ is selected from the group consisting of hydrogen, fluoro, chloro, bromo, methyl, cyclopropyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino, and dimethylamino.

In some embodiments, R ⁴ is hydrogen or fluoro.

In some embodiments, R ⁵ is carboxyl.

In some embodiments, R ⁶ is hydrogen.

In some embodiments, R ⁷ is hydrogen or methyl.

In a preferred embodiment, the compound of formula (I) is a compound of formula (I-a):

Where

M is selected from the group consisting of -CH ₂ -, -NH- and -NCH ₃ -;

R ¹ is C _2-4 alkyl;

R ² is independently selected from the group consisting of hydrogen, halogen, hydroxy, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, amino, N-(C _1-4 alkyl)amino, N,N-di(C _1-4 alkyl)amino, C _1-6 alkylcarbonylamino, C _3-6 cycloalkylcarbonylamino, N-(C _1-4 alkyl)aminocarbonyl, N,N-di(C _1-4 alkyl)aminocarbonyl, C _1-4 alkyloxycarbonylamino, C _1-4 alkylsulfonamido and C _3-6 cycloalkylsulfonamido;

n is equal to the integer 1 or 2;

R ³ is selected from the group consisting of hydrogen, halogen, C _1-4 alkyl, C _3-6 cycloalkyl, C _1-4 alkoxy, C _1-4 alkylthio, amino, N-(C _1-4 alkyl)amino and N,N-di(C _1-4 alkyl)amino;

R ⁴ represents hydrogen or fluorine;

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the compound of formula (I) is a compound of formula (I-b):

Where

M is selected from the group consisting of -CH ₂ -, -NH- and -NCH ₃ -;

R ¹ is n-propyl or n-butyl;

R ² is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, hydroxy, methoxy, amino, methylamino, dimethylamino, isopropylcarbonylamino, tert-butylcarbonylamino, tert-butylaminocarbonyl, tert-butoxycarbonylamino, methylsulfonamido and cyclopropylsulfonamido;

R ³ is selected from the group consisting of fluorine, chlorine, bromine, methyl, cyclopropyl, methoxy, ethoxy, methylthio, ethylthio, amino, methylamino and dimethylamino;

R ⁴ represents hydrogen or fluorine;

or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the compound of formula (I) is a compound of formula (Ib) as defined above, wherein R ² is selected from the group consisting of hydrogen, fluoro, chlorine, bromine, hydroxy and methoxy.

Preferred compounds of the invention are compounds of formula (Ib) as defined above, wherein M and R ¹ -R ⁴ are as specified in Table 1 below, or a pharmaceutically acceptable salt thereof:

In a particular embodiment, the compound of formula (I) is selected from the group consisting of

(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid;

(R)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid;

(S)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid;

(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid;

(S)-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid;

(R)-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid;

(Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid;

(R)-(Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid;

(S)-(Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; and

(Z)-3-((3-ethyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid;

or a pharmaceutically acceptable salt thereof.

As used in this document, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

As used herein, the term "C _1-6 alkyl" refers to a straight or branched chain alkyl group having from 1 to 6 carbon atoms, and the term "C _1-4 alkyl" refers to a straight or branched chain alkyl group having from 1 to 4 carbon atoms. Examples of C _1-4 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

As used herein, the term "C _1-4 haloalkyl" refers to a C _1-4 straight or branched chain alkyl group, as defined herein, in which one or more hydrogen atoms are replaced by a halogen. Examples of C _1-4 haloalkyl include chloromethyl, fluoroethyl, and trifluoromethyl.

As used herein, the terms "C _1-4 alkoxy" and "C _1-4 alkylthio" refer to a C _1-4 straight or branched chain alkyl group attached to the rest of the molecule through an oxygen or sulfur atom, respectively.

As used herein, the term " _C3-6 cycloalkyl" refers to a monocyclic saturated hydrocarbon ring having from 3 to 6 carbon atoms. Examples of _C3-6 cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term "aryl" means an aromatic monocyclic ring consisting of 6 carbon atoms or an aromatic bicyclic ring system consisting of 10 carbon atoms. Examples of aryl include phenyl, naphthyl, and azulenyl.

The term "amino" refers to the group _-NH2 . As used herein, the terms "N-( _C1-4 alkyl)amino" and "N,N-di( _C1-4 alkyl)amino" refer to an amino group in which one or both hydrogen atoms, respectively, are replaced by a _C1-4 straight-chain or branched-chain alkyl group. Examples of N-( _C1-4 alkyl)amino include methylamino, ethylamino, and tert-butylamino, and examples of N,N-di( _C1-4 alkyl)amino include dimethylamino and diethylamino.

As used herein, the term "N-(aryl-C _1-4 alkyl)amino" refers to an amino group in which a hydrogen atom has been replaced with an aryl-C _1-4 alkyl group. Examples of N-(aryl-C _1-4 alkyl)amino include benzylamino and phenylethylamino. The term "C _1-6 alkylcarbonylamino" refers to an amino group in which a hydrogen atom has been replaced with a C _1-6 alkylcarbonyl group. Examples of C _1-6 alkanoylamino include acetylamino and tert-butylcarbonylamino. The term "C _1-4 alkyloxycarbonylamino" refers to an amino group in which a hydrogen atom has been replaced with a C _1-4 alkyloxycarbonyl group. An example of C _1-4 alkyloxycarbonylamino is tert-butoxycarbonylamino. The terms "C _1-4 alkylsulfonamido" and "C _3-6 cycloalkylsulfonamido" refer to an amino group in which a hydrogen atom has been replaced by a C _1-4 alkylsulfonyl or C _3-6 cycloalkylsulfonyl group, respectively.

As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are suitable for pharmaceutical use in humans and that are generally safe, non-toxic and not biologically or otherwise undesirable.

As used herein, the term "about" refers to a value or parameter herein that includes (and describes) embodiments that relate to that value or parameter as such. For example, a description referring to "about 20" includes the description "20." Numeric ranges include the numbers defining the range. In general, the term "about" refers to the stated value of a variable and to all values of the variable that are within the experimental error of the stated value (e.g., within a 95% confidence interval for the mean) or within 10 percent of the stated value, whichever is greater.

The 1,5-benzothiazepine and 1,2,5-benzothiadiazepine compounds of formula (I) or pharmaceutically acceptable salts thereof are inhibitors of the apical sodium-dependent bile acid transporter (ASBT inhibitors), the hepatic bile acid transporter (LBAT inhibitors) or both the apical sodium-dependent bile acid transporter and the hepatic bile acid transporter (ASBT/LBAT dual inhibitors). They are therefore useful in the treatment or prevention of conditions, disorders and diseases in which inhibition of bile acid circulation is desirable, such as cardiovascular diseases, disorders of fatty acid metabolism and glucose utilization, gastrointestinal diseases and liver diseases.

Cardiovascular diseases and disorders of fatty acid metabolism and glucose utilization include, but are not limited to, hypercholesterolemia; disorders of fatty acid metabolism; diabetes mellitus type 1 and type 2; complications of diabetes including cataracts, micro- 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 arterial occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmias, and vascular restenosis; diabetes-related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated levels of fatty acids or glycerol in the blood, obesity, dyslipidemia, hyperlipidemia including hypertriglyceridemia, metabolic syndrome (syndrome X), atherosclerosis and hypertension; and to increase high-density lipoprotein levels.

Gastrointestinal diseases and disorders include constipation (including chronic constipation, functional constipation, chronic idiopathic constipation (CIC), intermittent/sporadic constipation, constipation due to diabetes mellitus, constipation due to stroke, constipation due to chronic kidney disease, constipation due to multiple sclerosis, constipation due to Parkinson's disease, constipation due to systemic sclerosis, drug-induced constipation, irritable bowel syndrome with constipation (IBS-C), irritable bowel syndrome mixed (IBS-M), functional constipation of childhood, and opioid-induced constipation); Crohn's disease; primary bile acid malabsorption; irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ileal inflammasome; and reflux disease and its complications such as Barrett's esophagus, bile reflux esophagitis and bile reflux gastritis.

Liver disease, as defined herein, is any disorder of the liver and associated organs such as the pancreas, portal vein, liver parenchyma, intrahepatic bile ducts, extrahepatic bile ducts, and gallbladder. In some cases, the liver disease is bile acid dependent liver disease. Liver diseases and disorders include, but are not limited to, inherited error of liver metabolism; congenital errors of bile acid synthesis; congenital anomalies of the bile ducts; biliary atresia; post-Kasai procedure biliary atresia; post-transplant biliary atresia; neonatal hepatitis; neonatal cholestasis; hereditary forms of cholestasis; cerebrotendinous xanthomatosis; secondary defect of bile acid synthesis (SDBS); Zellweger syndrome; cystic fibrosis-associated liver disease; alpha1-antitrypsin deficiency; Alagille syndrome (ALGS); Byler syndrome; primary defect of bile acid synthesis (BAS); progressive familial intrahepatic cholestasis (PFIC), including PFIC-1, PFIC-2, PFIC-3, and nonspecific PFIC, post-biliary diversion PFIC, and post-transplant PFIC; benign recurrent intrahepatic cholestasis (BRIC), including BRIC1, BRIC2, and nonspecific BRIC, post-biliary transplant BRIC, and post-liver transplant BRIC; autoimmune hepatitis; primary biliary cirrhosis (PBC); liver fibrosis; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); portal hypertension; cholestasis; cholestasis in Down syndrome; drug-induced cholestasis; intrahepatic cholestasis of pregnancy (jaundice in pregnancy); intrahepatic cholestasis; extrahepatic cholestasis; parenteral nutrition-associated cholestasis (PNAC); low phospholipid cholestasis; cholestasis syndrome with lymphedema 1 (LSC1); primary sclerosing cholangitis (PSC); immunoglobulin G4-associated cholangitis; primary biliary cholangitis; cholelithiasis (gallstones); biliary lithiasis; choledocholithiasis; gallstone pancreatitis; Caroli disease; malignancy of the bile ducts; malignancy causing obstruction of the biliary tree; biliary strictures; AIDS-related cholangiopathy; ischemic cholangiopathy; 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 disorders; congenital defect of bile acid synthesis type 1 (BAS type 1); drug-induced liver injury (DILI); liver fibrosis; congenital liver fibrosis; liver cirrhosis; Langerhans cell histiocytosis (LCH); neonatal ichthyosis sclerosing cholangitis (NISCH); erythropoietic protoporphyria (EPP); adult-onset idiopathic ductopenia (IAD); idiopathic neonatal hepatitis (INH); nonsyndromic interlobular bile duct insufficiency (NS PILBD); North American Indian childhood cirrhosis (NAIC); liver sarcoidosis; amyloidosis; necrotizing enterocolitis; serum bile acid toxicity, including cardiac arrhythmias (e.g., atrial fibrillation) in the presence of abnormal serum bile acid profiles, cardiomyopathy associated with liver cirrhosis ("cholecardia"), and skeletal muscle wasting associated with cholestatic liver disease; polycystic liver disease; viral hepatitis (including hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E); hepatocellular carcinoma (hepatoma); cholangiocarcinoma; bile acid-related gastrointestinal cancer; and cholestasis due to tumors and neoplasms of the liver, biliary tract, and pancreas. The compounds of formula (I) or pharmaceutically acceptable salts thereof are also useful for augmenting corticosteroid therapy in liver disease.

Other diseases that can be treated or prevented with the compounds of formula (I) or pharmaceutically acceptable salts thereof include hyperabsorption syndromes (including abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicron retention disease (CRD), and sitosterolemia); hypervitaminosis and osteopetrosis; hypertension; glomerular hyperfiltration; polycystic kidney disease (PKD), including autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD); and pruritus in renal failure. The compounds are also useful in protecting against kidney injury associated with liver disease or metabolic disease.

Bile acid transport in humans is controlled by the action of members of the SLC10 family of solute carrier proteins, in particular the Na+-taurocholate transporting polypeptide (NTCP, also called the hepatic bile acid transporter (LBAT); gene symbol SLC10A1), which is expressed in the sinusoidal membrane of hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT, also called the ileal bile acid transporter (IBAT), ISBT, ABAT, or NTCP2; gene symbol SLC10A2), which is expressed in the apical membrane of ileal enterocytes, proximal renal tubule cells, biliary epithelium, large cholangiocytes, and gallbladder epithelial cells. In the liver, bile acids are efficiently extracted from the portal blood by the hepatic bile acid transporter (LBAT) and resecreted across the canalicular membrane by the bile salt export pump (BSEP; gene symbol ABCB11). Bile acids are reabsorbed in the ileum by the apical sodium-dependent bile acid transporter (ASBT), where it is commonly referred to as the ileal bile acid transporter (IBAT). Both LBAT and ASBT act as electrogenic solute-sodium cotransporters that transport two or more Na ⁺ ions per solute molecule.

Xenobiotics and endobiotics, including bile acids, are taken up by the liver from the portal blood and secreted into bile by a variety of transport proteins with individual substrate specificities. Glycine- and taurine-conjugated bile acids exist in the anionic form and cannot cross membranes by diffusion and thus are entirely dependent on membrane transport proteins for entry or exit from the hepatocyte (Kosters and Karpen, Xenobiotica 2008, vol. 38, p.1043-1071). ASBT and LBAT prefer glycine- and taurine-conjugated bile salts over their unconjugated counterparts and show a higher affinity for dihydroxy bile salts than for trihydroxy bile salts. No non-bile acid substrates have yet been identified for ASBT, but LBAT has also been found to transport various steroid sulfates, hormones, and xenobiotics.

LBAT has not been as well characterized as ASBT in terms of drug inhibition requirements. Dong et al. identified FDA-approved drugs that inhibit human LBAT and compared the inhibition requirements of LBAT and ASBT. A series of LBAT inhibition studies were conducted using FDA-approved drugs coupled with the development of an iterative computational model. Screening studies identified 27 drugs as novel LBAT inhibitors, including irbesartan (Ki = 11.9 μM) and ezetimibe (Ki = 25.0 μM). A common pharmacophore feature suggests that two hydrophobes and one hydrogen bond acceptor are important for LBAT inhibition. Of the 72 drugs tested in vitro, a total of 31 drugs inhibited LBAT, while 51 drugs (i.e., more than half) inhibited ASBT. Therefore, despite the overlap of inhibitors, ASBT was unexpectedly more permissive to drug inhibition than LBAT, and this may be due to LBAT having fewer pharmacophoric properties (Dong et al., Mol. Pharm. 2013, vol. 10, p.1008-1019).

Vaz et al. describe the identification of LBAT deficiency as a novel inborn error of metabolism with a relatively mild clinical phenotype. The identification of LBAT deficiency confirms that this transporter is the major import system for conjugated bile salts into the liver, but also indicates that accessory transporters are able to maintain the enterohepatic cycle in its absence (Vaz et al., Hepatology 2015, vol. 61, p.260-267). These data support the hypothesis that LBAT inhibition is a safe mechanism of action, as hepatocytes still have the capacity to take up adequate amounts of bile acids.

Liu et al. describe the identification of a novel type of hypercholanemia that is associated with homozygosity for the p.Ser267Phe mutation in SLC10A1 (LBAT). The allele frequency of this mutation in the SLC10A1 gene varies among populations, with the highest frequency in southern China (8% and 12% in Chinese Han and Dai populations, respectively) and Vietnam (11%). This "hidden" hypercholanemia was thought to affect 0.64% of the Chinese southern Han population, 1.44% of the Dai population, and 1.21% of the Vietnamese population. Increased serum levels of conjugated and unconjugated LA were also observed in homozygous individuals. Liu et al. suggest that this finding is most likely due to decreased LA transport from the portal circulation to hepatocytes. This supports the hypothesis that the physiological function of enterohepatic circulation is not only to recycle bile acids but also to remove bile acids from the circulation to achieve homeostasis (Karpen and Dawson, Hepatology 2015, vol. 61, p.24-27). Alternatively, the liver may synthesize increased levels of bile acids to compensate for the reduced enterohepatic recycling in homozygous carriers. Since LBAT also transports unconjugated bile acids, the increase in unconjugated bile acids in this study was not unexpected (Liu et al., Scientific Reports 2017, 7: 9214, p.1-7).

LBAT has been found to be down-regulated in several forms of cholestatic liver injury and cholestasis, whereas ASBT is down-regulated in various gastrointestinal disorders such as Crohn's disease, primary bile acid malabsorption, inflammatory bowel disease, and ileal inflammasome but is up-regulated in cholestasis. LBAT also functions as a cellular entry receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV), which in turn are the major cause of liver disease and hepatocellular carcinoma.

ASBT inhibition has been studied to reduce plasma cholesterol levels and improve insulin resistance, as well as to reduce the hepatic bile acid load in cholestatic liver disease. In addition, ASBT inhibition has been found to restore insulin levels and normoglycemia, making ASBT inhibition a promising treatment for type 2 diabetes. ASBT inhibitors are also used to treat functional constipation.

Because ASBT is predominantly expressed in the ileum (where it is often referred to as IBAT), ASBT inhibitors do not necessarily need to be systemically available. On the other hand, ASBT is also expressed in proximal tubular cells of the kidney. Therefore, systemic ASBT inhibitors may also inhibit renal bile acid reuptake. This is expected to result in increased urinary bile acid levels and increased urinary bile acid removal. Therefore, systemically available ASBT inhibitors, which act not only on the ileum but also on the kidney, are expected to result in greater reductions in bile acid levels than non-systemically available ASBT inhibitors, which act only on the ileum.

Compounds with high ASBT inhibition activity are particularly suitable for the treatment of liver diseases causing cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), Alagille syndrome, biliary atresia and nonalcoholic steatohepatitis (NASH).

Biliary atresia is a rare liver disorder in children that involves partial or complete blockage (or even absence) of the large bile ducts. This blockage or absence causes cholestasis, which leads to a buildup of bile acids that damage the liver. In some embodiments, the bile acids accumulate in the extrahepatic bile ducts. In some embodiments, the bile acids accumulate in the intrahepatic bile ducts. The current standard of treatment is the Kasai procedure, which is a surgery to remove the blocked bile ducts and directly connect a portion of the small intestine to the liver. There are currently no approved medications to treat this disorder.

Provided herein are methods of treating biliary atresia in a subject in need thereof 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 the Kasai procedure prior to administering the compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is administered a compound of Formula (I) or a pharmaceutically acceptable salt thereof prior to undergoing the Kasai procedure. In some embodiments, treating biliary atresia reduces serum bile acid levels in the subject. In some embodiments, serum bile acid levels are determined, for example, using an ELISA assay or assays for 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, the serum bile acid level may be reduced, for example, by 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, or more than 90% of the serum bile acid level prior to administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, treating biliary atresia comprises treating pruritus.

PFIC is a rare genetic disorder that affects an estimated one in every 50,000 to 100,000 children born worldwide and causes progressive, life-threatening liver disease.

One of the manifestations of PFIC is itching, which often leads to a serious deterioration in quality of life. In some cases, PFIC leads to cirrhosis and liver failure. Current treatment options include partial external bile diversion (PEBD) and liver transplantation, but these options can carry a significant risk of postoperative complications, as well as psychological and social problems.

Three alternative gene defects have been identified that correlate with three distinct subtypes of PFIC, known as types 1, 2, and 3:

• PFIC type 1, sometimes called "Byler's disease," is caused by impaired bile secretion due to mutations in the ATP8B1 gene, which codes for a protein that helps maintain the proper balance of fats, known as phospholipids, in the cell membranes of the bile ducts. An imbalance of these phospholipids is associated with cholestasis and elevated bile acids in the liver. In individuals affected by PFIC type 1, cholestasis typically develops in the first months of life and, if untreated, progresses to cirrhosis and end-stage liver disease before the end of the first decade of life.

• PFIC type 2, sometimes called "Byler syndrome," is caused by defective bile salt secretion due to mutations in the ABCB11 gene, which codes for a protein known as the bile salt export pump, which removes bile acids from the liver. Individuals with PFIC type 2 often develop liver failure within the first few years of life and are at increased risk of developing a type of liver cancer known as hepatocellular carcinoma.

• PFIC type 3, which typically presents in early childhood with progressive cholestasis, is caused by mutations in the ABCB4 gene, which encodes a transporter that moves phospholipids across cell membranes.

In addition, mutations in the TJP2 gene, NR1H4 gene, or Myo5b gene are suspected to be the cause of PFIC. In addition, some individuals with PFIC do not have a mutation in any of the ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b genes. In these cases, the cause of the condition is unknown.

Representative mutations of the ATP8 B1 gene or resulting protein are listed in Tables 2 and 3, numbered based on the human wild-type ATP8 B1 protein (e.g., SEQ ID NO: 1) or gene (e.g., SEQ ID NO: 2). Representative mutations of the ABCB11 gene or resulting protein are listed in Tables 4 and 5, numbered based on the human wild-type ABCB11 protein (e.g., SEQ ID NO: 3) or gene (e.g., SEQ ID NO: 4).

Those skilled in the art will appreciate that the amino acid position in the reference protein sequence that corresponds to a particular amino acid position in SEQ ID NO: 1 or 3 can be determined by aligning the reference protein sequence with SEQ ID NO: 1 or 3 (e.g., using software such as ClustalW2). Changes in these residues (referred to herein as "mutations") can include single or multiple amino acid substitutions, insertions within or flanking sequences, and deletions within or flanking sequences. As those skilled in the art will appreciate, the nucleotide position in the reference gene sequence that corresponds to a particular nucleotide position in SEQ ID NO: 2 or 4 can be determined by aligning the reference gene sequence with SEQ ID NO: 2 or 4 (e.g., using software such as ClustalW2). Changes in these residues (referred to herein as “mutations”) may include substitutions of one or more nucleotides, insertions within or flanking sequences, and deletions within or flanking sequences. See also Kooistra, et al., “KLIFS: A structural kinase-ligand interaction database,” Nucleic Acids Res. 2016, vol. 44, no. Dl, pp. D365–D371, which is incorporated herein by reference in its entirety.

Canonical sequence of ATP8 B1 protein (SEQ ID NO: 1) Uniprot ID O43520

Canonical DNA sequence for ATP8 B1 (SEQ ID NO: 2)

References for Tables 2 and 3

¹ Folmer et al., Hepatology. 2009, vol. 50(5), p.1597-1605.

² Hsu et al., Hepatol Res. 2009, vol. 39(6), p.625-631.

³ Alvarez et al., Hum Mol Genet. 2004, vol. 13(20), p.2451-2460.

⁴ Davit-Spraul et al., Hepatology 2010, vol. 51(5), p.1645-1655.

⁵ Vitale et al., J Gastroenterol. 2018, vol. 53(8), p.945-958.

⁶ Klomp et al., Hepatology 2004, vol. 40(1), p.27-38.

⁷ Zarenezhad et al., Hepatitis Monthly: 2017, vol. 17(2); e43500.

⁸ Dixon et al., Scientific Reports 2017, vol. 7, 11823.

⁹ Painter et al., Eur J Hum Genet. 2005, vol. 13(4), p.435-439.

¹⁰ Deng et al., World J Gastroenterol. 2012, vol. 18(44), p.6504-6509.

¹¹ Giovannoni et al., PLoS One. 2015, vol. 10(12): e0145021.

¹² Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.Supplement 1, pp.S180. Abstract Number: OP284.

¹³ Togawa et al., Journal of Pediatric Gastroenterology and Nutrition 2018, vol. 67, Supp.Supplement 1, pp.S363. Abstract Number: 615.

¹⁴ Miloh et al., Gastroenterology 2006, vol. 130, No. 4, Suppl. 2, pp.A759-A760. Meeting Info.: Digestive Disease Week Meeting/107th Annual Meeting of the American-Gastroenterological-Association. Los Angeles, CA, USA. May 19.

¹⁵ Droge et al., Zeitschrift fur Gastroenterologie 2015, vol. 53, No. 12. Abstract Number: A3-27. Meeting Info: 32. Jahrestagung der Deutschen Arbeitsgemeinschaft zum Studium der Leber. Dusseldorf, Germany. 22 Jan 2016-23 Jan 2016

¹⁶ Mizuochi et al., Clin Chim Acta. 2012, vol. 413(15-16), p.1301-1304.

¹⁷ Liu et al., Hepatology International 2009, vol. 3, No. 1, p.184-185. Abstract Number: PE405. Meeting Info: 19th Conference of the Asian Pacific Association for the Study of the Liver. Hong Kong, China. 13 Feb 2009-16 Feb 2009

¹⁸ McKay et al., Version 2. F1000Res. 2013; 2: 32. DOI: 10.12688/fl000research.2-32. v2

¹⁹ Hasegawa et al., Orphanet J Rare Dis. 2014, vol. 9:89.

²⁰ Stone et al., J Biol Chem. 2012, vol. 287(49), p.41139-51.

²¹ Kang et al., J Pathol Transl Med. 2019 May 16. doi: 10.4132/jptm.2019.05.03. [Epub ahead of print]

²² Sharma et al., BMC Gastroenterol. 2018, vol. 18(1), p.107.

²³ Uegaki et al., Intern Med. 2008, vol. 47(7), p.599-602.

²⁴ Goldschmidt et al., Hepatol Res. 2016, vol. 46(4), p.306-311.

²⁵ Liu et al., J Pediatr Gastroenterol Nutr. 2010, vol. 50(2), p.179-183.

²⁶ Jung et al., J Pediatr Gastroenterol Nutr. 2007, vol. 44(4), p.453-458.

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

²⁸ Stolz et al., Aliment Pharmacol Ther. 2019, vol. 49(9), p.1195-1204.

²⁹ Ivashkin et al., Hepatology International 2016, vol. 10, No. 1, Supp.SUPPL. 1, pp.S461. Abstract Number: LBO-38. Meeting Info: 25th Annual Conference of the Asian Pacific Association for the Study of the Liver, APASL 2016. Tokyo, Japan. 20 Feb 2016-24 Feb 2016

³⁰ Blackmore et al., J Clin Exp Hepatol. 2013, vol. 3(2), p.159-161.

³¹ Matte et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.488-493.

³² Squires et al., J Pediatr Gastroenterol Nutr. 2017, vol. 64(3), p.425-430.

³³ Hayshi et al., EBioMedicine. 2018, vol. 27, p.187-199.

³⁴ Nagasaka et al., J Pediatr Gastroenterol Nutr. 2007, vol. 45(1), p.96-105.

³⁵ Wangetal., PLoS One. 2016; vol. 11(4): e0153114.

³⁶ Narchi et al., Saudi J Gastroenterol. 2017, vol. 23(5), p.303-305.

³⁷ Alashkar et al., Blood 2015, vol. 126, No. 23. Meeting Info.: 57th Annual Meeting of the American-Society-of-Hematology. Orlando, FL, USA. December 05 -08, 2015. Amer Soc Hematol.

³⁸ Ferreira et al., Pediatric Transplantation 2013, vol. 17, Supp.SUPPL. 1, pp.99. Abstract Number: 239. Meeting Info: IPTA 7th Congress on Pediatric Transplantation. Warsaw, Poland. 13 Jul 2013-16 Jul 2013.

³⁹ Pauli-Magnus et al., J Hepatol. 2005, vol. 43(2), p.342-357.

⁴⁰ Jericho et al., Journal of Pediatric Gastroenterology and Nutrition 2015, vol. 60(3), p.368-374.

⁴¹ van derWoerdetal.,PLoS One. 2013, vol. 8(11): e80553.

⁴² Copeland et al., J Gastroenterol Hepatol. 2013, vol. 28(3), p.560-564.

⁴³ Droge et al., J Hepatol. 2017, vol. 67(6), p.1253-1264.

⁴⁴ Chen et al., Journal of Pediatrics 2002, vol. 140(1), p.119-124.

⁴⁵ Jirsa et al., Hepatol Res. 2004, vol. 30(1), p.1-3.

⁴⁶ van der Woerd et al., Hepatology 2015, vol. 61(4), p.1382-1391.

In some embodiments, the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, 166IT, E665X, R930X, R952X, R1014X, and G1040R.

Canonical sequence of ABCB11 protein (SEQ ID NO: 3) - Uniprot ID O95342

Canonical DNA sequence ABCB11 (SEQ ID NO: 4)

References for Tables 4 and 5

¹ Noe et al., J Hepatol. 2005, vol. 43(3), p.536-543.

² Lam et al., Am J Physiol Cell Physiol. 2007, vol. 293(5), p.C1709-16.

³ Stindt et al., Liver Int. 2013, vol. 33(10), p.1527-1735.

⁴ Gao et al., Shandong Yiyao 2012, vol. 52(10), p.14-16.

⁵ Strautnieks et al., Gastroenterology. 2008, vol. 134(4), p.1203-1214.

⁶ Kagawa et al., Am J Physiol Gastrointest Liver Physiol. 2008, vol. 294(1), p.G58-67.

⁷ Byrne et al., Hepatology. 2009, vol. 49(2), p.553-567.

⁸ Chen et al., J Pediatr. 2008, vol. 153(6), p.825-832.

⁹ Davit-Spraul et al., Hepatology 2010, vol. 51(5), p.1645-1655.

¹⁰ Dröge et al., Sci Rep.2016, vol. 6: 24827.

¹¹ Lang et al., Pharmaeogenet Genomics. 2007, vol. 17(1), p.47-60.

¹² Ellinger et al., World J Gastroenterol. 2017, vol. 23(29), p.:5295-5303.

¹³ Vitale et al., J Gastroenterol. 2018, vol. 53(8), p.945-958.

¹⁴ Knisely et al., Hepatology. 2006, vol. 44(2), p.478-86.

¹⁵ Ellis et al., Hepatology. 2018, vol. 67(4), p.1531-1545.

¹⁶ Lam et al., J Hepatol. 2006, vol. 44(1), p.240-242.

¹⁷ Varma et al., Hepatology 2015, vol. 62(1), p.198-206.

¹⁸ Treepongkaruna et al., World J Gastroenterol. 2009, vol. 15(34), p.4339-4342. ¹⁹ Zarenezhad et al., Hepatitis Monthly: 2017, vol. 17(2); e43500.

²⁰ Hayashi et al., Hepatol Res. 2016, vol. 46(2), p.192-200.

²¹ Guorui et al., Linchuang Erke Zazhi 2013, vol. 31(10), 905-909.

²² van Mil et al., Gastroenterology. 2004, vol. 127(2), p.379-384.

²³ Anzivino et al., Dig Liver Dis. 2013, vol. 45(3), p.226-232.

²⁴ Park et al., World J Gastroenterol. 2016, vol. 22(20), p.4901-4907.

²⁵ Imagawa et al., J Hum Genet. 2018, vol. 63(5), p.569-577.

²⁶ Giovannoni et al., PLoS One. 2015, vol. 10(12): eO 145021.

²⁷ Hu et al., Mol Med Rep.2014, vol. 10(3), p.1264-1274.

²⁸ Lang et al. Drug Metab Dispos. 2006, vol. 34(9), p.1582-1599.

²⁹ Masahata et al. Transplant Proc. 2016, vol. 48(9), p.3156-3162.

³⁰ Holz et al., Hepatol Commun. 2018, vol. 2(2), p.152-154.

³¹ Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.Supplement 1, pp.S180. Abstract Number: OP284.

³² Francalanci et al., Laboratory Investigation 2011, vol. 91, Supp.SUPPL. 1, pp.360A. Abstract Number: 1526.

³³ Francalanci et al., Digestive and Liver Disease 2010, vol. 42, Supp.SUPPL. 1, pp.S16. Abstract Number: TN 5.

³⁴ Shah et al., J Pediatr Genet. 2017, vol. 6(2), p.126-127.

³⁵ Gao et al., Hepatitis Monthly 2017, vol. 17(10), e55087/l-e55087/6.

³⁶ Evason et al., Am J Surg Pathol. 2011, vol. 35(5), p.687-696.

³⁷ Davit-Spraul et al., Mol Genet Metab. 2014, vol. 113(3), p.225-229.

³⁸ Maggiore et al., J Hepatol. 2010, vol. 53(5), p.981-6.

³⁹ McKay et al., Version 2. FlOOORes. 2013; 2: 32. DOI: 10.12688/f1000research.2-32. v2

⁴⁰ Liu et al., Pediatr Int. 2013, vol. 55(2), p.138-144.

⁴¹ Waisbourd-Zinman et al., Ann Hepatol. 2017, vol. 16(3), p.465-468.

⁴² Griffin, et al., Canadian Journal of Gastroenterology and Hepatology 2016, vol. 2016. Abstract Number: A200. Meeting Info: 2016 Canadian Digestive Diseases Week, CDDW 2016. Montreal, QC, United States. 26 Feb 2016-29 Feb 2016

⁴³ Qiu et al., Hepatology 2017, vol. 65(5), p.1655-1669.

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

⁴⁵ Kang et al., J Pathol Transl Med. 2019 May 16. doi: 10.4132/jptm.2019.05.03. [Epub ahead of print]

⁴⁶ Takahashi et al., Eur J Gastroenterol Hepatol. 2007, vol. 19(11), p.942-6.

⁴⁷ Shimizu et al., Am J Transplant. 2011, vol. 11(2), p.394-398.

⁴⁸ Krawczyk et al., Ann Hepatol. 2012, vol. 11(5), p.710-744.

⁴⁹ Sharma et al., BMC Gastroenterol. 2018, vol. 18(1), p.107.

⁵⁰ Sattler et al., Journal of Hepatology 2017, vol. 66, No. 1, Suppl. S, pp.SI 77. Meeting Info.: International Liver Congress / 52nd Annual Meeting of the European-Association-for-the-Study-of-the-Liver. Amsterdam, NETHERLANDS. April 19-23, 2017. European Assoc Study Liver.

⁵¹ Jung et al., J Pediatr Gastroenterol Nutr. 2007, vol. 44(4), p.453-458.

⁵² Sciveres. Digestive and Liver Disease 2010, vol. 42, Supp.SUPPL. 5, pp.S329. Abstract Number: C018. Meeting Info: 17th National Congress SIGENP. Pescara, Italy. 07 Oct 2010-09 Oct 2010

⁵³ Sohn et al., Pediatr Gastroenterol Hepatol Nutr. 2019, vol. 22(2), p.201-206.

⁵⁴ Ho et al., Pharmacogenet Genomics. 2010, vol. 20(1), p.45-57.

⁵⁵ Wang et al., Hepatol Res. 2018, vol. 48(7), p.574-584.

⁵⁶ Shaprio et al., J Hum Genet. 2010, vol. 55(5), p.308-313.

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

⁵⁸ Stolz et al., Aliment Pharmacol Ther. 2019, vol. 49(9), p.1195-1204.

⁵⁹ Jankowska et al., J Pediatr Gastroenterol Nutr. 2014, vol. 58(1), p.92-95.

⁶⁰ Kim. Journal of Pediatric Gastroenterology and Nutrition 2016, vol. 62, Supp.SUPPL. 1, pp.620. Abstract Number: HP-045. Meeting Info: 49th Annual Meeting of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition, ESPGHAN 2016. Azatemz, Greece. 25 May 2016-28 May 2016.

⁶¹ Pauli-Magnus et al., Hepatology 2003, vol. 38, No. 4 Suppl. 1, pp.518A. print. Meeting Info.: 54th Annual Meeting of the American Association for the Study of Liver Diseases. Boston, MA, USA. October 24-28, 2003. American Association for the Study of Liver Diseases.

⁶² Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.Supplement 1, pp.S362. Abstract Number: PP0347. Meeting Info: 26th Annual Conference of the Asian Pacific Association for the Study of the Liver, APASL 2017. Shanghai, China. 15 Feb 2017-19 Feb 2017.

⁶³ Rumbo et al., Transplantation 2018, vol. 102, No. 7, Supp.Supplement 1, pp.S848. Abstract Number: P.752. Meeting Info: 27th International Congress of The Transplantation Society, TTS 2018. Madrid, Spain. 30 Jun 2018-05 Jul 2018.

⁶⁴ Lee et al., Pediatr Gastroenterol Hepatol Nutr. 2017, vol. 20(2), p.114-123.

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.

⁶⁶ Blackmore et al., J Clin Exp Hepatol. 2013, vol. 3(2), p.159-161.

⁶⁷ Matte et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.488-493.

⁶⁸ Lin et al., Zhongguo Dang Dai Er Ke Za Zhi. 2018, vol. 20(9), p.758-764.

⁶⁹ Harmanci et al., Experimental and Clinical Transplantation 2015, vol. 13, Supp.SUPPL. 2, pp.76. Abstract Number: P62. Meeting Info: 1st Congress of the Turkic World Transplantation Society. Astana, Kazakhstan. 20 May 2015-22 May 2015.

⁷⁰ Herbst et al., Mol Cell Probes. 2015, vol. 29(5), p.291-298.

⁷¹ Moghadamrad et al., Hepatology. 2013, vol. 57(6), p.2539-2541.

⁷² Holz et al., Zeitschrift fur Gastroenterologie 2016, vol. 54, No. 8. Abstract Number: KV275. Meeting Info: Viszeralmedizin 2016, 71. Jahrestagung der Deutschen Gesellschaft fur Gastroenterologie, Verdauungs- und Stoffwechselkrankheiten mit Sektion Endoskopie - 10. Herbsttagung der Deutschen Gesellschaft fur Allgemein- und Viszeralchirurgie. Hamburr, Germany. 21 Sep 2016-24 Sep 2016.

⁷³ Wangetal., PLoS One. 2016; vol. 11(4): e0153114.

⁷⁴ Hao et al., International Journal of Clinical and Experimental Pathology 2017, vol. 10(3), p.3480-3487.

⁷⁵ Arnell et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.494-499.

⁷⁶ Sharma et al., Indian Journal of Gastroenterology 2017, vol. 36, No. 1, Supp.Supplement 1, pp.A99. Abstract Number: M-20. Meeting Info: 58th Annual Conference of the Indian Society of Gastroenterology, ISGCON 2017. Bhubaneswar, India. 14 Dec 2017-17 Dec 2017.

⁷⁷ Beauséjour et al., Can J Gastroenterol. 2011, vol. 25(6), p.311-314.

⁷⁸ Imagawa et al., Journal of Pediatric Gastroenterology and Nutrition 2016, vol. 63, Supp.Supplement 2, pp.S51. Abstract Number: 166. Meeting Info: World Congress of Pediatric Gastroenterology, Hepatology and Nutrition 2016. Montreal, QC, Canada. 05 Oct 2016-08 Oct 2016.

⁷⁹ Peng et al., Zhonghua er ke za zhi (Chinese journal of pediatrics) 2018, vol. 56, No. 6, pp.440-444.

⁸⁰ Tibesar et al., Case Rep Pediatr. 2014, vol. 2014: 185923.

⁸¹ Ng et al., Journal of Pediatric Gastroenterology and Nutrition 2018, vol. 66, Supp.Supplement 2, pp.860. Abstract Number: HP-127. Meeting Info: 51st Annual Meeting of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition, ESPGHAN 2018. Geneva, Switzerland. 09 May 2018-12 May 2018.

⁸² Wonget al., Clin Chem. 2008, vol. 54(7), p.1141-1148.

⁸³ Pauli-Magnus et al., J Hepatol. 2005, vol. 43(2), p.342-357.

⁸⁴ Jericho et al., Journal of Pediatric Gastroenterology and Nutrition. 60, vol. 3, p.368-

374.

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

⁸⁶ Jaquotot-Haerranz et al., Rev Esp Enferm Dig. 2013, vol. 105(1), p.52-54.

⁸⁷ Khosla et al., American Journal of Gastroenterology 2015, vol. 110, No. Suppl. 1, pp.S397. Meeting Info.: 80th Annual Scientific Meeting of the American-College-of-Gastroenterology. Honolulu, HI, USA. October 16 -21, 2015.

⁸⁸ Dröge et al., J Hepatol. 2017, vol. 67(6), p.1253-1264.

⁸⁹ Liu et al., Liver International 2010, vol. 30(6), p.809-815.

⁹⁰ Chen et al., Journal of Pediatrics 2002, vol. 140(1), p.119-124.

⁹¹ US Patent No. 9295677

In some embodiments, the mutation in ABCB11 is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N59IS, A865V, G982R, R1153C, and R1268Q.

Provided are methods for treating PFIC (e.g., PFIC-1 and PFIC-2) in a subject, which comprise analyzing a sample obtained from the subject to determine whether the subject has a mutation associated with PFIC (e.g., an ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b mutation), and administering (e.g., specifically or selectively administering) a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, to the subject determined to have a mutation associated with PFIC. In some embodiments, the mutation is an ATP8B1 or ABCB11 mutation. For example, a mutation as set forth in any of Tables 1-4. In some embodiments, the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, 166IT, 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, N59IS, A865V, G982R, R1153ChR1268Q.

Also provided are methods of treating PFIC (e.g., PFIC-1 and PFIC-2) in a subject in need thereof, comprising: (a) detecting a mutation associated with PFIC (e.g., an 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, methods of treating PFIC may comprise administering a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, to a subject having a mutation associated with PFIC (e.g., an ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, or Myo5b mutation). In some embodiments, the mutation is an ATP8B1 or ABCB11 mutation. For example, a mutation as set forth in any of Tables 1-4. In some embodiments, the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, 166IT, 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.

In some embodiments of the invention, the subject is determined to have a mutation associated with PFIC in the subject or in a biopsy sample from the subject using any art-recognized tests, including next generation sequencing (NGS). In some embodiments of the invention, the subject is determined to have a mutation associated with PFIC using a regulatory agency-approved, such as an FDA-approved, test or assay for identifying a mutation associated with PFIC in the subject or in a biopsy sample from the subject, or by performing any of the non-limiting examples of assays described herein. Additional methods for diagnosing PFIC are described in Gunaydin, M. et al., Hepat Med. 2018, vol. 10, pp. 95-104, incorporated herein by reference in its entirety.

In some embodiments, treatment with PFIC (e.g., PFIC-1 or PFIC-2) reduces the level of serum bile acids in a subject. In some embodiments, the level of serum bile acids is determined, for example, using an ELISA or assays for measuring total bile acids, as described in Danese et al., PLoS One. 2017, vol. 12(6): e0179200, incorporated herein by reference in its entirety. In some embodiments, the level of serum bile acids can be reduced, for example, by 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, or greater than 90% of the level of serum bile acids before administration of the compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, treatment with PFIC comprises treating pruritus.

Because LBAT is expressed on hepatocytes, LBAT and dual ASBT/LBAT inhibitors should have at least some bioavailability and a free fraction in the blood. Because LBAT inhibitor compounds must be transported only from the intestine to the liver, the relatively low systemic exposure of such compounds is expected to be sufficient, thereby minimizing the potential risk of any adverse effects in the rest of the body. Inhibition of LBAT and ASBT is expected to have at least an additive effect in reducing intrahepatic bile acid concentrations. It is also expected that a dual ASBT/LBAT inhibitor may reduce bile acid levels without causing diarrhea, as is sometimes seen with ASBT inhibitors.

Compounds with high LBAT inhibition potency and sufficient bioavailability are expected to be particularly suitable for the treatment of hepatitis. Compounds with dual ASBT/LBAT inhibition activity and sufficient bioavailability are expected to be particularly suitable for the treatment of non-alcoholic steatohepatitis (NASH).

NASH is a common and serious chronic liver disease that has similarities to alcoholic liver disease but occurs in people who drink little or no alcohol. In patients with NASH, the accumulation of fat in the liver, known as nonalcoholic fatty liver disease (NAFLD) or steatosis, and other factors such as high LDL cholesterol and insulin resistance cause chronic inflammation in the liver and can lead to progressive tissue scarring known as fibrosis and cirrhosis, which ultimately leads to liver failure and death. Patients with NASH have been found to have significantly higher serum total bile acid concentrations than healthy subjects under fasting conditions (2.2- to 2.4-fold increase in NASH) and at all time points postprandial (1.7- to 2.2-fold increase in NASH). They are caused by elevated levels of primary and secondary bile acids conjugated with taurine and glycine. NASH patients demonstrated higher variability in their fasting and postprandial bile acid profiles. These results indicate that NASH patients have higher fasting and postprandial bile acid exposure, including more hydrophobic and cytotoxic secondary species. Increased bile acid exposure may be associated with liver injury and the pathogenesis of NAFLD and NASH (Ferslew et al., Dig Dis Sci. 2015, vol. 60, p.3318-3328). Therefore, it is likely that inhibition of ASBT and/or LBAT will be beneficial for the treatment of NASH.

NAFLD is characterized by hepatic steatosis without secondary causes of hepatic steatosis, including excessive alcohol consumption, other known liver diseases, or long-term use of steatogenic medications (Chalasani et al., Hepatology 2018, vol. 67(1), p.328-357). NAFLD can be divided into non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH). According to Chalasani et al., NAFL is defined as the presence of at least 5% hepatic steatosis without evidence of hepatocellular injury such as hepatocyte ballooning. NASH is defined as the presence of at least 5% hepatic steatosis and inflammation with hepatocyte injury (eg, ballooning), with or without liver fibrosis. NASH is also often associated with liver inflammation and 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, if present, may be associated with long-term consequences.

There are a variety of approaches used to assess whether a subject has NAFLD and, if present, the severity of the disease, including differentiating NAFLD into NAFL or NASH. In some embodiments, the severity of NAFLD can be assessed using NAS. In some embodiments, the processing of NAFLD can be assessed using NAS. In some embodiments, 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. See, for example, Table 6 for a simplified NAS schema adapted from Kleiner.

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

Additional approaches to determining and assessing NASH in a subject include determining one or more of the following: hepatic steatosis (e.g., accumulation of fat in the liver); liver inflammation; biomarkers indicative of one or more of liver injury, liver inflammation, liver fibrosis, and/or liver cirrhosis (e.g., serum markers and panels). Additional examples of physiological indicators of NASH may include liver morphology, liver stiffness, and the size or weight of the subject's liver. In some embodiments, NASH in a subject is confirmed by accumulation of liver fat and detection of a biomarker indicative of liver injury. For example, elevated serum ferritin and low serum autoantibody titers may be common features of NASH.

In some embodiments, methods for assessing NASH include magnetic resonance imaging using either spectroscopy or proton density lipid fraction (MRI-PDFF) to quantify steatosis, transient elastography (FIBROSCAN®), hepatic venous pressure gradient (HPVG), liver stiffness measurement using MRE to diagnose significant liver fibrosis and/or cirrhosis, and assessing histological features of a liver biopsy. In some embodiments, magnetic resonance imaging is used to detect one or more of steatohepatitis (NASH-MRI), liver fibrosis (Fibro-MRI), and steatosis. See, for example, U.S. Publication Nos. 2016/146715 and 2005/0215882, each of which is incorporated herein by reference in its entirety.

In some embodiments, treating NASH may include reducing one or more symptoms associated with NASH; reducing the degree of hepatic steatosis; reducing NAS; reducing liver inflammation; reducing the level of biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis; and reducing fibrosis and/or cirrhosis, not further progressing fibrosis and/or cirrhosis, or slowing the progression of fibrosis and/or cirrhosis in a subject following administration of one or more doses of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, treating NASH includes reducing one or more symptoms associated with NASH in a subject. Typical symptoms may include one or more of the following: enlarged liver, fatigue, pain in the right upper quadrant, bloating, enlarged blood vessels just under the surface of the skin, enlarged breasts in men, enlarged spleen, red palms, jaundice, and itching. In some embodiments, the subject is asymptomatic. In some embodiments, the subject's overall body weight does not increase. In some embodiments, the subject's overall body weight decreases. In some embodiments, the subject's body mass index (BMI) does not increase. In some embodiments, the subject's body mass index (BMI) decreases. In some embodiments, the subject's waist to hip (WTH) ratio does not increase. In some embodiments, the subject's waist to hip (WTH) ratio decreases.

In some embodiments, treatment of NASH can be assessed by measuring hepatic steatosis. In some embodiments of the invention, treatment of NASH includes reducing hepatic steatosis after administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof, as described herein. In some embodiments, hepatic steatosis is determined by one or more methods selected from the group consisting of ultrasonography, computed tomography (CT), magnetic resonance imaging, magnetic resonance spectroscopy (MRS), magnetic resonance elastography (MRE), transient elastography (TE) (e.g., FIBROSCAN®), measurement of liver size or mass, or liver biopsy (see, e.g., Di Lascio et al., Ultrasound Med Biol. 2018, vol. 44(8), p. 1585-1596; Lv et al., J Clin Transl Hepatol. 2018, vol. 6(2), p. 217-221; Reeder et al., J Magn Reson 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). A subject diagnosed with NASH may have hepatic steatosis of greater than about 5%, such as from about 5% to about 25%, from about 25% to about 45%, from about 45% to about 65%, or greater than about 65% hepatic steatosis. In some embodiments of the invention, a subject with hepatic steatosis of greater than about 5% to about 33% has stage 1 hepatic steatosis, a subject with hepatic steatosis of from about 33% to about 66% has stage 2 hepatic steatosis, and a subject with hepatic steatosis of greater than about 66% has stage 3 hepatic steatosis.

In some embodiments, the degree of liver steatosis is determined before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the degree of liver steatosis is determined during or after a period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, a decrease in the degree of liver steatosis during or after a period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof indicates treatment of NASH. For example, a decrease in the degree of liver steatosis from about 1% to about 50%, from about 25% to about 75%, or from about 50% to about 100% is indicative of treatment of NASH. In some embodiments, a reduction in the degree of hepatic steatosis by 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.

In some embodiments, the presence of liver inflammation is determined by one or more methods selected from the group consisting of biomarkers indicative of liver inflammation and a liver biopsy sample(s) from the subject. In some embodiments, the severity of liver inflammation is determined from the liver biopsy sample(s) from the subject. For example, liver inflammation in a liver biopsy sample can 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 incorporated herein by reference in their entireties. In some embodiments, the severity of liver inflammation is determined prior to administration of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the severity of liver inflammation is determined during or after a period of administration of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, a decrease in the severity of liver inflammation during or after a period of time of administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof is indicative of treatment of NASH. For example, a decrease in the severity of liver inflammation from about 1% to about 50%, from about 25% to about 75%, or from about 50% to about 100% is indicative of treatment of NASH. In some embodiments, a decrease in the severity of liver inflammation by 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% is indicative of treatment of NASH.

In some embodiments, treating NASH includes treating fibrosis and/or cirrhosis, such as reducing the severity of fibrosis, preventing further progression of fibrosis and/or cirrhosis, or slowing the progression of fibrosis and/or cirrhosis. In some embodiments, the presence of fibrosis and/or cirrhosis is determined by one or more methods selected from the group consisting of transient elastography (e.g., FIBROSCAN®), non-invasive markers of liver fibrosis, and histological characteristics of a liver biopsy. In some embodiments, the severity (e.g., stage) of fibrosis is determined by one or more methods selected from the group consisting of transient elastography (e.g., FIBROSCAN®), a fibrosis scoring system, liver fibrosis biomarkers (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, e.g., Angulo et al., Hepatology 2007, vol. 45(4), pp.846-54), the fibrosis scoring system in Brant 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 each of which are incorporated herein by reference in their entirety.

In some embodiments, the severity of fibrosis is determined before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the severity of fibrosis is determined during or after a period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments of the invention, a decrease in the severity of fibrosis during or after a period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof indicates treatment for NASH. In some embodiments, a decrease in the severity of fibrosis, a lack of 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, the severity of fibrosis is determined using a scoring system, such as any of the fibrosis scoring systems described herein, e.g., the score can indicate a stage of fibrosis, such as stage 0 (no fibrosis), stage 1, stage 2, stage 3, and stage 4 (cirrhosis) (see, e.g., Kleiner et al). In some embodiments, a decrease in the stage of fibrosis is a decrease in the severity of fibrosis. For example, a decrease of 1, 2, 3, or 4 stages is a decrease in the severity of fibrosis. In some embodiments, a decrease 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, is indicative of NASH treatment. In some embodiments, 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 the compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, 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 during the period of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, 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 a period of time of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, the presence of NASH is determined by one or more biomarkers indicative of one or more of the following: liver injury, inflammation, liver fibrosis, and/or liver cirrhosis, or scoring systems thereof. In some embodiments, the severity of NASH is determined by one or more biomarkers indicative of one or more of the following: liver injury, inflammation, liver fibrosis, and/or liver cirrhosis, or scoring systems thereof. The level of a biomarker can be determined, for example, by measuring, quantifying, and monitoring the expression level of a gene or mRNA encoding the biomarker and/or a peptide or protein of the biomarker. Non-limiting examples of biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis, and/or scoring systems thereof include aspartate aminotransferase (AST) to platelet ratio index (APRI); aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR); FIB-4 score, which is based on APRI, alanine aminotransferase (ALT) levels, and the subject's age (see, e.g., McPherson et al., Gut 2010, vol. 59(9), p.1265-9, which is incorporated herein by reference in its entirety); hyaluronic acid; proinflammatory cytokines; a panel of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma-glutamyl transpeptidase (GGT) in relation to the age and sex of the subject to measure fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consisting of bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin in relation to the age and sex of the subject (e.g., HEPASCORE®; see, e.g., Adams et al., Clin. Chem. 2005, vol. 51(10), p.1867-1873), and a panel of biomarkers consisting of tissue inhibitor of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g., FIBROSPECT®); a biomarker panel consisting of tissue inhibitor of metalloproteinase 1 (TIMP-1), procollagen type III amino-terminal propeptide (PIIINP), and hyaluronic acid (HA) (e.g., ELF assessment, see e.g., 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 determined by one or more of FIB-4, a panel of biomarkers consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma-glutamyl transpeptidase (GGT) in relation to the age and sex of the subject to measure fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consisting of bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin in relation to the age and sex of the subject (e.g., HEPASCORE®; see, e.g., Adams et al., Clin. Chem. 2005, vol. 51(10), pp. 1867-1873), and a panel of biomarkers consisting of tissue inhibitor metalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g., FIBROSPECT®); and a panel of biomarkers consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1), procollagen type III amino-terminal propeptide (PIIINP), and hyaluronic acid (HA) (e.g., Evaluation of Generalized Liver Fibrosis (ELF)). 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 the concentration of the enzyme, such as in the blood. For example, the AST or ALT level may be expressed in units/L.

In some embodiments, the severity of fibrosis is determined by one or more of a FIB-4 score, a biomarker panel consisting of α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma-glutamyl transpeptidase (GGT) in relation to the subject's age and sex to measure fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®), a biomarker panel consisting of bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin in relation to the subject's age and sex (e.g., HEPASCORE®; see, e.g., Adams et al., Clin. Chem. 2005, vol. 51(10), pp. 1867-1873, which is incorporated herein by reference in its entirety), and a biomarker panel consisting of tissue inhibitor of metalloproteinase-1, hyaluronic acid and α2-macroglobulin (e.g. FIBROSPECT®); and a biomarker panel consisting of tissue inhibitor of metalloproteinases 1 (TIMP-1), procollagen type III amino-terminal propeptide (PIIINP) and hyaluronic acid (HA) (e.g. Evaluation of Generalized Liver Fibrosis (ELF)).

In some embodiments, liver inflammation is determined by the level of biomarkers of liver inflammation, such as proinflammatory cytokines. Non-limiting examples of biomarkers indicative of liver inflammation include interleukin (IL)-6, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, monocyte chemotactic protein (MCP)-1, C-reactive protein (CRP), PAI-1, and collagen isoforms such as Collal, Colla2, and Col4a1 (see, e.g., 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 changes in CD68 expression levels. In some embodiments, liver inflammation can be determined by measuring or monitoring serum levels 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 chemotactic protein (MCP)-1, and C-reactive protein (CRP).

In some embodiments, the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis is determined for a sample from a subject prior to administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis is determined during or after a period of administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, a decrease in the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis during or after a period of administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof compared to before administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof is indicative of treatment for NASH. For example, a reduction in the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver 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% is indicative of NASH treatment. In some embodiments, the reduction in the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis following administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof is 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, the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis during a period of administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof is 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, the level of one or more biomarkers indicative of one or more of liver injury, inflammation, liver fibrosis, and/or liver cirrhosis after a period of time of administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof is 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, treatment with NASH reduces the level of serum bile acids in the subject. In some embodiments, the level of serum bile acids is determined, for example, using an ELISA or assays for 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, the level of serum bile acids can be reduced, for example, by 10-40%, 20-50%, 30-60%, 40-70%, 50-80%, or greater than 90% of the level of serum bile acids prior to administration of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, NASH is NASH with concomitant cholestasis. In cholestasis, the secretion of bile, including bile acids, from the liver is blocked. Bile acids can cause hepatocyte injury (see, e.g., Perez MJ, Briz O. World J. Gastroenterol. 2009, vol. 15(14), pp. 1677-1689), possibly causing or increasing the progression of fibrosis (e.g., cirrhosis) and increasing the risk of hepatocellular carcinoma (see, e.g., 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 by reference in its entirety). In some embodiments, treating NASH comprises treating pruritus. In some embodiments, treating NASH with concomitant cholestasis comprises treating pruritus. In some embodiments, a subject with NASH with associated cholestasis experiences pruritus.

Typical NASH biomarkers are presented in Table 7.

Table 7. Typical NASH biomarkers

Biomarkers of liver fibrosis

Aspartate Aminotransferase (AST) to Platelet Ratio Index (APRI)

Aspartate Aminotransferase (AST) to Alanine Aminotransferase (ALT) Ratio (AAR)

FIB-4 Score ¹

Thialuronic acid

Proinflammatory cytokines

A panel including α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma-glutamyl transpeptidase (GGT) in relation to the subject's age and gender to determine an index of fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST®, FIBROSURE®)

A panel including bilirubin, gamma-glutamyl transferase, hyaluronic acid, α2-macroglobulin in relation to the age and sex of the subject (e.g. HEPASCORE®2)

A panel including tissue inhibitor of metalloproteinases-1, hyaluronic acid and α2-macroglobulin (e.g. FIBROSPECT®)

Panel including tissue inhibitor of metalloproteinases 1 (TIMP-1), procollagen type III amino-terminal propeptide (PIIINP), and hyaluronic acid (HA) (e.g., Evaluation of Generalized Liver Fibrosis (ELF) ³ )

Biomarkers of liver inflammation ^4.5

Interleukin-(IL)6

Interleukin-(IL)1β

Tumor necrosis factor (TNF)-α

Transforming growth factor (TGF)-β

Monocyte chemotactic protein (MCP)-1

C-reactive protein (CRP)

PAI-1

Collagen isoforms (e.g. Colla1, Colla2, and Co14a1)

Changes in macrophage infiltration (eg, changes in CD68 expression levels)

References for Table 7

¹ McPherson et al., Gut. 2010, vol. 59(9), p.1265-1269.

² Adams, et al. Clin Chem. 2005, vol. 51(10), p.1867-1873.

³ Lichtinghagen, et al. J Hepatol. 2013, vol. 59(2), p.236-242.

⁴ Neuman, et al. Can J Gastroenterol Hepatol. 2014, vol. 28(11), p.607-618.

⁵ US Patent No. 9872844

Some compounds of formula (I) or pharmaceutically acceptable salts thereof may have a higher 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%.

Some compounds of formula (I) or pharmaceutically acceptable salts thereof may be excreted in the urine. In some embodiments, the proportion of the compound that is excreted in the 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%.

After absorption from the intestine, some compounds of formula (I) or pharmaceutically acceptable salts thereof may circulate through the enterohepatic circulation. In some embodiments, the proportion of the compound that circulates through the 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%.

Some compounds of formula (I) or pharmaceutically acceptable salts thereof may cause renal excretion of bile salts. In some embodiments, the proportion of circulating bile acids that is excreted by the renal route 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%.

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

Some compounds of formula (I) or pharmaceutically acceptable salts thereof may exhibit improved or optimal bioavailability. In some embodiments, the 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, the oral bioavailability is from about 10 to about 90%, such as from about 20 to about 80%, such as from about 30 to about 70%, or such as from about 40 to about 60%.

Some compounds of formula (I) or their pharmaceutically acceptable salts can be a substrate for the corresponding transporters in the kidneys.

Certain compounds of formula (I) or their pharmaceutically acceptable salts may cause increases in bile acid concentrations in the intestine, liver and serum that do not cause adverse gastrointestinal effects.

Certain compounds of formula (I) or their pharmaceutically acceptable salts can reduce the concentration of bile acids in the liver without causing gastrointestinal disturbances such as diarrhea.

As used herein, the terms "treating," "treating," and "treating" refer to regressing, alleviating, delaying the onset, or inhibiting the progression of a disease or disorder or one or more symptoms thereof, as described herein. In some embodiments, the treatment may be administered after one or more symptoms have developed. In other embodiments, the treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual before symptoms have developed (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

A suitable pharmaceutically acceptable salt of a compound of the invention is, for example, a base addition salt of a compound of the invention which is sufficiently acidic, such as an alkali metal salt (e.g. a sodium or potassium salt), an alkaline earth metal salt (e.g. a calcium or magnesium salt), an ammonium salt or a salt with an organic base which provides a physiologically acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

Some compounds of formula (I) or their pharmaceutically acceptable salts may have chiral centers and/or geometric isomeric centers (E- and Z-isomers). It is to be understood that the invention encompasses all such optical isomers, diastereoisomers and geometric isomers that have ASBT and/or LBAT inhibiting activity. The invention also encompasses any and all tautomeric forms of the compounds of formula (I) or their pharmaceutically acceptable salts that have ASBT and/or LBAT inhibiting activity. Some compounds of formula (I) or their pharmaceutically acceptable salts may exist in unsolvated as well as solvated forms, such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that have ASBT and/or LBAT inhibiting activity.

In another aspect, the 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, for example, include fillers, binders, disintegrants, lubricants and lubricants. In general, the pharmaceutical compositions can be prepared in a conventional manner using conventional excipients.

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

Examples of suitable binders 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 acacia and tragacanth), sodium alginate, cellulose derivatives (such as hydroxypropyl methylcellulose (or hypromellose), hydroxypropyl cellulose, and ethylcellulose), and synthetic polymers (e.g., acrylic acid-methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid copolymers, and polyvinylpyrrolidone (povidone)). In some embodiments, the binder is hydroxypropyl methylcellulose (hypromellose).

Examples of suitable disintegrants include, but are not limited to, dry starch, modified starch (such as (partially) pregelatinized starch, sodium starch glycolate, and sodium carboxymethyl starch), alginic acid, cellulose derivatives (such as sodium carboxymethylcellulose, hydroxypropylcellulose, and low-substituted hydroxypropylcellulose (L-HPC)) and cross-linked polymers (such as carmellose, croscarmellose sodium, carmellose calcium, and cross-linked PVP (crospovidone)). In some embodiments, the disintegrant is croscarmellose sodium.

Examples of suitable lubricants and glidants include, but are not limited to, talc, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, colloidal silicon dioxide, hydrous silicon dioxide, synthetic magnesium silicate, fumed silica, starch, sodium lauryl sulfate, boric acid, magnesium oxide, waxes (such as carnauba wax), hydrogenated oil, polyethylene glycol, sodium benzoate, polyethylene glycol, and mineral oil. In some embodiments, the lubricant or glidant is magnesium stearate or colloidal silicon dioxide.

The pharmaceutical composition can be typically coated with one or more coating layers. Enteric coating layers or coating layers for delayed or targeted release of the compound of formula (I) or its pharmaceutically acceptable salts are also provided. The coating layers can contain one or more coating agents and can optionally contain plasticizers and/or pigments (or dyes).

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

Examples of suitable plasticizers include, but are not limited to, triethyl citrate, glyceryl triacetate, tributyl citrate, diethyl phthalate,

acetyl tributyl citrate, dibutyl phthalate, dibutyl sebacate, and polyethylene glycol. In some embodiments, the plasticizer is polyethylene glycol.

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

The pharmaceutical composition may be in a form suitable for oral administration, for parenteral injection (including intravenous, subcutaneous, intramuscular and intravascular injection), for topical administration or for rectal administration. In a preferred embodiment, the pharmaceutical composition is in a form suitable for oral administration, such as a tablet or capsule.

The dosage required for therapeutic or prophylactic treatment will depend on the route of administration, the severity of the disease, the age and body weight of the patient and other factors normally considered by the attending physician in determining the appropriate treatment regimen and dosage level for a particular patient.

The amount of compound administered will vary for the patient being treated and may range from about 1 mcg/kg body weight to about 50 mg/kg body weight per day. A unit dosage form such as a tablet or capsule will typically contain from about 1 to about 250 mg of the active ingredient, such as from about 1 to about 100 mg, or from about 1 to about 50 mg, or from about 1 to about 20 mg, such as about 2.5 mg, or about 5 mg, or about 10 mg, or about 15 mg. The daily dose may be administered as a single dose or divided into one, two, three or more unit doses. The daily dose of the bile acid modulator administered orally is preferably from about 0.1 to about 250 mg, more preferably from about 1 to about 100 mg, such as from about 1 to about 5 mg, such as from about 1 to about 10 mg, such as from about 1 to about 15 mg or from about 1 to about 20 mg.

In another aspect, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use 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.

In another aspect, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in 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 also relates to a method for the treatment or prevention of any of the diseases listed herein in a subject, such as a human, comprising administering to a subject in need of such treatment or prevention a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Combination therapy

In one aspect of the invention, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with at least one other therapeutically active agent, such as one, two, three or more other therapeutically active agents. The compound of formula (I) or a pharmaceutically acceptable salt thereof and the at least one other therapeutically active agent may be administered simultaneously, sequentially or separately. Therapeutically active agents that are suitable for combination with the compounds of formula (I) include, but are not limited to, known active agents that are useful in the treatment of any of the above conditions, disorders and diseases.

In one embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with another ASBT inhibitor. Suitable ASBT inhibitors are described in WO 93/16055, WO 94/18183, WO 94/18184, WO 96/05188, WO 96/08484, WO 96/16051, WO 97/33882, WO 98/03818, WO 98/07449, WO 98/40375, WO 99/35135, WO 99/64409, WO 99/64410, WO 00/47568, WO 00/61568, WO 00/38725, WO 00/38726, WO 00/38727, WO 00/38728, WO 00/38729, WO 01/66533, WO 01/68096, WO 02/32428, WO 02/50051, WO 03/020710, WO 03/022286, WO 03/022825, WO 03/022830, WO 03/061663, WO 03/091232, WO 03/106482, WO 2004/006899, WO 2004/076430, WO 2007/009655, WO 2007/009656, WO 2011/137135, WO 2019/234077, WO 2020/161216, WO 2020/161217, DE 19825804, EP 864582, EP 489423, EP 549967, EP 573848, EP 624593, EP 624594, EP 624595, EP 624596, EP 0864582, EP 1173205, EP 1535913 and EP 3210977, all of which are incorporated herein by reference in their entirety.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a bile acid binder (also called a bile acid sequestrant or resin), such as colesevelam, cholestyramine or colestipol. In a preferred embodiment of such a combination, the bile acid binder is formulated as a colonic release formulation. 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.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a DPP-IV inhibitor, including gliptins such as sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, trelagliptin, alloliptin, omarigliptin, evogliptin, gosogliptin and dutogliptin, or pharmaceutically acceptable salts thereof.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an HMG CoA reductase inhibitor such as fluvastatin, lovastatin, pravastatin, simvastatin, atorvastatin, pitavastatin, cerivastatin, mevastatin, rosuvastatin, bervastatin or dalvastatin or a pharmaceutically acceptable salt thereof.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a cholesterol absorption inhibitor such as ezetimibe or a pharmaceutically acceptable salt thereof.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a PPAR-alpha agonist, including fibrates such as clofibrate, bezafibrate, ciprofibrate, clinofibrate, clofibride, fenofibrate, gemfibrozil, ronifibrate and simfribrate, or a pharmaceutically acceptable salt thereof.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a PPAR gamma agonist, including thiazolidinediones such as pioglitazone, rosiglitazone and lobeglitazone or pharmaceutically acceptable salts thereof.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a dual PPAR alpha/gamma agonist, including glitazaretes such as saroglitazar, aleglitazar, muraglitazar or tesaglitazar or pharmaceutically acceptable salts thereof.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a dual PPAR alpha/delta agonist such as elafibranor.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a PPAR pan-agonist (i.e., a PPAR agonist that has activity in all subtypes: α, γ and δ), such as IVA337.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with farnesoid X receptor (FXR) modulators, including FXR agonists such as cafestol, chenodeoxycholic acid, 6α-ethylchenodeoxycholic acid (obeticholic acid; INT-747), fexaramine, tropifexor, cilofexor and MET409.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a TGR5 receptor modulator, including TGR5 agonists such as 6α-ethyl-23(S)-methylcholic acid (INT-777).

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a dual FXR/TGR5 agonist such as INT-767.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with ursodeoxycholic acid (UDCA). In yet another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with nor-ursodeoxycholic acid (nor-UDCA).

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an FGF19 modulator such as NGM282.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an FGF21 agonist such as BMS-986036.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an integrin inhibitor such as PLN-74809 hPLN-1474.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a CCR2/CCR5 inhibitor such as cenicriviroc.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a caspase protease inhibitor such as emricasan.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a galectin-3 inhibitor such as GR-MD-02.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a stearoyl-CoA desaturase (SCD) inhibitor such as aramchol (arachidylamidocholanic acid).

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an apoptotic signal regulating kinase 1 (ASK1) inhibitor, such as selonsertib.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a LOXL2 inhibitor such as simtuzumab.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an ACC inhibitor such as GS-0976.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a thyroid hormone receptor-β agonist such as MGL3196.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a GLP-1 agonist such as liraglutide.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with dual glucagon-like peptide and glucagon receptor agonists such as SAR425899.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a mitochondrial pyruvate transporter inhibitor such as MSDC-0602K.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an antioxidant agent such as vitamin E.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an SGLT1 inhibitor, an SGLT2 inhibitor or a dual SGLT1 and SGLT2 inhibitor. Examples of such compounds are dapagliflozin, sotagliflozin, canagliflozin, empagliflozin, LIK066 and SGL5213.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a diacylglycerol O-acyltransferase 2 (DGAT2) inhibitor, such as DGAT2RX and PF-06865571.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a fatty acid synthase (FASN) inhibitor, such as TVB-2640.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an AMP-activated protein kinase (AMPK) activator, such as PXL-770.

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

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a cannabinoid receptor 1 (CB1) antagonist, such as IM102.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with Klothop (KLB) and a fibroblast growth factor receptor (FGFR) activator such as MK-3655 (formerly known as NGM-313).

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a chemokine (c-c-motif) ligand 24 (CCL24) inhibitor, such as CM101.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an A3 antagonist such as PBF-1650.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a P2x7 receptor antagonist such as SGM 1019.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with P2Y13 receptor agonists such as CER-209.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a sulfated oxysterol such as Dur-928.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a leukotriene D4 (LTD4) receptor antagonist such as MN-001.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a natural killer T cell type 1 (NKT1) inhibitor, such as GRI-0621.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an anti-lipopolysaccharide (LPS) compound such as IMM-124E.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a VAP1 inhibitor such as BI1467335.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an adenosine A3 receptor agonist such as CF-102.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a SIRT-1 activator such as NS-20.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a nicotinic acid receptor 1 agonist such as ARI-3037MO.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a TLR4 antagonist such as JKB-121.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a ketohexokinase inhibitor such as PF-06835919.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an adiponectin receptor agonist such as ADP-335.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with an autotaxin inhibitor such as PAT-505 and PF8380.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a chemokine (c-c-motif) receptor 3 (CCR3) antagonist such as bertilimumab.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a chloride channel stimulator such as cobiprostone and lubiprostone.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a heat shock protein 47 (HSP47) inhibitor, such as ND-L02-s0201.

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

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a biguanidine, such as metformin.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with insulin.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a glycogen phosphorylase inhibitor and/or a glucose-6-phosphatase inhibitor.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a sulfonylurea such as glipizide, glibenclamide and glimepiride.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a meglitinide, such as repaglinide, nateglinide and ormiglitinide.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a glucosidase inhibitor such as acarbose or miglitol.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a squalene synthase inhibitor such as TAK-475.

In another embodiment, the compounds of formula (I) or pharmaceutically acceptable salts thereof are administered in combination with a PTBB1 inhibitor such as trodusquemin, ertiprotafib, JTT-551 and claramine.

Getting connections

The compounds of the present invention can be prepared as the free acid or a pharmaceutically acceptable salt thereof by the processes described below. In the following description of such processes, it will be understood that, where appropriate, suitable protecting groups will be added and subsequently removed from the various reactants and intermediates in a manner that will be readily apparent to those skilled in the art of organic synthesis. Conventional procedures for the use of such protecting groups, as well as examples of suitable protecting groups, are described, for example, in Greene's Protective Groups in Organic Synthesis P.G.M Wutz and T.W. Greene, 4th Edition, John Wiley & Sons, Hoboken, 2006.

General methods

All solvents used were of analytical grade. Commercially available anhydrous solvents were generally used for reactions. Starting materials were available from commercial sources or were prepared according to literature procedures. Room temperature refers to 20-25°C. Solvent mixture compositions are given as volume percent or volume ratio.

LC-MS:

Instrument name: Agilent 1290 infinity II.

Method A: Mobile phase: A: 0.1% HCOOH in water: ACN (95:5), B: ACN; flow rate: 1.5 ml/min; column: ZORBAX XDB C-18 (50x4.6 mm, 3.5 µm).

Method B: Mobile phase: A: 10 mM _{NH4HCO3 in} water, B: ACN; flow rate: 1.2 ml/min; column: XBridge C8 (50x4.6 mm, 3.5 µm).

Method C: Mobile phase: A: 0.1% HCOOH in water: ACN (95:5), B: ACN; flow rate: 1.5 ml/min; column: ATLANTIS dC18 (50x4.6 mm, 5 µm).

Method D: Mobile phase: A: 10 mM NH ₄ OAC in water, B: ACN; flow rate: 1.2 ml/min; column: Zorbax Extend CI8 (50x4.6 mm, 5 µm).

Method E: Mobile phase: A: 0.1% TFA in water:ACN (95:5), B: 0.1% TFA in ACN; flow rate: 1.5 ml/min; column: XBridge C8 (50x4.6 mm, 3.5 μm).

UPLC:

Device name: waters Acquity I Class

Method A: Mobile phase: A: 0.1% HCOOH in water, B: 0.1% HCOOH in ACN; flow rate: 0.8 ml/min; column: Acquity UPLC HSS T3 (2.1x50 mm); 1.8 μm. HPLC:

Instrument Name: Agilent 1260 Infinity II Series instruments using % with UV detection (maxplot).

Method A: Mobile phase: A: 10 mM _{NH4HCO3 in} water, B: ACN; flow rate: 1.0 ml/min; column: XBridge C8 (50x4.6 mm, 3.5 µm).

Method B: Mobile phase: A: 0.1% TFA in water, B: 0.1% TFA in ACN; flow rate: 2.0 ml/min; column: XBridge C8 (50x4.6 mm, 3.5 μm).

Method C: Mobile phase: A: 10 mM NH ₄ OAC in Milli-q water, B: ACN; flow rate: 1.0 ml/min; column: Phenomenex Gemini C18 (150x4.6 mm, 3.0 µm).

Method D: Mobile phase: A: 0.1% TFA in water, B: ACN; flow rate: 1.0 ml/min; column: ATLANTIS dC18 (250x4.6 mm, 5.0 μm).

Chiral HPLC:

Instrument Name: Agilent 1260 Infinity II

Method A: Mobile phase: A: 0.1% TFA in n-hexane; B: ethanol, flow rate: 1.0 ml/min; Column: CHIRALPAK IA (250x4.6 mm, 5.0 µm).

Chiral SFC:

Device name: PIC SFX 10 (analytical)

The ratio between _CO2 and cosolvent is in the range between 60:40 and 80:20

Method A: Mobile phase: 0.5% isopropylamine in IP A; flow rate: 3 ml/min; column: YMC Amylose-SA (250x4.6 mm, 5 µm).

Method B: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 ml/min; column: Chiralpak AD-H (250x4.6 mm, 5 µm).

Method C: Mobile phase: 20 mM ammonia in methanol; flow rate: 3 ml/min; column: YMC Cellulose-SC (250x4.6 mm, 5 μm).

Method D: Mobile phase: methanol; flow rate: 3 ml/min; column: Lux Al (250x4.6 mm, 5 µm).

Method E: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 5 ml/min; column: Lux C4.

Method F: Mobile phase: 0.5% isopropylamine in methanol; Flow rate: 3 mL/min; Column: YMC Cellulose-SC.

Method G: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3 ml/min; column: Lux Al.

Method H: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 ml/min; column: Lux Al (250x4.6 mm, 5 µm).

Method I: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3 ml/min; column: Chiral CCS (250x4.6 mm, 5 µm).

Method J: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 5 mL/min; Column: YMC Cellulose-SC AD-H (250x4.6 mm, 5 µm).

Method K: Mobile phase: IP A; flow rate: 3 ml/min; column: YMC Cellulose-SC (250x4.6 mm, 5 µm).

Method L: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 4 ml/min; Column: YMC Cellulose-SC (250x4.6 mm, 5 µm).

Method M: Mobile phase: methanol; flow rate: 3 ml/min; column: YMC Cellulose-SC AD-H (250x4.6 mm, 5 µm).

Prep-HPLC:

Instrument Name: Agilent 1290 Infinity II

Method A: Mobile phase: A: 0.1% TFA in water; mobile phase; B: 0.1% TFA in ACN; flow rate: 2.0 ml/min; column: X-Bridge C8 (50x4.6 mm, 3.5 μm).

Method B: Mobile phase: A: 10 mM _NH4OAc in water; B: ACN; flow rate: 35 ml/min; column: X select C18 (30x150 mm, 5 µm).

Method C: Mobile phase: A: 10 mM _NH4HCO3 in water; B: ACN; _flow rate: 1.0 ml/min; column: XBridge C8 (50x4.6 mm, 3.5 µm).

Method D: Mobile phase: A: 0.1% HCOOH in water; B: ACN; flow rate: 1.0 ml/min; column: X-select C18 (30x150 mm, 5 µm).

Chiral preparative SFC:

Device name: PIC SFH 100 and PSC SFH 400

The ratio between _CO2 and cosolvent is in the range between 60:40 and 80:20

Method A: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 ml/min; column: YMC Amylose-SA (250x30 mm, 5 µm).

Method B: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 ml/min; column: Chiralpak AD-H (250x30 mm, 5 µm).

Method C: Mobile phase: 20 mM ammonia in methanol; flow rate: 3 ml/min; column: YMC Cellulose-SC (250x30 mm, 5 μm).

Method D: Mobile phase: methanol; flow rate: 3 ml/min; column: Chiral CCS (250x30 mm, 5 µm).

Method E: Mobile phase: methanol; flow rate: 3 ml/min; column: Lux Al (250x30 mm, 5 µm).

Method F: Mobile phase: 0.5% isopropylamine in IP A; flow rate: 3 ml/min; column: Lux Al (250x30 mm, 5 µm).

Method G: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3 ml/min; Column: Chiral CCS (250x30 mm, 5 µm).

Method H: Mobile phase: 0.5% isopropylamine in IP A, flow rate: 5 ml/min; column: YMC Amylose-SC (250x30 mm, 5 µm).

Chiral preparative HPLC:

Instrument Name: Agilent 1260 Infinity II

Method A: Mobile phase: A: 0.1% TFA in n-hexane; B: ethanol; flow rate: 15 ml/min; column: CHIRALPAK IA (250x19 mm, 5.0 µm).

Abbreviations

The invention will be further described by means of the following examples, which do not limit the invention in any respect. All cited documents and information sources are incorporated by reference.

EXAMPLES

Intermediate connection 1

2-(((2-Amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylhexanoic acid

To a stirred solution of 5-bromo-6-methoxybenzo[c1]thiazol-2-amine (63 g, 0.243 mol) in water (630 mL) was added KOH (218.2 g, 3.89 mol) and the reaction mixture was stirred for 16 h at 120 °C. After the reaction was complete (monitored by LC-MS), the reaction mixture was cooled to room temperature. 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 for 16 h at room temperature. After the reaction was complete (monitored by LC-MS), the reaction mixture was cooled to 0 °C and acidified with conc. HCl (pH ca. 2). The reaction mixture was extracted with EtOAc (2x350 mL) and the combined organic layer was washed with water (150 mL) and brine (150 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was carried as received to the next step without any further purification. Yield: 75 g (crude, brown gum).

LC-MS: (Method A) 376.1 (M ⁺ ), 378.0 (M ⁺⁺ 2), Rt: 2.44 min, 92.97% (max).

Intermediate connection 2

7-Bromo-3-butyl-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

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 were added dropwise triethylamine (60.4 g, 0.59 mol) and a solution of 1-propanephosphonic anhydride (50% in EtOAc, 95.1 g, 0.29 mol). The reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (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 layer was washed with brine (150 mL) and dried over _{anhydrous Na2SO4} . The organic portion was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 10-12% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 63% (45 g, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): 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). LC-MS: (Method A) 360.0 (M ⁺⁺ 2), Rt: 2.64 min, 97.14% (max).

Intermediate connection 3

7-Bromo-3-butyl-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 3-butyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a stirred solution of 7-bromo-3-butyl-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 2; 45 g, 0.12 mol) in iodobenzene (225 ml) were added copper(I) iodide (2.4 g, 0.012 mol) and _K2CO3 (34.6 g, 0.251 mol) and _the mixture was purged with nitrogen for 20 min to degas. Tris[2-(2-methoxyethoxy)ethyl]amine (8.11 g, 0.025 mol) was then added under nitrogen and the resulting reaction mixture was heated for 40 h at 135°C. After completion of the reaction (monitored by UPLC), the reaction mixture was filtered through Celite and the Celite pad was washed with EtOAc (200 mL). The filtrate was concentrated in vacuo and the resulting crude material was purified by recrystallization with MeOH to give the title compound. Yield: 88% (47.5 g, off-white solid).

LC-MS: (Method E) 434.1 (M ⁺ ) for 7-bromo compound and 482.1 (M ⁺⁺ H) for 7-iodo compound, Rt: 3.23 min, 99.31% (total for bromo and iodo compounds) (max).

Intermediate connection 4

7-Bromo-3-butyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 3-butyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-3-butyl-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 3-butyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 3; 47.5 g, 0.109 mol) in THF (475 mL) at 0 °C was added dropwise borane dimethyl sulfide (1 M in THF, 82 mL, 0.16 mol) and the reaction mixture was refluxed for 40 h at 75 °C. After completion of the reaction (monitored by UPLC), the reaction mixture was cooled to 0 °C and quenched with methanol (475 mL). The resulting solution was heated for 2 hours at 65°C and then cooled to room temperature and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 8-10% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 46 g (crude, colorless liquid).

LC-MS: (Method E) 421.8 (M ⁺ +2H), 467.8 (M ⁺ +H) Rt: 3.62 min, 54.71% (max).

Intermediate connection 5

1,1-Dioxide 7-bromo-3-butyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 1,1-dioxide 3-butyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-3-butyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 3-butyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine (Intermediate 4; 23 g, 0.05 mol) in acetic acid (230 ml) were added sodium tungstate (2.3 g, 10% w/w) and _H2O2 (30% in water, 18.6 ml, 0.16 mol) and _the reaction mixture was stirred for 5 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with water (200 mL) and the aqueous layer was extracted with EtOAc (2 x 200 mL). The combined organic layer was washed with water (150 mL) and brine (150 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 10-12% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 72% (18 g, yellowish solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 7.47 (s, 1H), 7.34 (s, 1H), 7.27-7.20 (m, 2H), 6.99-6.94 (m, 2H), 6.89-6.85 (m, 1H), 3.94 (s, 3H), 3.40-3.33 (m, 2H), 2.53 (s, 2H), 1.46-1.30 (m, 2H), 1.27-1.20 (m, 4H), 1.10-1.06 (m, 3H), 0.78 (t, J=6.80 Hz, 3H). LC-MS: (Method E) 454.1 (M ⁺ 2H) for 7-bromo compound and 500.1 (M ⁺ 2H) for 7-iodo compound, Rt: 3.25 min, 92.56% (max).

Intermediate connection 6

1,1-3-Butyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine dioxide

To a stirred solution of a mixture of 7-bromo-3-butyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and 3-butyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 5; 31 g, 0.07 mol) in DMF (310 mL) was added sodium thiomethoxide (24.01 g, 0.34 mol) at room temperature and the resulting mixture was stirred overnight at 65 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with water (150 mL) and the aqueous layer was extracted with EtOAc (2 x 300 mL). The combined organic layer was washed with brine (150 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by recrystallization with MeOH to give the title compound. Yield: 83% (23 g, off-white solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 10.60 (s, 1H), 7.32 (s, 1H), 7.18-7.14 (m, 2H), 6.86 (d, J=7.6 Hz, 2H), 6.77 (s, 1H), 6.76-6.73 (m, 1H), 3.27-3.15 (m, 2H), 2.54 (s, 2H), 2.26 (s, 3H), 1.47-1.29 (m, 2H), 1.27-1.20 (m, 4H), 1.18-1.12 (m, 3H), 0.81 (t, J=7.2 Hz, 3H). LC-MS: (Method E) 406.2 (M ⁺⁺ H), Rt: 2.98 min, 95.07% (max).

Intermediate connection 7

1,1-Dioxide (S)-3-butyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 1,1-dioxide (R)-3-butyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

The two enantiomers of racemic 3-butyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 6; 2.9 g, 0.01 mol) were separated by chiral SFC (Method M). This material was concentrated in vacuo at 40°C. The first eluting fraction corresponded to enantiomer 1 and the second eluting fraction corresponded to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Enantiomer 1: Yield: 41% (1.2 g, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.51 (s, 1H), 7.31 (s, 1H), 7.18-7.14 (m, 2H), 6.85 (d, J=8.0 Hz, 2H), 6.77-6.73 (m, 2H), 3.27-3.15 (m, 2H), 2.53 (s, 2H), 2.21 (s, 3H), 1.46-1.31 (m, 2H), 1.29-1.12 (m, 4H), 1.06-1.01 (m, 3H), 0.81 (t, J=7.2 Hz, 3H). LC-MS: (Method A) 406.1 (M ⁺⁺ H), Rt: 2.72 min, 97.0% (max). HPLC: (Method B) Rt: 5.61 min, 97.78% (max). Chiral SFC: (Method M) Rt: 2.36 min, 98.75% (max).

Enantiomer 2: Yield: 38% (1.1 g, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.57 (s, 1H), 7.31 (s, 1H), 7.18-7.14 (m, 2H), 6.85 (d, J=7.6 Hz, 2H), 6.77-6.73 (m, 2H), 3.27-3.15 (m, 2H), 2.53 (s, 2H), 2.21 (s, 3H), 1.48-1.31 (m, 2H), 1.29-1.18 (m, 4H), 1.12-1.01 (m, 3H), 0.81 (t, J=7.2 Hz, 3H). LC-MS: (Method A) 406.2 (M ⁺⁺ H), Rt: 2.85 min, 99.57% (max). HPLC: (Method B) Rt: 5.61 min, 99.83% (max). Chiral SFC: (Method M) Rt: 3.58 min, 100% (max).

Intermediate connection 8

Ethyl-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate

To a stirred solution of 3-butyl 8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 6; 0.3 g, 0.74 mmol) in DMA (3 mL) at 0 °C was added 60% NaH (57 mg, 2.40 mmol) portionwise and the reaction mixture was stirred for 30 min at 0 °C. Then a solution of ethyl 3-bromo-2,2-difluoropropanoate (0.401 g, 1.84 mmol) in DMA (1 mL) was added and the reaction mixture was heated for 3 h at 70 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with dilute HCl (1.5 N, pH ca. 4) and diluted with water (20 mL). The aqueous layer was extracted with EtOAc (2 x 15 mL). The combined organic layer was washed with brine (10 mL) and dried over anhydrous Na ₂ SO ₄ . The organic portion was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 15-20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 39% (0.15 g, white solid).

LC-MS: (Method A) 522.0 (M ⁺⁺ H), Rt: 3.32 min, 97.84% (max).

Intermediate connection 9

Ethyl-(R)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate and ethyl-(S)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiaz epin-8-yl)oxy)-2-fluoroacrylate

To a stirred solution of 3-butyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine enantiomer 1 (Intermediate 7; 9 g, 22.2 mmol) in DMA (75 mL) at 0 °C was added 60% NaH (2.88 g, 72.17 mmol) portionwise and the reaction mixture was stirred for 30 min at 0 °C. Then a solution of ethyl 3-bromo-2,2-difluoropropanoate (11.26 g, 55.5 mmol) in DMA (15 mL) was added and the reaction mixture was heated for 3 h at 65 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with 1.5 N HCl (pH ca. 4) and diluted with water (100 mL). The aqueous layer was extracted with EtOAc (2x75 mL). The combined organic layer was washed with brine (50 mL) and dried _over anhydrous _Na2SO4 . The organic portion was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 15-20% EtOAc PE; silica gel: 230-400 mesh) to afford enantiomer 1 of the title compound.

Enantiomer 2 of the title compound was prepared according to the same procedure starting from 1.10 g of enantiomer 2 of Intermediate 7. The absolute configuration of these two enantiomers is not known.

Enantiomer 1: Yield: 73% (8.4 g, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.68-7.62 (m, 2H), 7.26 (t, J=8.4 Hz, 2H), 7.06 (d, J=7.6 Hz, 2H), 6.91 (t, J=7.2 Hz, 1H), 6.82 (s, 1H), 4.27 (q, J=7.2 Hz, 2H), 3.61 (bs, 2H), 3.49 (s, 2H), 2.24 (s, 3H), 1.47-1.35 (m, 2H), 1.32-1.27 (m, 3H), 1.26-1.12 (m, 4H), 1.10-1.02 (m, 3H), 0.80-0.77 (m, 3H). LC-MS: (Method E) 522.2 (M ⁺⁺ H), Rt: 3.26 min, 97.33% (max).

Enantiomer 2: Yield: 46% (0.65 g, white solid). LC-MS: (Method E) 522.2 (M ⁺⁺ H), Rt: 3.27 min, 97.63% (max).

Intermediate connection 10

2-(((2-Amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylpentanoic acid

To a stirred solution of 5-bromo-6-methoxybenzo[d]thiazol-2-amine (19.0 g, 0.07 mol) in water (190 mL) was added KOH (65.81 g, 1.173 mol) and the reaction mixture was then stirred for 16 h at 120 °C. After completion of the reaction (monitored by LC-MS), the reaction mixture was cooled to room temperature. A solution of 2-(bromomethyl)-2-methylpentanoic acid (19.93 g, 0.09 mol) in THF (60 mL) was added dropwise at 0 °C and the reaction mixture was stirred for 16 h at room temperature. The reaction mixture was then heated for 16 h at 65 °C. After consumption of the starting material (monitored by LC-MS), the reaction mixture was poured into ice-cold water (50 mL) and acidified with conc. HCl (pH ca. 2). The aqueous layer was extracted with EtOAc (2x200 mL). The combined organic layer was washed with water (100 mL) and brine (100 mL) and dried _over anhydrous _Na2SO4 . The organic portion was concentrated in vacuo and the resulting crude material was carried as received to the next step without any further purification. Yield: 25 g (crude, brown gum).

LC-MS: (Method E) 361.8 (M ⁺⁺ H), Rt: 2.36 min, 97.93% (max).

Intermediate connection 11

7-Bromo-8-methoxy-3-methyl-3-propyl-2,3-dihydro-1,5-benzo-1,5-thiazepin-4(5H)-one

To a stirred solution of 2-(((2-amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylpentanoic acid (Intermediate 10; 25 g, 0.069 mol) in EtOAc (250 mL) at 0 °C were added dropwise triethylamine (28.8 mL, 0.207 mol) and a solution of 1-propanephosphonic anhydride (50% in EtOAc; 32.91 g, 0.103 mol) and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by LC-MS), the reaction mixture was quenched with water (100 mL) and the aqueous layer was extracted with EtOAc (2 x 200 mL). The combined organic layer was washed with brine (100 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 13%-100% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 49.6% (11.8 g, brown solid).

¹ H NMR (400 MHz, DMSO-d6): δ 9.63 (s, 1H), 7.33 (s, 1H), 7.13 (s, 1H), 3.83 (s, 3H), 2.99-2.95 (m, 2H), 1.50-1.40 (m, 2H), 1.24-1.16 (m, 5H), 0.77 (t, J=7.2 Hz, 3H). LC-MS: (Method E) 344.1 (M ⁺ ), Rt: 2.71 min, 99.73% (max).

Intermediate connection 12

7-Bromo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 7-iodo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a stirred solution of 7-bromo-8-methoxy-3-methyl-3-propyl-2,3-dihydro-1,5-benzo-1,5-thiazepin-4(5H)-one (Intermediate 11; 11.8 g, 34.27 mmol) in iodobenzene (118 ml) were added copper(II) iodide (0.65 g, 3.40 mmol) and _K2CO3 (9.47 g, _68.5 mmol) and the reaction mixture was purged with nitrogen for 20 min to degas. Tris[2-(2-methoxyethoxy)ethyl]amine (2.21 g, 6.85 mmol) was then added under nitrogen and the resulting reaction mixture was heated at 135°C for 16 h. After completion of the reaction (monitored by LC-MS), the reaction mixture was filtered through celite and the celite pad was washed with EtOAc (100 mL). The filtrate was washed with water (50 mL) and brine (50 mL) and dried _over anhydrous _Na2SO4 . The organic portion was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 10-12% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compounds. Yield: 92% (13.2 g, off-white solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 7.42-7.38 (m, 3H), 7.28-7.25 (m, 2H), 7.13-7.10 (m, 2H), 3.82 (s, 3H), 3.29-3.25 (m, 1H), 3.16-3.13 (m, 1H), 1.34-1.26 (m, 2H), 1.20-1.13 (m, 5H), 0.73-0.72 (m, 3H). LC-MS: (Method E) 420.1 (M ⁺⁺ H) for 7-bromo compound and 468.1 (M ⁺⁺ H) for 7-iodo compound, Rt: 3.13 min, 97.05% (max).

Intermediate connection 13

7-Bromo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 7-iodo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 7-iodo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 12; 13.2 g, 31.4 mmol) in THF (132 ml) at 0°C was added dropwise borane dimethyl sulfide (2 M in THF; 47.1 ml, 94.2 mmol) and the reaction mixture was refluxed for 16 h at 65°C. After completion of the reaction (monitored by UPLC), the reaction mixture was cooled to 0 °C, quenched with methanol (15 mL) and heated for 2 h at 65 °C. The resulting reaction mixture was then cooled to room temperature and concentrated in vacuo. The residue was diluted with water (100 mL) and the aqueous layer was extracted with DCM (2 x 200 mL). The combined organic layer was washed with water (100 mL) and brine (100 mL) and dried over anhydrous Na ₂ SO ₄ . The organic portion was concentrated in vacuo and the resulting crude material was carried as received to the next step without any further purification. Yield: 12.4 g (97%, white gum).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.25-7.13 (m, 3H), 7.06-7.02 (m, 1H), 6.83-6.77 (m, 2H), 6.75-6.72 (m, 1H), 3.84 (s, 3H), 2.81-2.70 (m, 2H), 2.64-2.60 (m, 2H), 1.23-1.16 (m, 4H), 0.89 (s, 3H), 0.74 (s, 3H). LC-MS: (Method E) 406.1 (M ⁺ +H) for 7-bromo compound and 454.1 (M ⁺ +H) for 7-iodo compound; Rt: 3.55 min, 97.03% (max).

Intermediate connection 14

7-bromo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and 7-iodo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide

To a stirred solution of a mixture of 7-bromo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 7-iodo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine (Intermediate 13; 12.4 g, 30.51 mmol) in a mixture of THF (87 mL) and water (37 mL) was added oxone (93.79 g, 30.5 mmol) at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by UPLC), the reaction mixture was filtered through a Buchner funnel and the filtrate was extracted with EtOAc (2 x 200 mL). The combined organic layer was washed with water (100 mL) and brine (100 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. This crude material was purified by Isolera column chromatography (eluent: 7% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compounds. Yield: 90% (7.0 g, off-white gum).

^1H NMR (400-400 MHz, DMSO- _d6 ): δ 7.47 (s, 1H), 7.34 (s, 1H), 7.28-7.20 (m, 2H), 6.98-6.94 (m, 2H), 6.89-6.85 (m, 1H), 3.85 (s, 3H), 3.52-3.48 (m, 2H), 3.39-3.36 (m, 2H), 1.52-1.41 (m, 1H), 1.29-1.25 (m, 3H), 1.00 (s, 3H), 0.74-0.73 (m, 3H). LC-MS: (Method E) 438.0 (M ⁺⁺ H) for 7-bromo compound and 485.7 (M ⁺⁺ H) for 7-iodo compound, Rt: 3.13 min, 95.69% (max).

Intermediate connection 15

1,1-Dioxide 8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and 7-iodo-8-methoxy-3-methyl-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 14; 5.0 g, 11.4 mmol) in DMF (50 mL) was added sodium thiomethoxide (3.99 g, 57 mmol) at room temperature and the reaction mixture was then stirred for 16 h at 65 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature and quenched with water (100 mL). The aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layer was washed with brine (50 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 89% (4.0 g, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.60 (s, 1H), 7.32 (s, 1H), 7.18-7.14 (m, 2H), 6.86-6.84 (m, 2H), 6.78-6.73 (m, 2H), 3.23-3.16 (m, 4H), 2.21 (s, 3H), 1.52-1.48 (m, 1H), 1.27-1.26 (m, 3H), 1.02 (s, 3H), 0.77-0.73 (m, 3H). LC-MS: (Method E) 392.2 (M ⁺⁺ H), Rt: 2.83 min, 97.89% (max).

Intermediate connection 16

tert-Butyl-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate

To a stirred solution of 8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 15; 300 mg, 0.76 mmol) in THF (5 ml) at room temperature were added DABCO (0.008 g, 0.076 mmol) and tert-butyl propiolate (0.144 g, 1.149 mmol) and the reaction mixture was then stirred for 3 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water (20 ml) and the aqueous layer was extracted with EtOAc (2 x 20 ml). The combined organic layer was washed with water (2 x 10 ml), dried over anhydrous Na ₂ SO ₄ and the organic portion was concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 16% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 83% (0.33 g, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.67 (d, J=12.0 Hz, 1H), 7.51 (s, 1H), 7.27 (t, J=8.4 Hz, 2H), 7.09 (d, J=8.0 Hz, 2H), 6.93 (t, J=7.2 Hz, 1H), 6.81 (s, 1H), 5.39 (d,.J=12.4 Hz, 1H), 3.70 (s, 2H), 3.41 (s, 1H), 3.36-3.33 (m, 1H), 2.22 (s, 3H), 1.44 (s, 9H), 1.26-1.16 (m, 4H), 1.02 (s, 3H), 0.73 (t, J=6.8 Hz, 3H). LC-MS: (Method E) 462.1 (M ⁺ - ^t Bu+H), Rt: 3.34 min, 97.13% (max).

Intermediate connection 17

Ethyl (Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate

To a stirred solution of 8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 15; 0.3 g, 0.76 mmol) in DMA (4 ml) at 0 °C was added 60% NaH (0.1 g, 2.49 mmol) portionwise and the reaction mixture was stirred for 30 min at 0 °C. Then a solution of ethyl 3-bromo-2,2-difluoropropanoate (0.42 g, 1.91 mmol) in DMA (1 ml) was added and the reaction mixture was heated for 3 h at 70 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with dilute HCl (1.5 N, pH ca. 4) and diluted with water (10 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL) and the combined organic layer was washed with brine (10 mL) and dried over anhydrous Na ₂ SO ₄ . The organic portion was concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 15-20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 36% (0.14 g, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.69 (s, 1H), 7.63 (d, J=9.6 Hz, 1H), 7.25 (t, J=8.4 Hz, 2H), 7.05 (d, J=7.2 Hz, 2H), 6.91 (t, J=7.2 Hz, 1H), 6.83 (s, 1H), 4.26 (q, J=6.8 Hz, 2H), 3.74 (s, 2H), 3.35 (s, 2H), 2.24 (s, 3H), 1.45-1.29 (m, 4H), 1.27-1.16 (m, 3H), 1.02 (s, 3H), 0.75-0.73 (m, 3H). LC-MS: (Method E) 508.2 (M ⁺⁺ H), Rt: 3.16 min, 96.26% (max).

Intermediate connection 18

2-(((2-Amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylbutanoic acid

To a stirred solution of 5-bromo-6-methoxybenzo[d]thiazol-2-amine (9 g, 34.7 mmol) in water (90 mL) were added KOH (31.2 g, 555.9 mmol) and _{Na2SO3 (} 4.3 g, 34.7 mmol) and the reaction mixture was stirred for 16 h at 120 °C. After completion of the reaction (monitored by LC-MS), the reaction mixture was cooled to room temperature. A solution of 2-(bromomethyl)-2-methylbutanoic acid (11.6 g, 52.0 mmol) in THF (20 mL) was added dropwise at 0 °C and the reaction mixture was stirred for 16 h at room temperature. The reaction mixture was then heated for 16 h at 65 °C. After completion of the reaction (monitored by LC-MS), the reaction mixture was poured into ice-cold water and acidified with conc. HCl (pH ca. 2). The aqueous layer was extracted with EtOAc (2x100 mL). The combined organic layer was washed with water (50 mL) and brine (50 mL) and dried over anhydrous _{Na2SO4 .} The organic portion was concentrated in vacuo and the resulting crude material was carried as received to the next step without any further purification. Yield: 10.5 g (crude, black liquid).

LC-MS: (Method E) 348.1 (M ⁺⁺ H), Rt: 2.21 min, 97.14% (max).

Intermediate connection 19

7-Bromo-3-ethyl-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a stirred solution of 2-(((2-amino-4-bromo-5-methoxyphenyl)thio)methyl)-2-methylbutanoic acid (Intermediate 18; 10.1 g, 29 mmol) in DCM (100 mL) at 0 °C were added dropwise triethylamine (7.81 mL, 58 mmol) and a solution of 1-propanephosphonic anhydride (50% in EtOAc, 18.4 g, 58 mmol) and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with water (50 mL) and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The resulting crude material was triturated with cold methanol to give the title compound. Yield: 54% (12 g, brown solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 9.64 (s, 1H), 7.34 (s, 1H), 7.13 (s, 1H), 3.83 (s, 3H), 3.06-2.94 (m, 2H), 1.57-1.46 (m, 2H), 1.21 (s, 3H), 0.76 (t, J=7.2 Hz, 3H). LC-MS: (Method E) 332.1 (M ⁺⁺ 2), Rt: 2.56 min, 90.58% (max).

Intermediate connection 20

7-Bromo-3-ethyl-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 3-ethyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a stirred solution of 7-bromo-3-ethyl-8-methoxy-3-methyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 19; 5.8 g, 17.5 mmol) in iodobenzene (58 ml) were added copper(I) iodide (0.33 g, 1.75 mmol) and _K2CO3 (4.83 g, 35 mmol) and _the reaction mixture was purged with nitrogen for 20 min to degas. Tris[2-(2-methoxyethoxy)ethyl]amine (1.13 g, 3.50 mmol) was then added under nitrogen atmosphere and the resulting reaction mixture was heated for 16 h at 135°C. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through Celite and the Celite pad was washed with EtOAc (50 mL). The filtrate was washed with water (25 mL) and brine (25 mL) and dried over anhydrous _Na2SO4 . The organic portion was concentrated in vacuo and the resulting crude material was triturated with petroleum ether to afford the title _compound . Yield: 98% (7 g, gray solid).

LC-MS: (Method E) 406.1 (M ⁺ ) for 7-bromo compound and 454.0 (M ⁺⁺ H) for 7-iodo compound, Rt: 3.00 min, 95.16% (max).

Intermediate connection 21

7-Bromo-3-ethyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 3-ethyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-3-ethyl-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 3-ethyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 20; 7 g, 17.2 mmol) in THF (70 ml) at 0°C was added dropwise borane dimethyl sulfide (2 M in THF; 26 ml, 51.6 mmol) and the reaction mixture was refluxed for 16 h at 65°C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with methanol (15 mL) and then heated for 2 h at 65 °C. The resulting reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting residue was diluted with water (50 mL) and the aqueous layer was extracted with DCM (2 x 100 mL). The combined organic layer was washed with water (50 mL) and brine (50 mL) and dried over anhydrous Na ₂ SO ₄ . The organic portion was concentrated in vacuo and the resulting crude material was carried as is to the next step without any further purification. Yield: 6.5 g (crude, colorless oil).

LC-MS: (Method E) 391.8 (M ⁺⁺ H) for 7-bromo compound and 439.7 (M ⁺⁺ H) for 7-iodo compound, Rt: 3.52 min, 95.99% (max).

Intermediate connection 22

1,1-Dioxide of 7-bromo-3-ethyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 1,1-dioxide of 3-ethyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-3-ethyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 3-ethyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine (Intermediate 21; 6.5 g, 16.5 mmol) in THF (46 ml) and water (20 ml) was added oxone (50.9 g, 16.56 mmol) at 0 °C. The resulting reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through a Buchner funnel. The filtrate was extracted with EtOAc (2x100 mL) and the combined organic layer was washed with water (50 mL) and brine (50 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 13% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 79% (5.5 g, white solid).

LC-MS: (Method E) 424.1 (M ⁺⁺ H) for 7-bromo compound and 473.1 (M ⁺⁺ 2) for 7-iodo compound, Rt: 3.03 min, 96.73% (max).

Intermediate connection 23

1,1-Dioxide 3-ethyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of a mixture of 7-bromo-3-ethyl-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and 3-ethyl-7-iodo-8-methoxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 22; 5.5 g, 12.9 mmol) in DMF (55 ml) was added sodium thiomethoxide (4.54 g, 64.8 mmol) at room temperature and the reaction mixture was then stirred for 16 h at 65 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature and quenched with water (15 ml). The aqueous layer was extracted with EtOAc (2x50 mL) and the combined organic layer was washed with brine (15 mL) and dried over anhydrous _Na2SO4 . The organic portion was concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 30% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 83% ( ₄ g, off-white solid).

^1H NMR (400 MHz, DMSO-d6: δ 10.60 (d,J=4.8 Hz, 1H), 7.32 (d, J=4.8 Hz, 1H), 7.15-7.14 (m, 2H), 6.84-6.71 (m, 4H), 3.83-3.72 (m, 2H), 3.18-3.13 (m, 2H), 2.22 (s, 3H), 1.56-1.49 (m, 1H), 1.31-1.28 (m, 1H), 0.99 (s, 3H), 0.81-0.80 (m, 3H): (Method E) 378.2 (M ⁺ +H), Rt: 2.71 min, 97.90% (max).

Intermediate connection 24

Ethyl (Z)-3-((3-ethyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate

To a suspension of 60% NaH (69 mg, 1.72 mmol) in DMF (1 ml) at 0 °C was added a solution of 3-ethyl-8-hydroxy-3-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 23; 200 mg, 0.52 mmol) in DMF (2 ml) and the reaction mixture was stirred for 30 min. Then 3-chloro-2,2-fluoroethyl propionate (228 mg, 1.32 mmol) was added and the reaction mixture was heated for 3 h at 60 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature, quenched with dilute HCl (1.5 N, 3 ml) and concentrated in vacuo. The resulting residue was partitioned between ice-cold water (5 mL) and EtOAc (5 mL). The aqueous layer was extracted with EtOAc (2x8 mL) and the combined organic layer was washed with ice-cold water (10 mL) and brine (10 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 16% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 76% (220 mg, pale yellow solid).

LC-MS: (Method A) 494.0 (M ⁺⁺ H), Rt: 2.84 min, 53.89% (max).

Example 1

(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid

To a stirred solution of ethyl (Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 8; 0.15 g, 0.29 mmol) in a mixture of 1,4-dioxane and water (4:1, 5 ml) was added lithium hydroxide (20 mg, 0.86 mmol) and the reaction mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, pH ca. 4) and diluted with ice-cold water (10 ml). The aqueous layer was extracted with EtOAc (2x10 mL) and the combined organic layer was washed with water (5 mL) and brine (5 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo to afford the title compound. Yield: 50% (70 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.51 (s, 1H), 7.61 (s, 1H), 7.57 (d, J=4.8 Hz, 1H), 7.25 (t, J=8.4 Hz, 2H), 7.05 (d, J=8.0 Hz, 1H), 6.90 (t, J=8.8 Hz, 1H), 6.83 (s, 1H), 3.40-3.36 (m, 4H), 2.24 (s, 3H), 1.47 (bs, 1H), 1.34-1.06 (m, 6H), 1.02 (s, 3H), 0.78 (t, J=6.8 Hz, 3H). LC-MS: (Method E) 494.0 (M ⁺⁺ H), Rt: 3.01 min, 99.84% (max). HPLC: (Method B) Rt: 5.71 min, 98.85% (max).

Examples 2 and 3

(R)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid and (S)-(Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid

To a stirred solution of enantiomer 1 ethyl (Z)-3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 9; 8.4 g, 16.1 mmol) in a mixture of 1,4-dioxane and water (7:3, 84 ml) was added lithium hydroxide (1.15 g, 48.3 mmol) and the reaction mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, pH ca. 4) and diluted with ice-cold water (25 ml). The aqueous layer was extracted with EtOAc (2x30 mL) and the combined organic layer was washed with water (15 mL) and brine (15 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo to give the title compound.

Enantiomer 2 of the title compound was prepared according to the same procedure starting from 0.65 g of enantiomer 2 of Intermediate 9. The absolute configuration of these two enantiomers is not known.

Enantiomer 1: Yield: 96% (7.6 g, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.56(s, 1H), 7.61-7.57(m, 2H), 7.25 (1,J=8.4 Hz, 2H), 7.05 (d, J=7.2 Hz, 2H), 6.90 (t, J=7.6 Hz, 1H), 6.82 (s, 1H), 3.62 (bs, 2H), 3.40 (s, 2H), 2.24 (s, 3H), 2.24 (bs, 1H), 1.32 (t, J=11.2 Hz, 1H), 1.28-1.05 (m, 4H), 1.01 (s, 3H), 0.78 (t, J=6.8 Hz, 3H). Analytical data: LC-MS: (Method E) 493.8 (M ⁺ +H), Rt: 3.23 min, 98.22% (max). HPLC: (Method B) Rt: 5.69 min, 99.32% (max). Analytical data: SFC: (Method H) Rt: 3.76 min, 100% (max).

Enantiomer 2: Yield: 88% (0.54 g, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.56 (s, 1H), 7.54-7.50 (m, 2H), 7.18 (t, J=7.6 Hz, 2H), 6.97 (d, J=7.6 Hz, 2H), 6.83 (t, J=7.2 Hz, 1H), 6.75 (s, 1H), 3.63 (bs, 2H), 3.33 (s, 2H), 2.17 (s, 3H), 1.39 (m, 1H), 1.24 (m, 1H), 1.16-1.02 (m, 4H), 0.94 (s, 3H), 0.70 (t, J=7.2 Hz, 3H). LC-MS: (Method E) 493.8 (M ⁺⁺ H), Rt: 2.96 min, 95.48% (max). HPLC: (Method B) Rt: 5.70 min, 98.38% (max). SFC: (Method H) Rt: 3.02 min, 98.36% (max).

Example 4

(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid

To a stirred solution of tert-butyl (E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 16; 0.33 g, 0.63 mmol) in DCM (10 ml) was added TFA (2 ml) at 0 °C and the reaction mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was poured into ice-cold water (15 ml) and the aqueous layer was extracted with DCM (2 x 20 ml). The combined organic layer was washed with water (10 ml) and brine (10 ml) and dried over anhydrous Na ₂ SO ₄ . The organic portion was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 50% EtOAc/PE; silica gel: 230-400 mesh). The resulting compound was repurified by prep-HPLC (Method A) to afford the title compound. Yield: 47% (140 mg, white solid).

^1H NMR (400 MHz, CDCl ₃ ): δ 7.78 (d, J=12.4 Hz, 1H), 7.68 (s, 1H), 7.36-7.32 (m, 2H), 7.13-7.05 (m, 3H), 6.67 (s, 1H), 5.55 (d, J=12.0 Hz, 1H), 3.90 (d, J=15.2 Hz, 1H), 3.67 (d, J=14.4 Hz, 1H), 3.29-3.16 (m, 2H), 2.18 (s, 3H), 1.55-1.50 (m, 1H), 1.34-1.22 (m, 3H), 1.14 (s, 3H), 0.83 (t, J=7.2 Hz, 3H). LC-MS: (Method A) 462.1 (M ⁺⁺ H), Rt: 2.56 min, 98.79% (max). HPLC: (Method B) Rt: 5.307 min, 98.20% (max).

Examples 5 and 6

(S)-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid and (R)-(E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid

The two enantiomers of racemic (E)-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid (Example 4; 0.1 g, 0.21 mmol) were separated by chiral SFC (Method H). This material was concentrated in vacuo at 40 °C. The first eluting fraction corresponded to enantiomer 1 and the second eluting fraction corresponded to enantiomer 2. Each of these compounds was dissolved in EtOAc (20 mL) and the EtOAc layer was washed with 1.5 N HCl (2 x 10 mL), water (10 mL), and brine (10 mL). The organic layer was dried over _{anhydrous Na2SO4} and concentrated in vacuo to give the title compounds. The absolute configuration of the two enantiomers is not known.

Analytical data for AS0616: Yield: 45% (45 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.26 (s, 1H), 7.71 (d, J=12.4 Hz, 1H), 7.51 (s, 1H), 7.26 (t, J=8.4 Hz, 2H), 7.08 (d, J=7.6 Hz, 2H), 6.92 (t, J=7.2 Hz, 1H), 6.83 (s, 1H), 5.46 (d, J=12.0 Hz, 1H), 3.85-3.60 (m, 2H), 3.45-3.35 (m, 2H), 2.23 (s, 3H), 1.54-1.48 (m, 1H), 1.33-1.16 (m, 3H), 1.02 (s, 3H), 0.75-0.73 (m, 3H). LC-MS: (Method A) 462.0 (M ⁺⁺ H), Rt: 2.59 min, 96.53% (max). HPLC: (Method B) Rt: 5.30 min, 97.11% (max). Chiral SFC: (Method H) Rt: 3.69 min, 99.03% (max).

Analytical data for AS0617: Yield: 43% (43 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.25 (s, 1H), 7.71 (d, J=12.0 Hz, 1H), 7.51 (s, 1H), 7.26 (t, J=8.4 Hz, 2H), 7.08 (d, J=7.6 Hz, 2H), 6.92 (t, J=7.2 Hz, 1H), 6.83 (s, 1H), 5.46 (d, J=12.4 Hz, 1H), 4.11-3.65 (m, 2H), 3.50-3.35 (m, 2H), 2.23 (s, 3H), 1.55-1.45 (m, 1H), 1.33-1.21 (m, 3H), 1.02 (s, 3H), 0.75-0.73 (m, 3H). LC-MS: (Method E) 461.8 (M ⁺⁺ H), Rt: 2.50 min, 97.16% (max). HPLC: (Method B) Rt: 5.30 min, 97.07% (max). Chiral SFC: (Method H) Rt: 4.83 min, 99.67% (max).

Example 7

(Z)-2-Fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid

To a stirred solution of ethyl (Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 17; 0.14 g, 0.27 mmol) in a mixture of 1,4-dioxane and water (4:1, 3 ml) was added lithium hydroxide (0.023 g, 0.55 mmol) and the reaction mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was acidified with dilute HCl (1.5 N, pH ca. 4) and diluted with ice-cold water (5 ml). The aqueous layer was extracted with EtOAc (2x10 mL) and the combined organic layer was washed with water (10 mL) and brine (10 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo to afford the title compound. Yield: 84% (0.11 g, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.59 (s, 1H), 7.58-7.56 (m, 1H), 7.53 (s, 1H), 7.25 (t, J=8.4 Hz, 2H), 7.04 (d, J=7.6 Hz, 2H), 6.90 (t, J=7.2 Hz, 1H), 6.83 (s, 1H), 3.69 (bs, 2H), 3.40 (s, 2H), 2.25 (s, 3H), 1.45 (bs, 1H), 1.31-1.24 (m, 3H), 1.02 (s, 3H), 0.75-0.73 (m, 3H). LC-MS: (Method E) 480.2 (M ⁺⁺ H), Rt: 2.85 min, 97.16% (max). HPLC: (Method B) Rt: 5.43 min, 95.69% (max).

Examples 8 and 9

(R)-(2)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid and (S)-(Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid

The two enantiomers of racemic (Z)-2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid (Example 7; 100 mg, 0.11 mmol) were separated by chiral SFC (Method H). This material was concentrated in vacuo at 40°C. The first eluting fraction corresponded to enantiomer 1 and the second eluting fraction corresponded to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Enantiomer 1: Yield: 38% (38 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.55 (s, 1H), 7.61-7.57 (m, 2H), 7.25 (t, J=8.0 Hz, 2H), 7.05 (d, J=7.6 Hz, 2H), 6.90 (t, J=7.6 Hz, 1H), 6.84 (s, 1H), 3.99-3.71 (m, 2H), 3.45-3.35 (m, 2H), 2.25 (s, 3H), 1.55-1.43 (m, 1H), 1.35-1.24 (m, 3H), 1.02 (s, 3H), 0.77-0.65 (m, 3H). LC-MS: (Method E) 479.8 (M ⁺ ), Rt: 2.45 min, 98.61% (max). HPLC: (Method B) Rt: 5.43 min, 97.96% (max). Chiral SFC: (Method E) Rt: 5.08 min, 100% (max).

Enantiomer 2: Yield: 30% (30 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.57 (s, 1H), 7.62 (m, 2H), 7.25 (t, J=8.0 Hz, 2H), 7.05 (d, J=7.2 Hz, 2H), 6.90 (t, J=7.6 Hz, 1H), 6.84 (s, 1H), 4.01-3.72 (m, 2H), 3.45-3.35 (m, 2H), 2.25 (s, 3H), 1.54-1.44 (m, 1H), 1.31-1.23 (m, 3H), 1.02 (s, 3H), 0.76-0.69 (m, 3H). LC-MS: (Method E) 479.8 (M ⁺ ), Rt: 2.45 min, 98.49% (max). HPLC: (Method B) Rt: 5.43 min, 96.61% (max). Chiral SFC: (Method E) Rt: 6.66 min, 100% (max).

Example 10

(Z)-3-((3-ethyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid

To a stirred solution of ethyl (Z)-3-((3-ethyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 24; 220 mg, 0.58 mmol) in a mixture of 1,4-dioxane and water (4:1, 5 ml) was added lithium hydroxide (50 mg, 1.17 mmol) and the reaction mixture was stirred for 1 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 2 ml, pH ca. 4) and concentrated in vacuo. The resulting residue was partitioned between ice-cold water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2x5 mL) and the combined organic layer was washed with ice-cold water (5 mL) and brine (5 mL). The organic portion was dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was triturated with hexane to afford the title compound. Yield: 22% (60 mg, pale yellow solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.59 (s, 1H), 7.63-7.58 (m, 2H), 7.24 (t, J=8.4 Hz, 2H), 7.02 (d, J=7.6 Hz, 2H), 6.90-6.86 (m, 2H), 3.75 (s, 2H), 3.17 (s, 2H), 2.26 (s, 3H), 1.65-1.48 (m, 1H), 1.34-1.29 (m, 1H), 1.00 (s, 3H), 0.79 (t, J=7.6 Hz, 3H). LC-MS: (Method E) 466.1 (M ⁺⁺ H), Rt: 2.36 min, 99.05% (max). HPLC: (Method B) Rt: 5.15 min, 96.39% (max).

BIOLOGICAL ANALYSES

IBAT Analysis Protocol (h/m)

10,000 cells (human or mouse IBAT overexpressing cells) were seeded in 96-well plates (Corning CLS3809) in 200 μl MEM-alpha medium (Gibco 12571-063) supplemented with 10% FBS (Gibco 10438026) containing puromycin (Gibco Al 113803) (10 μg/ml) and incubated at 37°C in 5% _CO2 for 48 h. After incubation, the medium was decanted from the wells and the cells were washed twice with 300 μl MEM-alpha base medium (without FBS). After decanting the MEM-alpha base medium, the plates were tapped on a paper towel each time to ensure maximum removal of residual medium. Dilutions of the test inhibitor (maximum test concentration 10 μM, 3-fold serial dilution, 10 points) prepared in DMSO (Sigma D2650) were added to the incubation mixture (maintaining a final DMSO concentration of 0.2%) containing 0.25 μM 3H-taurocholic acid (ARC ART-1368) and 5 μM chilled taurocholic acid (Sigma T4009). Then 50 μl of the incubation mixture containing the test inhibitors (in duplicate) were added to the wells and the plates were incubated for 20 min in a CO ₂ incubator at 37°C. After incubation, the reaction was stopped by keeping the plates on ice-water for 2-3 minutes, and then the incubation mixture was completely aspirated from the wells. Wells were washed twice with 250 µl of chilled unlabeled 1 mM taurocholic acid dissolved in HEPES (Gibco 15630080)-buffered (10 mM) HBSS (Gibco 14175079) (pH 7.4). Plates were tapped on a paper towel after each wash to ensure maximum removal of blocking buffer.

100 µl MicroScint-20 (PerkinElmer 6013621) was added to the wells and left overnight at room temperature, and then the plates were read on a PerkinElmer TopCount NXT™ Microplate Scintillation and Luminescence Counter using the 3H Test protocol (reading time per well set at 120 seconds).

LBAT Protocol (h/m)

20,000 cells (human or mouse LBAT overexpressing cells) were seeded in a 96-well plate (Corning CLS3809) in 100 μl MEM-alpha medium (Gibco 12571-063) supplemented with 10% FBS (Gibco 10438026) containing Geneticin (Gibco 10131-027) (1 mg/ml) 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 MEM-alpha base medium (without FBS). After decanting the MEM-alpha base medium, the plates were tapped on a paper towel each time to ensure maximum removal of residual medium. For human LBAT, the incubation mixture was prepared by adding dilutions of the test inhibitor (3-fold serial dilution in DMSO (Sigma D2650), 10 points) to MEM-alpha (without FBS) containing 0.3 μM 3H-taurocholic acid (ARC ART-1368) and 7.5 μM chilled taurocholic acid (Sigma T4009) (maintaining a final DMSO concentration of 0.2%). For mouse LBAT, the incubation mixture was prepared by adding dilutions of the test inhibitor (3-fold serial dilution in DMSO, 10 points) to MEM-alpha (without FBS) containing 0.3 μM 3H-taurocholic acid and 25 μM chilled taurocholic acid, maintaining a final DMSO concentration of 0.2%.

Then 50 μl of the incubation mixture containing the inhibitors to be tested (in duplicate) were added to the wells and the plates were incubated for 20 min in a _CO2 incubator at 37°C. After incubation, the reaction was stopped by keeping the plates on ice-water for 2-3 min, and then the incubation mixture was completely aspirated from the wells. The wells were washed twice with 250 μl of chilled unlabeled 1 mM taurocholic acid dissolved in HEPES (Gibco 15630080)-buffered (10 mM) HBSS (Gibco 14175079) (pH 7.4). The plates were tapped on a paper towel after each wash to ensure maximum removal of the blocking buffer.

100 µl MicroScint-20 (PerkinElmer 6013621) was added to the wells and left overnight at room temperature, then the plates were read on a PerkinElmer TopCount NXT™ Microplate Scintillation and Luminescence Counter using the 3H Test protocol (reading time per well was 120 seconds, with the plate in normal orientation).

Bidirectional permeability assay (Caco-2 cells)

Caco-2 cells (Evotec) were seeded at a density of 70,000 cells/well in 24-well Millicell® cell culture plates with inserts and incubated (37°C, 5% CC, 95% RH) for 21 days with medium change every other day.

Stock solutions (10 mM) of test compounds, atenolol (low permeability marker), propranolol (high permeability marker), and digoxin (substrate for the P-gp transport pathway) were prepared in dimethyl sulfoxide (DMSO). An intermediate stock solution (1 mM) was prepared by diluting 10 μl of 10 mM stock solution with 90 μl of neat DMSO. A working stock solution (10 μM) was prepared by diluting 50 μl of 1 mM in 4950 μl of FaSSIF buffer. After addition of compounds to FaSSIF, samples were sonicated for 2 h and centrifuged at 4000 rpm for 30 min at 37°C. 4 ml of the resulting supernatant was used directly in the assay. The final concentration of DMSO in the transport experiments was 1%.

On the day of analysis, Caco-2 monolayers were washed twice with transfer buffer (HBSS, pH 7.4) and pre-incubated for 30 min (37°C, 5% CO ₂ , 95% RH) in an incubator. The electrical resistance of the monolayers was measured using the Millicell® - ERS system. Monolayers with transepithelial electrical resistance (TEER) values greater than 350 Ohm⋅cm ² were selected for analysis.

The assay was performed in the absorption (A2B) and secretion (B2A) directions. Transfer experiments were initiated by adding assay transport buffer (FaSSIF buffer prepared in HBSS) consisting of compounds to the donor compartment (apical chamber A-B; basolateral chamber B-A) in duplicate (n=2) wells. Drug-free HBSS buffer (pH 7.4) containing 1% bovine serum albumin (BSA) was added to the receiver compartments (A-B-basolateral; B-A-apical). The volumes of the apical and basolateral compartments were 0.4 and 0.8 ml, respectively. After addition of the dosing solution, the plates were incubated for 120 min at 37°C. After 120 min, donor and recipient samples were collected and matrix matched (1:1, 30 µl test sample + 30 µl blank buffer) with the counter buffer. Pipetting samples were matrix matched (1:1, 30 µl test sample + 30 µl blank buffer) with the counter buffer. Samples were processed by adding acetonitrile containing the internal standard (60 µl test sample + 200 µl acetonitrile containing the internal standard tolbutamide, 500 ng/mL). Samples were vortexed and centrifuged at 4000 rpm for 10 min. The resulting supernatant (100 µl) was diluted with 100 µl water and transferred to fresh 96-well plates. Compound concentrations in samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a research-grade bioanalytical method where applicable.

The mean apparent permeability (Papp, x10 ^-6 cm/sec) of the test compounds, atenolol, propranolol and digoxin, was calculated as follows:

where dq/dt is the transfer rate (the rate of transfer of the compound into the receiving compartment), CO is the initial concentration in the donor compartment, and A is the surface area of the effective filter membrane.

HepaRG-based assay protocol

A cryopreserved flask of differentiated HepaRG cells (Biopredic International HPR116080) was thawed in HepaRG Thaw/Plant/General Purpose Medium (Biopredic International ADD670C) supplemented with 200 mM Glutamax (Gibco 35050061) according to the protocol provided by Biopredic International. 70,000 cells per well were seeded in a 96-well plate (Corning CLS3809) in 100 μl of HepaRG Thaw/Plant/General Purpose Medium supplemented with 200 mM Glutamax and incubated at 37°C in 5% _CO2 for 24 hours. After incubation, the seeding medium was replaced with HepaRG Maintenance/Metabolism Medium (Biopredic International ADD620C) and incubated for 6 days with fresh HepaRG Maintenance/Metabolism Medium every 48 hours. After 7 days of incubation after seeding, the incubation medium was discarded from the wells and the cells were washed twice with 250 μl of William's E Basal Medium (Gibco 12551032). Each time after decanting the William's E Basal Medium, the plates were tapped on a paper towel to ensure maximum removal of residual medium. The incubation mixture was prepared by adding dilutions of the inhibitor to be tested (3-fold serial dilution in DMSO (Sigma D2650)) to William's E (basal) medium containing 0.3 μM 3H-taurocholic acid (ARC ART-1368) and 7.5 μM chilled taurocholic acid (Sigma T4009) (maintaining a final concentration of 0.2% DMSO). Then, 50 μl of the incubation mixture containing the inhibitors to be tested were added to the wells (in duplicate), and the plates were incubated for 30 min in an incubator with 5% CO ₂ at 37°C. After incubation, the reaction was stopped by keeping the plates on ice-water for 2-3 min, and then the incubation mixture was completely aspirated from the wells. Wells were washed twice with 250 μl of chilled unlabeled 1 mM taurocholic acid dissolved in HEPES (Gibco 15630080)-buffered (10 mM) HBSS (Gibco 14175079) (pH 7.4). After each wash, plates were tapped on a paper towel to ensure maximum removal of blocking buffer.

100 µl MicroScint-20 (PerkinElmer 6013621) was added to the wells and incubated overnight at room temperature, then the plates were read on a PerkinElmer TopCount NXT™ Microplate Scintillation and Luminescence Counter using the 3H Test protocol (reading time per well was 120 seconds, with the plate in normal orientation).

Preparation of dilutions of test compounds

All test compounds were provided in powder form at room temperature. 10 mM stocks of test compounds were prepared in DMSO, aliquoted, and stored at -20°C. From the 10 mM DMSO stocks of compounds, 3-fold serial dilutions were prepared in DMSO to give a total of 10 dilutions of test compounds. 0.5 μl of this DMSO dilution was added to 250 μl of FBS-free basal medium containing 3H-taurocholic acid and chilled taurocholic acid to prepare the incubation mixture.

Bioavailability studies

Male mice (C57BL/6 or CD1) or Wistar rats aged 8-9 weeks were used. For each test compound, two groups of 3 animals each were used. One group was administered a single intravenous dose of 1 mg/kg (100% DMSO vehicle) via the tail vein, and the other group was administered a single oral dose of 10 mg/kg via a gastric needle. The group that received the oral dose was fasted overnight. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intravenous administration and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral administration. Blood was collected from the saphenous vein. 0.2% EDTA was used as an anticoagulant. Samples were analyzed using a research grade bioanalytical method designed to evaluate test compound in plasma using an LC-MS/MS system.

Results

Biological data for the Example compounds are shown in Table 8 below.

Bile acid excretion in urine in dogs

Male beagle dogs were used in this study. Animals were administered Example 2 once daily for three days at doses of 10 and 50 mg/kg, respectively. Baseline urine samples were collected prior to the study by cystocentesis. Urine samples were then collected at 2 h and 8 h post-dose on days 1 and 3. Urine analysis for bile acids and creatinine was performed in the clinical chemistry laboratory.

The results are shown in Fig. 1. It was found that the total amount of bile acids in urine increased by approximately 5-10 times compared to the baseline level after treatment with the compound of Example 2.

PD Model: Evaluation of the Effect of Test Compound on Total Bile Acid Levels in Male C57BL6 Mice

C57BL/6N Tac mice 8–9 weeks old were used to study the effects of bile acid modulators on bile acid levels. After completion of the quarantine and acclimation period, animals were randomized by body weight into x experimental groups: (1) control, vehicle, and (2) test compound at 1 mg/kg p.o. Animals were treated with the test compound for 7 days. On day 5 of the study, animals were individually placed in fresh cages. On day 7, feces were collected from each cage, followed by blood collection from each animal via the retro-orbital route. Animals were sacrificed to collect the liver and terminal ileum from each animal for further analysis. Body weight and food intake were measured twice weekly. Serum lipid profiles were analyzed in day 7 serum samples. Total serum bile acids were measured in day 7 serum samples. Faecal bile excretion is measured in the day 7 stool sample. Hepatic CYP7A1 and SHP expression are quantified in the day 7 liver samples. Hepatic triglycerides and total cholesterol are analyzed in the day 7 liver samples.

Urinary bile acid model: evaluation of test compounds on urinary bile acid levels in male C57BL/6 mice.

C57BL/6N Tac mice 8–9 weeks old were used to study the effects of bile acid modulators on bile acid levels. After completion of the quarantine and acclimation period, animals were randomized by body weight into x experimental groups: (1) control, vehicle, and (2) test compound at 1 mg/kg p.o. Animals were administered the test compound for 7 days. On day 6 of the study, animals were transferred to a metabolic cage. On day 7, feces and urine were collected from each metabolic cage, followed by blood collection from each animal via the retro-orbital route. Animals were sacrificed to collect kidneys from each animal for further analysis. Body weight was measured twice a week. Total serum bile acids were measured in day 7 serum samples. Fecal bile acid excretion was measured in day 7 stool sample. Urinary bile acid excretion was measured in day 7 sample. Renal expression of ASBT, OSTa, OSTAb and MRP2 was quantified in day 7 samples.

Claims (18)

1. A compound selected from the group consisting of: (Z) -3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; (R)- (Z) -3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; (S)- (Z) -3-((3-butyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; (E) -3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (S)- (E) -3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (R)- (E) -3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (Z) -2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (R)- (Z) -2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (S)- (Z) -2-fluoro-3-((3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-3-propyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; and (Z) -3-((3-ethyl-3-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition having the activity of inhibiting the apical sodium-dependent bile acid transporter (ASBT) and/or the hepatic bile acid transporter (LBAT), containing a therapeutically effective amount of the compound according to claim 1 and one or more pharmaceutically acceptable excipients. 3. Use of a compound according to claim 1 as a medicinal product having activity of inhibiting the apical sodium-dependent bile acid transporter (ASBT) and/or the hepatic bile acid transporter (LBAT). 4. The use according to claim 3, wherein the medicinal product is intended for the treatment or prevention of a cardiovascular disease or a disorder of fatty acid metabolism and glucose utilization, such as hypercholesterolemia; disorders of fatty acid metabolism; diabetes mellitus type 1 and 2; complications of diabetes, preferably cataract, micro- 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 arterial occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmias and vascular restenosis; diabetes-related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated levels of fatty acids or glycerol in the blood, obesity, dyslipidemia, hyperlipidemia, preferably hypertriglyceridemia, metabolic syndrome (syndrome X), atherosclerosis and hypertension; and to increase high-density lipoprotein levels. 5. The use according to claim 3, wherein the medicinal product is intended for the treatment or prevention of a disease or disorder of the gastrointestinal tract, such as constipation, preferably chronic constipation, functional constipation, chronic idiopathic constipation (CIC), intermittent/sporadic constipation, constipation due to diabetes mellitus, constipation due to stroke, constipation due to chronic kidney disease, constipation due to multiple sclerosis, constipation due to Parkinson's disease, constipation due to systemic sclerosis, drug-induced constipation, irritable bowel syndrome with constipation (IBS-C), mixed irritable bowel syndrome (IBS-M), childhood functional constipation and opioid-induced constipation; Crohn's disease; primary bile acid malabsorption; irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ileal reflux disease and its complications, preferably Barrett's esophagus, bile reflux esophagitis and bile reflux gastritis. 6. The use according to claim 3, wherein the medicinal product is intended for the treatment or prevention of a liver disease or disorder, such as an inherited disorder of liver metabolism; congenital errors of bile acid synthesis; congenital anomalies of the bile ducts; biliary atresia; biliary atresia after the Kasai operation; post-transplant biliary atresia; neonatal hepatitis; neonatal cholestasis; hereditary forms of cholestasis; cerebrotendinous xanthomatosis; secondary defect of bile acid synthesis (BA); Zellweger syndrome; liver disease associated with cystic fibrosis; alpha1-antitrypsin deficiency; Alagille syndrome (ALGS); Byler syndrome; primary defect of bile acid synthesis (BA); progressive familial intrahepatic cholestasis (PFIC), preferably PFIC-1, PFIC-2, PFIC-3 and non-specific PFIC, post-biliary diversion PFIC and post-transplant PFIC; benign recurrent intrahepatic cholestasis (BRIC), preferably BRIC1, BRIC2 and non-specific BRIC, post-biliary transplant BRIC and post-liver transplant BRIC; autoimmune hepatitis; primary biliary cirrhosis (PBC); liver fibrosis; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); portal hypertension; cholestasis; cholestasis in Down syndrome; drug-induced cholestasis; intrahepatic cholestasis of pregnancy (jaundice in pregnancy); intrahepatic cholestasis; extrahepatic cholestasis; parenteral nutrition-associated cholestasis (PNAC); Low phospholipid cholestasis; Cholestasis with lymphedema syndrome 1 (LCS1); Primary sclerosing cholangitis (PSC); Immunoglobulin G4-associated cholangitis; Primary biliary cholangitis; Cholelithiasis (gallstones); Biliary lithiasis; Choledocholithiasis; Gallstone pancreatitis; Caroli disease; Malignancy of the bile ducts; Malignancy causing obstruction of the biliary tree; Biliary strictures; AIDS-associated cholangiopathy; Ischemic cholangiopathy; Pruritus due to cholestasis or jaundice; Pancreatitis; Chronic autoimmune liver disease leading to progressive cholestasis; Hepatic steatosis; Alcoholic hepatitis; Acute fatty liver disease; Fatty liver of pregnancy; Drug-induced hepatitis; iron overload disorders; congenital defect of bile acid synthesis type 1 (BAS defect type 1); drug-induced liver injury (DILI); liver fibrosis; congenital liver fibrosis; liver cirrhosis; Langerhans cell histiocytosis (LCH); neonatal ichthyosis, sclerosing cholangitis (NISCH); erythropoietic protoporphyria (EPP); idiopathic ductopenia in adulthood (IAD); idiopathic neonatal hepatitis (INH); nonsyndromic interlobular bile duct insufficiency (NS PILBD); North American Indian childhood cirrhosis (NAIC); liver sarcoidosis; amyloidosis; necrotizing enterocolitis; Serum bile acid toxicity, preferably cardiac arrhythmia, more preferably atrial fibrillation, with abnormal serum bile acid profiles, cardiomyopathy associated with liver cirrhosis ("cholecardia"), and skeletal muscle wasting associated with cholestatic liver disease; polycystic liver disease; viral hepatitis, preferably hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E; hepatocellular carcinoma (hepatoma); cholangiocarcinoma; bile acid-related gastrointestinal cancer; and cholestasis due to tumors and neoplasms of the liver, biliary tract, and pancreas; or in augmentation of corticosteroid therapy in liver disease. 7. The use according to claim 3, wherein the medicinal product is intended for the treatment or prevention of hyperabsorption syndromes, preferably abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicron retention disease (CRD) and sitosterolemia; hypervitaminosis and osteopetrosis; hypertension; glomerular hyperfiltration; polycystic kidney disease (PKD), preferably autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD); and pruritus in renal failure; or in the protection against kidney damage associated with liver disease or metabolic disease.