RU2827704C1 - Benzothia(di)azepine compounds and use thereof as bile acid modulators - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, which have the activity of inhibiting apical sodium-dependent bile acid transporter (ASBT) and/or hepatic bile acid transporter (LBAT). In formula (I) M is selected from -CH₂- and -NR⁷-; R¹ is C_1-4alkyl; R² is independently selected from the group consisting of hydrogen and halogen; n is equal to integer 1, 2 or 3; R³ is selected from the group consisting of C_1-4alkoxy, C_3-6cycloalkyloxy, C_1-4alkylthio and C_3-6cycloalkylthio; one of R⁴ and R⁵ is carboxyl, and the other of R⁴ and R⁵ is selected from the group consisting of hydrogen and fluorine; R⁶ is hydrogen and R⁷ is selected from the group consisting of hydrogen and C_1-4alkyl. Invention also relates to a pharmaceutical composition containing said compound and use thereof as a drug possessing the activity of inhibiting apical sodium-dependent bile acid transporter (ASBT) and/or hepatic bile acid transporter (LBAT).

EFFECT: disclosed are benzothia(di)azepine compounds and use thereof as bile acid modulators.

14 cl, 4 dwg, 8 tbl, 62 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Indian Patent Application No. 201911049982, 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 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, pp. 1043–1071); Chiang (J. Lipid Res. 2009, vol. 50, pp. 1955–1966); and Dawson (Handb. Exp. Pharmacol. 2011, vol. 201, pp. 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, W O 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 serum bile acid (BA) levels in OATP KO mice following treatment with the compound of Example 3.

Fig. 2 shows serum bile acid (BA) levels in OATP KO mice following treatment with the compound of Example 6.

Fig. 3 shows serum bile acid (BA) levels in OATP KO mice following treatment with the compound of Example 13.

Fig. 4 shows serum bile acid (BA) levels in OATP KO mice following treatment with the compound of Example 15.

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 _1-6 alkylcarbonylamino, C _3-6 cycloalkylcarbonylamino, N-(C _1-4 alkyl)aminocarbonyl, N,N-di(C _1-4 alkyl)aminocarbonyl, C _1-4 alkyloxycarbonylamino, C _3-6 cycloalkyloxycarbonylamino, 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 selected from the group consisting of hydrogen, fluoro, chloro, bromo, hydroxy, methoxy, amino, methylamino, dimethylamino, isopropylcarbonylamino, tert-butylcarbonylamino, tert-butylaminocarbonyl, tert-butoxycarbonylamino, methylsulfonamido and cyclopropylsulfonamido. In a preferred embodiment, n is 1, i.e. the phenyl ring is substituted with only one R ² substituent. 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 a 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 C _2-4 alkyl, more preferably n-propyl or n-butyl;

R ² is independently selected from the group consisting of hydrogen, fluoro, 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.

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 pharmaceutically acceptable salts thereof:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(S)-(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(R)-(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(S)-(E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(R)-(E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(S)-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(R)-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

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

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

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

(E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

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

(E)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid;

(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid;

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

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

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

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

(Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid;

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

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

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

(Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; and

(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic 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 "C1-6alkyl" refers to a straight or branched chain alkyl group having from 1 to 6 carbon atoms, and the term " _C1-4alkyl " refers to a straight or branched chain alkyl group having from 1 to 4 carbon atoms. Examples of _C1-4alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and mpem-butyl.

As used herein, the term "C _1-4 haloalkyl" refers to a straight or branched chain C _1-4 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 straight or branched chain C _1-4 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-(C _1-4 alkyl)amino" and "N,N-di(C _1-4 alkyl)amino" refer to an amino group in which one or both hydrogen atoms, respectively, are replaced by a C _1-4 straight-chain or branched-chain alkyl group. Examples of N-(C _1-4 alkyl)amino include methylamino, ethylamino, and tert-butylamino, and examples of N,N-di(C _1-4 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 stricture; 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 setting 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 liver 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, pp. 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. Elevated 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, pp. 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, pp. 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 includes 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 may 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 ATP8 B1 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, the NR1H4 gene, or the Myo5b gene have been suggested to cause 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 ATP8B1 gene or resulting protein are listed in Tables 2 and 3, numbered based on the human wild-type ATP8B1 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. D1, pp. D365–D371, which is incorporated herein by reference in its entirety.

Canonical sequence of ATP8B1 protein (SEQ ID NO: 1) - Uniprot ID 043520

Canonical DNA sequence for ATP8B1 (SEQ ID NO: 2)

^A Mutation in "X" denotes an early stop codon.

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In some embodiments, the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, I66IT, E665X, R930X, R952X, R1014X, and G1040R.

Canonical sequence of ABCB11 protein (SEQ ID NO: 3) - Uniprot ID 095342

Canonical DNA sequence ABCB11 (SEQ ID NO: 4)

^A Mutation in "X" denotes an early stop codon.

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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, I661T, E665X, R930X, R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11 is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N59IS, A865V, G982R, R1153C, and R1268Q.

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, I661T, E665X, R930X, R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11 is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V, G982R, R1153C, and R1268Q.

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, fat accumulation 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 beneath 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 comprises 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), pp. 1585-1596; Lv et al., J Clin Transl Hepatol. 2018, vol. 6(2), pp. 217-221; Reeder et al., J Magn Reson Imaging. 2011, vol. 34(4), spcone; and de Ledinghen 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 greater than about 5% hepatic steatosis, 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, a subject with greater than about 5% to about 33% hepatic steatosis has stage 1 hepatic steatosis, a subject with from about 33% to about 66% hepatic steatosis has stage 2 hepatic steatosis, and a subject with greater than about 66% hepatic steatosis 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 with 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 Brunt et al., Am. J. Gastroenterol. 1999, vol. 94, pp. 2467-2474, the fibrosis scoring system in Kleiner et al., Hepatology 2005, vol. 41(6), pp. 1313-1321, and the ISHAK fibrosis scoring system (see Ishak et al., J. Hepatol. 1995, vol. 22, pp. 696-699), the contents of 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., Evaluation of Generalized Liver Fibrosis (ELF), 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 levels of liver inflammation biomarkers, 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 Col1a1, Col1a2, 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 a change in macrophage infiltration, such as a change in the expression level of CD68. 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 NASH treatment. 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 the 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 serum bile level 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

Hyaluronic 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 the 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. Col1a1, Col1a2, and Col4a1)

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%.

Following 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%.

Certain compounds of formula (I) or pharmaceutically acceptable salts thereof may exhibit improved or optimal permeability. Permeability can be measured in Caco2 cells and the values are given as Papp (apparent permeability) values 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. 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 AS BT 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, 6a-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 and PLN-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 inhibitor of SGLT1 and SGLT2. 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 Klothoβ (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:

Device name: Agilent 1290 infinity P.

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 (50×4.6 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 (50×4.6 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 (50×4.6 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 C18 (50×4.6 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 (50×4.6 mm), 3.5 μm.

Method F: Mobile phase: A: 0.1% TFA in water, B: 0.1% TFA in ACN; flow rate: 0.8 ml/min; column: ZORBAX ECLIPSE PLUS C18 (50×2.1 mm), 1.8 µm.

Method G: Mobile phase: A: 0.1% TFA in water, B: 0.1% TFA in ACN; flow rate: 0.8 mL/min; column: Acquity UPLC BEH C18 (2.1×50 mm), 1.7 µm.

Method H: Mobile phase: A: 10 mM _NH4OAc , B: 100% ACN; flow rate: 0.8 ml/min; column: Acquity UPLC BEH C18 (2.1×50) mm; 1.7 µm.

Method I: Mobile phase: A: 0.1% HCOOH in water: ACN (95:5), B: ACN; flow rate: 0.8 ml/min; column: ZORBAX ECLIPSE PLUS C18 (2.1×50) mm, 1.8 µm.

Method J: Mobile phase: A: 0.1% TFA in water, B: ACN; flow rate: 1.0 mL/min; Column: Zorbax Extend C18 (50×4.6 mm), 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 (2.1×50) 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 (50×4.6 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 (50×4.6 mm, 3.5 μm).

Method C: Mobile phase: A: 10 mM _NH4OAc in Milli-q water, B: ACN; flow rate: 1.0 ml/min; column: Phenomenex Gemini C18 (150×4.6 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 (250×4.6 mm, 5.0 μm).

Method E: Mobile phase: A: 0.1% TFA in water, B: ACN, flow rate: 2.0 ml/min; column: X-Bridge C8 (50×4.6 mm, 3.5 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 IPA; flow rate: 3 mL/min; Column: YMC Amylose-SA (250×4.6 mm, 5 μm).

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

Method C: Mobile phase: 20 mM ammonia in methanol; flow rate: 3 ml/min; column: YMC Cellulose-SC (250×4.6 mm, 5 μm).

Method D: Mobile phase: methanol; flow rate: 3 ml/min; column: Lux A1 (250×4.6 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 A1.

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

Method I: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3 ml/min; column: Chiral CCS (250×4.6 mm, 5 μm).

Method J: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 5 mL/min; Column: YMC Cellulose-SC AD-H (250×4.6 mm, 5 μm).

Method K: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 4 ml/min; column: (R,R)-Whelk-01 (250×4.6 mm, 5 μm).

Method L: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 ml/min; column: Chiralcel OX-H (250×4.6 mm, 5 µm).

Method M: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 5 mL/min; Column: YMC Cellulose-SC (250×4.6 mm, 5 μm).

Method N: Mobile phase: methanol, flow rate: 5 ml/min; column: Chiralcel OX-H (250×4.6 mm, 5 µm).

Method O: Mobile phase: 0.1% isopropylamine in IPA:methanol (1:1), flow rate: 3 ml/min; Column: Chiralpak AS-H (250×4.6 mm, 5 µm).

Method P: Mobile phase: 0.5% isopropylamine in methanol, flow rate: 3 ml/min; column: Chiralpak AS-H (250×4.6 mm, 5 µm).

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

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

Method S: Mobile phase: 0.5% isopropylamine in methanol, flow rate: 3 ml/min; column: Chiralpak OX-H (250×4.6 mm, 5 µm).

Method T: Mobile phase: 0.5% isopropylamine in IPA, flow rate: 4 ml/min; Column: YMC Cellulose-SB (250×4.6 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 (50×4.6 mm, 3.5 μm).

Method B: Mobile phase: A: 10 mM NH ₄ OAC in water; B: ACN; flow rate: 35 ml/min; column: X select C18 (30×150 mm, 5 μm).

Method C: Mobile phase: A: 10 mM _NH4HCO3 in water; B: ACN; flow rate: 1.0 ml _/ min; column: XBridge C8 (50×4.6 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 (30×150 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 (250×30 mm, 5 μm).

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

Method C: Mobile phase: 20 mM ammonia in methanol; flow rate: 3 ml/min; column: YMC Cellulose-SC (250×30 mm, 5 μm).

Method D: Mobile phase: methanol; flow rate: 3 ml/min; column: Chiral CCS (250×30 mm, 5 µm).

Method E: Mobile phase: methanol; flow rate: 3 ml/min; column: Lux A1 (250×30 mm, 5 µm).

Method F: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 ml/min; column: Lux A1 (250×30 mm, 5 µm).

Method G: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3 ml/min; Column: Chiral CCS (250×30 mm, 5 µm).

Method H: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 5 ml/min; column: Chiralcel OX-H (250×30 mm, 5 µm).

Abbreviations

ACN acetonitrile

BOC tert-butoxycarbonyl

DABCO 1,4-diazabicyclo[2.2.2]octane

DCM dichloromethane

DMA dimethylacetamide

DMF dimethylformamide

IPA isopropyl alcohol

LC-MS liquid chromatography - mass spectrometry

HPLC high performance liquid chromatography

PE petroleum ether

SFC supercritical fluid chromatography

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

UPLC ultra-performance liquid chromatography

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 sources of information are incorporated by reference.

EXAMPLES

Intermediate connection 1

2-(((2-Amino-5-methoxyphenyl)thio)methyl)hexanoic acid

To a stirred solution of 6-methoxybenzo[d]thiazol-2-amine (15.0 g, 0.08 mol) in water (300 mL) was added KOH (74.7 g, 1.33 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. Then, a solution of 2-(bromomethyl)hexanoic acid (20.0 g, 0.095 mol) in THF (100 mL) was added dropwise and the reaction mixture was stirred for 48 h at room temperature. After the consumption of the starting material (monitored by LC-MS), the reaction mixture was concentrated to remove THF. The reaction mixture was then cooled to 0 °C and acidified with concentrated HCl (pH ca. 2). The aqueous layer was extracted with DCM (2 x 200 mL) and _the combined organic layer was washed with water (100 mL) and brine (100 mL). The organic portion was dried over anhydrous _Na2SO4 and then concentrated in vacuo. The resulting crude material was carried as received to the next step without any further purification. Yield: 19.8 g (crude, brown gum).

LC-MS: (Method A) 284.2 (M ⁺⁺ H), Rt: 1.89 min, 86.69% (max).

Intermediate connection 2

3-Butyl-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a stirred solution of 2-(((2-amino-5-methoxyphenyl)thio)methyl)hexanoic acid (Intermediate 1; 19.8 g, 0.069 mol) in EtOAc (200 mL) at 0 °C were added dropwise triethylamine (19.5 mL, 0.139 mol) and a solution of 1-propanephosphonic anhydride (50% in EtOAc; 31.1 g, 0.09 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 (2×200 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 15% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 64% (12.0 g, brown liquid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.09 (d, J=2.8 Hz, 1H), 7.09-7.01 (m, 1H), 6..971-6.94 (m, 1H), 3.75 (s, 3H), 3.46 (q, J=5.6 Hz, 1H), 2.88 (t, J=12.0 Hz, 1H), 1.69-1.67 (m, 1H), 1.23-1.11 (m, 6H ), 0.79 (t, J=6.8 Hz, 3H). LC-MS: (Method A) 266.2 (M ⁺ +H), Rt: 2.31 min, 98.96% (max).

Intermediate connection 3

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

To a stirred solution of 3-butyl-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 2; 10 g, 0.03 mol) in ACN (50 mL) and DCM (50 mL) at 0 °C was added N-bromosuccinimide (8.04 g, 0.045 mol) and the resulting reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by UPLC), the reaction mixture was concentrated in vacuo. The resulting crude material was quenched with water (100 mL) and the aqueous layer was extracted with EtOAc (2×200 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 10% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 70% (9.16 g, brown resinous solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 9.63 (s, 1H), 7.30 (s, 1H), 7.23 (s, 1H), 3.85 (s, 3H), 3.48 (q, J=6, 0 Hz, 1H), 2.93 (t, J=12.4 Hz, 1H), 2.49-2.49 (m, 1H), 1.75-1.66 (m, 1H), 1.25-1.07 (m, 5H), 0.80 (t , J=6.8 Hz, 3H). LC-MS: (Method A) 344.1 (M ⁺ ), Rt: 2.53 min, 97.48% (max).

Intermediate connection 4

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

To a stirred solution of 7-bromo-3-butyl-8-methoxy-2,3-dihydro-1,5-benzo-1,5-thiazepin-4(5H)-one (Intermediate 3; 5.5 g, 0.015 mol) in iodobenzene (55 ml) were added copper(I) iodide (0.304 g, 0.001 mol) and K ₂ CO ₃ (4.41 g, 0.031 mol) and the solution was purged with nitrogen for 20 min to degas. Tris[2-(2-methoxyethoxy)ethyl]amine (1.0 g, 0.003 mol) was then added under nitrogen and the resulting reaction mixture was heated for 16 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 (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: 6% EtOAc/PE; silica gel: 230-400 mesh) to afford a mixture of the title compounds. Yield: 66% (4.43 g, brown gum; overall yield).

LC-MS: (Method A) 420.0 (M ⁺ ), Rt: 3.09 min, 49.30% (max) for 7-bromo compound and 468.0 (M ⁺⁺ H), Rt: 3.15 min, 47.54% (max) for 7-iodo compound.

Intermediate connection 5

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

To a stirred solution of a mixture of 7-bromo-3-butyl-8-methoxy-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 3-butyl-7-iodo-8-methoxy-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 4; 7.0 g, 0.016 mol) in THF (70 ml) at 0°C was added dropwise borane dimethyl sulfide (2 M in THF; 25 ml, 0.049 mol) 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 resulting 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 is to the next step without any further purification. Yield: 7.2 g (crude, white gum). LC-MS: (Method A) 406.1 (M ⁺ ), Rt: 2.47 min, 57.90% (max) for 7-bromo compound, 454.1 (M ⁺⁺ H), Rt: 2.66 min, 40.08% (max) for 7-iodo compound.

Intermediate connection 6

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

To a stirred solution of a mixture of 7-bromo-3-butyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 3-butyl-7-iodo-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine (Intermediate 5; 7.2 g, 0.02 mol) in THF (50 mL) and water (22 mL) was added oxone (54.45 g, 0.177 mol) 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 (2×200 mL). The combined organic layer was washed with water (100 mL) and brine (100 mL), dried over _{anhydrous Na2SO4} and then concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 7% EtOAc/PE; silica gel: 230-400 mesh) to give a mixture of the title compounds. Yield: 90% (7.0 g, off-white gum, overall yield).

LC-MS: (Method E) 438.0 (M ⁺ ) for 7-bromo compound and 486.0 (M ⁺⁺ H) for 7-iodo compound, Rt: 2.75 min, 92.14% (max).

Intermediate connection 7

1,1-3-Butyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetra-1,5-hydrobenzothiazepine dioxide

To a stirred solution of a mixture of 7-bromo-3-butyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and 3-butyl-7-iodo-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 6; 3.0 g, 0.006 mol) in DMF (30 mL) was added sodium thiomethoxide (2.39 g, 0.034 mol) 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 (2×200 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: 18% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 93% (2.5 g, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.75 (s, 1H), 7.37 (s, 1H), 7.15-7.11 (m, 2H), 6.99 (s, 1H), 6.65 (t, J=7.2 Hz, 1H), 6.52 (d, J=8.4 Hz, 2H), 4. 19 (d, J=14.8 Hz, 1H), 3.37 (s, 1H), 3.25-3.18 (m, 1H), 3.07-3.01 (m, 1H), 2.34 (s, 3H), 3.00-2.22 (m, 1H), 1.39-1.28 (m, 6H), 0.91 (t, J=7.20 Hz, 3H). LC-MS: (Method A) 392.2 (M ⁺⁺ H), Rt: 2.62 min, 96.86% (max).

Intermediate connection 8

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

These two enantiomers of racemic 3-butyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 7; 500 mg, 1.27 mmol) were separated by chiral preparative SFC (Instrument: Pic SFC 100); mobile phase: _CO2 :methanol; flow rate: 4 ml/min; column: YMC Amylose-C; wavelength: 280 nm; cycle time: 5 min; back pressure: 100 bar. 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: 45% (500 mg, light brown solid). LC-MS: (Method E) 392.1 (M ⁺⁺ H), Rt: 3.048 min, 97.00% (max). SFC: (Method F) Rt: 1.77 min, 96.55% (max).

Enantiomer 2: Yield: 45% (500 mg, light brown solid). LC-MS: (Method E) 392.1 (M ⁺⁺ H), Rt: 2.907 min, 90.95% (max). SFC: (Method F) Rt: 3.65 min, 96.89% (max).

Intermediate connection 9

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

To a stirred solution of 3-butyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 7, 0.2 g, 0.51 mmol) in THF (5 mL) were added DABCO (0.06 g, 0.05 mmol) and ethyl propiolate (0.075 g, 0.76 mmol) at 0 °C and the reaction mixture was stirred for 1 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was poured into ice-cold water and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 8-9% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound: Yield: 90% (0.23 g, colorless gum).

LC-MS: (Method E) 490.2 (M ⁺⁺ H), Rt: 2.75 min, 90.08% (max).

Intermediate connection 10

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

To a stirred solution of 3-butyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (intermediate 7; 300 mg, 0.76 mmol) in DMF (5 ml) at 0 °C was added portionwise 60% NaH (99 mg, 2.49 mmol) and the reaction mixture was stirred for 15 min. Then ethyl 3-bromo-2,2-difluoropropanoate (374 mg, 1.72 mmol) was added and the reaction mixture was heated for 16 h at 60 °C. After completion of the reaction (monitored by TLC), the reaction mass was cooled to 0 °C, quenched with dilute HCl (1.5 N, pH ca. 4) and diluted with water (15 ml). The aqueous layer was extracted with EtOAc (2×50 mL), the combined organic layer was washed _with brine (20 mL) and dried over anhydrous _Na2SO4 . The organic portion was concentrated in vacuo to give the crude title compound, which was carried as received to the next step without any further purification. Yield: 0.17 g (crude, brown gummy solid).

LC-MS: (Method A) 508.1 (M ⁺⁺ H), Rt: 2.95 min, 39.38% (max).

Intermediate connection 11

Ethyl (S)-(Z)-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate and ethyl (R)-(2)-3-((3-butyl-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-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine enantiomer 1 (Intermediate 8; 500 mg, 1.27 mmol) in DMF (8 mL) at 0 °C was added 60% NaH (166 mg, 4.1 mmol) portionwise and the reaction mixture was stirred for 15 min. Then ethyl 3-bromo-2,2-difluoropropanoate (0.41 mL, 3.19 mmol) 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 1.5 N HCl, and the mixture was heated at 70 °C for 3 h. HCl (pH ca. 4) and diluted with water (15 mL). The aqueous layer was extracted with EtOAc (2×25 mL). The combined organic layer was washed with water (20 mL) and brine (20 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: 7-15% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound.

Enantiomer 2 of the title compound was prepared according to the same procedure starting from 500 mg of enantiomer 2 of Intermediate 8. The absolute configuration of these two enantiomers is not known.

Enantiomer 1: Yield: 650 mg (crude, white solid). LC-MS: (Method A) 508.2 (M ⁺⁺ H), Rt: 2.96 min, 92.04% (max).

Enantiomer 2: Yield: 92% (600 mg, white solid). LC-MS: (Method E) 508.2 (M ⁺⁺ H), Rt: 3.42 min, 96.34% (max).

Intermediate connection 12

2-(((2-Amino-5-methoxyphenyl)thio)methyl)butanoic acid

To a solution of 6-methoxybenzo[c1]thiazol-2-amine (13 g, 0.072 mol) in water (260 mL) was added KOH (64.7 g, 1.15 mol) and the reaction mixture was heated for 48 h at 120 °C. The reaction mixture was then cooled to 10 °C. A solution of 2-(bromomethyl)butanoic acid (10.4 g, 0.06 mol) in THF (60 mL) was added dropwise and the reaction mixture was heated for 16 h at 70 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched, then acidified using conc. HCl and the aqueous layer was extracted with DCM (2×150 mL). _The combined organic layer was washed with ice-cold water (150 mL) and brine (150 mL) and dried over anhydrous _Na2SO4 . The organic layer was filtered and the resulting solution was concentrated in vacuo to afford the title compound. Yield: 14.5 g (crude, brown liquid).

LC-MS: (Method E) 256.2 (M ⁺⁺ H), Rt: 1.50 min, 50.35% (max).

Intermediate connection 13

3-Ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a solution of 2-(((2-amino-5-methoxyphenyl)thio)methyl)butanoic acid (Intermediate 12; 14.9 g, 0.06 mol) in EtOAc (150 ml) was added triethylamine (16.3 ml, 0.12 mol) and the solution was cooled to 0 °C. Then a solution of 1-propanephosphonic anhydride (50% in EtOAc; 26 g, 0.08 mol) was added dropwise 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 ice-cold water (50 ml) and diluted with EtOAc (100 ml). The organic portion was washed with ice-cold 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 purified by Isolera column chromatography (eluent: 18% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 44% (6.2 g, yellow solid).

LC-MS: (Method A) 238.1 (M ⁺⁺ H), Rt: 1.96 min, 87.42% (max).

Intermediate connection 14

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

To a solution of 3-ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 13; 6.2 g, 0.03 mol) in DCM (30 ml) and ACN (30 ml) at 0 °C was added portionwise N-bromosuccinimide (5.58 g, 0.03 mol) and the reaction mixture was stirred for 4 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting residue was dissolved in EtOAc (100 ml). The organic layer was washed with ice-cold water (70 ml) and brine (100 ml) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 10% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 34% (2.8 g, pale brown solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.66 (s, 1H), 7.31 (s, 1H), 7.24 (s, 1H), 3.86 (s, 3H), 3.53-3.48 (m, 1H), 2.97-2.91 (m, 1H), 2.39-2.33 (m, 1H), 1.72-1.65 (m, 1H), 1.32-1.27 (m, 1H), 0.78-0.73 (m, 3H). LC-MS: (Method E) 315.9 (M ⁺ ), Rt: 2.44 min, 50.63% (max).

Intermediate connection 15

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

To a degassed solution of 7-bromo-3-ethyl-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 14; 1.6 g, 5.05 mmol) in iodobenzene (16 ml) were added K ₂ CO ₃ (1.4 g, 10.2 mmol), tris[2-(2-methoxyethoxy)ethyl]amine (0.32 g, 1.01 mmol) and CuI (0.1 g, 0.5 mmol) and the reaction mixture was heated for 16 h at 135 °C. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting residue was diluted with EtOAc (50 ml). The organic layer was washed with water (50 mL) and brine (50 mL) and dried over _{anhydrous Na2SO4} . The organic portion was filtered, concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 10% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 54% (1.9 g, brown gum).

LC-MS: (Method A) 394.0 (M ⁺⁺ 2), Rt: 2.82 min, 34.92% (max) for 7-bromo compound and 439.9 (M ⁺⁺ H), Rt: 2.88 min, 31.38% (max) for 7-iodo compound.

Intermediate connection 16

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

To a solution of a mixture of 7-bromo-3-ethyl-8-methoxy-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one and 3-ethyl-7-iodo-8-methoxy-5-phenyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 15; 1.9 g, 4.84 mmol) in THF (20 ml) at 0 °C was added borane dimethyl sulfide (2 M solution in THF, 7.2 ml, 14.5 mmol) and the reaction mixture was heated for 16 h at 75 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0 °C. Then methanol (10 ml) was added dropwise and the mixture was heated for 2 h at 60 °C. The reaction mixture was then cooled to room temperature and concentrated in vacuo. The resulting residue was partitioned between water (30 mL) and EtOAc (30 mL) and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with water (50 mL) and brine (50 mL) and dried over _{anhydrous Na2SO4} . The organic portion was filtered and concentrated in vacuo to afford the title compound. Yield: 2.5 g (crude, brown gum).

LC-MS: (Method A) 378.1 (M ⁺ ), Rt: 3.50 min, 14.77% (max) for 7-bromo compound and 426.1 (M ⁺⁺ H), Rt: 3.62 min, 11.01% (max) for 7-iodo compound.

Intermediate connection 17

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

To a solution of a mixture of 7-bromo-3-ethyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine and 3-ethyl-7-iodo-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine (Intermediate 16; 2.5 g, 6.60 mmol) in a mixture of THF and water (8:2, 25 ml) was added oxone (20.3 g, 66.0 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 extracted with EtOAc (2×30 ml). The combined organic layer was washed with ice-cold 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: 55% (1.5 g, brown gum).

LC-MS: (Method A) 410.0 (M ⁺ ), Rt: 2.76 min, 43.90% (max) for 7-bromo-substituted compound and 457.9 (M ⁺⁺ H), Rt: 2.81 min, 38.27% (max) for 7-iodo-substituted compound.

Intermediate connection 18

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

To a solution of a mixture of 7-bromo-3-ethyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and 3-ethyl-7-iodo-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 17; 1.5 g, 3.6 mmol) in DMF (15 mL) was added sodium thiomethoxide (1.3 g, 18.2 mmol) and the reaction mixture was stirred for 12 h at 80 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with ice-cold water (30 mL) and the aqueous layer was extracted with EtOAc (2×30 mL). _The combined organic layer was washed with water (30 mL) and brine (30 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: 20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 68% (0.9 g, off-white solid).

LC-MS: (Method E) 364.0 (M ⁺⁺ H), Rt: 2.67 min, 78.14% (max).

Intermediate connection 19

Ethyl (Z)-3-((3-ethyl-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-ethyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 18; 0.3 g, 0.82 mmol) in DMA (6 ml) at 0 °C was added 60% NaH (0.107 g, 2.68 mmol) portionwise and the reaction mixture was stirred for 30 min at 0 °C. Then a solution of ethyl 3-bromo-2,2-difluoropropanoate (0.6 ml, 2.06 mmol) in DMA (1 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 dilute HCl (1.5 N, pH ca. 4) and diluted with water (10 mL). The aqueous layer was extracted with EtOAc (2 x 15 mL). The combined organic layer was then 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: 16% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 33% (0.13 g, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.78-7.73 (m, 1H), 7.68 (s, 1H), 7.22-7.16 (m, 3H), 6.74 (t, J=7.2 Hz, 1H), 6.65 (d, J=8.0 Hz, 2H), 4.28 (q, J=7.2 Hz, 2H), 3.53-3.49 (m, 2H), 3.30-3.24 (m, 2H), 2.43 (s, 3H), 2.25-2.23 (m, 1H), 1.49-1.39 (m, 2H), 1.37-1.26 (m, 3H), 1.01-0.97 (m, 3H). LC-MS: (Method E) 480.0 (M ⁺⁺ H), Rt: 2.98 min, 94.13% (max).

Intermediate connection 20

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

To a stirred solution of 3-ethyl-8-hydroxy-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 18, 300 mg, 0.82 mmol) in THF (5 mL) were added DABCO (0.01 g, 0.08 mmol) and tert-butyl propiolate (0.16 g, 1.23 mmol) at room temperature and the reaction mixture was 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 concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 12% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 89% (0.36 g, white solid).

LC-MS: (Method E) 433.9 (M ⁺ - ^t Bu+H), Rt: 3.17 min, 89.46% (max).

Intermediate connection 21

2-((tert-Butoxycarbonyl)amino)hexanoic acid

To a solution of 2-aminohexanoic acid (25 g, 0.19 mol) in water (250 mL) and THF (250 mL) were added _NaHCO3 (48 g, 0.57 mol) and Boc anhydride (52.2 mL, 0.23 mol) and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was cooled, quenched and acidified with 1.5 N HCl. The reaction mixture was extracted with EtOAc (2×150 mL). The combined organic layer was washed with ice-cold water (150 mL) and brine (150 mL), dried over anhydrous _Na2SO4 and concentrated to give the title compound. Yield: 45.5 _g (crude, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 5.10-4.98 (m, 1H), 4.33-4.30 (m, 1H), 1.95-1.81 (m, 1H), 1.75-1.55 (m, 1H), 1.46 (s, 10H), 1.45-1.31 (m, 4H), 0.93 (t, J=7.2 Hz, 3H). LC-MS: (Method E) 132.2 (M ⁺ -Boc+H), Rt: 2.36 min, 99.98% (max).

Intermediate connection 22

tert-Butyl-(1-oxo-1-(phenylamino)hexan-2-yl)carbamate

To a stirred solution of 2-((tert-butoxycarbonyl)amino)hexanoic acid (Intermediate 21; 45.5 g, 0.196 mol) in DMF (150 mL) was added triethylamine (54.83 mL, 0.39 mol) and the reaction mixture was cooled to 0 °C. A solution of 1-propanephosphonic anhydride (50% in EtOAc; 75.1 g, 0.23 mol) and aniline (18 g, 0.196 mol) were added 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 ice-cold water (50 mL) and diluted with EtOAc (200 mL). The aqueous layer was washed with ice-cold water (200 mL) and brine (200 mL), then dried over _{anhydrous Na2SO4} and filtered. The organic portion was concentrated in vacuo to afford the title compound. Yield: 82% (49.7 g, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.93 (s, 1H), 7.60 (d, J=7.6 Hz, 2H), 7.30 (t, J=8.0 Hz, 2H), 7.06-6.99 (m, 2H), 4.07-4.02 (m, 1H), 1.63-1.56 (m, 2H), 1.46-1.28 (m, 13H), 0.86 (t, J=6.8 Hz, 3H). LC-MS: (Method E) 207.0 (M ⁺ -Boc+H), Rt: 2.69 min, 91.20% (max).

Intermediate connection 23

2-Amino-N-phenylhexanamide

To a solution of tert-butyl (1-oxo-1-(phenylamino)hexan-2-yl)carbamate (Intermediate 22; 49.7 g, 0.162 mol) in 1,4-dioxane (500 ml) at 0 °C was added a solution of HCl in 1,4-dioxane (4 M, 199 ml) and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting residue was quenched with saturated NaHCO ₃ solution. The aqueous layer was extracted with EtOAc (2×200 ml). The combined organic layer was washed with brine (200 ml) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo to yield the title compound. Yield: 95% (32 g, colorless resin).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.20-9.66 (m, 1H), 7.64 (dd, J=4.8, 2.4 Hz, 2H), 7.32-7.27 (m, 2H), 7.06-7.02 (m, 1H), 3.29-3.26 (m, 1H), 1.67-1.61 (m, 1H), 1.46-1.27 (m, 5H), 0.89-0.85 (m, 3H). LC-MS: (Method B) 207.2 (M ⁺ +H), Rt: 2.03 min, 84.36% (max).

Intermediate connection 24

N1-Pheni lhexane-1,2-diamine

To a solution of 2-amino-N-phenylhexanamide (Intermediate 23; 32 g, 0.15 mol) in THF (320 ml) at 0 °C was added borane dimethyl sulfide (2 M solution in THF, 117 ml, 0.23 mol) and the reaction mixture was heated for 16 h at 75 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to 0 °C, quenched with methanol (150 ml) and then heated for 2 h at 60 °C. The reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting residue was partitioned between water (50 ml) and EtOAc (50 ml). The aqueous layer was extracted with EtOAc (2×250 ml). The combined organic layer was washed with water (250 ml) and brine (250 ml). The organic portion was dried over _{anhydrous Na2SO4} , filtered and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 10% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 93% (28 g, yellow oil). LC-MS: (Method B) 193.3 (M ⁺⁺ H), Rt: 1.98 min, 76.9% (max).

Intermediate connection 25

2,4-Dibromo-5-methoxy-N-(1-(phenylamino)hexan-2-yl)benzenesulfonamide

To a solution of 2,4-dibromo-5-methoxybenzenesulfonyl chloride (3.2 g, 8.79 mmol) in THF (50 mL) at 0 °C were added N1-phenylhexane-1,2-diamine (Intermediate 24; 1.3 g, 6.76 mmol) and triethylamine (2.8 mL, 20.3 mmol) and the reaction mixture was stirred for 4 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting residue was dissolved in EtOAc (60 mL). The organic layer was washed with water (50 mL) and brine (50 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 18% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 97% (3.5 g, brown gum). LC-MS: (Method E) 521.0 (M ⁺⁺ H), Rt: 3.14 min, 93.57% (max).

Intermediate connection 26

1,1-7-bromo-3-butyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazenine dioxide

To a solution of 2,4-dibromo-5-methoxy-N-(1-(phenylamino)hexan-2-yl)benzenesulfonamide (Intermediate 25; 3.5 g, 6.72 mmol) in DMF (30 mL) were added K ₂ CO ₃ (1.73 g, 12.5 mmol) and copper powder (0.42 g, 6.72 mmol). The reaction mixture was degassed for 5 min under N ₂ and the reaction mixture was then heated for 16 h at 115 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with ice-cold water (25 mL) and the aqueous layer was extracted with a 1:1 mixture of EtOAc and PE (2×50 mL). The combined organic layer was washed with brine (30 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 25% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 51% (1.5 g, brown gum). LC-MS: (Method A) 439.0 (M ⁺⁺ H), Rt: 2.83 min, 82.06% (max).

Intermediate connection 27

1,1-Dioxide 7-bromo-3-butyl-8-methoxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a solution of 7-bromo-3-butyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 26; 1.25 g, 2.84 mmol) in N-methyl-2-pyrrolidone (15 ml) was added _Cs2CO3 (1.4 g, 4.27 mmol) and the reaction mixture was stirred for 15 min at room temperature. Then p-methoxybenzyl _bromide (0.5 ml, 3.41 mmol) was added 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 ice-cold water (25 ml) and the precipitated solid was filtered off. The obtained crude material was purified by Isolera column chromatography (eluent: 30% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 90% (1.4 g, white solid).

LC-MS: (Method E) 558.9 (M ⁺⁺ H), Rt: 3.36 min, 90.75% (max).

Intermediate connection 28

1,1-Dioxide 3-butyl-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a stirred solution of 7-bromo-3-butyl-8-methoxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 27; 1.4 g, 2.57 mmol) in DMF (10 mL) was added sodium thiomethoxide (0.9 g, 12.8 mmol) and the reaction mixture was heated for 16 h at 60 °C. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with EtOAc (30 mL) and the organic layer was washed with water (2×30 mL) and brine (30 mL). The organic portion was dried over anhydrous Na ₂ SO ₄ and 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: 68% (0.9 g, off-white solid). LC-MS: (Method E) 513.0 (M ⁺⁺ H), Rt: 3.12 min, 90.12% (max).

Intermediate connection 29

tert-Butyl-(E)-3-((3-butyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro- 1,2,5-benzothiadiazepin-8-yl)oxy)acrylate

To a stirred solution of 3-butyl-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (intermediate 28; 0.2 g, 0.39 mmol) in THF (5 ml) at 0 °C were added DABCO (0.005 g, 0.04 mmol) and tert-butyl propiolate (0.08 g, 0.58 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 poured into ice-cold water (10 ml) and the aqueous layer was extracted with EtOAc (2×15 ml). The combined organic layer was washed with brine (10 mL), dried over _{anhydrous Na2SO4} 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 afford the title compound. Yield: 80% (0.2 g, white gum).

LC-MS: (Method E) 583.0 (M+-'Bu+H), Rt: 3.49 min, 86.23% (max)

Intermediate connection 30

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

To a stirred solution of 3-butyl 8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (intermediate 28; 0.5 g, 0.97 mmol) in DMA (5 ml) at 0 °C was added 60% NaH (0.127 g, 3.16 mmol) portionwise and the reaction mixture was stirred for 30 min at 0 °C. Then a solution of ethyl 3-bromo-2,2-difluoropropanoate (0.47 g, 2.18 mmol) in DMA (2.5 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 and then 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 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: 50% (0.3 g, off-white solid). LC-MS: (Method E) 629.2 (M ⁺⁺ H), Rt: 3.37 min, 90.09% (max).

Intermediate connection 31

(Z)-3-((3-Butyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

To a stirred solution of ethyl (Z)-3-((3-butyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 30; 0.3 g, 0.48 mmol) in a mixture of 1,4-dioxane and water (4:1, 5 ml) was added lithium hydroxide (0.06 g, 1.43 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 (2×15 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. The resulting crude material was purified by Isolera column chromatography (eluent: 100% EtOAc; silica gel: 230-400 mesh) to afford the title compound. Yield: 77% (0.22 g, brown solid). LC-MS: (Method E) 600.8 (M ⁺⁺ H), Rt: 3.10 min, 41.73% (max).

Intermediate connection 32

1,1-Dioxide 7-bromo-3-butyl-8-methoxy-2-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a stirred solution of 7-bromo-3-butyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 26; 4.5 g, 10.24 mmol) in N-methyl-2-pyrrolidone (20 ml) were added _Cs2CO3 (6.7 g, 20.5 mmol) and then iodomethane (3.2 ml, 51.2 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 (25 ml) and the aqueous layer was extracted with a mixture of EtOAc and PE (30%, 2×75 ml). The combined organic layer was washed with brine (100 mL), 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: 4.5 g (crude, pale brown solid).

LC-MS: (Method E) 452.8 (M ⁺⁺ H), Rt: 3.19 min, 95.26% (max).

Intermediate connection 33

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

To a solution of 7-bromo-3-butyl-8-methoxy-2-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 32; 4.5 g, 9.92 mmol) in DMF (50 mL) was added sodium thiomethoxide (3.5 g, 49.6 mmol) and the reaction mixture was heated for 16 h at 80 °C. After completion of the reaction (monitored by UPLC), the reaction mixture was quenched with ice-cold water (25 mL) and the aqueous layer was extracted with EtOAc (4 x 50 mL). The combined organic layer was washed with water (2 x 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 purified by Isolera column chromatography (eluent: 26% EtOAc PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 77% (3.1 g, off-white solid).

^1H NMR (400 MHz, DMSO-d ₆ ): δ 10.73 (s, 1H), 7.29 (s, 1H), 7.12-7.16 (m, 2H), 6.99 (s, 1H), 6.67 (t, J= 7.2 Hz, 1H), 6.54 (d, J=8.0 Hz, 2H), 4.06-3.89 (m, 2H), 3.21-3.09 (m, 1H), 2.42 (s, 3H), 2.34 (s , 3H), 1.68-1.55 (m, 1H), 1.54-1.42 (m, 1H), 1.41-1.25 (m, 4H), 0.93-0.90 (m, 3H). LC-MS: (Method A) 406.9 (M ⁺⁺ H), Rt: 2.65 min, 92.05% (max).

Intermediate connection 34

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

These two enantiomers of racemic 3-butyl-8-hydroxy-2-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 33; 10.0 g, 24.59 mmol) were separated by means of an SFC apparatus (Method E). 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 these two enantiomers is not known.

Enantiomer 1: Yield: 42% (4.3 g, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.69 (s, 1H), 7.28 (s, 1H), 7.13 (t, J=10.8 Hz, 2H), 6.99 (s, 1H), 6.66 (t, J=9.6 Hz, 1H), 6.53 (d, J=10.8 Hz, 2 H), 4.00-3.89 (m, 2H), 3.19-3.14 (m, 1H), 2.42 (s, 3H), 2.34 (s, 3H), 1.68-1.55 (m, 1H), 1.55-1.45 (m, 1H), 1.40-1.25 (m, 4H), 0.95-0.85 (m, 3H). LC-MS: (Method A) 407.1 (M ⁺⁺ H), Rt: 2.56 min, 98.41% (max). HPLC: (Method E) Rt: 5.43 min, 98.58% (max). Chiral SFC: (Method D) Rt: 1.83 min, 100% (max).

Enantiomer 2: Yield: 34% (4.2 g, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.58 (s, 1H), 7.27 (s, 1H), 7.13 (t, J=10.0 Hz, 2H), 6.98 (s, 1H), 6.66 (t, J=10.0 Hz, 1H), 6.53 (d, J=10.8 Hz, 2H), 4.10-3.80 (m, 2H), 3.25-3.05 (m, 1H), 2.42 (s, 3H), 2.34 (s, 3H), 1.68-1.45 (m, 2H), 1.45-1.25 (m, 4H), 0.98-0.85 (m, 3H). LC-MS: (Method A) 407.2 (M ⁺⁺ H), Rt: 2.56 min, 98.25% (max). HPLC: (Method E) Rt: 5.43 min, 97.49% (max). Chiral SFC: (Method D) Rt: 3.06 min, 99.76% (max).

Intermediate connection 35

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

To a stirred solution of 3-butyl 8-hydroxy-2-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (intermediate 33; 0.5 g, 1.23 mmol) in DMA (7.5 ml) at 0 °C was added portionwise NaH (60%, 0.16 g, 3.99 mmol) and the reaction mixture was stirred for 30 min at 0 °C. Then a solution of ethyl 3-bromo-2,2-difluoropropanoate (0.6 g, 2.78 mmol) in DMA (2.5 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 HCl, pH ca. 4) and diluted with water (10 mL). The aqueous layer was extracted with EtOAc (2 x 10 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: 94% (0.6 g, off-white solid).

¹ NMR (400 MHz, DMSO-d ₆ ): δ 7.83 (m, 1H), 7.60 (s, 1H), 7.18 (t, J=7.6 Hz, 3H), 6.74 (t, J=10.0 Hz, 1H), 6.66 (d, J=10.8 Hz, 2H), 4.28 (q, J=9.2 Hz, 2H), 4.09-4.02 (m, 1H), 3.87-3.82 (m, 1H) , 3.34-3.31 (m, 1H), 2.49 (s, 3H), 2.41 (m, 3H), 1.62-1.53 (m, 2H), 1.51-1.49 (m, 3H), 1.35-1.24 (m, 4H), 0.93-0.91 (m, 3H). LC-MS: (Method E) 523.1 (M ⁺⁺ H), Rt: 3.18 min, 97.01% (max) .

Intermediate connection 36

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

To a stirred solution of 3-butyl-8-hydroxy-2-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine enantiomer 1 (Intermediate 34; 2.0 g, 4.91 mmol) in DMA (20 ml) at 0 °C was added NaH (60%; 640 mg, 15.98 mmol) and the reaction mixture was stirred for 1 h at room temperature. Then ethyl 3-bromo-2,2-difluoropropanoate (2.66 g, 12.29 mmol) was added at 0 °C and the resulting reaction mixture was stirred for 12 h at 90 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with dilute HCl (1.5 N, 20 mL), diluted with water (50 mL) and the aqueous layer was extracted with EtOAc (2×100 mL). The combined organic layer was washed with water (50 mL) and brine (50 mL) and dried over anhydrous _Na2SO4 . The organic portion was filtered, 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 2.0 g of enantiomer 2 of Intermediate 34. The absolute configuration of these two enantiomers is not known.

Enantiomer 1: Yield: 76% (1.95 g, off-white solid). LC-MS: (Method E) 523.0 (M ⁺⁺ H), Rt: 3.19 min, 97.85% (max). HPLC: (Method B) Rt: 6.25 min, 97.32% (max).

Enantiomer 2: Yield: 59% (1.51 g, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.84 (d, J=18.8 Hz, 1H), 7.60 (s, 1H), 7.20-7.16 (m, 3H), 6.77-6.65 (m, 3H), 4.31-4.25 (m, 2H), 4.08 (m, 1H), 3.95-3.70 (m, 1H), 3.30-3.15 (m, 1H), 2.51 (s, 3H), 2.42 (s, 3H), 1.70-1.50 (m, 2H), 1.50-1.32 (m, 7H), 0.95-0.85 (m, 3H). LC-MS: (Method E) 523.0 (M ⁺⁺ H), Rt: 3.19 min, 98.36% (max). HPLC: (Method B) Rt: 6.26 min, 97.97% (max).

Intermediate connection 37

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

To a stirred solution of 3-butyl-8-hydroxy-2-methyl-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (intermediate 33; 0.5 g, 1.23 mmol) in THF (7.5 ml) at 0 °C were added DABCO (0.02 g, 0.12 mmol) and tert-butyl propiolate (0.23 g, 1.84 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 poured into ice-cold water (15 ml) and the aqueous layer was extracted with EtOAc (2×15 ml). The combined organic layer was washed with brine (10 mL), dried over _{anhydrous Na2SO4} 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 afford the title compound. Yield: 92% (0.6 g, off-white gum).

Intermediate connection 38

1,1-Dioxide 3-butyl-7-(ethylthio)-8-hydroxy-2-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a solution of 7-bromo-3-butyl-8-methoxy-2-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (0.3 g, 0.66 mmol) in N-methyl-2-pyrrolidone (5 mL) was added sodium ethanethiolate (0.27 g, 3.31 mmol) and the reaction mixture was stirred for 12 h at 100 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with ice-cold water (25 mL) and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with ice-cold 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 purified by Isolera column chromatography (eluent: 30% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 69% (0.2 g, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.63 (s, 1H), 7.42 (s, 1H), 7.23-7.21 (m, 2H), 6.83 (t, J=7.6 Hz, 1H), 6.68-6.64 (m, 3H), 4.05-4.01 (m, 2H), 3.23-3.21 (m, 1H), 2.80 (q, J=7.2 Hz, 2H), 2.60 (s, 3H), 1.57-1.31 (m, 6H), 1.27 (t, J=7.20 Hz, 3H), 0.96 (t, J=7.20 Hz, 3H). LC-MS: (Method E) 421.0 (M ⁺⁺ H), Rt: 3.04 min, 95.23% (max).

Intermediate connection 39

tert-Butyl-(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2, 5-benzothiadiazepin-8-yl)oxy)acrylate

To a stirred solution of 3-butyl-7-(ethylthio)-8-hydroxy-2-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 38; 0.15 g, 0.36 mmol) in THF (5 ml) were added tert-butyl propiolate (67 mg, 0.53 mmol) and DABCO (20 mg, 0.18 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 concentrated in vacuo and the resulting residue was partitioned between water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2×5 mL) and the combined organic layer was washed with brine (5 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: 70% (140 mg, white solid).

LC-MS: (Method E) 490.9 (M ⁺ -'Bu+H), Rt: 3.43 min, 98.33% (max).

Intermediate connection 40

tert-Butyl (1-((4-fluorophenyl)amino)-1-oxohexan-2-yl)carbamate

To a stirred solution of 2-((tert-butoxycarbonyl)amino)hexanoic acid (93.0 g, 402 mmol) in DMF (930 mL) at 0 °C was added dropwise triethylamine (112 mL, 804 mmol). Then 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide (307 g, 483 mmol) was added and the reaction mixture was stirred for 10 min at 0 °C. Then 4-fluoroaniline (44.7 g, 402 mmol) was added dropwise and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with ice-cold water (500 mL) and the solid product precipitated. The resulting solid was filtered, washed with water (2 x 100 mL) and dried in vacuo to afford the title compound. The resulting crude material was carried as received to the next step without any further purification. Yield: 69% (90 g, light yellow solid).

LC-MS: (Method K) 225.2 (M ⁺ +H-Boc), Rt: 2.66 min, 99.41% (max).

Intermediate connection 41

2-Amino-N-(4-fluorophenyl)hexanamide

To a stirred solution of tert-butyl (1-((4-fluorophenyl)amino)-1-oxohexan-2-yl)carbamate (Intermediate 40; 90 g, 277 mmol) in 1,4-dioxane (900 ml) was added HCl in dioxane (4 M in 1,4-dioxane, 360 ml, 1280 mmol) at 0 °C and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting residue was basified with 10% NaHCO ₃ solution (400 ml). The aqueous layer was extracted with EtOAc (2 x 500 ml) and the combined organic layer was washed with water (2 x 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: 91% (60 g, light brown liquid).

LC-MS: (Method K) 225.1 (M ⁺⁺ H), Rt: 1.97 min, 94.23% (max).

Intermediate connection 42

N1-(4-fluorophenyl)hexane-1,2-diamine

To a stirred solution of 2-amino-N-(4-fluorophenyl)hexanamide (Intermediate 41; 60 g, 268 mmol) in THF (300 mL) was added borane dimethyl sulfide (1 M in THF, 401 mL, 401 mmol) at 0 °C and the reaction mixture was heated for 24 h at 75 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with methanol (200 mL) at 0 °C and the reaction mixture was heated for 2 h at 75 °C. The reaction mixture was concentrated in vacuo and the resulting residue was dissolved in DCM (500 mL). The organic layer was washed with water (2 x 200 mL), dried over anhydrous Na ₂ SO ₄ and then concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 5-20% MeOH in DCM; silica gel: 230-400 mesh) to afford the title compound. Yield: 87% (53 g, brown liquid).

LC-MS: (Method K) 211.2 (M ⁺⁺ H), Rt: 1.97 min, 92.44% (max). Intermediate 43

2,4-Dibromo-N-(1-((4-fluorophenyl)amino)hexan-2-yl)-5-methoxybenzenesulfonamide

To a stirred solution of N1-(4-fluorophenyl)hexane-1,2-diamine (Intermediate 42; 20 g, 95 mmol) in THF (200 mL) were added 2,4-dibromo-5-methoxybenzenesulfonyl chloride (41.6 g, 114 mmol) and triethylamine (39.8 mL, 285 mmol) at 0 °C and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with EtOAc (400 mL). The organic layer was washed with water (2×100 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% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 86% (45.5 g, brown solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.02-7.90 (m, 2H), 7.55 (s, 1H), 6.82 (m, 2H), 6.31-6.26 (m, 2H), 5.45-5.30 ( m, 1H), 3.90 (s, 3H), 3.30-3.15 (m, 1H), 3.05-2.82 (m, 2H), 1.60-1.40 (m, 1H), 1.40-1.30 (m, 1H), 1.30- 1.20 (m, 1H), 1.15-0.98 (m, 3H), 0.73 (t, J=8.80 Hz, 3H). LC-MS: (Method A) 538.9 (M ⁺⁺ H), Rt: 3.04 min, 85.57% (max).

Intermediate connection 44

1,1-7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine dioxide

To a stirred solution of 2,4-dibromo-N-(1-((4-fluorophenyl)amino)hexan-2-yl)-5-methoxybenzenesulfonamide (Intermediate 43; 45 g, 84 mmol) in DMF (450 ml) were added potassium carbonate (23.11 g, 167 mmol) and copper iodide (1.59 g, 8.36 mmol) at room temperature. The reaction mixture was degassed for 15 min with nitrogen. Then tris[2-(2-methoxyethoxy)ethyl]amine (5.41 g, 16.72 mmol) was added at room temperature and the reaction mixture was heated for 16 h at 130 °C. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through a pad of celite and the pad of celite was washed with EtOAc (50 mL). The filtered portion was concentrated in vacuo. The resulting residue was diluted with EtOAc (400 mL) and the organic layer was washed with water (2×100 mL). The organic portion was dried over anhydrous Na ₂ SO ₄ and the resulting crude material was purified by Isolera column chromatography (eluent: 12% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 55% (22.0 g, white solid).

^1H NMR (400 MHz, DMSO-d ₆ ): δ 7.57 (s, 1H), 7.50-7.42 (d, J=6.6 Hz, 2H), 7.02 (t, J=9.0 Hz, 2H) , 6.70-6.50 (m, 2H), 4.38-4.22 (m, 1H), 3.95 (s, 3H), 3.33 (m, 1H), 2.95-2.80 (m, 1H), 1.65-1.15 (m, 6H) , 0.89 (t, J=9.2 Hz, 3H). LC-MS: (Method A) 455.0 (M+-H), Rt: 2.71 min, 95.55% (max).

Intermediate connection 45

1,1-Dioxide 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2-methyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a stirred solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 44; 5.8 g, 12.68 mmol) in N-methyl-2-pyrrolidone (24 ml) was added _Cs2CO3 (8.26 g, 25.4 mmol) at 0 °C and _the reaction mixture was stirred for 15 min. Then methyl iodide (1.58 ml, 25.4 mmol) was added dropwise at 0 °C and the reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with EtOAc (100 ml) and the organic layer was washed with water (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: 15% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 75% (5.1 g, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.47 (s, 1H), 7.41 (s, 1H), 6.98-6.94 (m, 2H), 6.72-6.69 (m, 2H), 3.99-3.92 (m, 5H), 3.40-3.22 (m, 1H), 2.63 (s, 3H), 1.68-1.60 (m, 1H), 1.50-1.35 (m, 5H), 0.95 (t, J=7.2 Hz, 3H). LC-MS: (Method A) 471.0 (M ⁺ +H), Rt: 2.89 min, 88.26% (max).

Intermediate connection 46

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

To a stirred solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2-methyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 45; 2.0 g, 4.24 mmol) in DMF (20 mL) was added sodium thiomethoxide (1.48 g, 21.21 mmol) at room temperature and the reaction mixture was stirred for 16 h at 100 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature and quenched with water (20 mL). The aqueous layer was extracted with EtOAc (2×30 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: 81% (1.6 g, light yellow solid).

^1H NMR (400 MHz, DMSO-d ₆ ): δ 10.72 (s, 1H), 7.27 (s, 1H), 6.97 (t, J=11.2 Hz, 3H), 6.55-6.50 (m, 2H) , 3.98-3.86 (m, 2H), 3.30-3.05 (m, 1H), 2.42 (s, 3H), 2.34 (s, 3H), 1.70-1.45 (m, 2H), 1.40-1.25 (m, 4H) , 1.00-0.80 (m, 3H). LC-MS: (Method A) 425.2 (M ⁺ +H), Rt: 2.56 min, 90.64% (max).

Intermediate connection 47

tert-Butyl-(E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro- 1,2,5-benzothiadiazepin-8-yl)oxy)acrylate

To a stirred solution of 3-butyl-5-(4-fluorophenyl)-8-hydroxy-2-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 46; 50 mg, 0.118 mmol) in anhydrous THF (2 ml) were added tert-butyl propiolate (22 mg, 0.18 mmol) and DABCO (6.61 mg, 0.06 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 concentrated in vacuo and the resulting residue was partitioned between water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2×5 mL) and the combined organic layer was washed with brine (5 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: 9% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 80% (55 mg, colorless gum).

^1H NMR (400 MHz, CDCl ₃ ): δ 7.62-7.59 (m, 2H), 7.01-6.97 (m, 2H), 6.88 (s, 1H), 6.81-6.78 (m, 2H), 5.55 (4 J =12.0 Hz, 1H), 3.97-3.93 (m, 2H), 3.55-3.30 (m, 1H), 2.67 (s, 3H), 2.31 (s, 3H), 1.70-1.60 (m, 2H), 1.50 (s, 9H), 1.45-1.35 (m, 4H), 1.00-0.90 (m, 3H). LC-MS: (Method A) 495.0 (M+-'Bu+H), Rt: 3.18 min, 94.92% (max).

Intermediate connection 48

1,1-Dioxide 3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2-methyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a stirred solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2-methyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 45; 1.0 g, 2.12 mmol) in DMF (5 mL) was added sodium ethanethiolate (0.89 g, 10.61 mmol) at room temperature and the reaction mixture was stirred for 16 h at 100 °C. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature and quenched with water (10 mL). The aqueous layer was extracted with EtOAc (2×20 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% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 74% (0.8 g, light yellow solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 10.66 (s, 1H), 7.28 (s, 1H), 7.10-6.90 (m, 3H), 6.62-6.40 (m, 2H), 4.02-3.80 ( m, 2H), 3.00-3.10 (m, 1H), 3.00-2.78 (m, 2H), 2.43 (s, 3H), 1.70-1.45 (m, 2H), 1.45-1.28 (m, 4H), 1.20 ( t,J=9.60 Hz, 3H), 0.98-0.82 (m, 3H). LC-MS: (Method K) 439.1 (M ⁺⁺ H), Rt: 3.07 min, 85.59% (max).

Intermediate connection 49

tert-Butyl-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro- 1,2,5-benzothiadiazepin-8-yl)oxy)acrylate

To a stirred solution of 3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2-methyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 48; 60 mg, 0.137 mmol) in anhydrous THF (1 ml) were added tert-butyl propiolate (26 mg, 0.20 mmol) and DABCO (7.67 mg, 0.06 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 concentrated in vacuo and the resulting residue was partitioned between water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2×5 mL) and the combined organic layer was washed with brine (5 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: 20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 74% (60 mg, colorless gum).

¹ H NMR (300 MHz, DMSO-d ₆ ): δ 7.74 (d, J=12.0 Hz, 1H), 7.49 (s, 1H), 7.17 (s, 1H), 7.04 (t, J=8.4 Hz, 2H), 6.75-6.73 (m, 2H), 5.48 (d, J=12.3 Hz, 1H), 4.06-4.01 (m, 1H), 3.79 (m, 1H), 2.94-2.92 (m, 3H), 2.58 (s, 3H), 1.60-1.50 (m, 2H), 1.50-1.40 (m, 10H), 1.40-1.30 (m, 3H), 1.20-1.15 (m, 3H), 0.95-0.85 (m, 3H). LC-MS: (Method A) 509.1 (M ⁺ -'Bu+H), Rt: 3.17 min, 94.65% (max).

Intermediate connection 50

3-Butyl-7-chloro-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one

To a stirred solution of 3-butyl-7-chloro-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (6 g, 20.01 mmol) in 1-bromo-4-fluorobenzene (88 g, 500 mmol) were added copper(I) iodide (0.38 g, 2.00 mmol) and K ₂ CO ₃ (5.53 g, 40.0 mmol), and the solution was purged with nitrogen for 20 min to degas. Tris[2-(2-methoxyethoxy)ethyl]amine (1.30 g, 4.00 mmol) was then added under nitrogen, and the resulting reaction mixture was heated at 135°C for 40 h. After completion of the reaction (monitored by UPLC), the reaction mixture was filtered through Celite and the Celite pad was washed with EtOAc (100 mL). The filtrate 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: 64% (5.5 g, off-white solid).

LC-MS: (Method E) 394.0 (M ⁺⁺ H), Rt: 3.19 min, 91.57%.

Intermediate connection 51

3-Butyl-7-chloro-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of 3-butyl-7-chloro-5-(4-fluorophenyl)-8-methoxy-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (Intermediate 50; 5.5 g, 13.96 mmol) in THF (60 mL) at 0 °C was added dropwise borane dimethyl sulfide (140 mL, 140 mmol) and the reaction mixture was heated under reflux 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 (100 mL). The resulting solution was heated for 2 h at 65 °C, then cooled to room temperature and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 15-20% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 77% (4.5 g, colorless liquid).

LC-MS: (Method A) 380.0 (M+) Rt: 3.61 min, 90.76% (max) Intermediate 52

1,1-3-Butyl-7-chloro-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazepine dioxide

To a stirred solution of 3-butyl-7-chloro-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazepine (Intermediate 51; 4.5 g, 11.84 mmol) in THF (45 mL) and water (10 mL) was added oxone (36.4 g, 59.2 mmol) and the reaction mixture was stirred for 24 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was filtered through a Buchner funnel and the filtrate was extracted with EtOAc (2×200 mL). The combined organic layer was washed with water (50 mL) and brine (50 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. This crude material was purified by Isolera column chromatography (eluent: 10-13% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 84% (4.1 g, colorless solid). LC-MS: (Method A) 412.1 (M ⁺⁺ H), Rt: 2.83 min, 96.78% (max).

Intermediate connection 53

1,1-Dioxide 3-butyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazepine

To a stirred solution of 3-butyl-7-chloro-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 52; 1 g, 2.42 mmol) in DMF (12 ml) was added sodium thiomethoxide (0.85 g, 12.14 mol) at room temperature and the resulting mixture was stirred overnight at 100 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with water (20 ml) and the aqueous layer was extracted with EtOAc (2×20 ml). The combined organic layer was washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 30-35% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 30% (0.300 g, off-white solid).

^1H NMR (400 MHz, CDCl ₃ ): δ 7.66 (s, 1H), 7.27 (s, 1H), 6.94-6.94 (m, 2H), 6.62-6.64 (m, 2H), 4.14-4.16 (m, 1H), 3.41-3.42 (m, 1H), 2.96-2.96 (m, 1H), 2.50 (s, 1H), 2.40 (s, 3H), 1.37-1.37 (m, 4H), 1.29-1.30 (m, 4H), 0.95 (t, J=7.20 Hz, 3H). LC-MS: (Method A) 408.2 (M+-H), Rt: 2.54 min, 98.39% (max).

Intermediate connection 54

tert-Butyl(E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepine -8-yl)oxy)acrylate

To a stirred solution of 3-butyl-5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 53; 50 mg, 0.12 mmol) in anhydrous THF (3 ml) were added tert-butyl propiolate (23 mg, 0.18 mmol) and DABCO (6.85 mg, 0.06 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 concentrated and the resulting residue was partitioned between water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2×5 ml) and the combined organic layer was washed with brine (5 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: 12% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 95% (65 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.72 (d, J=12.0 Hz, 1H), 7.56 (s, 1H), 7.17 (s, 1H), 7.04 (t, J=9, 2 Hz, 2H), 6.71-6.67 (m, 2H), 5.46 (d, J=12.0 Hz, 1H), 4.22-4.19 (m, 1H), 3.51-3.48 (m, 1H), 3.33-3.24 (m, 1H), 3.18-3.13 (m, 1H), 2.40 (s, 3H), 2.30-2.20 (m, 1H), 1.46-1.43 (m, 9H), 1.39-1.34 (m, 3H), 1.31-1.28 (m, 3H), 0.9 (t, J=7.2 Hz, 3H). LC-MS: (Method A) 480.1 (M ⁺ -'Bu+ H), Rt: 3.03 min, 95.09% (max).

Intermediate connection 55

1,1-Dioxide 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2-(4-methoxybenzyl)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a stirred solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 44; 5 g, 10.93 mmol) in N-methyl-2-pyrrolidone (50 ml) was added Cs ₂ CO ₃ (7.12 g, 21.86 mmol) and the reaction mixture was stirred for 30 min at 0 °C. Then 1-(bromomethyl)-4-methoxybenzene (3.30 g, 16.40 mmol) was added and the 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 pad of Celite and the pad was washed with EtOAc (2 x 20 mL). The filtrate was concentrated in vacuo and the crude material was purified by Isolera column chromatography (eluent: 15-20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 73% (6 g, off-white solid).

LC-MS: (Method A) 578.1 (M ⁺⁺ H), Rt: 3.49 min, 75.44%.

Intermediate connection 56

1,1-Dioxide 3-butyl-5-(4-fluorophenyl)-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine

To a stirred solution of 7-bromo-3-butyl-5-(4-fluorophenyl)-8-methoxy-2-(4-methoxybenzyl)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 55; 1 g, 1.23 mmol) in DMF (20 mL) was added sodium thiomethoxide (0.43 g, 6.14 mmol) at room temperature and the resulting mixture was stirred for 16 h at 100 °C. 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 (2×50 mL). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 10-15% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 66% (500 g, off-white solid).

LC-MS: (Method B) 529.1 (M+-H), Rt: 3.20 min, 85.70% (max).

Intermediate connection 57

tert-Butyl-(E)-3-((3-butyl-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-2,3,4, 5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate

To a stirred solution of 3-butyl-5-(4-fluorophenyl)-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 56; 60 mg, 0.113 mmol) in anhydrous THF (2 ml) were added tert-butyl propiolate (21.40 mg, 0.17 mmol) and DABCO (6.34 mg, 0.06 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 concentrated in vacuo and the resulting residue was partitioned between water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2×5 mL) and the combined organic layer was washed with brine (5 mL) and then dried over _{anhydrous Na2SO4} . The organic portion was concentrated in vacuo and 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: 81% (60 mg, colorless gum).

LC-MS: (Method A) 601.1 (M ⁺ -'Bu+H), Rt: 3.44 min, 94.98% (max).

Intermediate connection 58

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

To a solution of 3-butyl-7-chloro-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (1 g, 2.54 mmol) in DMF (25 mL) was added sodium ethanethiolate (1.07 g, 12.69 mmol) and the reaction mixture was stirred for 16 h at 120 °C. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with ice-cold water (25 mL) and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with ice-cold 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 purified by Isolera column chromatography (eluent: 30% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 52% (0.56 g, off-white solid).

LC-MS: (Method A) 406.1 (M ⁺⁺ H), Rt: 3.11 min, 96.20% (max).

Intermediate connection 59

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

To a stirred solution of 3-butyl-7-(ethylthio)-8-hydroxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 58; 100 mg, 0.24 mmol) in anhydrous THF (4 ml) were added tert-butyl propiolate (46.7 mg, 0.37 mmol) and DABCO (13.83 mg, 0.12 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 concentrated in vacuo and the resulting residue was partitioned between water (5 ml) and EtOAc (5 ml). The aqueous layer was extracted with EtOAc (2×5 mL) and the combined organic layer was washed with brine (5 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 the title compound. Yield: 55% (72 mg, off-white solid).

¹ H NMR (300 MHz, DMSO-d ₆ ): δ 7.72 (d, J=12.30 Hz, 1H), 7.58 (s, 1H), 7.16-7.19 (m, 3H), 6.65-6.68 (m, 3H), 5.46 (d,J=12.30 Hz, 1H), 4.25 (m, 1H), 3.47 (s, 1H), 3.15 (s, 1H), 2.96 (d, J=7.50 Hz, 2H ), 2.28 (m, 2H), 1.45 (s, 9H), 1.17-1.19 (m, 6H), 1.22-1.38 (m, 3H), 0.90 (t, J=6.90 Hz, 3H). LC-MS: (Method A) 476.1 (M ⁺ -'Bu), Rt: 3.18 min, 98.54% (max).

Intermediate connection 60

tert-Butyl (E)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate

To a stirred solution of 3-butyl-7-(ethylthio)-8-hydroxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (50 mg, 0.12 mmol) in THF (5 mL) were added tert-butyl propiolate (16 mg, 0.12 mmol) and DABCO (14 mg, 0.12 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 concentrated in vacuo and the resulting residue was partitioned between water (5 mL) and EtOAc (5 mL). The aqueous layer was extracted with EtOAc (2×5 mL) and the combined organic layer was washed with brine (5 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: 20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 30% (20 mg, off-white solid).

LC-MS: (Method E) 477.1 (M ⁺ -'Bu+H), Rt: 3.23 min, 99.30% (max).

Intermediate connection 61

tert-Butyl (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate

The title compound was prepared from 3-butyl 7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and tert-butyl propiolate according to the same procedure as described for Intermediate 60.

Intermediate connection 62

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

The title compound was prepared from 3-butyl 5-(4-fluorophenyl)-8-hydroxy-7-(methylthio)-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 53) and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 63

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

The title compound was prepared from 3-butyl 7-(ethylthio)-8-hydroxy-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide (Intermediate 58) and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 64

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

The title compound was prepared from 3-butyl 7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2,3,4,5-tetrahydro-1,5-benzothiazepine 1,1-dioxide and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 65

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

The title compound was prepared from 3-butyl 5-(4-fluorophenyl)-8-hydroxy-2-methyl-7-(methylthio)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 46) and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 66

Ethyl (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate

The title compound was prepared from 3-butyl 7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2-methyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 48) and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10. Intermediate 67

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

The title compound was prepared from 3-butyl 7-(ethylthio)-8-hydroxy-2-methyl-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 38) and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 68

Ethyl-(Z)-3-((3-butyl-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-2,3,4,5- tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate

The title compound was prepared from 3-butyl 5-(4-fluorophenyl)-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide (Intermediate 56) and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 69

(Z)-3-((3-Butyl-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 68) according to the procedure described for Intermediate 31.

Intermediate connection 70

Ethyl (Z)-3-((3-butyl-(ethylthio)-(4-methoxybenzyl)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate

The title compound was prepared from 3-butyl 7-(ethylthio)-8-hydroxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 71

(Z)-3-((3-Butyl-7-(ethylthio)-2-(4-methoxybenzyl)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-7-(ethylthio)-2-(4-methoxybenzyl)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 70) according to the procedure described for Intermediate 31.

Intermediate connection 72

Ethyl (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate

The title compound was prepared from 3-butyl 7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2-(4-methoxybenzyl)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide and ethyl 3-bromo-2,2-difluoropropanoate according to the same procedure as described for Intermediate 10.

Intermediate connection 73

(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 72) according to the procedure described for Intermediate 31.

Intermediate connection 74

tert-Butyl-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

The title compound was prepared from 3-butyl 7-(ethylthio)-5-(4-fluorophenyl)-8-hydroxy-2-(4-methoxybenzyl)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine 1,1-dioxide and tert-butyl propiolate according to the same procedure as described for Intermediate 57.

Example 1

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

To a stirred solution of ethyl (E)-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 9; 0.23 g, 0.45 mmol) in a mixture of 1,4-dioxane and water (4 ml; 3:1) was added lithium hydroxide (97 mg, 2.29 mmol) and the resulting mixture was stirred for 12 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting residue was acidified with dilute HCl (1.5 N, 2 ml). The aqueous portion was extracted with EtOAc (2×15 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. The resulting crude material was purified by Isolera column chromatography (eluent: 2-3% MeOH/DCM; silica gel: 230-400 mesh) to afford the title compound. Yield: 59% (125 mg, off-white solid).

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 12.29 (s, 1H), 7.76 (d, J=12.0 Hz, 1H), 7.56 (s, 1H), 7.18 (s, 1H), 7.18 (t, j=7.2 Hz, 2H), 6.73 (t, J=7.2 Hz, 1H), 6.65 (d, j=8.0 Hz, 2H), 5.52 (d, J=12.4 Hz, 1H), 4.26-4.22 (m, 1H), 3.49-3.46 (m, 1H), 3.32-3.27 (m, 1H), 3.15-3.08 (m, 1H), 2.41 (s, 3H), 2.27-2.26 (m, 1H), 1.40-1.23 (m, 6H), 0.98-0.88 (m, 3H). LC-MS: (Method A) 462.2 (M ⁺⁺ H), Rt: 2.56 min, 94.94% (max). HPLC: (Method B) Rt: 5.42 min, 95.08% (max).

Examples 2 and 3

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

The two enantiomers of racemic (E)-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid (Example 1; 0.12 g, 0.26 mmol) were separated by chiral preparative SFC (Method A); mobile phase: _CO2 :0.5% isopropylamine in IPA (70:30); wavelength: 280 nm; cycle time: 5 min; back pressure: 100 bar. 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: 10% (15 mg, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.36 (s, 1H), 7.74 (d, J=12.0 Hz, 1H), 7.56 (s, 1H), 7.22 (s, 1H), 7.18 (t, J=7.2 Hz, 2H), 6.73 (t, J=7.2 Hz, 1H ), 6.65 (d, J=8.4 Hz, 2H), 5.53 (d, J=12.4 Hz, 1H), 4.27-4.23 (m, 1H), 3.49-3.46 (m, 1H), 3.33-3.31 (m, 1H), 3.28-3.13 (m, 1H), 2.41 (s, 3H), 2.31-2.27 (m, 1H), 1.41-1.29 (m, 6H), 0.9 (t, J=7.2 Hz, 3H). LC-MS: (Method E) 462.1 (M ⁺⁺ H), Rt: 2.45 min, 97.2% (max). HPLC: (Method B) Rt: 5.42 min, 97.78% (max). SFC: (Method A) Rt: 2.79 min, 98.25%.

Enantiomer 2: Yield: 12% (23 mg, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.29 (s, 1H), 7.75 (d, J=12.0 Hz, 1H), 7.57 (s, 1H), 7.22 (s, 1H), 7.18 (t, J=7.2 Hz, 2H), 6.73 (t, J=7.2 Hz, 1H), 6.65 (d, J=8.4 Hz, 2H), 5.53 (d, J=12.0 Hz, 1H), 4.27-4.21 (m, 1H), 3.49-3.33 (m, 1H), 3.33-3.31 (m, 1H), 3.28-3.13 (m, 1H), 2.41 (s, 3H), 2.33-2.28 (m, 1H), 1.42-1.31 (m, 6H), 0.97-0.95 (m, 3H). LC-MS: (Method E) 462.1 (M ⁺⁺ H), Rt: 2.45 min, 96.57% (max). HPLC: (Method B) Rt: 5.42 min, 97.12% (max). SFC: (Method A) Rt: 4.51 min, 99.75%.

Example 4

(Z)-3-((3-Butyl-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-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 10; 170 mg, 0.33 mmol) in a mixture of 1,4-dioxane and water (5 ml, 4:1) was added lithium hydroxide (28 mg, 0.66 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 then diluted with water (5 ml). The aqueous layer was extracted with EtOAc (2 x 20 mL) and _the combined organic layer was washed with water (10 mL) and brine (10 mL), dried over anhydrous _Na2SO4 and concentrated in vacuo. The resulting crude material was purified by prep. HPLC (Method D) to afford the title compound. Yield: 43% (70 mg, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.58 (s, 1H), 7.50 (d, J=18.8 Hz, 1H), 7.19-7.16 (m, 3H), 6.72 (t, J=7.2 Hz, 1H), 6.63 (d, J=8.0 Hz, 2H), 4.24 (d, J=14.8 Hz, 1H), 3.51-3.46 (m, 1H), 3.28-3.24 (m, 1H), 3.12-3.09 (m, 1H), 2.42 (s, 3H), 2.33-2.20 (m, 1H), 1.41-1.24 (m, 6H), 0.91 (t, J=7.20 Hz, 3H). LC-MS: (Method E) 480.1 (M ⁺⁺ H), Rt: 2.51 min, 96.23% (max). HPLC: (Method B) Rt: 5.54 min, 94.04% (max).

Examples 5 and 6

(S)-(Z)-3-((3-Butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid and (R)-(S)-3-((3-butyl-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-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 11; 650 mg, 1.28 mmol) in a mixture of 1,4-dioxane and water (10 ml, 4:1) was added lithium hydroxide (107 mg, 2.56 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 then diluted with water (5 ml). The aqueous layer was extracted with EtOAc (2×20 ml). The combined organic layer was washed with water (10 mL) and brine (10 mL), dried over _{anhydrous Na2SO4} and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 3-4% MeOH/DCM; silica gel: 230-400 mesh) to afford the title compounds.

Enantiomer 2 of the title compound was prepared according to the same procedure starting from 600 mg of enantiomer 2 of Intermediate 11. The absolute configuration of these two enantiomers is not known.

Enantiomer 1: Yield: 36% (224 mg, light brown solid) ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.60 (s, 1H), 7.69 (s, 1H), 7.64 (d, J=3.6 Hz, 1H), 7.20-7.16 (m, 3H), 6.73 (t, J=7.6 Hz, 1H), 6.64 (d, J=8.0 Hz, 2H), 4.25 (d, J=14.4 Hz, 1H), 3.51-3.47 (m, 1H), 3.30-3.26 (m, 1H), 3.13-3.07 (m, 1H), 2.43 (s, 3H), 2.34-2.21 (m, 1H), 1.44-1.28 (m, 6H), 0.91 (t, J=7.20 Hz, 3H). LC-MS: (Method E) 480.0 (M ⁺⁺ H), Rt: 2.93 min, 99.97% (max). HPLC: (Method B) Rt: 5.54 min, 97.21% (max). Chiral HPLC: (Method E) Rt: 4.83 min, 100% (max)

Enantiomer 2: Yield: 51% (290 mg, light brown solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.76 (s, 1H), 7.62 (t, J=8.4 Hz, 2H), 7.20-7.16 (m, 3H), 6.73 (t, J=7.2 Hz, 1H), 6.64 (d, J=8.0 Hz, 2H), 4.25 (d , J=15.2 Hz, 1H), 3.51-3.46 (m, 1H), 3.33-3.27 (m, 1H), 3.13-3.07 (m, 1H), 2.43 (s, 3H), 2.34-2.29 (m, 1H), 1.44-1.28 (m, 6H), 0.91 (t, J=7.2 0 Hz, 3H). LC-MS: (Method E) 480.0 (M ⁺⁺ H), Rt: 2.93 min, 96.76% (max). HPLC: (Method B) Rt: 5.54 min, 98.69% (max). Chiral HPLC: (Method E) Rt: 4.02 min, 100% (max).

Example 7

(Z)-3-((3-Ethyl-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-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 19; 0.13 g, 0.27 mmol) in a mixture of 1,4-dioxane and water (4:1, 5 ml) was added lithium hydroxide (0.02 g, 0.54 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 (2 x 20 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. The resulting crude material was purified by prep. HPLC (method B) to afford the title compound. Yield: 12% (15 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.46 (s, 1H), 7.19-7.15 (m, 3H), 7.10-7.06 (m, 1H), 6.71 (t, J=7.2 Hz, 1H), 6.62 (d, J=8.4 Hz, 2H), 4.26-4.23 (m, 1H), 3.51-3.41 (m, 2H), 3.11-3.09 (m, 1H), 2.41 (s, 3H) , 2.33-2.23 (m, 1H), 1.48-1.36 (m, 2H), 1.01-0.99 (m, 3H). LC-MS: (Method D) 452.0 (M ⁺⁺ H), Rt: 2.11 min, 99.01% (max). HPLC: (Method C) Rt: 4.50 min, 97.92% (Max).

Example 8

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

To a stirred solution of tert-butyl (E)-3-((3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 20; 0.36 g, 0.73 mmol) in DCM (10 ml) at 0 °C was added TFA (2 ml) and the reaction mixture was then 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 (2×20 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 layer was concentrated in vacuo and the resulting crude material was purified by prep-HPLC (Method B) to afford the title compound. Yield: 23% (75 mg, white solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 12.30 (s, 1H), 7.77 (d, J=12.4 Hz, 1H), 7.57 (s, 1H), 7.23-7.16 (m, 3H) , 6.73 (t, J=7.2 Hz, 1H), 6.66 (d, J=8.0 Hz, 2H), 5.53 (d, J=12.4 Hz, 1H), 4.28-4.24 (m, 1H ), 3.51-3.48 (m, 1H), 3.39-3.33 (m, 1H), 3.15-3.09 (m, 1H), 2.42 (s, 3H), 2.28-2.22 (m, 1H), 1.48-1.37 (m, 2H), 1.01-0.97 (m, 3H). LC-MS: (Method D) 434.1 (M ⁺ +H), Rt: 2.51 min, 99.14% (max) . HPLC: (Method B) Rt: 4.47 min, 99.54% (max).

Example 9

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

To a stirred solution of tert-butyl (Z)-3-((3-butyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 29; 0.2 g, 0.31 mmol) in toluene (5 ml) at 0°C were added triphenylamine (0.15 g, 0.62 mmol) and TFA (0.5 ml, 6.2 mmol). The reaction mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by LC-MS), the reaction mixture was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 30-50% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 14% (20 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.26 (s, 1H), 7.74 (d, J=12.40 Hz, 1H), 7.52 (s, 1H), 7.39 (d, J=9.2 Hz, 1H), 7.20-7.16 (m, 3H), 6.75 (t, J=7.20 Hz, 1H), 6.67 (d, J=8.40 Hz, 2H), 5.46 (d, J=12.40 Hz, 1H), 4.39-4.35 (m, 1H), 3.51-3.49 (m, 1H), 2.95-2.91 (m, 1H), 2.40 (s, 3H), 1.61-1.58 (m, 2H), 1.48-1.39 (m, 2H), 1.36-1.29 (m, 2H), 0.95-0.92 (m, 3H). LC-MS: (Method E) 462.9 (M ⁺ +H), Rt: 2.76 min, 94.28% (max). HPLC: (Method B) Rt: 5.24 min, 94.22% (max).

Example 10

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

To a stirred solution of (Z)-3-((3-butyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Intermediate 31; 0.22 g, 0.37 mmol) in toluene (4 ml) were added triphenylamine (0.18 g, 0.73 mmol) and TFA (0.6 ml, 7.3 mmol) at 0°C and the resulting mixture was stirred for 16 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 30-50% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. The resulting compound was re-purified by prep. HPLC (method A) to afford the title compound. Yield: 14% (25 mg, off-white solid).

^1H NMR (400 MHz, DMSO-d ₆ ): δ 7.65 (s, 1H), 7.60 (d, J=3.6 Hz, 1H), 7.39 (d, J=9.2 Hz, 1H), 7.20 -7.16 (m, 3H), 6.74 (t, J=7.2 Hz, 1H), 6.66 (d, J=8.0 Hz, 2H), 4.38-4.35 (m, 1H), 3.45-3.33 (m , 1H), 2.95-2.92 (m, 1H), 2.42 (s, 3H), 1.61-1.58 (m, 2H), 1.48-1.42 (m, 2H), 1.41-1.31 (m, 2H), 0.95-0.92 (m, 3H). LC-MS: (Method E) 481.1 (M ⁺⁺ H), Rt: 2.81 min, 99.52% (max). HPLC: (Method B) Rt: 5.36 min, 98.40% (max).

Example 11

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

To a stirred solution of ethyl (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 34; 0.6 g, 1.15 mmol) in 1,4-dioxane/water (4:1, 10 ml) was added lithium hydroxide (0.141 g, 3.44 mmol) and the reaction mixture was stirred for 2 h at RT. 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 (2×15 mL), then _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. The resulting crude material was purified by Isolera column chromatography (eluent: 1-5% MeOH/DCM; silica gel: 230-400 mesh). Yield: 27% (0.15 g, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.73 (d, J=18.4 Hz, 1H), 7.56 (s, 1H), 7.18 (t, J=8.4 Hz, 3H), 6.75 (t, J=7.2 Hz, 1H), 6.67 (d, J=8.4 Hz, 2H), 4.08-4.03 (m, 1H), 3.88-3.86 (m, 1H), 3.34-3.31 (m , 1H), 2.51 (s, 3H), 2.46 (m, 3H), 1.64-1.52 (m, 2H), 1.51-1.31 (m, 4H), 0.94-0.92 (m, 3H). LC-MS: (Method E) 495.1 (M ⁺⁺ H), Rt: 2.88 min, 98.16% (max). HPLC: (Method B) Rt: 6.85 min, 96.53% (Max).

Examples 12 and 13

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

To a stirred solution of enantiomer 1 ethyl (Z)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 36; 1.95 g, 3.73 mmol) in a mixture of 1,4-dioxane and water (3:1, 20 ml) was added LiOH.H ₂ O (313 mg, 7.47 mmol) at 0°C. 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, 20 mL), diluted with water (30 mL) and the aqueous layer was extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL) and dried over _{anhydrous Na2SO4} . The organic portion was filtered, concentrated in vacuo and the resulting crude material was purified by Isolera column chromatography (eluent: 30-50% 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 1.5 g of enantiomer 2 of Intermediate 36. This crude material was purified by trituration with n-hexane (2 x 10 ml) to give the title compound. The absolute configuration of the two enantiomers is not known.

Enantiomer 1: Yield: 93% (1.72 g, white solid). ¹ H ЯМР (400 МГц, DMSO-d ₆ ): δ 13.58 (bs, 11H), 7.76 (d, J=18,8 Гц, 1H), 7.56 (s, 1H), 7.20-7.16 (m, 3Н), 6.74 (t, J=7,2 Гц, 1H), 6.65 (d, J=8,4 Гц, 2H), 4.08-4.04 (m, 1H), 3.87-3.86 (m, 1H), 3.25 (bs, 1H), 2.52 (s, 3H), 2.42 (s, 3H), 1.64-1.61 (m, 1H), 1.53-1.50 (m, 1H), 1.37-1.30 (m, 4H), 0.92 (t, J=6.80 Hz, 3H). LC-MS: (Method E) 495.0 (M ⁺ +H), Rt: 2.94 min, 99.38% (max). HPLC: (Method B) Rt: 5.53 min, 97.59% (max). Chiral SFC: (Method H) Rt: 1.83 min, 99.93% (max).

Enantiomer 2: Yield: 60% (850 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 13.63 (bs, 1H), 7.77 (d, J=18.8 Hz, 1H), 7.57 (s, 1H), 7.20-7.16 (m, 3H), 6.75 (t, J=7.2 Hz, 1H), 6.66 (d, J=8.0 G ts, 2H), 4.06-4.03 (m, 1H), 3.88-3.86 (m, 1H), 3.24 (bs, 1H), 2.53 (s, 3H), 2.46 (s, 3H), 1.66-1.61 (m, 1H), 1.53-1.51 (m, 1H), 1.39-1.31 (m, 4H), 0.92 (t, J=6.80 Hz, 3H). LC-MS: (Method E) 495.0 (M ⁺ +H), Rt: 2.94 min, 99.54% (max). HPLC: (Method B) Rt: 5.55 min, 99.08% (max). Chiral SFC: (Method H) Rt: 2.76 min, 100% (max).

Example 14

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

To a stirred solution of tert-butyl (E)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 37; 0.6 g, 1.13 mmol) in DCM (6 ml) at 0 °C was added TFA (1.0 ml) and the resulting mixture was stirred for 2 h at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo and the obtained crude material was purified by Isolera column chromatography (eluent: 2-3% MeOH/DCM; silica gel: 230-400 mesh). The obtained residue was purified by prep. HPLC (Method A) to afford the title compound. Yield: 28% (150 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.30 (s, 1H), 7.81 (d, J=12.4 Hz, 1H), 7.50 (s, 1H), 7.20-7.17 (m, 3H) , 6.75 (t, J=7.6 Hz, 1H), 6.68 (d, J=8.0 Hz, 2H), 5.54 (d, J=12.0 Hz, 1H), 4.09-4.05 (m, 1H ), 3.87-3.85 (m, 1H), 3.34-3.31 (m, 1H), 2.51 (s, 3H), 2.40 (s, 3H), 1.66-1.53 (m, 2H), 1.52-1.30 (m, 4H), 0.92 (t, J=6.80 Hz, 3H). LC-MS: (Method E) 476.9 (M ⁺⁺ H), Rt: 2.87 min, 98, 40% (max). HPLC: (Method B) Rt: 5.41 min, 99.27% (max).

Examples 15 and 16

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

The two enantiomers of racemic (E)-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid (Example 14; 120 mg, 0.25 mmol) were separated by chiral preparative SFC (Method B). 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: 14% (20 mg, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.76 (d, J=12.0 Hz, 1H), 7.48 (s, 1H), 7.21-7.17 (m, 3H), 6.75 (t, J=7.2 Hz, 1H), 6.68 (d, J=8.4 Hz, 2H), 5.55 (d, J=12.0 Hz, 1H), 4.08-4.04 (m, 1H), 3.86-3.85 (m, 1H), 3.34-3.31 (m, 1H), 2.51 (s, 3H), 2.40 (s, 3H), 1.64-1.53 (m, 2H), 1.52-1.30 (m, 4H), 0.95-0.92 (m, 3H). LC-MS: (Method E) 477.1 (M ⁺⁺ H), Rt: 2.85 min, 99.84% (max). HPLC: (Method B) Rt: 5.48 min, 98.34% (max). SFC: (Method H) Rt: 1.71 min, 99.79%.

Enantiomer 2: Yield: 14% (20 mg, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.75 (d, J=12.0 Hz, 1H), 7.48 (s, 1H), 7.21-7.17 (m, 3H), 6.75 (t, J=7.2 Hz, 1H), 6.68 (d, J=8.0 Hz, 2H), 5.55 (d, J=12.0 Hz, 1H), 4.08-4.05 (m, 1H), 3.87-3.86 (m, 1H), 3.34-3.31 (m, 1H), 2.51 (s, 3H), 2.41 (s, 3H), 1.64-1.53 (m, 2H), 1.52-1.30 (m, 4H), 0.95-0.92 (m, 3H). LC-MS: (Method E) 477.1 (M ⁺⁺ H), Rt: 2.85 min, 99.88% (max). HPLC: (Method B) Rt: 5.48 min, 99.0% (max). SFC: (Method H) Rt: 3.47 min, 99.41%.

Example 17

(E)-3-((3-Butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

To a stirred solution of tert-butyl (E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 39; 0.14 g, 0.25 mmol) in DCM (5 mL) at 0 °C was added TFA (0.39 mL, 5.12 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 cold water (5 mL) and the aqueous layer was extracted with DCM (2×5 mL). The combined organic layer was washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo. The resulting crude material was purified by prep. HPLC purification (Method A). The fraction was concentrated in vacuo, dissolved in ethyl acetate (5 mL), and the organic portion was washed with water (2 x 5 mL) and brine (5 mL). The organic portion was dried over anhydrous Na2SU4 and concentrated in vacuo to afford the title compound. Yield: 45% (60 mg, white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.27 (s, 1H), 7.80 (d, J=12.4 Hz, 1H), 7.50 (s, 1H), 7.23-7.18 (m, 3H) , 6.77 (t, J=6.8 Hz, 1H), 6.69 (d, J=8.0 Hz, 2H), 5.53 (d, J=12.0 Hz, 1H), 4.09-4.05 (m, 1H ), 3.90-3.80 (m, 1H), 2.98-2.93 (m, 2H), 2.51 (s, 3H), 2.50-2.90 (m, 1H), 1.70-1.50 (m, 2H), 1.40-1.25 (m, 4H), 1.20 (t, J=7.20 Hz, 3H), 0.95-0.85 (m, 3H). LC-MS: (Method E) 490.9 (M ⁺ +H), Rt: 3.02 min, 97.10% (max). HPLC: (Method E) Rt: 5.66 min, 95.37% (max).

Examples 18 and 19

(S)-(E)-3-((3-Butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid and (R)-(E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,314,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

The two enantiomers of racemic (E)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid (Example 17; 50 mg, 0.102 mmol) were separated by chiral SFC (Method S). 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: 40% (20 mg, white solid). ^1H NMR (400 MHz, DMSO-Ju): δ 12.26 (s, 1H), 7.80 (d, J=12.0 Hz, 1H), 7.50 (s, 1H), 7.23-7.17 (m, 3H), 6.76 (t, J=7.2 Hz, 1H), 6.69 (d, J=8.4 Hz, 2H), 5.52 (d, J=12.4 Hz, 1H), 4.09-4.05 (m, 1H), 3.90-3.75 (m, 1H), 2.98-2.92 (m, 2H), 2.51 (s, 3H), 2.50-2.40 (m, 1H), 1.75-1.50 (m, 2H), 1.40-1.32 (m, 4H), 1.20 (t, J=7.60 Hz, 3H), 0.9 (t, J=6.8 Hz, 3H). LC-MS: (Method A) 491.0 (M ⁺ +H), Rt: 2.71 min, 99.40% (max). HPLC: (Method E) Rt: 5.61 min, 99.72% (max). Chiral SFC: (Method S) Rt: 2.99 min, 98.37% (max).

Enantiomer 2: Yield: 40% (30 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.29 (s, 1H), 7.81 (d, J=12.0 Hz, 1H), 7.50 (s, 1H), 7.24-7.17 (m, 3H), 6.76 (t, J=7.2 Hz, 1H), 6.69 (d, J=8.0 G ts, 2H), 5.53 (d, J=12.0 Hz, 1H), 4.07 (m, 1H), 3.92-3.80 (m, 1H), 3.42-3.38 (m, 1H), 2.98-2.93 (m, 2H), 2.51 (s, 3H), 1.75-1.48 (m, 2H), 1.48-1.30 (m, 4H), 1.20 (t, J=7.20 Hz, 3H), 0.9 (t, J=6.8 Hz, 3H). LC-MS: (Method A) 491.2 (M ⁺⁺ H), Rt: 2.71 min, 98.99% (max). HPLC: (Method E) Rt: 5.61 min, 99.18% (max). Chiral SFC: (Method S) Rt: 3.73 min, 98.28% (max).

Example 20

(E)-3-((3-Butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,314,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

To a stirred solution of tert-butyl (E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 47; 40 mg, 0.07 mmol) in DCM (2 ml) at 0 °C was added TFA (0.6 ml, 2.18 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 quenched with cold water (5 ml) and the aqueous layer was extracted with DCM (2×5 ml). The combined organic layer was washed with water (5 ml) and brine (5 ml) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 35-40% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 91% (32 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.30 (s, 1H), 7.80 (d, J=12.4 Hz, 1H), 7.48 (s, 1H), 7.15 (s, 1H), 7.03 (t, J=8.8 Hz, 2H), 6.72-6.69 (m, 2H), 5.53 (d, J=12.4 Hz, 1H), 4.05-4.01 (m, 1H), 3.83-3.82 (m , 1H), 3.30-3.25 (m, 1H), 2.51 (s, 3H), 2.40 (s, 3H), 1.70-1.45 (m, 2H), 1.45-1.20 (m, 4H), 0.91 (t, J=6.80 Hz, 3H). LC-MS: (Method A) 495.1 (M ⁺ +H), Rt: 2.50 min, 93.89% (max). HPLC: (Method E) Rt: 5.56 min, 94.80% (max).

Examples 21 and 22

(S)-(E)-3-((3-Butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid and (R)-(E)-3-((3-Butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

The two enantiomers of racemic (E)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid (Example 20; 35 mg, 0.07 mmol) were separated by chiral SFC (Method S). 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: 29% (10 mg, off-white solid). ¹ Н ЯМР (400 МГц, DMSO-d ₆ ): δ 12.27 (s, 1H), 7.80 (d, J=12,0 Гц, 1H), 7.49 (s, 1H), 7.15 (s, 1H), 7.03 (t, J=8,8 Гц, 2H), 6.73-6.70 (m, 2H), 5.53 (d, J=12,0 Гц, 1H), 4.10-3.98 (m, 1H), 3.90-3.75 (m, 1H), 3.30-3.25 (m, 1H), 2.53 (s, 3H), 2.40 (s, 3H), 1.68-1.45 (m, 2H), 1.42-1.30 (m, 4H), 0.91 (t, J=6.80 Hz, 3H). LC-MS: (Method A) 495.2 (M ⁺ +H), Rt: 2.46 min, 93.75% (max). HPLC: (Method E) Rt: 5.43 min, 94.52% (max). Chiral SFC: (Method S) Rt: 2.29 min, 98.89% (max).

Enantiomer 2: Yield: 28% (10 mg, off-white solid). ¹ Н ЯМР (400 МГц, DMSO-d ₆ ): δ 12.27 (s, 1Н), 7.80 (d, J=12,4 Гц, 1H), 7.49 (s, 1H), 7.15 (s, 1H), 7.03 (t, J=8,4 Гц, 2H), 6.80-6.50 (m, 2H), 5.53 (d, J=12,0 Гц, 1H), 4.10-3.95 (m, 1H), 3.90-3.75 (m, 1H), 3.35-3.30 (m, 1H), 2.58 (s, 3H), 2.40 (s, 3H), 1.78-1.45 (m, 2H), 1.42-1.30 (m, 4H), 0.95-0.88 (m, 3H). LC-MS: (Method A) 495.1 (M ⁺ +H), Rt: 2.472 min, 97.27% (max). HPLC: (Method E) Rt: 5.434 min, 97.10% (max). Chiral SFC: (Method S) Rt: 4.79 min, 97.64% (max).

Example 23

(E)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

To a stirred solution of tert-butyl (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 49; 60 mg, 0.10 mmol) in DCM (1.5 ml) at 0 °C was added TFA (0.16 ml, 2.12 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 quenched with cold water (5 ml) and the aqueous layer was extracted with DCM (2×5 ml). The combined organic layer was washed with water (5 ml) and brine (5 ml) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo. The resulting crude material was purified by Isolera column chromatography (eluent: 35-40% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 37% (20 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.27 (s, 1H), 7.79 (d, J=12.4 Hz, 1H), 7.49 (s, 1H), 7.18 (s, 1H), 7.04 (t, J=8.8 Hz, 2H), 6.75-6.71 (m, 2H), 5.51 (d, J=12.0 Hz, 1H), 4.05-4.01 (m, 1H), 3.82-3.80 (m , 1H), 3.30 (m, 1H), 2.96-2.90 (m, 2H), 2.46 (s, 3H), 1.61-1.51 (m, 2H), 1.34-1.29 (m, 4H), 1.21 (m, 3H), 0.92-0.89 (m, 3H). LC-MS: (Method A) 509.2 (M ⁺ +H), Rt: 2.62 min, 95.13% (max ). HPLC: (Method E) Rt: 5.62 min, 96.69% (max).

Examples 24 and 25

(S)-(E)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid and (R)-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

The two enantiomers of racemic (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid (Example 23; 35 mg, 0.078 mmol) were separated by chiral SFC (Method S). 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: 40% (14 mg, off-white solid), δ 12.28 (s, 1H), 7.79 (d, J=12.0 Hz, 1H), 7.49 (s, 1H), 7.18 (s, 1H), 7.04 (t, J=8.8 Hz, 2H), 6.73-6.70 (m, 2H), 5.52 (d, J=12.0 Hz, 1H), 4.05-4.01 (m, 1H), 3.90-3.72 (m, 1H), 3.45-3.38 (m, 1H), 2.94 (q, J=6.40 Hz, 2H), 2.55 (s, 3H), 1.68-1.45 (m, 2H), 1.42-1.25 (m, 4H), 1.19 (t, J=7.60 Hz, 3H), 0.9 (t, J=6.8 Hz, 3H). LC-MS: (Method A) 509.1 (M ⁺⁺ H), Rt: 3.03 min, 95.96% (max). HPLC: (Method E) Rt: 5.75 min, 97.05% (max). Chiral SFC: (Method S) Rt: 2.75 min, 100% (max).

Enantiomer 2: Yield: 37% (13 mg, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.28 (s, 1H), 7.79 (d, J=12.0 Hz, 1H), 7.49 (s, 1H), 7.18 (s, 1H), 7.04 (t, J=8.8 Hz, 2H), 6.74-6.70 (m, 2H), 5 .52 (d, J=12.0 Hz, 1H), 4.1-4.0 (m, 1H), 3.88-3.72 (m, 1H), 3.45-3.35 (m, 1H), 2.94 (q, J=6.8 Hz, 2H), 2.55 (s, 3H), 1.70-1.45 (m, 2H), .28 (m, 4H), 1.19 (t, J=7.20 Hz, 3H), 0.91 (t, J=6.40 Hz, 3H). LC-MS: (Method B) 508.9 (M ⁺⁺ H), Rt: 2.09 min, 96.57% (max). HPLC: (Method E) Rt: 5.76 min, 94.92% (max). Chiral SFC: (Method S) Rt: 3.42 min, 97.83% (max).

Example 26

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

To a stirred solution of tert-butyl (E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 54; 65 mg, 0.12 mmol) in DCM (3 ml) at 0 °C was added TFA (1 ml, 12.98 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 quenched with cold water (5 ml) and the aqueous layer was extracted with DCM (2×5 ml). The combined organic layer was washed with water (5 ml) and brine (5 ml) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 35-40% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 69% (42 mg, white solid).

^1H NMR (400 MHz, DMSO- _d6 ): δ 12.28 (s, 1H), 7.76 (d, J=12.0 Hz, 1H), 7.56 (s, 1H), 7.17 (s, 1H), 7.04 (t, J=8.8 Hz, 2H), 6.70-6.68 (m, 2H), 5.52 (d, J=12.4 Hz, 1H), 4.22-4.19 (m, 1H), 3.50-4.47 (m , 1H), 3.21-3.10 (m, 2H), 2.41 (s, 3H), 2.30-2.25 (m, 1H), 1.40-1.28 (m, 6H), 0.90 (t, J=7.20 Hz, 3H). LC-MS-Q-TOF: (Method J) 480.1 (M ⁺⁺ H), Rt: 2.88 min, 97.00% (max). HPLC: ( Method E) Rt: 5.35 min, 96.21% (max).

Examples 27 and 28

(S)-(E)-3-((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid and (R)-(E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid

The two enantiomers of racemic (E)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid (Example 26; 38 mg, 0.079 mmol) were separated by chiral SFC (Method T). 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: 34% (13 mg, white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.30 (s, 1H), 7.75 (d, J=12.4 Hz, 1H), 7.56 (s, 1H), 7.17 (s, 1H), 7.03 (t, J=9.2 Hz, 2H), 6.69-6.66 (m, 2H), 5 .52 (d, J=12.0 Hz, 1H), 4.21 (d, J=15.2 Hz, 1H), 3.51-3.47 (m, 1H), 3.34-3.28 (m, 1H), 3.20-3.05 (m, 1H), 2.41 (s, 3H), 2.30-2.18 (m, 1H), 1.50-1.20 (m, 6H), 0.90 (t, J=7.20 Hz, 3H). LC-MS: (Method A) 480.1 (M ⁺⁺ H), Rt: 2.43 min, 96.73% (max). HPLC: (Method E) Rt: 5.34 min, 96.74% (max). Chiral SFC: (Method T) Rt: 3.53 min, 100% (max).

Enantiomer 2: Yield: 32% (12 mg, off-white solid). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.23 (s, 1H), 7.75 (d, J=12.4 Hz, 1H), 7.56 (s, 1H), 7.17 (s, 1H), 7.06-7.01 (m, 2H), 6.71-6.68 (m, 2H), (d, J=12.0 Hz, 1H), 4.21 (d, J=14.4 Hz, 1H), 3.51-3.46 (m, 1H), 3.40-3.30 (m, 1H), 3.22-3.12 (m, 1H), 2.41 (s, 3H), 2.30-2.20 (m, 1H), 1.45-1.35 (m, 4H), 1.32-1.28 (m, 2H), 0.9 (t, J=8.4 Hz, 3H). LC-MS: (Method A) 480.1 (M ⁺ +H), Rt: 2.43 min, 97.29% (max). HPLC: (Method E) Rt: 5.34 min, 97.07% (max). Chiral SFC: (Method T) Rt: 5.07 min, 99.37% (max).

Example 29

(E)-3-((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

To a stirred solution of tert-butyl (E)-3-((3-butyl-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 57; 60 mg, 0.09 mmol) in toluene (3 ml) at 0 °C were added triphenylamine (67.2 mg, 0.27 mmol) and TFA (0.11 ml, 1.46 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 cold water (5 ml) and the aqueous layer was extracted with ethyl acetate (2×5 ml). The combined organic layer was washed with water (5 mL) and brine (5 mL) and dried over _{anhydrous Na2SO4} . The organic portion was filtered and concentrated in vacuo. The resulting crude material was purified by prep. HPLC purification (Method D) to afford the title compound. Yield: 50% (22 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.3 (s, 1H), 7.73 (d, J=12.0 Hz, 1H), 7.51 (s, 1H), 7.41 (d, J=9.2 Hz, 1H), 7.15 (s, 1H), 7.03 (t, J=8.8 Hz, 2H), 6.72-6.69 (m, 2H), 5.41 (d, J=12.4 Hz, 1H), 4.31 (m, 1H), 3.27 (m, 1H), 2.50 (m, 1H), 2.33 (s, 3H), 1.57-1.56 (m, 1H), 1.43-1.32 (m, 3H), 1.29-1.24 (m, 2H), 0.91-0.88 (m, 3H). LC-MS: (Method A) 479.0 (M ⁺ -H), Rt: 2.80 min, 95.41% (max) . HPLC: (Method E) Rt: 5.30 min, 97.48% (max).

Example 30

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

To a stirred solution of tert-butyl (E)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 59; 72 mg, 0.135 mmol) in DCM (5 mL) at 0 °C was added TFA (1 mL, 12.98 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 quenched with cold water (5 mL) and the aqueous layer was extracted with DCM (2×5 mL). The combined organic layer was washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na ₂ SO ₄ . The organic portion was filtered and concentrated in vacuo. The obtained crude material was purified by Isolera column chromatography (eluent: 30-35% EtOAc/PE; silica gel: 230-400 mesh) to give the title compound. Yield: 46% (30 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.26 (s, 1H), 7.75 (d, J=12.40 Hz, 1H), 7.58 (s, 1H), 7.26 (s, 1H), 7.17 -7.19 (m, 2H), 6.75 (t, J=7.20 Hz, 1H), 6.67 (d, J=8.00 Hz, 2H), 5.51 (d, J=12.40 Hz, 1H), 4.25 (m, 1H), 3.49 (m, 1H), 3.41 (m, 1H), 3.15 (m, 1H), 2.94-2.96 (m, 2H), 2.34-2.34 (m, 1H), 1.36-1.38 (m, 5H), 1.30-1.32 (m, 1H), 1.18-1.20 (m, 3H), 0.05-0.89 (m, 3H). LC-MS: (Method A) 476.1 (M ⁺ +H), Rt: 2.68 min, 98.13% (max). HPLC: (Method B) Rt: 5.65 min, 99.73% (max).

Example 31

(E)-3-((3-Butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

To a stirred solution of tert-butyl (E)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylate (Intermediate 60; 40 mg, 0.07 mmol) in DCM (5 mL) at 0 °C was added TFA (0.11 mL, 1.50 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 quenched with cold water (5 mL) and the aqueous layer was extracted with DCM (2×5 mL). The combined organic layer was washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na2SO4. The organic portion was filtered and concentrated in vacuo. The resulting crude material was purified by prep. HPLC purification (Method A) to afford the title compound. Yield: 56% (10 mg, off-white solid).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.28 (s, 1H), 7.74 (d, J=12.0 Hz, 1H), 7.53 (s, 1H), 7.41 (d, J=9.2 Hz, 1H), 7.25 (s, 1H), 7.19 (t, J=8.4 Hz, 2H), 6.75 (t, J=7.2 Hz, 1H), 6.67 (d, J=8.4 Hz, 2H), 5.44 (d, J=12.4 Hz, 1H), 4.39-4.35 (m, 1H), 3.45-3.35 (m, 2H), 2.98-2.92 (m, 2H), 1.65-1.55 (m, 1H), 1.50-1.25 (m, 5H), 1.19 (t, J=7.20 Hz, 3H), 0.9 (t, J=7.6 Hz, 3H). LC-MS: (Method B) 474.9 (M ⁺ -H), Rt: 2.03 min, 94.04% (max). HPLC: (Method E) Rt: 5.36 min, 95.06% (max).

Example 32

(E)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid

The title compound was prepared from tert-butyl (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylate (Intermediate 61) according to the same procedure as described in Example 29.

Example 33

(Z)-3-((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepine-8- yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 62) according to the procedure described for Example 4.

Examples 34 and 35

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

The two enantiomers of racemic (Z)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 33) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 36

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

The title compound was prepared from ethyl (Z)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 63) according to the procedure described for Example 4.

Examples 37 and 38

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

These two enantiomers of racemic (Z)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid

(Example 36) was separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1, and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of these two enantiomers is not known.

Example 39

(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 64) according to the procedure described for Example 4.

Examples 40 and 41

(S)-(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid and (R)-(Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid

The two enantiomers of racemic (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 39) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 42

(Z)-3-((3-Butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2, 5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 65) according to the procedure described for Example 4.

Examples 43 and 44

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

The two enantiomers of racemic (Z)-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 42) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 45

(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 66) according to the procedure described for Example 4.

Examples 46 and 47

(S)-(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid and (R)-(Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

These two enantiomers of racemic (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 45) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of these two enantiomers is not known.

Example 48

(Z)-3-((3-Butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from ethyl (Z)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylate (Intermediate 67) according to the procedure described for Example 4.

Examples 49 and 50

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

The two enantiomers of racemic (Z)-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 48) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 51

(Z)-3-((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine- 8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from (Z)-3-((3-butyl-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Intermediate 69) according to the procedure described for Example 10.

Examples 52 and 53

(S)-(Z)-3-((3-Butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2, 5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid and (R)-(Z)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1- dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The two enantiomers of racemic (Z)-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 51) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 54

(Z)-3-((3-Butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from (Z)-3-((3-butyl-7-(ethylthio)-2-(4-methoxybenzyl)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Intermediate 71) according to the procedure described for Example 10.

Examples 55 and 56

(S)-(Z)-3-((3-Butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid and (R)-(Z)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The two enantiomers of racemic (Z)-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 54) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 57

(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The title compound was prepared from (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Intermediate 73) according to the procedure described for Example 10.

Examples 58 and 59

(S)-(Z)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid and (R)-(Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid

The two enantiomers of racemic (Z)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid (Example 57) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of the two enantiomers is not known.

Example 60

(E)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

The title compound was prepared from tert-butyl (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-(4-methoxybenzyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid (Intermediate 74) according to the procedure described for Example 29.

Examples 61 and 62

(S)-(E)-3-((3-Butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid and (R)-(E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid

These two enantiomers of racemic (E)-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid (Example 60) were separated by chiral SFC. This material was concentrated in vacuo at 40°C. The first eluting fraction corresponds to enantiomer 1 and the second eluting fraction corresponds to enantiomer 2. The absolute configuration of these two enantiomers is not known.

BIOLOGICAL ANALYSES

IBAT Analysis Protocol (h/m)

10,000 cells (human or mouse IBAT overexpressing cells) were seeded in a 96-well plate (Corning CLS3809) in 200 μl MEM-alpha medium (Gibco 12571-063) supplemented with 10% FBS (Gibco 10438026) containing puromycin (Gibco A1113803) (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 standing 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 maintained in an incubator (37°C, 5% _CO2 , 95% relative humidity (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 DMSO concentration 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. Electrical resistance of 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 transfer 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 in the receiving compartment), C ₀ is the initial concentration in the donor compartment, and A is the surface area of the effective filter membrane.

HepaRG-based analysis 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, replenished 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 the compounds, 3-fold serial dilutions were prepared in DMSO to give a total of 10 dilutions of the 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.

Effect of LBAT-selective compounds on serum bile acid levels in OATP KO mice

Male OATP KO mice (Oatp1a/1b cluster knockout mice, Taconic, USA) aged 8 to 9 weeks were used in these studies. After completion of the quarantine and acclimatization period, the animals were randomized by body weight into experimental groups (4 mice each). On the day of the experiment, a baseline blood sample was collected, followed by oral administration of vehicle (0.5% CMC, 0.1% Tween 80) or compound of Examples 3, 6, 13, or 15 at the indicated doses. Blood samples were then collected at various time points after dosing (indicated in each graph). Blood was allowed to clot, serum was collected by centrifugation at 3000 g, 4 °C for 15 minutes, and stored at -80 °C for further analysis. Total bile acid concentration in each serum sample was determined using the CELL BIOLABS Total Bile Acids kit (STA631) according to the manufacturer's instructions. Mean ± SEM, n=4.

The results are shown in Figs. 1-4. Serum bile acid levels before dosing and during vehicle treatment ranged from 1 to 5 μmol/L. Oral administration of Example 13 increased serum bile acid levels in a dose-dependent manner. Within one hour after dosing, serum bile acid levels increased to approximately 100 μmol/L in response to 30 mg/kg of Example 13. The compounds of Examples 3, 6, and 15 also increased serum bile acid levels.

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) vehicle control, 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 the effects 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) vehicle control, and (2) test compound at 1 mg/kg p.o. Animals were treated with 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 weekly. Total serum bile acids were measured from day 7 serum samples. Fecal bile acid excretion was measured from day 7 stool sample. Urinary bile acid excretion was measured from day 7 sample. Renal expression of ASBT, OSTa, OSTAb, and MRP2 was quantified in day 7 samples.

--->

SEQUENCE LIST

<110>ALBIREO AB

<120> BENZOTHIUM(DI)AZEPINE COMPOUNDS AND THEIR USE AS

BILE ACIDS MODULATORS

<130> NP0500WO

<150> IN 201911049982

<151> 2019-12-04

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1251

<212> PRT

<213> Homo sapiens

<400> 1

Met Ser Thr Glu Arg Asp Ser Glu Thr Thr Phe Asp Glu Asp Ser Gln

1 5 10 15

Pro Asn Asp Glu Val Val Pro Tyr Ser Asp Asp Glu Thr Glu Asp Glu

20 25 30

Leu Asp Asp Gln Gly Ser Ala Val Glu Pro Glu Gln Asn Arg Val Asn

35 40 45

Arg Glu Ala Glu Glu Asn Arg Glu Pro Phe Arg Lys Glu Cys Thr Trp

50 55 60

Gln Val Lys Ala Asn Asp Arg Lys Tyr His Glu Gln Pro His Phe Met

65 70 75 80

Asn Thr Lys Phe Leu Cys Ile Lys Glu Ser Lys Tyr Ala Asn Asn Ala

85 90 95

Ile Lys Thr Tyr Lys Tyr Asn Ala Phe Thr Phe Ile Pro Met Asn Leu

100 105 110

Phe Glu Gln Phe Lys Arg Ala Ala Asn Leu Tyr Phe Leu Ala Leu Leu

115 120 125

Ile Leu Gln Ala Val Pro Gln Ile Ser Thr Leu Ala Trp Tyr Thr Thr

130 135 140

Leu Val Pro Leu Leu Val Val Leu Gly Val Thr Ala Ile Lys Asp Leu

145 150 155 160

Val Asp Asp Val Ala Arg His Lys Met Asp Lys Glu Ile Asn Asn Arg

165 170 175

Thr Cys Glu Val Ile Lys Asp Gly Arg Phe Lys Val Ala Lys Trp Lys

180 185 190

Glu Ile Gln Val Gly Asp Val Ile Arg Leu Lys Lys Asn Asp Phe Val

195 200 205

Pro Ala Asp Ile Leu Leu Leu Ser Ser Ser Glu Pro Asn Ser Leu Cys

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Tyr Val Glu Thr Ala Glu Leu Asp Gly Glu Thr Asn Leu Lys Phe Lys

225 230 235 240

Met Ser Leu Glu Ile Thr Asp Gln Tyr Leu Gln Arg Glu Asp Thr Leu

245 250 255

Ala Thr Phe Asp Gly Phe Ile Glu Cys Glu Glu Pro Asn Asn Arg Leu

260 265 270

Asp Lys Phe Thr Gly Thr Leu Phe Trp Arg Asn Thr Ser Phe Pro Leu

275 280 285

Asp Ala Asp Lys Ile Leu Leu Arg Gly Cys Val Ile Arg Asn Thr Asp

290 295 300

Phe Cys His Gly Leu Val Ile Phe Ala Gly Ala Asp Thr Lys Ile Met

305 310 315 320

Lys Asn Ser Gly Lys Thr Arg Phe Lys Arg Thr Lys Ile Asp Tyr Leu

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Met Asn Tyr Met Val Tyr Thr Ile Phe Val Val Leu Ile Leu Leu Ser

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Ala Gly Leu Ala Ile Gly His Ala Tyr Trp Glu Ala Gln Val Gly Asn

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Ser Ser Trp Tyr Leu Tyr Asp Gly Glu Asp Asp Thr Pro Ser Tyr Arg

370 375 380

Gly Phe Leu Ile Phe Trp Gly Tyr Ile Ile Val Leu Asn Thr Met Val

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Pro Ile Ser Leu Tyr Val Ser Val Glu Val Ile Arg Leu Gly Gln Ser

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His Phe Ile Asn Trp Asp Leu Gln Met Tyr Tyr Ala Glu Lys Asp Thr

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Pro Ala Lys Ala Arg Thr Thr Thr Leu Asn Glu Gln Leu Gly Gln Ile

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His Tyr Ile Phe Ser Asp Lys Thr Gly Thr Leu Thr Gln Asn Ile Met

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Thr Phe Lys Lys Cys Cys Ile Asn Gly Gln Ile Tyr Gly Asp His Arg

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Asp Ala Ser Gln His Asn His Asn Lys Ile Glu Gln Val Asp Phe Ser

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Trp Asn Thr Tyr Ala Asp Gly Lys Leu Ala Phe Tyr Asp His Tyr Leu

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Ile Glu Gln Ile Gln Ser Gly Lys Glu Pro Glu Val Arg Gln Phe Phe

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Phe Leu Leu Ala Val Cys His Thr Val Met Val Asp Arg Thr Asp Gly

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Gln Leu Asn Tyr Gln Ala Ala Ser Pro Asp Glu Gly Ala Leu Val Asn

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Ala Ala Arg Asn Phe Gly Phe Ala Phe Leu Ala Arg Thr Gln Asn Thr

565 570 575

Ile Thr Ile Ser Glu Leu Gly Thr Glu Arg Thr Tyr Asn Val Leu Ala

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Ile Leu Asp Phe Asn Ser Asp Arg Lys Arg Met Ser Ile Ile Val Arg

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Thr Pro Glu Gly Asn Ile Lys Leu Tyr Cys Lys Gly Ala Asp Thr Val

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Ile Tyr Glu Arg Leu His Arg Met Asn Pro Thr Lys Gln Glu Thr Gln

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Asp Ala Leu Asp Ile Phe Ala Asn Glu Thr Leu Arg Thr Leu Cys Leu

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Cys Tyr Lys Glu Ile Glu Glu Lys Glu Phe Thr Glu Trp Asn Lys Lys

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Phe Met Ala Ala Ser Val Ala Ser Thr Asn Arg Asp Glu Ala Leu Asp

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Lys Val Tyr Glu Glu Ile Glu Lys Asp Leu Ile Leu Leu Gly Ala Thr

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Ala Ile Glu Asp Lys Leu Gln Asp Gly Val Pro Glu Thr Ile Ser Lys

705 710 715 720

Leu Ala Lys Ala Asp Ile Lys Ile Trp Val Leu Thr Gly Asp Lys Lys

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Glu Thr Ala Glu Asn Ile Gly Phe Ala Cys Glu Leu Leu Thr Glu Asp

740 745 750

Thr Thr Ile Cys Tyr Gly Glu Asp Ile Asn Ser Leu Leu His Ala Arg

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Met Glu Asn Gln Arg Asn Arg Gly Gly Val Tyr Ala Lys Phe Ala Pro

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Pro Val Gln Glu Ser Phe Phe Pro Pro Gly Gly Asn Arg Ala Leu Ile

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Ile Thr Gly Ser Trp Leu Asn Glu Ile Leu Leu Glu Lys Lys Thr Lys

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Arg Asn Lys Ile Leu Lys Leu Lys Phe Pro Arg Thr Glu Glu Glu Arg

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Arg Met Arg Thr Gln Ser Lys Arg Arg Leu Glu Ala Lys Lys Glu Gln

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Arg Gln Lys Asn Phe Val Asp Leu Ala Cys Glu Cys Ser Ala Val Ile

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Cys Cys Arg Val Thr Pro Lys Gln Lys Ala Met Val Val Asp Leu Val

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Lys Arg Tyr Lys Lys Ala Ile Thr Leu Ala Ile Gly Asp Gly Ala Asn

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Asp Val Asn Met Ile Lys Thr Ala His Ile Gly Val Gly Ile Ser Gly

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Gln Glu Gly Met Gln Ala Val Met Ser Ser Asp Tyr Ser Phe Ala Gln

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Phe Arg Tyr Leu Gln Arg Leu Leu Leu Val His Gly Arg Trp Ser Tyr

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Ile Arg Met Cys Lys Phe Leu Arg Tyr Phe Phe Tyr Lys Asn Phe Ala

945 950 955 960

Phe Thr Leu Val His Phe Trp Tyr Ser Phe Phe Asn Gly Tyr Ser Ala

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Gln Thr Ala Tyr Glu Asp Trp Phe Ile Thr Leu Tyr Asn Val Leu Tyr

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Thr Ser Leu Pro Val Leu Leu Met Gly Leu Leu Asp Gln Asp Val Ser

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Asp Lys Leu Ser Leu Arg Phe Pro Gly Leu Tyr Ile Val Gly Gln

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Arg Asp Leu Leu Phe Asn Tyr Lys Arg Phe Phe Val Ser Leu Leu

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His Gly Val Leu Thr Ser Met Ile Leu Phe Phe Ile Pro Leu Gly

1040 1045 1050

Ala Tyr Leu Gln Thr Val Gly Gln Asp Gly Glu Ala Pro Ser Asp

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Tyr Gln Ser Phe Ala Val Thr Ile Ala Ser Ala Leu Val Ile Thr

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Val Asn Phe Gln Ile Gly Leu Asp Thr Ser Tyr Trp Thr Phe Val

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Asn Ala Phe Ser Ile Phe Gly Ser Ile Ala Leu Tyr Phe Gly Ile

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Met Phe Asp Phe His Ser Ala Gly Ile His Val Leu Phe Pro Ser

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Ala Phe Gln Phe Thr Gly Thr Ala Ser Asn Ala Leu Arg Gln Pro

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Tyr Ile Trp Leu Thr Ile Ile Leu Ala Val Ala Val Cys Leu Leu

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Pro Val Val Ala Ile Arg Phe Leu Ser Met Thr Ile Trp Pro Ser

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Glu Ser Asp Lys Ile Gln Lys His Arg Lys Arg Leu Lys Ala Glu

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Glu Gln Trp Gln Arg Arg Gln Gln Val Phe Arg Arg Gly Val Ser

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Thr Arg Arg Ser Ala Tyr Ala Phe Ser His Gln Arg Gly Tyr Ala

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Asp Leu Ile Ser Ser Gly Arg Ser Ile Arg Lys Lys Arg Ser Pro

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Leu Asp Ala Ile Val Ala Asp Gly Thr Ala Glu Tyr Arg Arg Thr

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Gly Asp Ser

1250

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cgtctgaaaa aaaatgattt tgttccagct gacattctcc tgctgtctag ctctgagcct 660

aacagcctct gctatgtgga aacagcagaa ctggatggag aaaccaattt aaaatttaag 720

atgtcacttg aaatcacaga ccagtacctc caaagagaag atacattggc tacatttgat 780

ggttttattg aatgtgaaga acccaataac agactagata agtttacagg aacactattt 840

tggagaaaca caagttttcc tttggatgct gataaaattt tgttacgtgg ctgtgtaatt 900

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aagaatagtg ggaaaaccag atttaaaaga actaaaattg attacttgat gaactacatg 1020

gtttacacga tctttgttgt tcttattctg ctttctgctg gtcttgccat cggccatgct 1080

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gagccagaag tacgacagtt cttcttcttg ctcgcagttt gccacacagt catggtggat 1620

aggactgatg gtcagctcaa ctaccaggca gcctctcccg atgaaggtgc cctggtaaac 1680

gctgccagga actttggctt tgccttcctc gccaggaccc agaacaccat caccatcagt 1740

gaactgggca ctgaaaggac ttacaatgtt cttgccattt tggacttcaa cagtgaccgg 1800

aagcgaatgt ctatcattgt aagaacccca gaaggcaata tcaagcttta ctgtaaaggt 1860

gctgacactg ttatttatga acggttacat cgaatgaatc ctactaagca agaaacacag 1920

gatgccctgg atatctttgc aaatgaaact cttagaaccc tatgcctttg ctacaaggaa 1980

attgaagaaa aagaatttac agaatggaat aaaaagttta tggctgccag tgtggcctcc 2040

accaaccggg acgaagctct ggataaagta tatgaggaga ttgaaaaaga cttaattctc 2100

ctgggagcta cagctattga agacaagcta caggatggag ttccagaaac catttcaaaa 2160

cttgcaaaag ctgacattaa gatctgggtg cttactggag acaaaaagga aactgctgaa 2220

aatataggat ttgcttgtga acttctgact gaagacacca ccatctgcta tggggaggat 2280

attaattctc ttcttcatgc aaggatggaa aaccagagga atagaggtgg cgtctacgca 2340

aagtttgcac ctcctgtgca ggaatctttt tttccacccg gtggaaaccg tgccttaatc 2400

atcactggtt cttggttgaa tgaaattctt ctcgagaaaa agaccaagag aaataagatt 2460

ctgaagctga agttcccaag aacagaagaa gaaagacgga tgcggaccca aagtaaaagg 2520

aggctagaag ctaagaaaga gcagcggcag aaaaactttg tggacctggc ctgcgagtgc 2580

agcgcagtca tctgctgccg cgtcaccccc aagcagaagg ccatggtggt ggacctggtg 2640

aagaggtaca agaaagccat cacgctggcc atcggagatg gggccaatga cgtgaacatg 2700

atcaaaactg cccacattgg cgttggaata agtggacaag aaggaatgca agctgtcatg 2760

tcgagtgact attcctttgc tcagttccga tatctgcaga ggctactgct ggtgcatggc 2820

cgatggtctt acataaggat gtgcaagttc ctacgatact tcttttacaa aaactttgcc 2880

tttactttgg ttcatttctg gtactccttc ttcaatggct actctgcgca gactgcatac 2940

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ggaatacatg ttctctttcc atctgcattt caatttacag gcacagcttc aaacgctctg 3420

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catcgcaagc ggttgaaggc ggagggagcag tggcagcgac ggcagcaggt gttccgccgg 3600

ggcgtgtcaa cgcggcgctc ggcctacgcc ttctcgcacc agcggggcta cgcggacctc 3660

atctcctccg ggcgcagcat ccgcaagaag cgctcgccgc ttgatgccat cgtggcggat 3720

ggcaccgcgg agtacaggcg caccggggac agctga 3756

<210> 3

<211> 1321

<212> PRT

<213> Homo sapiens

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Met Ser Asp Ser Val Ile Leu Arg Ser Ile Lys Lys Phe Gly Glu Glu

1 5 10 15

Asn Asp Gly Phe Glu Ser Asp Lys Ser Tyr Asn Asn Asp Lys Lys Ser

20 25 30

Arg Leu Gln Asp Glu Lys Lys Gly Asp Gly Val Arg Val Gly Phe Phe

35 40 45

Gln Leu Phe Arg Phe Ser Ser Ser Thr Asp Ile Trp Leu Met Phe Val

50 55 60

Gly Ser Leu Cys Ala Phe Leu His Gly Ile Ala Gln Pro Gly Val Leu

65 70 75 80

Leu Ile Phe Gly Thr Met Thr Asp Val Phe Ile Asp Tyr Asp Val Glu

85 90 95

Leu Gln Glu Leu Gln Ile Pro Gly Lys Ala Cys Val Asn Asn Thr Ile

100 105 110

Val Trp Thr Asn Ser Ser Leu Asn Gln Asn Met Thr Asn Gly Thr Arg

115 120 125

Cys Gly Leu Leu Asn Ile Glu Ser Glu Met Ile Lys Phe Ala Ser Tyr

130 135 140

Tyr Ala Gly Ile Ala Val Ala Val Leu Ile Thr Gly Tyr Ile Gln Ile

145 150 155 160

Cys Phe Trp Val Ile Ala Ala Ala Arg Gln Ile Gln Lys Met Arg Lys

165 170 175

Phe Tyr Phe Arg Arg Ile Met Arg Met Glu Ile Gly Trp Phe Asp Cys

180 185 190

Asn Ser Val Gly Glu Leu Asn Thr Arg Phe Ser Asp Asp Ile Asn Lys

195 200 205

Ile Asn Asp Ala Ile Ala Asp Gln Met Ala Leu Phe Ile Gln Arg Met

210 215 220

Thr Ser Thr Ile Cys Gly Phe Leu Leu Gly Phe Phe Arg Gly Trp Lys

225 230 235 240

Leu Thr Leu Val Ile Ile Ser Val Ser Pro Leu Ile Gly Ile Gly Ala

245 250 255

Ala Thr Ile Gly Leu Ser Val Ser Lys Phe Thr Asp Tyr Glu Leu Lys

260 265 270

Ala Tyr Ala Lys Ala Gly Val Val Ala Asp Glu Val Ile Ser Ser Met

275 280 285

Arg Thr Val Ala Ala Phe Gly Gly Glu Lys Arg Glu Val Glu Arg Tyr

290 295 300

Glu Lys Asn Leu Val Phe Ala Gln Arg Trp Gly Ile Arg Lys Gly Ile

305 310 315 320

Val Met Gly Phe Phe Thr Gly Phe Val Trp Cys Leu Ile Phe Leu Cys

325 330 335

Tyr Ala Leu Ala Phe Trp Tyr Gly Ser Thr Leu Val Leu Asp Glu Gly

340 345 350

Glu Tyr Thr Pro Gly Thr Leu Val Gln Ile Phe Leu Ser Val Ile Val

355 360 365

Gly Ala Leu Asn Leu Gly Asn Ala Ser Pro Cys Leu Glu Ala Phe Ala

370 375 380

Thr Gly Arg Ala Ala Ala Thr Ser Ile Phe Glu Thr Ile Asp Arg Lys

385 390 395 400

Pro Ile Ile Asp Cys Met Ser Glu Asp Gly Tyr Lys Leu Asp Arg Ile

405 410 415

Lys Gly Glu Ile Glu Phe His Asn Val Thr Phe His Tyr Pro Ser Arg

420 425 430

Pro Glu Val Lys Ile Leu Asn Asp Leu Asn Met Val Ile Lys Pro Gly

435 440 445

Glu Met Thr Ala Leu Val Gly Pro Ser Gly Ala Gly Lys Ser Thr Ala

450 455 460

Leu Gln Leu Ile Gln Arg Phe Tyr Asp Pro Cys Glu Gly Met Val Thr

465 470 475 480

Val Asp Gly His Asp Ile Arg Ser Leu Asn Ile Gln Trp Leu Arg Asp

485 490 495

Gln Ile Gly Ile Val Glu Gln Glu Pro Val Leu Phe Ser Thr Thr Ile

500 505 510

Ala Glu Asn Ile Arg Tyr Gly Arg Glu Asp Ala Thr Met Glu Asp Ile

515 520 525

Val Gln Ala Ala Lys Glu Ala Asn Ala Tyr Asn Phe Ile Met Asp Leu

530 535 540

Pro Gln Gln Phe Asp Thr Leu Val Gly Glu Gly Gly Gly Gln Met Ser

545 550 555 560

Gly Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Ala Leu Ile Arg Asn

565 570 575

Pro Lys Ile Leu Leu Leu Asp Met Ala Thr Ser Ala Leu Asp Asn Glu

580 585 590

Ser Glu Ala Met Val Gln Glu Val Leu Ser Lys Ile Gln His Gly His

595 600 605

Thr Ile Ile Ser Val Ala His Arg Leu Ser Thr Val Arg Ala Ala Asp

610 615 620

Thr Ile Ile Gly Phe Glu His Gly Thr Ala Val Glu Arg Gly Thr His

625 630 635 640

Glu Glu Leu Leu Glu Arg Lys Gly Val Tyr Phe Thr Leu Val Thr Leu

645 650 655

Gln Ser Gln Gly Asn Gln Ala Leu Asn Glu Glu Asp Ile Lys Asp Ala

660 665 670

Thr Glu Asp Asp Met Leu Ala Arg Thr Phe Ser Arg Gly Ser Tyr Gln

675 680 685

Asp Ser Leu Arg Ala Ser Ile Arg Gln Arg Ser Lys Ser Gln Leu Ser

690 695 700

Tyr Leu Val His Glu Pro Pro Leu Ala Val Val Asp His Lys Ser Thr

705 710 715 720

Tyr Glu Glu Asp Arg Lys Asp Lys Asp Ile Pro Val Gln Glu Glu Val

725 730 735

Glu Pro Ala Pro Val Arg Arg Ile Leu Lys Phe Ser Ala Pro Glu Trp

740 745 750

Pro Tyr Met Leu Val Gly Ser Val Gly Ala Ala Val Asn Gly Thr Val

755 760 765

Thr Pro Leu Tyr Ala Phe Leu Phe Ser Gln Ile Leu Gly Thr Phe Ser

770 775 780

Ile Pro Asp Lys Glu Glu Gln Arg Ser Gln Ile Asn Gly Val Cys Leu

785 790 795 800

Leu Phe Val Ala Met Gly Cys Val Ser Leu Phe Thr Gln Phe Leu Gln

805 810 815

Gly Tyr Ala Phe Ala Lys Ser Gly Glu Leu Leu Thr Lys Arg Leu Arg

820 825 830

Lys Phe Gly Phe Arg Ala Met Leu Gly Gln Asp Ile Ala Trp Phe Asp

835 840 845

Asp Leu Arg Asn Ser Pro Gly Ala Leu Thr Thr Arg Leu Ala Thr Asp

850 855 860

Ala Ser Gln Val Gln Gly Ala Ala Gly Ser Gln Ile Gly Met Ile Val

865 870 875 880

Asn Ser Phe Thr Asn Val Thr Val Ala Met Ile Ile Ala Phe Ser Phe

885 890 895

Ser Trp Lys Leu Ser Leu Val Ile Leu Cys Phe Phe Pro Phe Leu Ala

900 905 910

Leu Ser Gly Ala Thr Gln Thr Arg Met Leu Thr Gly Phe Ala Ser Arg

915 920 925

Asp Lys Gln Ala Leu Glu Met Val Gly Gln Ile Thr Asn Glu Ala Leu

930 935 940

Ser Asn Ile Arg Thr Val Ala Gly Ile Gly Lys Glu Arg Arg Phe Ile

945 950 955 960

Glu Ala Leu Glu Thr Glu Leu Glu Lys Pro Phe Lys Thr Ala Ile Gln

965 970 975

Lys Ala Asn Ile Tyr Gly Phe Cys Phe Ala Phe Ala Gln Cys Ile Met

980 985 990

Phe Ile Ala Asn Ser Ala Ser Tyr Arg Tyr Gly Gly Tyr Leu Ile Ser

995 1000 1005

Asn Glu Gly Leu His Phe Ser Tyr Val Phe Arg Val Ile Ser Ala

1010 1015 1020

Val Val Leu Ser Ala Thr Ala Leu Gly Arg Ala Phe Ser Tyr Thr

1025 1030 1035

Pro Ser Tyr Ala Lys Ala Lys Ile Ser Ala Ala Arg Phe Phe Gln

1040 1045 1050

Leu Leu Asp Arg Gln Pro Pro Ile Ser Val Tyr Asn Thr Ala Gly

1055 1060 1065

Glu Lys Trp Asp Asn Phe Gln Gly Lys Ile Asp Phe Val Asp Cys

1070 1075 1080

Lys Phe Thr Tyr Pro Ser Arg Pro Asp Ser Gln Val Leu Asn Gly

1085 1090 1095

Leu Ser Val Ser Ile Ser Pro Gly Gln Thr Leu Ala Phe Val Gly

1100 1105 1110

Ser Ser Gly Cys Gly Lys Ser Thr Ser Ile Gln Leu Leu Glu Arg

1115 1120 1125

Phe Tyr Asp Pro Asp Gln Gly Lys Val Met Ile Asp Gly His Asp

1130 1135 1140

Ser Lys Lys Val Asn Val Gln Phe Leu Arg Ser Asn Ile Gly Ile

1145 1150 1155

Val Ser Gln Glu Pro Val Leu Phe Ala Cys Ser Ile Met Asp Asn

1160 1165 1170

Ile Lys Tyr Gly Asp Asn Thr Lys Glu Ile Pro Met Glu Arg Val

1175 1180 1185

Ile Ala Ala Ala Lys Gln Ala Gln Leu His Asp Phe Val Met Ser

1190 1195 1200

Leu Pro Glu Lys Tyr Glu Thr Asn Val Gly Ser Gln Gly Ser Gln

1205 1210 1215

Leu Ser Arg Gly Glu Lys Gln Arg Ile Ala Ile Ala Arg Ala Ile

1220 1225 1230

Val Arg Asp Pro Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Ala

1235 1240 1245

Leu Asp Thr Glu Ser Glu Lys Thr Val Gln Val Ala Leu Asp Lys

1250 1255 1260

Ala Arg Glu Gly Arg Thr Cys Ile Val Ile Ala His Arg Leu Ser

1265 1270 1275

Thr Ile Gln Asn Ala Asp Ile Ile Ala Val Met Ala Gln Gly Val

1280 1285 1290

Val Ile Glu Lys Gly Thr His Glu Glu Leu Met Ala Gln Lys Gly

1295 1300 1305

Ala Tyr Tyr Lys Leu Val Thr Thr Gly Ser Pro Ile Ser

1310 1315 1320

<210> 4

<211> 3966

<212> DNA

<213> Homo sapiens

<400> 4

atgtctgact cagtaattct tcgaagtata aagaaatttg gagaggagaa tgatggtttt 60

gagtcagata aatcatataa taatgataag aaatcaaggt tacaagatga gaagaaaggt 120

gatggcgtta gagttggctt ctttcaattg tttcggtttt cttcatcaac tgacatttgg 180

ctgatgtttg tgggaagttt gtgtgcattt ctccatggaa tagcccagcc aggcgtgcta 240

ctcatttttg gcacaatgac agatgttttt attgactacg acgttgagtt acaagaactc 300

cagattccag gaaaagcatg tgtgaataac accattgtat ggactaacag ttccctcaac 360

cagaacatga caaatggaac acgttgtggg ttgctgaaca tcgagagcga aatgatcaaa 420

tttgccagtt actatgctgg aattgctgtc gcagtactta tcacaggata tattcaaata 480

tgcttttggg tcattgccgc agctcgtcag atacagaaaa tgagaaaatt ttactttagg 540

agaataatga gaatggaaat agggtggttt gactgcaatt cagtggggga gctgaataca 600

agattctctg atgatattaa taaaatcaat gatgccatag ctgaccaaat ggcccttttc 660

attcagcgca tgacctcgac catctgtggt ttcctgttgg gatttttcag gggttggaaa 720

ctgaccttgg ttattatttc tgtcagccct ctcattggga ttggagcagc caccattggt 780

ctgagtgtgt ccaagtttac ggactatgag ctgaaggcct atgccaaagc aggggtggtg 840

gctgatgaag tcatttcatc aatgagaaca gtggctgctt ttggtggtga gaaaagagag 900

gttgaaaggt atgagaaaaa tcttgtgttc gcccagcgtt ggggaattag aaaaggaata 960

gtgatgggat tctttactgg attcgtgtgg tgtctcatct ttttgtgtta tgcactggcc 1020

ttctggtacg gctccacact tgtcctggat gaaggagaat atacaccagg aacccttgtc 1080

cagattttcc tcagtgtcat agtaggagct ttaaatcttg gcaatgcctc tccttgtttg 1140

gaagcctttg caactggacg tgcagcagcc accagcattt ttgagacaat agacaggaaa 1200

cccatcattg actgcatgtc agaagatggt tacaagttgg atcgaatcaa gggtgaaatt 1260

gaattccata atgtgacctt ccattatcct tccagaccag aggtgaagat tctaaatgac 1320

ctcaacatgg tcattaaacc aggggaaatg acagctctgg taggacccag tggagctgga 1380

aaaagtacag cactgcaact cattcagcga ttctatgacc cctgtgaagg aatggtgacc 1440

gtggatggcc atgacattcg ctctcttaac attcagtggc ttagagatca gattgggata 1500

gtggagcaag agccagttct gttctctacc accattgcag aaaatattcg ctatggcaga 1560

gaagatgcaa caatggaaga catagtccaa gctgccaagg aggccaatgc ctacaacttc 1620

atcatggacc tgccacagca atttgacacc cttgttggag aagggaggagg ccagatgagt 1680

ggtggccaga aacaaagggt agctatcgcc agagccctca tccgaaatcc caagattctg 1740

cttttggaca tggccacctc agctctggac aatgagagtg aagccatggt gcaagaagtg 1800

ctgagtaaga ttcagcatgg gcacacaatc atttcagttg ctcatcgctt gtctacggtc 1860

agagctgcag ataccatcat tggttttgaa catggcactg cagtggaaag agggacccat 1920

gaagaattac tggaaaggaa aggtgtttac ttcactctag tgactttgca aagccaggga 1980

aatcaagctc ttaatgaaga ggacataaag gatgcaactg aagatgacat gcttgcgagg 2040

acctttagca gagggagcta ccaggatagt ttaagggctt ccatccggca acgctccaag 2100

tctcagcttt cttacctggt gcacgaacct ccattagctg ttgtagatca taagtctacc 2160

tatgaagaag atagaaagga caaggacatt cctgtgcagg aagaagttga acctgcccca 2220

gttaggagga ttctgaaatt cagtgctcca gaatggccct acatgctggt agggtctgtg 2280

ggtgcagctg tgaacgggac agtcacaccc ttgtatgcct ttttattcag ccagattctt 2340

gggacttttt caattcctga taaagaggaa caaaggtcac agatcaatgg tgtgtgccta 2400

ctttttgtag caatgggctg tgtatctctt ttcacccaat ttctacaggg atatgccttt 2460

gctaaatctg gggagctcct aacaaaaagg ctacgtaaat ttggtttcag ggcaatgctg 2520

gggcaagata ttgcctggtt tgatgacctc agaaatagcc ctggagcatt gacaacaaga 2580

cttgctacag atgcttccca agttcaaggg gctgccggct ctcagatcgg gatgatagtc 2640

aattccttca ctaacgtcac tgtggccatg atcattgcct tctcctttag ctggaagctg 2700

agcctggtca tcttgtgctt cttccccttc ttggctttat caggagccac acagaccagg 2760

atgttgacag gatttgcctc tcgagataag caggccctgg agatggtggg acagattaca 2820

aatgaagccc tcagtaacat ccgcactgtt gctggaattg gaaagggagag gcggttcatt 2880

gaagcacttg agactgagct ggagaagccc ttcaagacag ccattcagaa agccaatatt 2940

tacggattct gctttgcctt tgcccagtgc atcatgttta ttgcgaattc tgcttcctac 3000

agatatggag gttacttaat ctccaatgag gggctccatt tcagctatgt gttcagggtg 3060

atctctgcag ttgtactgag tgcaacagct cttggaagag ccttctctta caccccaagt 3120

tatgcaaaag ctaaaatatc agctgcacgc ttttttcaac tgctggaccg acaaccccca 3180

atcagtgtat acaatactgc aggtgaaaaa tgggacaact tccaggggaa gattgatttt 3240

gttgattgta aatttacata tccttctcga cctgactcgc aagttctgaa tggtctctca 3300

gtgtcgatta gtccagggca gacactggcg tttgttggga gcagtggatg tggcaaaagc 3360

actagcattc agctgttgga acgtttctat gatcctgatc aagggaaggt gatgatagat 3420

ggtcatgaca gcaaaaaagt aaatgtccag ttcctccgct caaacattgg aattgtttcc 3480

caggaaccag tgttgtttgc ctgtagcata atggacaata tcaagtatgg agacaacacc 3540

aaagaaattc ccatggaaag agtcatagca gctgcaaaac aggctcagct gcatgatttt 3600

gtcatgtcac tcccagagaa atatgaaact aacgttgggt cccaggggtc tcaactctct 3660

agaggggaga aacaacgcat tgctattgct cgggccattg tacgagatcc taaaatcttg 3720

ctactagatg aagccacttc tgccttagac acagaaagtg aaaagacggt gcaggttgct 3780

ctagacaaag ccagagaggg tcggacctgc attgtcattg cccatcgctt gtccaccatc 3840

cagaacgcgg atatcattgc tgtcatggca cagggggtgg tgattgaaaa ggggacccat 3900

gaagaactga tggcccaaaa aggagcctac tacaaactag tcaccactgg atcccccatc 3960

agttga 3966

<---

Claims (68)

1. 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 and halogen; n is an integer 1, 2, or 3; R ³ is selected from the group consisting of C _1-4 alkoxy, C _3-6 cycloalkyloxy, C _1-4 alkylthio and C _3-6 cycloalkylthio; 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 and fluorine; R ⁶ is hydrogen and R ⁷ is selected from the group consisting of hydrogen and C _1-4 alkyl; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, wherein R ¹ is C _2-4 alkyl. 3. The compound according to claim 1 or 2, wherein R ¹ is n -butyl. 4. A compound according to any one of claims 1-3, wherein R ² is independently selected from the group consisting of hydrogen, fluorine, chlorine and bromine. 5. A compound according to any one of claims 1 to 4, wherein R ³ is selected from the group consisting of methoxy, ethoxy, methylthio and ethylthio. 6. A compound according to any one of claims 1 to 5, wherein R ⁴ is hydrogen or fluorine. 7. A compound according to any one of claims 1 to 6, wherein R ⁵ is carboxyl. 8. A compound according to claim 1, selected from the group consisting of: ( E )-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (R)-( E )-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (S)-( E )-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; ( Z )-3-((3-butyl-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-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-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( E )-3-((3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( Z )-3-((3-butyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; (S)-( Z )-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; (R)-( Z )-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( E )-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (S)-( E )-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (R)-( E )-3-((3-butyl-2-methyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (S)-( E )-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (R)-( E )-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (S)-( E )-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (R)-( E )-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (S)-( E )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; (R)-( E )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (S)-( E )-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; (R)-( E )-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; ( E )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)acrylic acid; ( Z )-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,5-benzothiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-5-(4-fluorophenyl)-2-methyl-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-2-methyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-7-(ethylthio)-2-methyl-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-5-(4-fluorophenyl)-7-(methylthio)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-7-(ethylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid; ( Z )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)-2-fluoroacrylic acid and ( E )-3-((3-butyl-7-(ethylthio)-5-(4-fluorophenyl)-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acrylic acid; or a pharmaceutically acceptable salt thereof. 9. 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 a compound according to any one of claims 1-8 and one or more pharmaceutically acceptable excipients. 10. Use of a compound according to any one of claims 1 to 8 as a medicinal product having activity of inhibiting the apical sodium-dependent bile acid transporter (ASBT) and/or the hepatic bile acid transporter (LBAT). 11. The use according to claim 10, where the medicinal product is intended for the treatment or prevention of a cardiovascular disease, or a fatty acid metabolism disorder, or a glucose utilization disorder, such as hypercholesterolemia; a fatty acid metabolism disorder; diabetes mellitus type 1 and 2; complications of diabetes, including 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 including hypertriglyceridemia, metabolic syndrome (syndrome X), atherosclerosis and hypertension; and to increase high-density lipoprotein levels. 12. The use according to claim 10, wherein the medicinal product is intended for the treatment or prevention of a disease or disorder of the gastrointestinal tract, such as 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), 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 such as Barrett's esophagus, bile reflux esophagitis and bile reflux gastritis. 13. The use according to claim 10, 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), including PFIC-1, PFIC-2, PFIC-3, and non-specific PFIC, post-biliary diversion PFIC, and post-transplant PFIC; benign recurrent intrahepatic cholestasis (BRIC), including 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 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-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 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; or in augmentation of corticosteroid therapy in liver disease. 14. The use according to claim 10, wherein the medicinal product is intended for the treatment or prevention of 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; or for use in protecting against kidney injury associated with liver disease or metabolic disease.