Fluorinated bile acid derivatives

Abstract

To provide compounds useful for the treatment or prevention of diseases and conditions including nonalcoholic steatohepatitis and primary biliary cholangitis.SOLUTION: The present invention provides a compound of formula (I) or a salt or isotopic variant thereof, where R2a, R2b, R3a, and R3b independently represent H or F; at least one of R2b and R3b is F; R5 is CR6aR6bR8, OR8, SR8 or NR6aR8; R6a, R6b and R8 independently represent H or methyl; Y is a bond or a C1-4 alkylene or C2-4 alkenylene linker group; R7 is C(O)NR17S(O)2R15, NR17C(O)NR18S(O)2R15 or the like; R15 is a 5-10 membered aryl or heteroaryl ring, optionally substituted with C1-6 alkyl, C1-6 haloalkyl, halo or the like; R17 and R18 independently represent H or methyl.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compounds which are derivatives of bile acids and can be used in the treatment of liver diseases. In particular, the present invention relates to selective agonists of the farnesoid X receptor and, therefore, to non-agonists. Compounds used to treat diseases such as nonalcoholic steatohepatitis (NASH) and primary biliary cholangitis The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention. [Background technology]

[0002] BACKGROUND OF THEINVENTION Nonalcoholic fatty liver disease (NAFLD) is one of the most prominent causes of chronic liver disease worldwide. and is rapidly becoming the leading indication for liver transplantation (Bellentani et al. 2017). can range from simple lipid accumulation in the liver (steatosis) to a condition characterized by both hepatic lobular inflammation and hepatocellular injury. Representing the spectrum of physiological symptoms of nonalcoholic steatohepatitis (NASH) (Haas, Francqu In Western Europe, NAFLD is a significant public health burden, with It is believed to affect an estimated 30% of the country's population (Dyson, Anstee and McPherson, 2013). The increasing prevalence of NAFLD mirrors the increasing prevalence of obesity and type 2 diabetes. Thus, NAFLD is considered to be the hepatic manifestation of the metabolic syndrome (Cave et al., 2016).

[0003] At least 10-20% of NAFLD patients progress to NASH, which is a condition characterized by fibrosis and cirrhosis. , leading to both intrahepatic and extrahepatic complications, such as hepatocellular carcinoma (HCC) and cardiovascular disease. Although poorly understood, the pathological progression from steatosis to NASH is , which consists of multiple "hits" involving lipotoxicity, oxidative stress, and endoplasmic reticulum stress, affecting the liver. This sensitization leads to additional damage mediated by the innate immune defense system, resulting in cytokine induction. It is believed that this leads to induced cell damage (Pacana & Sanyal, 2015). The "hit" involves the activation of the hepatic endothelial cell, which is activated by hyperinsulinemia and a carbohydrate-rich diet. Furthermore, insulin resistance results in dysfunctional adipocytes. Lipolysis in the cells is not inactivated, leading to leakage of free fatty acids (FFA) into the circulation. These FFAs accumulate in ectopic tissues, such as the liver, where they are stored as triglycerides. However, excess FFA accumulation beyond the triglyceride storage threshold will eventually result in triglyceride storage. Lipotoxicity occurs due to toxic metabolites derived from triglycerides. Decreased liver function and decreased export of very low density lipoprotein (VLDL) contribute to the accumulation of fat in the liver. These events contribute to the oxidation of fatty acids by mitochondria and peroxidation. Although it is suppressed by cytoplasmic endothelium, it ultimately leads to damage of these organelles and their activation. This leads to the overproduction of oxygen species (ROS) and damage-associated molecular pattern (DAMP) molecules. In "HIT," lipid peroxidation and activation of inflammatory cytokines by FFA and ROS were shown to be involved in inflammation. and apoptosis, and activates the natural immune defense system via Toll-like receptors. It can also worsen insulin resistance and initiate fibrillation, which can contribute to the progression of NASH. (Cusi 2012) Furthermore, recent evidence suggests that the gut-liver axis may play a role in the progression of the disease. In patients with NAFLD, changes in the composition of the intestinal microbiota and leaky gut are associated with Increased levels of inflammatory cytokines were observed, and inflammasome-mediated dysbiosis also promotes NASH progression. It is thought that (Henao-Mejia et al., 2012; Mouzaki et al., 2013). In other words, fat Toxic hepatic events may be the indirect consequence of inflammatory mediators from adipose tissue, the intestine and the immune system. In addition to the specific effects of oxidative stress, this study highlights the complex multifactorial and cross-system nature of this metabolic disorder and provides important insights into the mechanism of action of oxidative stress. Importantly, these findings reflect multiple potential therapeutic targets for NASH (Haas, Francque, and Staels, References, 2016).

[0004] Currently, there are no effective medical treatments for the treatment or prevention of NASH. Weight loss and improved insulin sensitivity through diet and lifestyle changes are recommended, but Many patients are unable to initiate or maintain these changes, so pharmacological, long-term solutions are required. Solutions are needed (Neuschwander-Tetri et al., 2015). We focused on potential molecular targets for the treatment of NASH, including several nuclear hormone receptors. These receptors, particularly the farnesoid X receptor (FXR), have been implicated in the pathogenesis of this disease. due to their fundamental role in several pathways involved in the pathogenesis of disease. It's an attractive target.

[0005] Like all nuclear receptors, the farnesoid X receptor (FXR) mediates epigenetic changes that control transcription. It acts as a ligand-activated transcription factor that regulates the cellular mechanisms underlying cellular transformation. Two genes that control the expression of FXRα (NR1H4), which is highly conserved in many species, and a pseudogene in humans The FXRα gene is expressed as a cytoplasmic marker, and is expressed as a cytoplasmic marker, whereas the FXRβ gene is expressed as a cytoplasmic marker. It encodes four distinct isoforms that arise from alternative mRNA splicing combinations. All four isoforms have a highly conserved ligand-binding domain. This suggests that FXR ligands bind to any isoform in a nonselective manner and that they differentially It has been shown that the expression of the two isoforms is localized primarily in hepatocytes and Some are expressed in cells with steroid metabolic activity, others are involved in the colon, intestine, and enterohepatic circulation. It is expressed in other cells that receive it (Huber et al., 2002; Vaquero et al., 2013). Some FXR target genes are more responsive to certain isoforms than others. The overall pattern of isoform expression may explain the sensitivity and / or specificity of a given tissue to FXR ligands. and transcriptional responses (Zhang, Kast-woelbern, & Edwards, 2003). years; Vaquero et al., 2013 ).

[0006] FXR represents a typical nuclear receptor, both in structural organization and activation. Specifically, FXR contains an N-terminal DNA-binding domain (DBD), which is a consensus hormone response domain. Two ZnS molecules recognize and are involved in binding to this element. 2+ Composed of fingers It is connected to the C-terminal ligand-binding domain (LBD) via a variable hinge region. exists as a hydrophobic pocket necessary for identifying and accessing small molecule ligands (C (Hiang et al., 2013). Like other nuclear receptors, FXR is an obligate partner. It binds to DNA as a heterodimer with the retinoid X receptor (RXR). In its absence, FXR is inactive. Generally, FXR / RXR heterodimers form a coreplex. AGGTCA inverted repeat response elements of their target genes in complex with a surpeptide (Neuschwander-Tetri 2012). When activated by ligand binding, The receptor undergoes a conformational change, which releases the corepressor complex and inhibits ligand binding. The hydrophobic groove of the pocket exposes a binding site for the LXXLL coactivation motif (Copple and (Langl et al., 2016). The “charge clamp” is a mechanism that mobilizes the LBD surface and the core charge mobilized at the site. This is formed by hydrogen bonds between the two ends of the target protein. This induces changes in the chromatin structure of targeted genes, and activates general transcription factors and RNA polymerase allows genes to access their promoters and thus initiate their transcription. (Ananthanarayanan et al., 2004).

[0007] The classical endogenous ligands for FXR are the bile acids, which are secreted in mammalian bile. The steroid acids found in the human body are cholic acid, chenodeoxycholic acid, lithocholic acid, and deoxycholic acid. These include compounds such as cholic acid, all of which are also found in humans.

[0008] Below is the general numbering system for steroids and the carbon atoms of chenodeoxycholic acid. The numbering of [ka]

[0009] FXR functions as a master regulator of bile acid metabolism. The main role of FXR is in response to feeding state and Facilitating nutrient and energy transfer along the enterohepatic axis in both fasted and fed states. (Evans and Mangelsdorf, 2014). When stimulated after a meal, bile acids inhibit lipid absorption. This allows the release of nutrients from the intestine and activates the FXR-mediated signaling pathway. and promotes uptake of fibroblast growth factor 19 (FGF19), a FXR target gene, into the liver. In addition to intestinal FGF19, FXR is a transcriptional target of Expression of short heterodimer partner (SHP) in the liver downregulates de novo bile acid synthesis. downregulation, tightly controlling the enterohepatic bile acid pool according to metabolic needs In a study of patients with NASH, FXR and the bile acid biosynthetic enzymes, cholesterol 7-alpha-hyaluronan, The expression of both sterol 27 hydroxylase (CYP7A1) and sterol 27 hydroxylase (CYP27A1) was It has been shown that hepatic FGF19 response is decreased in proportion to disease severity; NAFLD patients also show a decreased hepatic FGF19 response. It has also been shown that people with cerebral palsy may show impaired cognitive functioning (Yang, Shen, & Sun, 2010; Min et al., 2013; ave et al., 2016 ).

[0010] More recently, FXR has been implicated as a key regulator of lipid, glucose and cholesterol homeostasis. It is a key player in hepatic lipid biosynthesis, VLDL synthesis, insulin sensitivity, and other nuclear A genetic disorder that is involved in gluconeogenesis and glycogen synthesis, due in part to interactions with the endoreceptor It has been shown that the GABA receptor regulates the expression of the GABA receptor (Kast et al., 2001; Watanabe et al., 2003). (Hsien et al., 2004; Ma et al., 2006; Zhang et al., 2006). SHP signaling cascade By acting through FXR, sterol regulatory element-binding protein 1 (SREBP1) is activated. Downregulates fatty acid synthesis while decreasing peroxisome proliferator activity Upregulating PPARα, which promotes mitochondrial β-oxidation It increases fatty acid catabolism and, therefore, reduces hepatic fatty acid accumulation. FXR also mediates VLD. Apolipoprotein C2 (APOC2) and the VLDL receptor are involved in the hydrolysis and clearance of VLDL. It also acts to increase the expression levels of FXR. Studies using FXR-deficient mice have shown that it significantly reduces liver Similar to human NASH patients, including elevated triglyceride levels, increased circulating FFAs, and increased hepatic steatosis The phenotype was demonstrated (Maloney et al., 2000; Zhang et al., 2004). FXR activation by steroids and synthetic agonists improves plasma triglyceride levels in rodents. (Kast et al., 2001) By lowering triglyceride and FFA levels, FXR activation also induces mild glucose intolerance and impaired insulin signaling in both liver and muscle. Similar to that observed in FXR-deficient mice, which showed suppressed cell death in both liver and peripheral tissues, It also appears to improve insulin sensitivity (Ma et al., 2006). Consistently, insulin sensitivity is increased in both human subjects with NASH and animal models of obesity. In this study, the semi-synthetic FXR agonist obeticholic acid (OCA) improved the condition (Ciprian 2010; Mudaliar et al., 2013). Similarly, FXR is involved primarily in the regulation of FGF19 signaling. It has been proposed that it plays an important role in glucose homeostasis through It lowers plasma glucose levels and inhibits the three major gluconeogenic enzymes, phosphoenzymes, Norpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (F BP1) and glucose-6-phosphatase (G6Pase) expression and activity, and cAM P regulatory element binding protein (CREB) and downstream PPAR gamma coactivator 1-alpha (PGC1 α) and promotes the storage of glucose as glycogen (Zhang et al., 2013). However, conflicting results from animal models suggest that the involvement of FXR is a function of the receptor and pathway. It also suggests that it may be just part of a complex network (Watanabe et al., 2013). (2011).

[0011] Notably, FXR has been implicated in suppressing hepatitis, as it mediates the expression of certain nuclear factor kappa B (NF-κB) targets. It is involved in an elaborate pathway that leads to the negative regulation of genes and proinflammatory cytokines (Wang et al. Furthermore, although the underlying mechanisms are still poorly understood, FXR has Plays an important role in intestinal protection and maintaining the intestinal barrier against intestinal flora Inagaki et al., 2006, FXR knockout mice fed a high-fat diet The disease is caused by the overgrowth of intestinal bacteria, as well as tumor necrosis factor alpha (TNFα), metalloproteinases, and the like. Inflammatory factors such as tissue inhibitors of myocarditis (TIMPs) and transforming growth factor beta (TGFβ-1) showed increased levels of proinflammatory and profibrotic mediators (Kong et al. Furthermore, preclinical evidence indicates that FXR agonists mediate the induction of inflammatory bowel disease in primary hepatocytes. Inhibition of NFκB expression improves the inflammatory microenvironment and fibrosis in mouse models of inflammatory and nonalcoholic steatohepatitis (NASH). These results suggest that the effects of immunization on the immune system have been improved (Kong et al., 2009; Ma et al., 2013).

[0012] Further supporting its role in NASH, and particularly HCC, FXR regulates the tumor suppressor gene FXR agonists have been shown to downregulate expression in mouse liver xenograft tumor models. It has been shown to reduce tumor growth and metastasis in mice (Deuschle et al., 2012; Jiang et al., 2013). This evidence therefore supports its central role in the gut-liver fat axis. maintenance of intestinal barrier integrity, suppression of inflammation, and bile acid, glucose and lipid metabolism. In addition to regulation of FXR, dysregulation of FXR contributes to the pathogenesis of NASH. These findings further validate FXR as an ideal target for the treatment of NASH.

[0013] Many FXR agonists are known, including a variety of nonsteroidal compounds. Bile acid analogs with agonistic activity have been developed. These include those disclosed in International Publication WO02 / 07259 8 and obeticholic acid (OCA; INT-747) described in EP 1568706. OCA analogs and The medical uses of OCA and analogs and methods for their preparation are described in International Publication WO2005 / 092925 and International Publication WO2005 / 092925. WO2005 / 089316, International Publication WO2006 / 122977, International Publication WO2007095174, International Publication WO2008 / 00257 3. International Publication WO2008 / 091540, International Publication WO2010 / 014836, International Publication WO2010 / 059853, International Publication WO2010 / 059859, International Publication WO2013 / 192097, International Publication WO2014 / 066819, International Publication WO2015 / 0854 74, International Publication WO2014 / 184271, International Publication WO2016 / 127019, International Publication WO2016 / 144946, International Publication WO2016 / 164413, WO2016 / 176208, WO2016 / 205475, WO2017 / 01 9524, International Publication WO2017 / 027396, International Publication WO2017 / 053428, International Publication WO2017 / 053826, International Publication WO2017 / 062763, International Publication WO2017 / 079062, International Publication WO2017 / 111979 and International Publication WO201 7 / 156024 (all from Intercept Pharmaceuticals, Inc.).

[0014] Additionally, 6-alkyl bile acid analogs with modified side chains are disclosed in International Publication WO2016 / 073767 , International Publication WO2016 / 086115, International Publication WO2016 / 086134, International Publication WO2016 / 086169, International Publication W WO2016 / 086218, International Publication WO2016 / 130809, International Publication WO2016 / 161003, WO2017 / 147137, International Described in publication WO2017 / 147159 and international publication WO2017 / 147174 (all from Enanta Pharmaceuticals, Inc.) It has been done.

[0015] Other documents related to similar compounds include Chinese Patent Publication No. 105646634 and International Publication WO2016 / 1735. 24, International Publication WO2016 / 173397, Chinese Patent Application Publication No. 105348365, US Patent Application Publication No. 2014 / 02066 Includes 57.

[0016] The inventor's earlier applications, International Publication WO2016 / 079518, International Publication WO2016 / 079518, International Publication WO2016 / 079519, International Publication WO2016 / 079520, International Publication WO2017 / 199036, International Publication WO2017 / 1990 39 and International Publication WO2017 / 199033 describe methods for preparing these bile acid analogs and their synthesis. This relates to intermediates in the synthesis of

[0017] One problem with the bile acid analogs described in the prior art is their limited efficacy as FXR agonists. In addition to their activity, they are also modulators of the G protein-coupled receptor TGR5. This is a member of the rhodopsin-like superfamily of G protein-coupled receptors. It is a key player in the bile acid signaling network. For example, OCA use One of the side effects involved is pruritus, which is thought to be due to OCA activation of off-target receptors such as TGR5. This is thought to result from (Alemi et al., 2013).

[0018] Xiao et al. (2017) reported the synthesis and characterization of OCA and a series of its derivatives as FXR agonists. In these derivatives, the carboxylic acid group is replaced by various alternative groups. The authors reported that all compounds tested showed low to moderate TGR5 potency, with the best results obtained. The highest selectivity was 30-fold higher than FXR, which was achieved with a tetrazole derivative. The compound with the highest liver:plasma concentration ratio has a CH2CH2C(O)NH-S(O)2CH3 side chain. It was compound 18. Summary of the Invention

[0019] The present invention relates to a method for the preparation of a FXR agonist having enhanced selectivity for FXR over TGR5 while maintaining FXR agonist activity. This relates to new compounds that have been

[0020] Further, fluorinated at the 2-position and / or 4-position and an arylsulfonamide or an aryl Bile acid derivatives with sulfonylurea side chains have been shown to have a greater ability to bind to FXR than known bile acid derivatives. The present inventors have demonstrated that this increased agonist activity The activity of the FXR ligand-binding domain is determined by the canonical and allosteric pockets. However, the compound of the present invention The validity of is not affected by whether this assumption is correct or not.

[0021] (Summary of the invention) Thus, in accordance with the present invention, a compound of general formula (I): [ka] (In the formula, Each R 2a , R 2b , R 3a and R 3b are independently H or F, but R 2b and R 3b At least One is F; R 5 is CR 6a R 6b R 8 , OR 8 , S.R. 8 Or NR 6a R 8 and; Each R 6a , R 6b and R 8 are independently H or methyl; Y is a bond or C 1-4 Alkylene or C 2-4Alkenylene linker groups are Either R is 1 or more 10 is optionally substituted with; Each R 10 are independently halo or OH; R 7 is C(O)NR 17 S(O)2R 15 , N.R. 17 C(O)NR 18 S(O)2R 15 , N.R. 17 C(S)NR 18 S(O)2R 15 and N.R. 17 C(NR 20 )NR 18 S(O)2R 15 Selected from; R 15 is optional, C 1-6 Alkyl, C 1-6 Haloalkyl, Halo, O(C 1-6 alkyl) and O(C 1-6 Ha 5-10 membered aryl or is a heteroaryl ring; Each R 17 and R 18 are independently H or methyl; R 20 is H, methyl or CN; or a salt or isotopic variant thereof is provided.

[0022] Several fluorinated bile acid derivatives are known. See, e.g., Roda et al. (1995); Honori (2006), U.S. Patent No. 5,175,320, and International Publication WO 97 / 44043 all disclose ursodeoxycholic acid. On the other hand, International Publication WO2014 / 160441 relates to 6,6-difluoro analogues of 6-fluorocholic acid. Sato et al. (2008) described 7-fluorolithocholic acid derivatives. Regarding conductors, Sievanen et al. (2008) and Cushman et al. (1995) EP 3290 discloses 3,3-difluorocholan-24-oic acid and its methyl ester, respectively. 429 discloses bile acid derivatives that are said to be useful in the treatment of FXR-mediated diseases. The compounds studied include several 4-fluoro bile acid derivatives. Clerici et al. (2006) and Macchiarulo et al. (2008) both report 3α-6α-dihydroxy-7α-fluoro-5β- Regarding cholanoate, it is said to be useful in the treatment of liver diseases. International Publication WO2016 / 154216 relates to 3- and 7-fluorinated derivatives of UDCA for use in the treatment of neurodegenerative disorders. International Publication WO2016 / 173493 relates to bile acid derivatives which have modified side chains and which are XR and / or TGR5 modulators. Regarding substituted bile acid derivatives, they are said to be useful in the treatment of FXR-mediated diseases. However, compounds of general formula (I) are not taught in the prior art.

[0023] The compounds of general formula (I) are selective FXR agonists and therefore non-alcoholic fatty Hepatitis (NASH); Primary biliary cirrhosis (PBC); Primary sclerosing cholangitis; Biliary atresia; Cholestatic liver disease Hepatitis C infection; alcoholic liver disease; fibrosis; and liver damage resulting from fibrosis. It is useful in treating the symptoms of

[0024] Without being bound by this theory, the inventors believe that the presence of fluorine in the ring enhances the 3α-hydrogen This alters the hydrogen bonding ability of the oxy group, resulting in the compound's ability to bind to the FXR and TGR5 receptors. In addition, the addition of fluoro groups to the 2- or 4-position of the steroid ring system was speculated to affect the activity and selectivity of the steroids. It has also been found that compounds having the formula (I) may have increased metabolic stability. The present inventors have also discovered that the side chain group (-YR 7 ) properties of FXR agonist We found that it significantly affected the activity of

[0025] In this specification, when the context requires a different meaning by words or necessary implicatures, Except as otherwise specified, the term "include" or variations thereof such as "includes" or "including" are inclusive. That is, the presence of the stated features is clearly indicated, but various implementations of the invention may be used. The presence or addition of further features in the embodiments is not excluded.

[0026] As used herein, the reference "medicinal use" refers to the use of a compound or a pharmaceutical agent for the treatment or prevention of a disease or medical condition. , for administration to humans or animals, particularly humans or mammals, such as domestic or livestock mammals. The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, and "Pharmaceutically acceptable" refers to an agent that is suitable for use in a pharmaceutical composition. Other similar terms include: It should be interpreted accordingly. In the context of this specification, the term "plurality" refers to two or more.

[0027] All publications, including but not limited to patents and patent applications, cited in this specification. are incorporated herein by reference as if each individual publication were fully described herein. Each of the above-mentioned references is incorporated herein by reference as if specifically and individually indicated to be incorporated by reference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Invention In this application, the term "C 1-6 "Alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms. Refers to a fully saturated hydrocarbon group. This term includes methyl, ethyl, n-propyl, isopropyl, Other alkyl groups include n-butyl, isobutyl, sec-butyl and t-butyl. 1-4 a Rukill, C 1-3 Alkyl or C 1-2 Alkyl is as above, but with a different number of carbon atoms. Includes children.

[0029] The term "alkylene" refers to a straight or branched fully saturated hydrocarbon chain. Rukylen is C 1-4 Alkylene, C 1-3 Alkylene or C 1-2 It is an alkylene. Examples of groups are -CH-, -CHCH-, -CH(CH)-CH-, -CHCH(CH)-, -CHCHCH-, and -CHCH(CH 2CH3)-included.

[0030] The term "alkenylene" refers to a linear or branched alkyl group containing at least one carbon-carbon double bond. Suitably, alkenylene refers to a C 2-4 Alkenylene or C 2-3 Arken Examples of alkenylene groups are -CH=CH-, -CH=C(CH3)-, -CH2CH=CH-, -CH=CHCH2-, Includes -CH2CH2CH=CH- and -CH2CH=C(CH3)-.

[0031] The terms "aryl" and "aromatic" refer to aryl groups having 6 to 14 ring carbon atoms (unless otherwise specified, e.g., A cyclic group having aromaticity, for example having 6 to 10 ring carbon atoms, containing up to 3 rings. When an aryl group contains more than one ring, not all of the rings need be aromatic. Examples include phenyl, naphthyl, and anthracenyl, as well as tetrahydronaphthyl. Further examples of aryl groups include partially saturated systems such as 1, 2,3,4-tetrahydronaphthalene.

[0032] The terms "heteroaryl" and "heteroaromatic" refer to heteroaryls having 5 to 14 ring atoms (unless otherwise specified). A cyclic group having aromaticity, for example, 5 to 10 ring atoms, at least One of the heteroatoms is selected from N, O, and S, and the ring contains up to three rings. When an heteroaryl group contains more than one ring, not all of the rings need be aromatic. Examples of aryl groups include pyridine, pyrimidine, indole, benzofuran, benzimidine, Further examples of heteroaryl groups include quinoline and isoxazole. Including Norrin. The term "halogen" refers to fluorine, chlorine, bromine, or iodine, and the term "halo" refers to fluorine, chlorine, bromine, or iodine. It refers to a perfluoro, chloro, bromo, or iodo group.

[0033] The term “C 1-6 "Haloalkyl" means a straight or branched alkyl group having 1 to 6 carbon atoms as defined above. substituted with one or more halo atoms, up to perhalo-substituted; Examples include trifluoromethyl, chloroethyl and 1,1-difluoroethyl. Other haloalkyl groups, such as C 1-5 Haloalkyl, C 1-4 Haloalkyl, C 1-3 Hello Aruki Ru or C 1-2 Haloalkyl is as previously described but containing different numbers of carbon atoms. The term "side chain" refers to a -YR 7 In the case of UDCA, -YR 7 is -CH2CH2-C(O)OH, The reference to the ant side chain is other -YR 7 Refers to the ingredients.

[0034] The term "isotopically-labeled" refers to a compound that is isotopically labeled with one or more atoms. has an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature Substituted with atoms having mass numbers or atomic masses that are less common in nature Formula (I) except that the proportion of atoms having mass numbers is high (the latter concept is called "isotopically enriched"). Examples of isotopes that can be incorporated into the compounds of the invention are 2 H (deuterium), 3 H, 11 C. 13 C. 14 C. 18 F, 123 I or 125 I etc. (e.g. 3 H, 11 C. 14 C. 18 F, 12 3 I or 125 I) isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine, These may be natural or non-natural isotopes.

[0035] The salt of the compound of general formula (I) is a suitable pharma- ceutically acceptable salt. Pharmaceutically unacceptable salts may also be used, for example, in the preparation of compounds of formula (I). as compounds which are intermediates in the preparation of compounds of general formula (I) having alternative side chains, It can be used to carry out reactions for the purpose of

[0036] Suitable salts of the compounds described herein include sodium, potassium, calcium, ammonium, and ammonium salts. Base addition salts such as salts of aluminum, zinc, magnesium and other metals, as well as choline, Diethanolamine, Ethanolamine, Ethyldiamine, Meglumine and Paulekuhn J. Med. Chem. 2007, 50, 6665-6672, and / or those skilled in the art may These include other well known base addition salts which are known in the art.

[0037] As mentioned above, in the compounds of general formula (I), each R 2a , R 2b , R 3a and R 3b is independent H Or F but R 2b and R 3b At least one of is F. In some compounds of general formula (I), R 3b is F, and each R 2a , R 2b and R 3a is H or F In other compounds of general formula (I), R 2b is F, and each R 2a , R 3a and R 3b H or is F.

[0038] Some suitable compounds of the present invention are monofluorinated. In some monofluorinated compounds, R 3b is F, and each R 3a , R 2a and R 2b is H That is, the compound of general formula (I) has a 4β-fluoro substituent. For other monofluorinated compounds, R 2b is F, and each R 2a , R 3a and R 3bis H, That is, the compounds of general formula (I) have 2β-fluoro substituents.

[0039] Some suitable compounds of formula (I) are difluorinated. In the difluorinated compounds of R 3b is F and R 3a is H and R 2a and R 2b One of them is F, Remaining R 2a and R 2b is H, i.e. the compound of general formula (I) has a 2-fluoro substituent and a 4β-fluoro It has a fluoro substituent. In another suitable difluorinated compound of general formula (I), the compound of general formula (I) is a 4,4-difluoro group. R 3a and R 3b are both F and R 2a and R 2b are both H It is.

[0040] The compound of formula (I) is R 2a , R 2b , R 3a and R 3b Three of them are F and the rest are H. R may be a refluorinated compound. 2a , R 2b , R 3a and R 3b Tetrafluorinated compounds in which all of It can also be an object. In the compounds of general formula (I), suitably R 6a and R 6b are both hydrogen, and / or R 8 is methyl.

[0041] In some suitable compounds, R 5 is ethyl, OH or methoxy, especially ethyl or methoxy There are, especially ethyl. In some more suitable compounds, R 5 is methoxy. Particularly suitable compounds include R 5 is ethyl.

[0042] In some suitable compounds of formula (I), Y is a bond. In other suitable compounds of formula (I), Y is C 1-4 Alkylene or C 2-4 Alkenylene Linkers - groups, any of which may optionally be one or more R 10 Replaced by is.

[0043] More suitably in these compounds, Y is C 1-4 , C 1-3 Or C 1-2 The alkylene linker group Optionally, one or more R 10 is substituted with a group. In some compounds of formula (I), Y is unsubstituted.

[0044] In other compounds of formula (I), Y is one or more substituents R 10 has been replaced by In some cases, R 10 is a halo. In other cases, R 10 is OH.

[0045] Examples of suitable linkers Y include a bond, -CH-, -CHCH-, -CH(OH)-CH-, -CH=CH- or -CH=C (CH3)-, in particular a bond, -CH2-, -CH2CH2-, -CH=CH- or -CH=C(CH3)-, especially -CH2-, -CH2CH2- , -CH=CH- or -CH=C(CH3)-.

[0046] More suitably, Y is a bond or a C1-3 Alkylene phosphorus More suitably, Y is a C alkyl group optionally substituted with one or more OH groups. 1-3 Alki is a linker group. In some particularly suitable compounds, Y is unsubstituted C 1-3 It is an alkylene linker. Particularly suitable compounds of formula (I) are those in which Y is -CH2- or -CH2-CH2-, especially -CH2-CH2-. .

[0047] In the compounds of the present invention, R 7 is more appropriately C(O)NR 17 S(O)2R 15 Or NR 17 C(O)NR 18 S( O)2R 15 and R 15 , R 17 and R 18 is as defined in general formula (I). More appropriately, each R 17 and R 18 is H (if present).

[0048] In some suitable compounds of formula (I), R 15 Phenyl and 5- or 6-membered heterocyclic rings aryl, for example pyridyl, pyrimidinyl or pyrrolyl, any of which may be selected from It may be unsubstituted or may contain one or more substituents as defined above, in particular 1 to 3 substituents, more usually 1 or 2. may be substituted with two substituents, in particular one substituent. More suitably, R 15 Is Feni a 6-membered heteroaryl group, either of which may be unsubstituted or as defined above; one or more substituents, especially 1 to 3 substituents, more usually 1 or 2 substituents, especially 1 substituent It may be substituted with a group.

[0049] R 15 Particularly suitable substituents for the moiety are fluoro, C 1-4 Alkyl, C 1-4 Fluoroal Kill, O(C 1-4 alkyl) and O(C 1-4 fluoroalkyl). R of the compound of formula (I) 15 Exemplary groups include unsubstituted phenyl or substituted with one substituent. Phenyl, the substituents of which are fluoro, C 1-4 Alkyl, C 1-4 Fluoroalkyl, O(C 1-4 alkyl) and O(C 1-4 fluoroalkyl), in particular fluoro, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, trifluoromethyl, methoxy and trifluoro The moiety is selected from fluoromethoxy.

[0050] R 7 NR 17 C(O)NR 18 S(O)2R 15 and R 15 When is a phenyl having one substituent, The substituents in may be at any position on the ring, but are most suitably m- or p-substituents, especially In particular, R 7 NR 17 C(O)NR 18 S(O)2R 15 And R 15 Phenyl substituted with CF3 In this case, the CF3 substituent is preferably in the m- or p-position.

[0051] Examples of compounds of general formula (I) include: N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-p-Toluenesulfonylurea (Compound 1); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-Benzenesulfonylurea (compound 2); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-4-(tert-butyl)benzenesulfonylurea (compound 3); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-m-Toluenesulfonylurea (compound 4); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-o-toluenesulfonylurea (compound 5); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-p-Fluorobenzenesulfonylurea (compound 6); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-m-Fluorobenzenesulfonylurea (compound 7); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-o-fluorobenzenesulfonylurea (compound 8); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-p-(trifluoromethyl)benzenesulfonylurea (compound 9); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-m-(trifluoromethyl)benzenesulfonylurea (compound 10); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-o-(trifluoromethyl)benzenesulfonylurea (compound 11); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-4-(trifluoromethoxy)benzenesulfonylurea (compound 12); N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl )-p-Methoxybenzenesulfonylurea (compound 13); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-trifluoro Fluoromethoxybenzenesulfonamide (compound 14); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-fluoro bromobenzenesulfonamide (compound 15); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-3-fluoro phenylsulfonamide (compound 16); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-2-fluoro phenylsulfonamide (compound 17); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-4-trifluoroacetate Fluoromethylphenylsulfonamide (compound 18); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-3-trifluoroacetate Fluoromethylphenylsulfonamide (compound 19); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-2-trifluoroacetate fluoromethylphenylsulfonamide (compound 20); N,N'-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-24-nor-5β-cholan-23-yl (III)-Benzenesulfonylurea (Compound 21) N-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholan-24-oyl)benzene sulphonamide (compound 22); and salts and isotopic variants thereof.

[0052] R 7 NHC(O)N(R 18 )S(O)2R 15 The compound of general formula (I), [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 40 Protect The compound can be prepared by deprotection of the aryl group (the protected OH group).

[0053] Suitably, the protecting group is one which can be removed by treatment with a base. Examples of OH groups include R 41 C(O)O, R 41 is C 1-6 alkyl or benzyl, especially methyl Silyl ether protecting groups can also be used. Other suitable OH protecting groups are well known to those skilled in the art. (See PGM Wuts and TW Greene, 2006).

[0054] The compound of general formula (II) may be a compound of general formula (III): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 40 is general As defined in formula (II), Sulfonamides of general formula (IV): [ka] (In the formula, R 15 and R 18 is as defined in general formula (I)) with 1,8-diazabicyclo[5. 4.0] In the presence of a catalyst such as undec-7-ene (DBU), the reaction takes place in a suitable organic solvent such as toluene. It may also be prepared by The sulfonamides of formula (IV) are known and readily available or can be prepared by known methods. It can be prepared.

[0055] The compound of general formula (III) may be a compound of general formula (V): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 40 is general (as defined in formula (II)) in an inert atmosphere, for example under an argon atmosphere. It may also be prepared by heating. Suitably, the compound of general formula (V) is heated to about 100-150°C, typically to about 125°C. do.

[0056] The compound of general formula (V) can be prepared by reacting a compound of general formula (VI): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 40 is general (II) with diphenylphosphoryl azide under basic conditions, It may be prepared, for example, by reaction in the presence of triethylamine. The reaction is suitably carried out in an inert atmosphere, for example under an argon atmosphere.

[0057] The compound of general formula (VI) can be prepared by reacting a compound of general formula (VII): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), It may also be prepared by For example, a protected OH group R 40 When is acetoxy, the compound of formula (VII) is a mild The reaction may be carried out under basic conditions, for example in the presence of sodium bicarbonate with acetic anhydride. The reaction is suitably carried out in an inert atmosphere, for example under an argon atmosphere.

[0058] The compound of general formula (VII) may be a compound of general formula (VIII): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 47 is C 1-6 It may also be prepared by hydrolysis of a cycloalkyl group (which may be alkyl or benzyl). This hydrolysis can be carried out, for example, with an alkali metal hydroxide, such as sodium hydroxide or lithium hydroxide. The reaction may be base catalysed hydrolysis using an alkoxysilane such as methanol. It is carried out in a coal solvent.

[0059] R 3b The compound of general formula (VIII) in which is F can be prepared by the suitable compound of general formula (XX): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 47 is general It may also be prepared by reduction of the compound (VIII) Suitable reducing agents include hydrides, such as sodium borohydride. Preferably, the reaction is carried out in an organic solvent such as tetrahydrofuran under an inert atmosphere such as argon. do.

[0060] R 2b The preparation methods of the compound of general formula (XX) in which is F and the compound of general formula (VIII) are described below. In a separate step, R 7 NHC(O)N(R 18 )S(O)2R 15 The compound of general formula (I) is represented by the general formula (XII) It can be prepared by deprotection of the compound: [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5are as defined in general formula (I), R 45 and R 46 are independently protected OH groups).

[0061] Appropriately, each R 45 and R 46 are independently a silyl ether, e.g., tert-butyldi Methylsilyloxy or trimethylsilyloxy. R 45 and R 46 is silyl ether In these cases, they may be the same or different and may be reacted with reagents such as tetrabutylammonium fluoride (TBAF). Deprotection can be achieved by treating the compound with an alternative protecting group, such as the group in the above general formula (II). R 41 C(O)O may also be used. Other protecting groups are well known in the art (PGMWuts and TWG (see Reene (2006)).

[0062] The compound of general formula (XII) may be a compound of general formula (XIII): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 45 and R 4 6 is as defined in general formula (XII), the sulfonyl group of general formula (IV) can be obtained as described above. with amide in the presence of a catalyst such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and Suitably it may be prepared by reaction in an organic solvent such as toluene.

[0063] The compound of general formula (XIII) may be a compound of general formula (XIV): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 45 and R 4 6 is as defined in general formula (XII) in an inert atmosphere, for example, in an argon atmosphere. It may also be prepared by heating in air.

[0064] The compound of general formula (XIV) can be prepared by reacting a compound of general formula (XV): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 45 and R 4 6 is as defined in the general formula (XII), and diphenylphosphoryl azide is reacted with It may also be prepared by reaction under conditions such as in the presence of triethylamine.

[0065] The compound of general formula (XV) can be prepared by reacting a compound of general formula (XVI): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 45 and R 4 6is as defined in general formula (XII), and R 47 is as defined in general formula (VIII) It may also be prepared by hydrolysis of Suitably the hydrolysis is carried out using an alkali metal hydroxide such as, for example, sodium hydroxide or lithium hydroxide. The reaction is carried out under basic conditions by treatment with an alcoholic hydroxide. It is carried out in a solvent.

[0066] The compound of general formula (XVI) can be prepared by reacting a compound of general formula (XVII): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 46 is general As defined in formula (XII), and R 47 is as defined in general formula (VIII). The compound may be prepared by protection of the compound.

[0067] Protected OH group R 45 is a silyl ether, this protection is achieved by converting the compound of general formula (XVII) to This can be achieved by reacting with a compound of formula (XVIII): (R 50 )3Si-R 48 (XVIII) (In the formula, R 48 is a leaving group, typically a halide, e.g., fluoride, chloride or or bromide, etc., or a sulfonyl group such as triflyl, tosyl, or mesyl; And each R 50 are independent, e.g. C 1-6 alkyl or phenyl, etc., and represented by the general formula (XVI) Group R in the compound 45 (R50 )3Si-O). The reaction is carried out under an inert atmosphere in the presence of a base such as 2,6-lutidine at about -5 to 5°C, typically The reaction can be carried out at a temperature of about 0° C. Suitable solvents include organic solvents such as dichloromethane. Contains a medium.

[0068] The compound of general formula (XVII) can be prepared by reacting a compound of general formula (XIX): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 46 is general As defined in formula (XII), and R 47 is as defined in general formula (VIII). It may also be prepared by reduction of Suitable reducing agents include hydrides, such as sodium borohydride. Preferably, the reaction is carried out in an inert atmosphere, such as argon, at a temperature of about 15-25° C., typically at room temperature. It is carried out in an organic solvent such as tetrahydrofuran.

[0069] R 3b The compound of general formula (XIX) in which is F can be synthesized by the compound of general formula (XX): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R 47 is general The compound (VIII) may be prepared by protecting the compound (VIII). Protected OH group R46 If is a silyl ether, protection can be achieved by the addition of a base such as n-butyllithium. In the presence of an appropriate silyl halide or silyl sulfonate (e.g., triflate, This may be achieved by reacting with a carboxylate or tosylate. 46 Trimethylsilyl In the case of silyl, the compound of formula (XX) can be reacted with trimethylsilyl chloride. Cut.

[0070] R 7 C(O)N(R 17 )S(O)2R 15 The compound of general formula (I), [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b , R 5 , R 15 and R 17 is as defined in general formula (I) , R 46 can be prepared by deprotection of the compound (XII). Protected OH group R 46 If is a silyl ether, it can be deprotected by treatment with a base such as TBAF. The reaction is suitably carried out under an inert atmosphere, for example argon, in an anhydrous solvent such as tetrahydrofuran. It is carried out in an organic solvent.

[0071] The compound of general formula (XXII) may be a compound of general formula (XXIII): [ka] (In the formula, Y, R 2a , R 2b , R 3a , R 3b and R 5 is as defined in general formula (I), R46 is general As defined in formula (XII), with sulfonamides of general formula (XXIV): [ka] (In the formula, R 15 and R 17 is as defined in general formula (I), Coupling agents such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDCI) and di It may also be prepared by reaction in the presence of a base such as methylaminopyridine.

[0072] Compounds of general formula (XXIII) may be prepared by hydrolysis of compounds of general formula (XVII), Suitably under basic conditions, for example an alkali metal hydroxide such as sodium hydroxide or lithium hydroxide It may also be prepared by treatment with a hydroxide. Suitably the reaction is carried out in an alcoholic solvent. will be done.

[0073] R 2b is F and R 3a and R 3b is H and R 7 C(O)N(R 17 )S(O)2R 15 of general formula (I) For compounds, R 2b is F and R 3a and R 3b Starting from a compound of general formula (VII) in which A similar route can be used.

[0074] As described above, the compound of general formula (XVII) can be produced by converting the compound of general formula (XX) into the compound of general formula (XIX). It can be prepared via The compound of general formula (XX) may be a compound of general formula (XXa), (XXb), (XXc), (XXd) or (XXe). stomach: [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) (As shown).

[0075] The compound of general formula (XXa) can be prepared by the reaction of a compound of general formula (XXX): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) (as it is); (i) A strong base such as n-BuLi or lithium diisopropylamide (LDA) and, for example, trimethyl Treatment with a trialkylsilyl halide such as silyl chloride, followed by (ii) For example, Selectfluor® (1-chloromethyl-4-fluoro-1,4- Diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate): [ka] The compound can be prepared by a process comprising electrophilic fluorination of the product of step (i) with When using Selectfluor®, the reaction is suitably carried out at a temperature of 15-25° C., typically It is typically carried out at room temperature in a polar organic solvent such as acetonitrile. Suitable bases include lithium diisopropylamide (LDA) and n-butyllithium. However, it is typically used in combination with trimethylsilyl chloride.

[0076] Compounds of general formula (XXb) can be prepared from compounds of general formula (XXa) by further fluorination, It is typically prepared in a manner similar to that described above for compounds of general formula (XXa) So, that is: (i) a strong base such as n-BuLi or LDA and a trialkyl chloride such as trimethylsilyl chloride Treatment with arylsilyl halide, followed by (ii) electrophilic fluorination of the product of step (i), for example with Selectfluor® The compound can be prepared by a method comprising the steps of:

[0077] The compound of the general formula (XXc) can be produced by converting the compound of the general formula (XXb) into trimethylsilyl chloride or the like. Treatment with a strong base, typically LDA, used in combination with a trialkylsilyl halide. It can be prepared by polymerization, followed by fluorination with Selectfluor®. Compounds of general formula (XXd) can be prepared from compounds of general formula (XXc), typically by the process of Selectfluor® ) and further fluorination with a weak base such as triethylamine.

[0078] The compound of general formula (XXe) can be produced by reacting the compound of general formula (XXa) with the compound of formula (XXXI): R 51 -Si(R 13 )3(XXXI) (Wherein, each R 13 is independently C 1-6 alkyl and phenyl; R 51 Hello (e.g., chloro) in the presence of a strong base such as lithium diisopropylamide. followed by fluorination, typically with Selectfluor®. Cut.

[0079] The compound of general formula (XXX) may be a compound of general formula (XXXII): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) Typically, (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl It may also be prepared by oxidation with reagents such as (TEMPO) or (diacetoxyiodo)benzene (BAIB). good.

[0080] Compounds of general formula (XXXII) can be prepared by reacting a carboxylic acid of general formula (XXXIII): [ka] (Wherein, Y and R 5 is as defined in general formula (I), 47 With -OH alcohol, For example, by reaction in the presence of an acid such as p-toluenesulfonic acid or methanesulfonic acid, It may also be prepared by termination. The compound of the general formula (XXXIII) is known. 5 is ethyl and Y is -CH2CH2- The compound XXXIII) is obeticholic acid (see International Publication WO02 / 072598).

[0081] The applicant's earlier applications, International Publication WO2016 / 079518, International Publication WO2016 / 079518, International Publication WO2016 / 079519, International Publication WO2016 / 079520, International Publication WO2017 / 199036 and International Publication WO2017 / 19 No. 9033 describes a method for preparing compounds of general formula (XXXIII) according to Scheme 1; Scheme 1 [ka] (wherein Y is as defined in general formula (I)).

[0082] Some compounds of the general formulae (XL) and (XLI) are known and are described, for example, in Uekawa et al. ) is the synthesis of (22E)-3-oxo-4,22-choladien-24-oic acid ethyl ester from stigmasterol: [ka] and subsequent synthesis of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid ethyl ester: [ka] Uekawa et al. then described its conversion to (6α,7α,22E)-6, The conversion of 7-epoxy-3-oxo-4,22-co-radien-24-oic acid to ethyl ester is also described. There are.

[0083] Other compounds of general formula (XL) and (XLI) are phytosterols similar to stigmasterol. It may also be prepared by a similar method from Stigmasterol and other phytosterols are plant sterols and are readily available. It is possible or can be prepared by known routes.

[0084] R 2b is F and R 2a , R 3a and R 3b The compound of general formula (VIII) in which all are H can be represented by general formula (XLV) Compounds of: [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47is defined by the general formula (VIII) It may also be prepared by reduction of Suitable reducing agents include, for example, hydrides such as sodium borohydride, and the reaction proceeds as follows: For example, it is carried out under an inert atmosphere such as argon. The product is obtained as a mixture with the 3β-hydroxy isomer.

[0085] The compound of general formula (XLV) can be prepared by the reaction of a compound of general formula (XLVI): [ka] (Wherein, Y and R 5 is as defined in general formula (I), good. Suitably, the esterification is carried out by esterifying an alkyl or benzyl group R 47 Halides of, for example, iodide In the presence of a base such as cesium carbonate, the reaction is carried out in an alcoholic solvent such as methanol. It is carried out within.

[0086] The compound of general formula (XLVI) can be prepared by the reaction of a compound of general formula (XLVII): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) It may also be prepared by epimerization of This epimerization can be carried out by reacting a compound of formula (XLVII) with a strong base such as sodium hydroxide. This may be achieved by treating 47 Hydrolysis of the components to OH occurs. The resulting product is a 60:40 mixture of the compound of formula (XLVI) and its 2α-isomer. The mixture is then used without further separation in the re-esterification step as described above, but This is because the ester compound of the general formula (XLV) is more easily reacted with the 2α-isomer than the carboxylic acid compound (XLVI). This is because it can be separated from the

[0087] The compound of general formula (XLVII) can be prepared by reacting a compound of general formula (XLVIII): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) Suitable oxidizing agents include the Dess-Martin periodontium phosphate oxidizing agent. Jinan included.

[0088] The compound of general formula (XLVIII) can be synthesized by the method of the present invention by the compound of general formula (XLIX): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) (As shown below), It is prepared by reaction with HF-pyridine (70%) complex under an inert atmosphere, e.g. argon. Suitably, the reaction is carried out in a halogenated solvent such as dichloromethane at low temperature, e.g. The reaction is typically carried out at a temperature of -10°C to 10°C, typically about 0°C.

[0089] The compound of general formula (XLIX) can be prepared by reacting a compound of general formula (L): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47is defined by the general formula (VIII) The dashed lines indicate that one of the two bonds is a single bond and the other is a double bond. ); from m-perchlorobenzoic acid in a halogenated organic solvent such as dichloromethane. Suitably, the reaction is carried out at a temperature of 15 to 25° C., typically It is carried out at room temperature.

[0090] The compound of general formula (L) may be a compound of general formula (LI): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) (The formula is as follows:) and then dehydration with a suitable dehydrating agent such as trifluoromethanesulfonic anhydride. Alternatively, the compound may be prepared by dehydration in the presence of a base such as dimethylaminopyridine. Typically, this reaction is carried out in a halogenated organic solvent such as dichloromethane at temperatures between 15 and 25°C. Typically, this is carried out at room temperature.

[0091] A compound of general formula (LI) can be prepared by reacting a compound of general formula (LII): [ka] (Wherein, Y and R 5 is as defined in general formula (I), R 47 is defined by the general formula (VIII) (Lithium tri-sec-butylborohydride) They may also be prepared by reaction with a reducing agent followed by treatment with hydrogen peroxide. Suitably, the reaction with L-Selectride® is carried out under an inert atmosphere, such as argon, The reaction is carried out at low temperature, typically -78°C. The reaction with hydrogen peroxide is suitably carried out at a temperature of about 0°C. It will be carried out.

[0092] The compound of general formula (LII) can be prepared by reacting a compound of general formula (LIII): [ka] (Wherein, Y and R 5 is as defined in general formula (I), good. Suitably, the esterification is carried out in the presence of an acid containing a leaving group, such as, for example, p-toluenesulfonic acid. So, Alcohol R 47 This reaction is carried out at temperatures of about 20-40°C, e.g. The reaction may be carried out at a temperature of, for example, about 30°C.

[0093] Compounds of formula (LIII) are known and can be synthesized by methods known to those skilled in the art, for example, by methods described in International Publication WO2016 / 079 518, International Publication WO2016 / 079518, International Publication WO2016 / 079519, International Publication WO2016 / 079520, International Publication It may also be prepared by the methods taught in International Publication WO2017 / 199036 and International Publication WO2017 / 199033.

[0094] The compounds of general formula (I) are FXR agonists and are therefore useful in the treatment or administration of FXR mediated diseases and conditions. is useful for prevention. Thus, in a further aspect of the invention there is provided a compound of general formula (I) for use in medicine Provided. The compounds may be used in human or veterinary medicines, suitably for the treatment of mammals, particularly humans. It can be used for.

[0095] The compounds of general formula (I) are useful for treating non-alcoholic steatohepatitis (NASH); primary biliary cirrhosis; Sclerosing cholangitis; Biliary atresia; Cholestatic liver disease; Hepatitis C infection; Alcoholic liver disease; Fibrosis and are particularly useful for the treatment or prevention of metabolic syndrome, including liver damage resulting from fibrosis. be.

[0096] The present invention also relates to a method for treating non-alcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing bile duct disease; inflammation; biliary atresia; cholestatic liver disease; hepatitis C infection; alcoholic liver disease; fibrosis; or fibrosis In the preparation of a medicament for the treatment or prevention of liver damage resulting from Provide use.

[0097] Similarly, nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; Biliary atresia; cholestatic liver disease; hepatitis C infection; alcoholic liver disease; fibrosis; or fibrosis? A method for treating or preventing liver damage resulting from the administration of an effective amount of a compound of general formula (I) to a patient in need thereof. Fibrosis includes liver, kidney and intestinal fibrosis.

[0098] Liver fibrosis is associated with NASH, alcoholic liver disease, or nonalcoholic fatty liver disease. Hepatic fibrosis may also be caused by hepatitis, particularly hepatitis B or C, or parasitic liver disease. Other causes of liver fibrosis include Wilson's disease, Gaucher's disease, and Hemochromatosis, glycogen storage disorder, Zellweger syndrome and congenital hepatic fibrosis This includes damage caused by congenital disorders such as liver fibrosis. Liver fibrosis can also be caused by chlorpromazine. Drugs such as azithromycin, tolbutamide, methotrexate, isoniazid and methyldopa This includes those caused by

[0099] Renal fibrosis is associated with diabetic nephropathy, hypertensive nephrosclerosis, glomerulonephritis, interstitial nephritis, and transplantation. These include those associated with diseases such as glomerulopathy and polycystic kidney disease. Intestinal fibrosis also includes that associated with intestinal disorders. Bowel disorders include irritable bowel syndrome, Crohn's disease and ulcerative colitis.

[0100] The compounds of general formula (I) are suitably provided as pharmaceutical compositions, in accordance with a further aspect of the present invention. A pharmaceutical composition comprising a compound of general formula (I) and a pharma- ceutical acceptable excipient or carrier. Things are provided.

[0101] The particular excipient or carrier used will depend upon the route of administration chosen and the nature of the formulation. acceptable in the sense of being compatible with the other ingredients of the product and not deleterious to the recipient Must be. The compositions of the present invention may also contain one or more additional compounds suitable for treating one or more of the above diseases and conditions. It may contain active ingredients.

[0102] Formulations include oral, enteral, nasal, bronchial (inhalation), topical (ophthalmic, intrabuccal and lingual) suitable for oral, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration and may be prepared by any method well known in the pharmaceutical art.

[0103] The composition may be prepared by combining the active agent defined above with a carrier. The formulations comprise a mixture of the active agent and liquid carriers or finely divided solid carriers, or both. The product is prepared by uniformly and intimately bringing into association the copolymer, and then, if necessary, shaping the product. The present invention relates to a compound of general formula (I) in a pharma- ceutical or veterinarily acceptable excipient or carrier. The present invention also covers a method for preparing a pharmaceutical composition comprising linking or binding to

[0104] The oral dosage forms of the present invention include: capsules, sachets, or the like, each containing a predetermined amount of the active agent. or tablets; powders or granules; active substances in aqueous or non-aqueous liquids solutions or suspensions of active ingredients; or oil-in-water liquid emulsions or water-in-oil liquid emulsions or as a bolus, etc.

[0105] For compositions for oral administration (e.g., tablets and capsules), the term "acceptable carrier" is used herein. " includes vehicles such as common excipients, i.e., syrup, acacia, gelatin, soybean oil, etc. Lubitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethyl Cellulose, Sodium Carboxymethylcellulose, Hydroxypropyl Methylcellulose Binders such as sugar, sucrose and starch; e.g. corn starch, gelatin, lactose cellulose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate fillers and carriers such as sodium chloride and alginic acid; and magnesium stearate. sodium stearate and other metallic stearates, glycerol stearate Lubricants such as talc, stearic acid, silicone fluid, talc wax, oils and colloidal silica Contains peppermint, wintergreen oil, cherry flavor, and other fragrances. Flavoring agents may also be used. Coloring agents may be desirably added to make the dosage form easily identifiable. In some cases, it may be preferable to coat the tablets by methods well known in the art. Cut.

[0106] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compacted tablets contain an active ingredient in a free-flowing form, such as a powder or granules, optionally with a binder, lubricant, inert diluent, or the like. It is mixed with a diluent, preservative, surfactant or dispersant and compressed in a suitable machine. Molded tablets can be made by mixing the powdered compound moistened with an inert liquid diluent. The tablets can be prepared by molding the tablet in a suitable machine. The capsule may be scored or scored and may be formulated to provide slow or controlled release of the active agent. It is also possible.

[0107] Other formulations suitable for oral administration include flavored bases, usually sucrose and anacid. Lozenges containing active ingredients in glycerin or tragacanth; inert bases such as gelatin and glycerin or a drop containing the active agent in sucrose and acacia; and a suitable liquid carrier. This includes mouthwashes that contain active agents.

[0108] For topical application to the skin, the compounds of general formula (I) or (II) may be formulated as creams, ointments, jellies, or the like. The drug may be administered in the form of a cream or softener. Ointment formulations are described in standard textbooks on medicines, for example the British Pharmacopoeia. These are conventional formulations well known in the art. Parenteral formulations will generally be sterile. [Brief description of the drawings]

[0109] (drawing) In the figures, * represents P value < 0.05, ** represents P value < 0.01, and *** represents P value < 0.001. . [Figure 1]FIG. 1 shows the change in SHP expression after incubation of the human hepatoma cell line Huh7 with control, OCA (at EC50 and EC90), and Compound 2 (at EC50 and EC90) for 24 hours. [Diagram 2] FIG. 2 shows the change in OSTα expression after incubating the human hepatoma cell line Huh7 with control, OCA (at EC50 and EC90), and Compound 2 (at EC50 and EC90) for 24 hours. [Diagram 3] FIG. 3 shows the change in CYP7A1 expression after incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC50 and EC90), and Compound 2 (at EC50 and EC90) for 24 hours. [Figure 4] FIG. 4 shows the change in TGFβ1 expression after incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC50 and EC90), and Compound 2 (at EC50 and EC90) for 24 hours.

[0110] [Diagram 5] FIG. 5 shows the change in SHP expression after incubation of the human hepatic cell line Huh7 with control, OCA (at EC50 and EC90), and compound 14 (at EC50 and EC90) for 24 hours. [Figure 6] FIG. 6 shows the change in OSTα expression after incubation of the human hepatic cell line Huh7 with control, OCA (at EC50 and EC90), and compound 14 (at EC50 and EC90) for 24 hours. [Figure 7] FIG. 7 shows the change in CYP7A1 expression after incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC50 and EC90), and compound 14 (at EC50 and EC90) for 24 hours. [Figure 8] FIG. 8 shows the change in TGFβ1 expression after incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC50 and EC90), and compound 14 (at EC50 and EC90) for 24 hours. EXAMPLES

[0111] (Example) The following abbreviations are used in the examples: [Table 1]

[0112] Example 1 3α-hydroxyl-4β-fluoro-6α- with sulfonylurea-substituted side chains Synthesis of Ethyl-7α-hydroxyl-5β-cholanic Acid Analogues (A. Methyl 6α-ethyl-3α,7α-dihydroxyl-5β-cholan-24-oate) [ka] A solution of OCA (23.5 g, 55.87 mmol) in MeOH (540 mL) was added with para-toluenesulfonic acid (1 The reaction was completed after adding 1.0001 mg of 1,000 sucrose (0.02 mg, 5.59 mmol, ~0.1 equiv.) and sonicating for 3 hours at 30 °C. After this, the reaction mixture was concentrated under reduced pressure in vacuum. The residue was dissolved in chloroform (500 mL) and saturated The mixture was washed with NaHCO3 solution (500 mL), H2O (500 mL), and brine (500 mL), and dried over MgSO4. The mixture was then filtered and concentrated under vacuum to give the title compound as a white solid in quantitative yield. The solid obtained was used without further purification. [ka] LRMS(ESI + ) m / z: 452.4 [M+NH4] + ,100%.

[0113] (B. Methyl 6α-ethyl-7α-hydroxyl-3-oxo-5β-cholan-24-oate) [ka] Methyl 6α-ethyl-3α,7α-dihydroxyl-5β-cholesterol obtained in step A with stirring To a solution of 2-methyl-24-oxoate (9.53 g, 21.9 mmol) in HO (22 mL) and tert-butanol (88 mL) At room temperature, KBr (5.22 g, 43.9 mmol, ~2.0 equiv.), KHCO3 (22.0 g, 219 mmol, ~10 equiv.) and TEMPO (4.45 g, 28.5 mmol, ∼1.3 equiv.) was added. The reaction mixture was cooled to 0 °C and NaClO( 28 mL, 32.9 mmol, ~1.5 equiv) was added dropwise at a rate of 4 mL / hr over 7 hours. Upon completion, the reaction was quenched by slow addition of 1:1 saturated Na2S2O3 solution (250 mL) and EtOAc ( The organic phase was removed and the aqueous phase was then back extracted with EtOAc (3×150 mL). The phases were combined, dried over MgSO4, filtered, and concentrated under vacuum to give 14.2 g of crude material as an orange extract. The oil was purified by column chromatography (a gradient of acetone in PE). Purification by elution 40-60, 0-20%) afforded the title compound as a white solid (8.48 g, 89%). [ka] LRMS(ESI + ) m / z: 450.3 [M+NH4] + ,100%.

[0114] (C. Methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholan-24-ol (Rate) [ka] Pre-cooled diisopropylamine (0.78 mL, 5.54 mmol, ∼12 equiv.) in -78 To a solution of dry THF (6.9 mL) at 37 °C, n-BuLi (1.44 mL, 2.31 mmol, ~5.0 equiv.) in hexane was added. The mixture was added dropwise over 0.25 hours under an argon atmosphere. After the addition, trimethylsilyl chloride (0 The methyl 6 from step B was added and stirred for 1 h. α-Ethyl-7α-hydroxyl-3-oxo-5β-cholan-24-oate (200 mg, 0.46 mmol) A solution of dry THF (3 mL) and triethylamine (1.16 mL, 8.32 mmol, ~18 equiv) was then added. After the addition, the reaction was gradually warmed to -20°C and stirred for 2 hours. The reaction was quenched by dropwise addition of saturated NaHCO3 solution (5 mL) and allowed to warm to room temperature over 2 h. The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3×10 mL). The organic phases were combined and washed with brine (30 1 mL), dried over MgSO4, filtered, and concentrated under vacuum to give 271 mg of crude material as a yellow residue. Obtained as a residue.

[0115] Selectfluor® was added to a stirred solution of the resulting crude material in MeCN (13 mL) and mixed. The mixture was stirred for 16 hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure in vacuo. Dissolved in EtOAC (20 mL) and acidified with 2M HCl (30 mL). The organic phase was removed and the aqueous phase was diluted with EtOAc (3 The combined organic phases were washed with brine (100 mL), dried over MgSO4, and Filtration and concentration under vacuum reduced pressure gave 196 mg of crude material as a green solid. Purification by HPLC The title compound and methyl-2β-fulveolar Inseparable mixture of 3-oxo-6α-ethyl-7α-hydroxyl-5β-cholan-24-oates The product was obtained as a colorless oil (79 mg, 0.18 mmol, 37% of the title compound, 1 H NMR shows that methyl -2β-fluoro-3-oxo-6α-ethyl-7α-hydroxyl-5β-cholan-24-oate 1% (It is believed that [ka] LRMS(ESI + ) m / z: 468.4 [M+NH4] + ,100%.

[0116] (D. Methyl 6α-ethyl-4β-fluoro-(3α,7α)-dihydroxyl-5β-cholan-24-ol to) [ka] Methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-acetate obtained in step C with stirring A solution of oxo-5β-cholan-24-oate (75 mg, 0.17 mmol) in dry THF (6.7 mL) at room temperature was added. NaBH4 (19 mg, 0.50 mmol, ∼3.0 equiv.) was added and stirred under an argon atmosphere for 16 h. Upon completion, the reaction was quenched by dropwise addition of H2O (8 mL) and diluted with EtOAc (10 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3×50 mL). The organic phases were combined and diluted with H2O (100 mL), dried over MgSO4, filtered, and concentrated under vacuum to give 77 mg of crude material. The product was obtained as a colored residue. It was purified by HPLC using hexane / acetone (90 / 10) as the eluent. This afforded the title compound as a colourless oil (55 mg, 0.12 mmol, 74%). [ka] LRMS(ESI + ) m / z: 470.4 [M+NH4] + ,100%.

[0117] (E. 3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholanic acid) [ka] Methyl 6α-ethyl-4β-fluoro-(3α,7α)-dihydroxyl-5β-cholane-24 during stirring To a solution of 2-amino-3-hydroxybenzoate (58 mg, 0.13 mmol) in MeOH (5 mL) was added NaOH (250 mg, 5% solution) at room temperature. After the reaction was complete, the reaction mixture was concentrated under reduced pressure in vacuo and the residue was diluted with 1M The mixture was acidified to pH 2 with HCl and diluted with EtOAc (20 mL). The organic phase was removed and the aqueous phase was diluted with EtOAc (3 × 50 mL). The organic phases were combined, washed with brine (100 mL), dried over MgSO4, and filtered. Concentration under reduced pressure in vacuo afforded 76 mg of crude material as a yellow oil. Using ethanol / acetone (70 / 30) as the eluent, the title compound was obtained as a colorless oil (41 mg, 0. 09 mmol, 72%). [ka] LRMS(ESI + ) m / z: 456.2, [M+NH4] + ,100%.

[0118] (Synthesis of compounds with sulfonylurea-substituted side chains) The method below is shown for the 4β-fluoro derivative, but 2β-fluorinated, 4,4-difluorinated Alternatively, 2,4-difluoroinated compounds may also be used.

[0119] (F. 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholanic acid) [ka] 3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5α-dimethylformamide obtained in step E with stirring A solution of β-cholanic acid (2.08 g, 4.74 mmol) in dry THF (160 mL) was added at room temperature under an argon atmosphere. , NaHCO3 (2.04 g, 23.7 mmol, ~5.0 equiv.) and Ac2O (2.29 mL, 23.7 mmol, ~5.0 equiv.) were added to 5 mL of After the addition, the reaction mixture was heated at 70° C. for 16 hours. The reaction was complete. The reaction mixture was then cooled to room temperature and quenched by dropwise addition of H2O (100 mL) and 1M HCl (20 The organic phase was removed and the aqueous phase was diluted with EtOAc (3×150 mL). The combined organic phases were washed with brine (400 mL), dried over MgSO4, filtered, and Concentration under reduced pressure in vacuum gave a yellow oil, which was purified by column chromatography (CH2Cl Purification by gradient elution of MeOH in 2 (0-3%) afforded the title compound as a white solid (660 mg, 29%). [ka] LRMS(ESI + ) m / z: 498.2, [M+NH4] + ,100%.

[0120] (G. 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholan-24-oyl Ruazide) [ka] 3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydrogenase obtained in step F with stirring A solution of xyl-5β-cholanic acid (200 mg, 0.42 mmol) in dry THF (4 mL) was added at room temperature under an argon atmosphere. Et3N (0.12 mL, 0.83 mmol, ~2.0 equiv) was added dropwise under air. After addition, the reaction mixture was cooled to 0 °C. The mixture was cooled to rt and diphenylphosphoryl azide (0.13 mL, 0.62 mmol, ∼1.5 equiv) was added dropwise. After the addition, the reaction mixture was stirred behind a blast shield for 3 hours. The reaction was complete. The reaction was then quenched with brine (5 mL) and diluted with CH2Cl2 (5 mL). The organic phase was removed and the aqueous phase The extract was back-extracted with CH2Cl2 (3 x 5 mL). The organic phases were combined, dried over MgSO4, filtered, and reduced in vacuo. Concentration under reduced pressure at 0° C. gave a yellow oil which was used without further purification. [ka]

[0121] (H. 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholane- 23-yl isocyanate) [ka] The lipophobic 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxybutyrate obtained in step G is stirred. Hydroxyl-5β-cholan-24-oyl azide (105 mg, estimated 0.42 mmol) in dry toluene (3.1 The solution was heated to 125° C. under an argon atmosphere. After 5 h, the reaction was allowed to warm to room temperature. Allow to cool and the resulting solution was used without further purification. [ka]

[0122] General Procedure 1 for the Conversion of Isocyanates to Sulfonylureas [ka] Stirring the soluble 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxide obtained in step H. A toluene solution of 24-nor-5β-cholan-23-yl isocyanate was added to the sulfonamide. Add 1.5 eq. of dimethylformamide (~1.5 eq.) and DBU (~1.5 eq.) and stir for 16 h. The mixture was quenched by dropwise addition of 1M HCl (2 mL) and diluted with EtOAc (5 mL). The organic phase was removed. The aqueous phase was back-extracted with EtOAc (3×5 mL). The organic phases were combined, dried over MgSO4, and filtered. The resulting residue was purified by column chromatography (acetone in PE). The desired sulfonylurea was obtained by elution with a gradient of 40-60, 5-20%.

[0123] (N,N'-(3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholesterol) (23-yl)-p-toluenesulfonylurea (Intermediate 1) [ka] Intermediate 1 was prepared according to general procedure 1 using 53.4 mg of p-toluenesulfonamide. Obtained as a yellow oil (51.7 mg, 38%). [ka] LRMS(ESI + ) m / z: 666.4, [M+NH4] +,100%.

[0124] (N,N'-(3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholesterol) (23-yl)-benzenesulfonylurea (intermediate 2) [ka] Prepared according to general procedure 1 using 49.0 mg of benzenesulfonamide to give intermediate 2 in yellow. Obtained as a coloured oil (49.9 mg, 38%). [ka] LRMS(ESI + ) m / z: 652.3, [M+NH4] + ,100%.

[0125] (N,N'-(3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholesterol) (23-yl)-4-(tert-butyl)benzenesulfonylurea (Intermediate 3) [ka] Prepared according to general procedure 1 using 53.2 mg of 4-(tert-butyl)benzenesulfonamide This afforded intermediate 3 as a colorless oil (81.6 mg, 71%). [ka] LRMS(ESI + ) m / z: 708.4, [M+NH4] + ,100%.

[0126] (N,N'-(3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholesterol) (23-yl)-m-toluenesulfonylurea (Intermediate 4) [ka] Intermediate 4 was prepared according to general procedure 1 using 42.6 mg of m-toluenesulfonamide. Obtained as a colorless oil (85.7 mg, 80%). [ka] LRMS(ESI + ) m / z: 666.3, [M+NH4] + ,100%.

[0127] (N,N'-(3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholesterol) (23-yl)-o-toluenesulfonylurea (Intermediate 5) [ka] Intermediate 5 was prepared according to general procedure 1 using 42.6 mg of o-toluenesulfonamide. Obtained as a colorless oil (55.4 mg, 51%). [ka] LRMS(ESI + ) m / z: 666.3, [M+NH4] + ,100%.

[0128] (General procedure 2 for deprotection of 3α-acetate sulfonylureas) [ka] Add 10 ml of NaOH in MeOH (5% solution) to the flask containing the protected sulfonylurea. L) solution was added and stirred for 16 hours. Upon completion of the reaction, the reaction was acidified with 1M HCl to pH The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3×10 mL). The combined organic phases were washed with NaHCO3 solution (50 mL), dried over MgSO4, filtered and reduced in vacuo. The mixture was concentrated under reduced pressure. The resulting residue was purified by column chromatography (MeOH in CH2Cl2 gradient). Purification by elution with 0-5% acetonitrile afforded the deprotected sulfonylurea.

[0129] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (p-toluenesulfonylurea (compound 1)) [ka] Prepared according to general procedure 2 using 49.7 mg of intermediate 1 to give compound 1 as a colorless residue. (18.6 mg, 40%). [ka] LRMS(ESI + ) m / z: 624.4, [M+NH4] + ,100%.

[0130] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (III)-Benzenesulfonylurea (Compound 2) [ka] Prepared according to general procedure 2 using 44.8 mg of intermediate 2 to give compound 2 as a colorless residue. (28.5 mg, 64%). [ka] LRMS(ESI + ) m / z: 610.2, [M+NH4] + ,100%.

[0131] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (4-(tert-butyl)benzenesulfonylurea (compound 3)) [ka] Prepared according to general procedure 2 using 79.6 mg of intermediate 3 to give compound 3 as a colorless residue. (50.7 mg, 65%). [ka] LRMS(ESI + ) m / z: 666.4, [M+NH4] + ,100%.

[0132] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) m-Toluenesulfonylurea (Compound 4) [ka] Prepared according to general procedure 2 using 83.7 mg of intermediate 4 to give compound 4 as a colorless residue. (29.0 mg, 37%). [ka] LRMS(ESI + ) m / z: 624.3, [M+NH4] + ,100%.

[0133] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (I)-o-toluenesulfonylurea (Compound 5) [ka] Prepared according to general procedure 2 using 53.4 mg of intermediate 5 to give compound 5 as a colorless residue. (24.2 mg, 48%). [ka] LRMS(ESI + ) m / z: 624.3, [M+NH4] + ,100%.

[0134] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (III)-p-fluorobenzenesulfonylurea (compound 6) [ka] This compound was prepared by methods similar to those described above for compounds 1-5. [ka]

[0135] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) m-Fluorobenzenesulfonylurea (Compound 7) [ka] This compound was prepared by methods similar to those described above for compounds 1-5. [ka]

[0136] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (III)-o-fluorobenzenesulfonylurea (Compound 8) [ka] This compound was prepared by methods similar to those described above for compounds 1-5. [ka]

[0137] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (p-(trifluoromethyl)benzenesulfonylurea (compound 9)) [ka] This compound was prepared by methods similar to those described above for compounds 1-5. [ka]

[0138] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (III)-m-(trifluoromethyl)benzenesulfonylurea (Compound 10) [ka] This compound was prepared by methods similar to those described above for compounds 1-5. [ka]

[0139] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (11)-o-(trifluoromethyl)benzenesulfonylurea [ka] This compound was prepared by methods similar to those described above for compounds 1-5. [ka]

[0140] (N,N'-(3α,7α-dihydroxyl-6α-ethyl-24-nor-5β-cholan-23-yl)-benzenesulfonyl Sulfonylurea (Comparative Compound A) [ka] This compound was prepared by methods similar to those described above for compounds 5-9. [ka]

[0141] Example 2 Alternative synthesis of compounds with sulfonylurea substituted side chains The method below is shown for the 4β-fluoro derivative, but 2β-fluorinated, 4,4-difluorinated Alternatively, 2,4-difluoroinated compounds may also be used.

[0142] (A. Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3-oxo-5β-cholane-24 -Oart) [ka] Pre-cooled diisopropylamine (0.78 mL, 5.54 mmol, ∼12 equiv.) in -78 To a solution of dry THF (6.9 mL) at 37 °C, n-BuLi (1.44 mL, 2.31 mmol, ~5.0 equiv.) in hexane was added. The mixture was added dropwise over 0.25 hours under an argon atmosphere. After the addition, trimethylsilyl chloride (0 0.29 mL, 2.31 mmol, ~5.0 equiv) was added and stirred for 1 h. Methyl 6α-ethyl-7α-hydroxyl-3-oxo-5β-cholan-24-oate (200 mg, 0.46 A solution of 1.2 mmol) in dry THF (3 mL) and triethylamine (1.16 mL, 8.32 mmol, ~18 equiv.) After the addition, the reaction was gradually warmed to -20°C and stirred for 2 hours. After this time, the reaction was quenched by dropwise addition of saturated NaHCO3 solution (5 mL) and allowed to warm to room temperature over 2 h. The organic phase was removed and the aqueous phase was back extracted with EtOAc (3×10 mL). The organic phases were combined and Wash with brine (30 mL), dry over MgSO4, filter, and concentrate under vacuum to give 271 mg of crude material. was obtained as a yellow residue.

[0143] To a solution of the crude material (1.16 g, 2.3 mmol) in dry MeCN (55 mL) was added Selectfluor® (1.23 g, 3.47 mmol) was added. After stirring at room temperature for 14.5 hours, the mixture was diluted with ethyl acetate (100 mL). The aqueous phase was washed with a mixture of 5% NaHCO3 (100 mL) and 10% NaCl (50 mL). The aqueous phase was diluted with ethyl acetate (3× 100 mL) and the combined organic phases were dried over MgSO4, filtered and concentrated under vacuum to give An orange / yellow oil was obtained. The crude material was purified by column chromatography (SiO2, 0-40% EtOAc in heptane). c) to give the title compound as a colourless oil (319.5 mg).

[0144] (B) Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β- COLAN-24-OART) [ka] Step A from the phobic methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3-oxo 5β-Cholan-24-oate (319.5 mg, 0.71 mmol) was added to TH F (28 mL). NaBH4 (80.5 mg, 2.13 mmol) was charged and the reaction was stirred at room temperature for 16.5 h. The mixture was stirred for an additional 4.5 hours, after which additional NaBH4 (0.24 g, 6.38 mmol) was charged. After stirring for 1 h, water (20 μL) was added and the mixture was stirred for ∼60 h. The reaction was then (15 mL) and diluted with EtOAc (50 mL). The two phases were separated and the aqueous phase was diluted with EtOAc (50 mL). (3×50 mL). The combined extracts were dried over MgSO4, filtered, and concentrated in vacuo. A clear syrup was obtained (0.34 g). This crude material was purified by column chromatography (SiO 2, 0-40% EtOAc in heptane) to give the title compound as a clear oil (162.3 mg).

[0145] (C. Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethyl Tyrsilyl-5β-cholan-24-oate [ka] The methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxybenzoate obtained in step B Dissolve hydroxyl-5β-cholan-24-olate (0.48 g, 0.92 mmol) in dry DCM (12 mL). The mixture was cooled to 0°C under argon atmosphere while stirring. 2,6-lutidine (1.1 mL, 9.17 mmol) was added The reaction was allowed to warm to room temperature and then TBMDS-OTf (0.32 mL, 1.38 mmol) was added dropwise. The mixture was stirred for 1 h, then cooled to 0° C. and quenched by the dropwise addition of 10% citric acid (5 mL). The layers were separated and the aqueous phase was extracted with DCM (3×5 mL). The combined extracts were washed with 10% citric acid (5 mL), NaH Wash with aqueous CO3 (5 mL) and water (5 mL), dry over MgSO4, filter, and concentrate under reduced pressure in vacuo. A yellow oil was obtained (0.69 g). This crude material was purified by column chromatography (SiO2, 0-20% heptane in EtOAc) to give the title compound as a clear oil (0.58 g).

[0146] (D. 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl 5-aryl-5β-cholanic acid [ka] Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O- tert-Butyldimethylsilyl-5β-cholan-24-olate (0.58 g) was added to IPA (5.8 mL) with stirring. 0.5 M NaOH (5.8 mL) was charged and the reaction was stirred at room temperature for 15 hours. The mixture was concentrated under reduced pressure to half its volume, then water (5 mL) was charged and the solution was added 2 M H2SO4. The mixture was neutralized with hexanes and diluted with EtOAc (10 mL). The mixture was acidified to pH 1 with 2 M H2SO4 and the two phases were separated. The aqueous phase was extracted with EtOAc (10 mL), and the combined extracts were washed with water (5 mL) and brine (5 mL). The extract was dried over MgSO4, filtered, and concentrated in vacuo to give a white foam (0.52 g). The crude material was purified by column chromatography (SiO2, 0-50% acetone in toluene) and the title compound was The compound was obtained as a white solid (0.41 g, 72%).

[0147] (E. 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl Lyl-5β-cholan-24-oyl azide) [ka] 6α-Ethyl-4β-fluoro-7α-trimethylsiloxy-3α- O-tert-Butyldimethylsilyl-5β-cholanic acid (197 mg, 0.32 mmol) dissolved in dry THF (3.2 mL) To the solution was added Et3N (0.09 mL, 0.64 mmol, ∼2.0 equiv) dropwise at room temperature under an argon atmosphere. After the addition, the reaction mixture was cooled to 0 °C and diphenylphosphoryl azide (0.1 mL, 0.48 mmol, ~1 After the addition, the reaction mixture was stirred behind a blast shield for 2.5 hours. Upon completion of the reaction, the reaction was quenched with brine (3 mL) and extracted with DCM (3×5 mL). The combined organic phase was dried over MgSO4, filtered, and concentrated under vacuum at 0 °C. The resulting oil was and used without further purification.

[0148] General Procedure 3 for the Formation of Sulfonylureas [ka] Stirring of the phobic 6α-ethyl-4β-fluoro-7α-trimethyl ether obtained in step E of Example 2 Siloxy-3α-O-tert-butyldimethylsilyl-5β-cholan-24-oyl azide (69 mg) The solution in toluene (2.1 mL) was heated to 125° C. under an argon atmosphere. After 5 h, the reaction was The solution was allowed to cool to room temperature and was used without further purification. The mixture was stirred under nitrogen atmosphere, and sulfonamide (1.5 equivalents) and DBU (1.5 equivalents) were added. Upon completion, the reaction was quenched by the dropwise addition of 1M HCl (1 mL) and diluted with EtOAc (5 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3×5 mL). The combined organic phase was washed with brine (3 mL). The mixture was washed, dried over MgSO4, filtered and concentrated under reduced pressure in vacuo (231.7 mg). Purification by column chromatography afforded the desired sulfonylurea from an inseparable crude mixture. And obtained.

[0149] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) N,N'-(3α,7α- Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzyl benzenesulfonylurea (compound 13) Compounds 12 and 13 were prepared according to General Procedure 3 above to give the isocyanatophobic product and 4-( trifluoromethoxy)benzenesulfonamide, and the isocyanatophobic product with 4-(methyl) It was prepared by reaction with (methoxy)benzenesulfonamide. To obtain the pure products, the crude compounds 12 and 13 were reacted with the functional group-protected materials (intermediates 12 and 13). 3), which was purified and then deprotected to regenerate compounds 12 and 13. This method is described below. Please refer to the following.

[0150] (N,N'-(3α,7α-di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor-5 β-Cholan-23-yl)-4-(trifluoromethoxy)benzenesulfonylurea (Intermediate 12) [ka] N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl Dry D)-4-(trifluoromethoxy)benzenesulfonylurea (24.3 mg, 0.036 mmol) The mixture was dissolved in CM (1 mL) and cooled to 0°C under argon atmosphere with stirring. 2,6-Lutidine (0. 0.04 mL, 0.36 mmol) was added, followed by the dropwise addition of TBMDS-OTf (0.02 mL, 0.108 mmol). The reaction was allowed to warm to room temperature and stirred for 1.5 h, then cooled to 0° C. and 10% citric acid (1 mL) was added dropwise. The two phases were separated and the aqueous phase was extracted with DCM (3 x 1 mL). Wash with 10% citric acid (1 mL), aqueous NaHCO3 (1 mL) and water (1 mL), dry over MgSO4 and filter. The crude material was purified by column chromatography (SiO2, 0 Purification with 50% EtOAc in heptane gave the title compound as a clear oil (9.4 mg, 33%).

[0151] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (trifluoromethoxy)benzenesulfonylurea (compound 12) [ka] N,N'-(3α,7α-di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor 5β-Cholan-23-yl)-4-(trifluoromethoxy)benzenesulfonylurea (9.4 mg) was added to The mixture was dissolved in dry THF (1 mL) with stirring under argon. 1 M TBAF in THF (31 μL, 0. A solution of 0.03 mmol) was added and the reaction was stirred at room temperature for 6 days. The solution was dried over silica gel and The title compound was purified by column chromatography (SiO2, 50-100% EtOAc in heptane) to give Obtained (1 mg).

[0152] (N,N'-(3α,7α-di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor-5 β-Cholan-23-yl)-p-methoxybenzenesulfonylurea (Intermediate 13) [ka] N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl (26.1 mg, 0.042 mmol) was dissolved in dry DCM (1 mL). The mixture was cooled to 0°C under stirring under an argon atmosphere. 2,6-lutidine (0.05 mL, 0.419 mM mol) was added, followed by the dropwise addition of TBMDS-OTf (0.03 mL, 0.126 mmol). The reaction was allowed to warm to room temperature. The mixture was warmed and stirred for 16 h, then cooled to 0° C. and quenched by the dropwise addition of 10% citric acid (1 mL). The two phases were separated and the aqueous phase was extracted with DCM (3 × 1 mL). The combined extracts were washed with 10% citric acid (1 mL ), washed with aqueous NaHCO3 (1 mL) and water (1 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude material was purified by column chromatography (SiO2, 0–80% D Purification by column chromatography (SiO2, 0-50% acetone in toluene) Re-purification at rt gave the title compound (7.8 mg).

[0153] (N,N'-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl) (p-Methoxybenzenesulfonylurea (Compound 13)) [ka] N,N'-(3α,7α-di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor 5β-Cholan-23-yl)-p-methoxybenzenesulfonylurea (7.8 mg) was added under an argon atmosphere. The mixture was dissolved in dry THF (1 mL) while stirring at RT. A 1 M solution of TBAF in THF (28 μL, 0.03 mmol) was The reaction was stirred at room temperature for 17 hours. The eluate was dried over silica gel and then purified by column chromatography. The mixture was purified by column chromatography (SiO2, 0-80% acetone in toluene) to give the title compound (3.4 mg, 5 9.6%). [ka]

[0154] Example 3 Synthesis of Compounds with Sulfonamide-Substituted Side Chains The method below is shown for the 4β-fluoro derivative, but 2β-fluorinated, 4,4-difluorinated Alternatively, 2,4-difluoroinated compounds can also be used. Step A and B are the same as in Example 2.

[0155] (C. 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholane acid) [ka] The methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxybenzoate obtained in step B Hydroxyl-5β-cholan-24-oate (162.3 mg) was dissolved in IPA (1.6 mL) with stirring. 0.5 M NaOH (1.6 mL) was charged and the reaction was stirred at room temperature for 16 hours. The reaction mixture was cooled to room temperature under reduced pressure. The mixture was concentrated to half its volume using a 500 ml_ flask, then water (5 mL) was added and the solution was neutralized with 2 M H2SO4. The mixture was acidified to pH 1 with 2 M H2SO4, the two phases were separated, and the aqueous phase was diluted with Et The combined extracts were washed with water (3 mL) and brine (5 mL) and concentrated with MgSO4. Drying, filtration, and concentration under reduced pressure gave a white foam (151.1 mg). Purification by column chromatography (SiO2, 0-80% EtOAc in heptane) afforded the title compound as a clear solid. Obtained as an oil (164.1 mg).

[0156] (General Procedure 4 for the Formation of Acyl Sulfonamide Side Chains) [ka] 6α-Ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholanic acid (5 EDCI (43.7 mg, 0.23 mmol) and DMAP ( 27.8 mg, 0.23 mmol) followed by the appropriate sulfonamide (3 equiv.). After stirring overnight at room temperature, (5 mL), the two phases were separated and the aqueous phase was extracted with DCM (2×5 mL). M HCl (2 mL) and brine (2 mL), dried over MgSO4, filtered, and concentrated in vacuo. The crude material was obtained as an off-white solid.

[0157] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-trimethyl Fluoromethoxybenzenesulfonamide (Compound 14) [ka] N-(6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholesterol) Trifluoromethoxybenzenesulfonamide (81.6 mg) was added to trifluoro General procedure 4 was followed using 2-methoxybenzenesulfonamide and The mixture was dissolved in dry THF (5 mL) under stirring under atmospheric pressure. 1 M TBAF in THF (0.48 mL, 0.48 mm The reaction was stirred at room temperature for 17.5 hours. The soluble solution was dried over silica gel and Purification was performed by column chromatography (SiO2, 0-100% EtOAc in heptane). The combined fractions were concentrated in vacuo, dissolved in EtOAc (5 mL) and diluted with 2 M HCl (5 mL). The aqueous phase was extracted with EtOAc (2×5 mL) and the combined extracts were dried over MgSO4 and filtered. The resulting white solid was purified by column chromatography (SiO2, 0–25 % acetone in toluene) to give the title compound as a clear residue (4.9 mg). [ka]

[0158] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-fulv Orobenzenesulfonamide (Compound 15) [ka] This was prepared by a similar route to that used for compound 14 above. [ka]

[0159] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-m-fluoro Orophenylsulfonamide (Compound 16) [ka] This was prepared by a similar route to that used for compound 14 above. [ka]

[0160] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-o-fluoro Orophenylsulfonamide (Compound 17) [ka] This was prepared by a similar route to that used for compound 14 above. [ka]

[0161] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-4-trimethylol Fluoromethylphenylsulfonamide (Compound 18) [ka] This was prepared by a similar route to that used for compound 14 above. [ka]

[0162] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-3-trimethyl Fluoromethylphenylsulfonamide (Compound 19) [ka] This was prepared by a similar route to that used for compound 14 above. [ka]

[0163] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-2-trimethylol Fluoromethylphenylsulfonamide (Compound 20) [ka] This was prepared by a similar route to that used for compound 14 above. [ka]

[0164] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-cyclo Propylsulfonamide (Comparative Compound B) [ka] N-(3α-hydroxyl-4β-fluoro-6α-ethyl-7α-trimethylsiloxy-5β-cholane- 24-yl)-cyclopropylsulfonamide (50 mg) was added to the flask under stirring under an argon atmosphere. A 1 M solution of TBAF in THF (0.3 mL, 0.3 mmol) was added and the reaction was stirred at room temperature for 2 h. The reaction was stirred for 3 h. The reaction was diluted with EtOAc (20 mL) and washed with water (10 mL) and 10% aqueous NaCl. The solution was dried on silica gel and purified by column chromatography (SiO2, 0-50% toluene). in acetone) to give the title compound (5.4 mg). Rf value: 0.65 (EtOAc / heptane, 50:50). [ka]

[0165] (N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-methyl Sulfonamide (Comparative Compound C) [ka] N-(3α-hydroxyl-4β-fluoro-6α-ethyl-7α-trimethylsiloxy-5β-cholane- 24-yl)-methylsulfonamide (50 mg) was dissolved in dry THF under argon atmosphere with stirring. A 1 M solution of TBAF in THF (0.3 mL, 0.3 mmol) was charged and the reaction was stirred at room temperature for 16 h. The reaction was diluted with EtOAc (5 mL) and washed with brine (3 mL). The eluate was precipitated on silica gel. The mixture was dried at 40°C and purified by column chromatography (SiO2, 0-50% acetone in toluene). The product-containing fractions were combined, concentrated under reduced pressure, dissolved in CDCl3, and diluted with 2 M HCl and water. The mixture was washed with 100 ml of ethyl acetate, filtered through a PTFE filter pad, and concentrated under reduced pressure to give the title compound. (7.1 mg). [ka]

[0166] Example 4 4,4-difluoro-3α,7-dimers bearing sulfonylurea and acylsulfonamide side chains Synthesis of α-dihydroxyl-6α-ethyl-5β-cholanic acid analogues (A) Methyl 6α-ethyl-4,4-difluoro-7α-hydroxyl-3-oxo-5β-cholan-24-ol art) [ka] Pre-cooled methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholane to a solution of 1,2-dihydro-2,4-trimethylaminopropionate (product of Example 1C; 7.30 g, 16.0 mmol) in dry THF (300 mL) at -78 °C. LDA (21.1 mL, 21.1 mmol, ∼1.3 equiv.) in hexane was added under argon atmosphere for 0.25 h. After the addition, trimethylsilyl chloride (2.70 mL, 21.1 mmol, ~1.3 equiv. ) was added as a solution in dry THF (150 mL) and stirred for 1 h. The reaction was quenched by dropwise addition of saturated NaHCO3 solution (300 mL) and allowed to warm to room temperature over 0.25 h. The organic phase was removed and the aqueous phase was back extracted with dichloromethane (2×150 mL). The organic phases were combined and Wash with brine (300 mL), dry over MgSO4, filter, and concentrate under reduced pressure to give the crude material ( 19 F.N.M. According to R, methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholane-24 The resulting residue was subjected to purification without further purification. It was then used in the next reaction. [ka]

[0167] To a stirred solution of the resulting crude material in MeCN (360 mL) was added Selectfluor® (11.4 g, 32.0 mL). (mmol, ~2.0 equiv.) was added and stirred for 16 h. Upon completion of the reaction, the reaction mixture was reduced in vacuum. The residue was dissolved in dichloromethane (500 mL) and H2O (500 mL). The aqueous phase was back-extracted with dichloromethane (2×250 mL). The organic phases were combined and washed with brine ( 250 mL), dried over MgSO4, filtered, and concentrated under reduced pressure to remove the crude material to a yellow residue. The resulting residue was used in the next reaction without purification. [ka] LRMS(ESI + ) m / z: 486.6, [M+NH4] + ,100%.

[0168] (B) Methyl 4,4-difluoro-(3α,7α)-dihydroxyl-6α-ethyl-5β-cholan-24-ol (Rate) [ka] Stirring the phosphatidyl 6α-ethyl-4,4-difluoro-7α-hydroxybenzoate obtained in step A Dimethyl-3-oxo-5β-cholan-24-olate (7.51 g, estimated 16.0 mmol) in dry methanol (500 To the solution of 1 mL of 1000 mM NaBH4 (3.03 g, 80.1 mmol, ~5.0 equiv.) was added at room temperature under argon atmosphere. The reaction was stirred for 72 hours. Upon completion, the reaction was concentrated under reduced pressure in vacuo. The residue was dissolved in dichloromethane. The organic phase was removed and the aqueous phase was dissolved in dichloromethane (2 The organic phases were combined, washed with brine (250 mL), dried over MgSO4, and Filtration over SiO2 and concentration under reduced pressure gave 7.63 g of crude material as a colorless residue. By flash column chromatography (Biotage SNAP KP-Sil 100 g cartridge), The title compound, methionine, was purified using hexane / acetone (100 / 0 to 80 / 20) as the eluent. 4,4-difluoro-(3α,7α)-dihydroxyl-6α-ethyl-5β-cholan-24-oate , as a colorless residue (3.09 g, 6.57 mmol, 41% over three steps). [ka] LRMS(ESI + ) m / z: 488.6, [M+NH4] + ,100%.

[0169] (C. 3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholanic acid) [ka] Methyl 4,4-difluoro-(3α,7α)-dihydroxyl-6α obtained in step B during stirring 1.77 g, 3.75 mmol, 1.0 equiv. of 1,4-dioxane (95 To a solution of concentrated (37%) hydrochloric acid (11 mL, 9:3:1 ratio) was added at room temperature. After refluxing, the reaction mixture was cooled to room temperature and neutralized with saturated NaHCO3 solution (50 mL). The organic phase was removed. The organic phases were combined and washed with brine (200 ml). L), dried over MgSO4, filtered, and concentrated under vacuum to give 1.84 g of crude material as a brown oil. The product was obtained by flash column chromatography (Biotage SNAP KP-Sil 50 g cartridge). The title compound was obtained by purifying the product using hexane / acetone (100 / 0 to 90 / 10) as the eluent. The product, 3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholanic acid, was dissolved in water in a colorless oil. (1.51 g, 3.30 mmol, 88%) [ka] LRMS(ESI + ) m / z: 474.6, [M+NH4] + ,100%.

[0170] (D. 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholanic acid) [ka] 3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl- obtained in step C with stirring A solution of 5β-cholanic acid (400 mg, 0.88 mmol, 1.0 equiv.) in dry THF (50 mL) was added to the flask and cooled to room temperature with hydrogen carbonate. Sodium (370 mg, 4.38 mmol, ~5.0 equiv.) and acetic anhydride (0.41 mL, 4.38 mmol, ~5.0 equiv.) After 16 h at 70 °C, the reaction mixture was cooled to room temperature and H2O (50 mL) was added slowly. The organic phase was removed and the aqueous phase was back extracted with EtOAc (2×50 mL). The combined phases were washed with saturated NaHCO3 solution (100 mL), dried over MgSO4, filtered and concentrated in vacuo. Concentration under reduced pressure gave 462 mg of crude material as a yellow oil. Flash column chromatography - (Biotage SNAP KP-Sil Ultra 25g cartridge) was used to measure hexane / acetone (100 / 0~80 / 20) as the eluent, and the title compound, 3α-acetoxy-4,4-difluoro- 6α-Ethyl-7α-hydroxyl-5β-cholanic acid was obtained as a colorless oil (270 mg, 0.54 mmol, 62%). [ka] LRMS(ESI + ) m / z: 516.5, [M+NH4] + ,100%.

[0171] (E. 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholan-24-ol Ile-azide) [ka] Pre-cooled 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxybutyrate obtained in step D A solution of hydroxyl-5β-cholanic acid (240 mg, 0.48 mmol, 1.0 equiv.) in dry THF (5.3 mL) at 0 °C. to triethylamine (0.14 mL, 0.96 mmol, ~2.0 equiv.) and diphenylphosphoryl azide (0. After 3 h, the reaction mixture was slowly diluted with brine (10 mL). The organic phase was removed and the aqueous phase was back-extracted with dichloromethane (3×20 mL). The organic phases were combined, dried over MgSO4, filtered, and concentrated under vacuum at 0 °C to remove the crude materials. Obtained as a pale yellow oil, which was used for further purification in the next reaction. [ka]

[0172] (F. 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholesterol 23-phenyl isocyanate [ka] Stirring the lipophobic 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-formaldehyde obtained in step E Hydroxyl-5β-cholan-24-oyl azide (252 mg, estimated 0.48 mmol) in dry toluene (7 The solution (0.5 mL) was heated to 125° C. under an argon atmosphere. After 4 h, the reaction was cooled to room temperature. The resulting solution was used without further purification. [ka]

[0173] (G. N,N'-(3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5 β-Cholan-23-yl)-benzenesulfonylurea [ka] The title compound was prepared according to general procedure 1 using 113 mg of benzenesulfonamide. N,N'-(3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5β (-cholan-23-yl)-benzenesulfonylurea was obtained as a white residue (235 mg, 0.36 mmol , 75%). [ka] LRMS(ESI + ) m / z: 635.8, [M+NH4] + ,100%.

[0174] (N,N'-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-24-nor-5β-cholane-23- (yl)-benzenesulfonylurea (compound 21) [ka] 210 mg of N,N'-(3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24- Prepared according to general procedure 2 using (5β-cholan-23-yl)-benzenesulfonylurea, The title compound, N,N'-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-24-nor 5β-Cholan-23-yl)-benzenesulfonylurea was obtained as a white solid (102 mg, 0.17 mmol , 52%). [ka] LRMS(ESI + ) m / z: 593.6, [M+NH4] + ,100%.

[0175] (N-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholan-24-oyl)-benzoyl Zenesulfonamide (Compound 22) [ka] 400 mg of 3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5-aminopropyl ether obtained in step C The title compound, N-(3α,7α-dihydro- Roxyl-4,4-difluoro-6α-ethyl-5β-cholan-24-oyl)-benzenesulfonamide was obtained as a white solid (161 mg, 0.27 mmol, 31%). [ka] LRMS(ESI + ) m / z: 613.6, [M+NH4] + ,100%.

[0176] Example 5: 6α-Ethyl-2α / β,4β-diphenyl ether with sulfonylurea and sulfonamide side chains Synthesis of fluoro-(3α,7α)-dihydroxyl-5β-cholanic acid analogues (A. Methyl 6α-ethyl-2α / β,4β-difluoro-7α-hydroxyl-3-oxo-5β-cholane -24-Oart) [ka] A stirring, pre-cooled solution of 1 M LDA in THF / hexanes (1.63 mL, 1.625 mmol) and TMS-Cl (0.21 mL, 1.626 mmol) in dry THF (6 mL) at -78 °C. 1-7α-hydroxyl-3-oxo-5β-cholan-24-oate (product of Example 1C; 170 mg, 0.3 A solution of 25 mmol) in dry THF (2.5 mL) was added dropwise over 10 min. After the addition, the reaction was slowly cooled to room temperature. The mixture was gradually warmed to room temperature and stirred for 20 hours. After the reaction was complete, the reaction mixture was cooled to 0°C and saturated The reaction mixture was quenched by dropwise addition of NaHCO3 solution (5 mL) and diluted with H2O (5 mL). The organic phase was removed. The aqueous phase was back-extracted with EtOAc (3×5 mL). The combined organics were washed with NaHCO3 (5 mL), H2O (5 mL), and brine (5 mL). The combined organic phase was dried over MgSO4, filtered and concentrated under vacuum. The resulting syrup was used in the next reaction without further purification. Used. [ka]

[0177] Methyl-3-trimethylsilyl-6α-ethyl-4β-fluoro-7α-hydroxyl-5 To a solution of β-cholan-2-en-24-oate (estimated 0.19 g, 0.325 mmol) in dry MeCN (10 mL), Electrofluor® (0.17 g, 0.488 mmol) was added portionwise and the reaction was stirred at room temperature for 16 h. Upon completion of the reaction, the reaction was diluted with EtOAc (5 mL) and saturated NaHCO3 (3 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3×5 mL). The organic phases were combined and dried over MgSO4. It was then filtered and concentrated under reduced pressure to give a yellow oil. The product of Step A was subjected to the procedures described above in Examples 2D and 2E to obtain methyl 6α-enolate. To obtain ethyl-2α / β,4β-difluoro-(3α,7α)-dihydroxyl-5β-cholan-24-oate . This compound may be prepared in a similar manner using general procedures 1-4 as described in Examples 1-3 above. Can be converted to the sulfonylurea or sulfonamide analogs.

[0178] (Synthesis of 2β-fluoro compounds) The 2α-fluoro and 4α-fluoro derivatives of obeticholic acid were prepared as follows.

[0179] Example 6 2β-Fluoro-3α,7α-dihydropyridine with sulfonylurea and sulfonamide side chains Synthesis of hydroxyl-6α-ethyl-5β-cholanic acid analogues (A. Methyl-3,7-dioxo-6α-ethyl-5β-cholan-24-oate) [ka] (6α,5β,7α)-6-ethyl-7-hydroxybenzoate prepared as described in International Publication WO2016 / 079520 -3,7-dioxo-cholan-24-oic acid (36.0 g, 87.7 mmol, 1.0 equiv.) in methanol (800 mL) Then, para-toluenesulfonic acid (1.67 g, 8.78 mmol, ~0.1 equiv.) was added at room temperature and the mixture was stirred at 30° C. for 4 hours. The reaction mixture was concentrated under reduced pressure in vacuo after TLC showed the reaction was complete. The residue was dissolved in chloroform (400 mL) and washed with saturated NaHCO3 solution (400 mL) and brine (400 mL). 1 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give 37.6 g of the crude material as a white solid. Obtained as a solid (87.3 mmol, 99%) and used without further purification. [ka] LRMS(ESI + ) m / z: 448.3 [M+NH4] + ,100%.

[0180] (B. Methyl-3β-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl -3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate [ka] Step A Methyl-3,7-dioxo-6α-ethyl-5β-cholan-24-oate (10.0 g, 23.2 (mmol, 1.0 equiv.) in tetrahydrofuran (340 mL) at -78 °C under argon atmosphere. L-Selectride (35.0 mL, 34.8 mmol, ∼2.5 equiv) was added dropwise over 15 min. After 10 min, To the reaction mixture was added a solution of hydrogen peroxide (40 mL, 30% v / v) and 2M sodium hydroxide (40 mL). A solution of water (400 mL) was added at 0° C. After an additional 10 min, the reaction mixture was added with 2 M hydrochloric acid (130 mL) at room temperature. The aqueous phase was separated and extracted with ethyl acetate (2×250 mL) and the combined organic fractions were washed with water (50 0 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give 11.0 g of crude material. The product was obtained as a yellow oil by flash column chromatography (Biotage SNAP KP-Sil 100 g column). The product was purified by column chromatography (HPLC) using PE 40-60 / acetone (90 / 10-80 / 20) as the eluent. The compounds methyl-3β-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl Inseparable mixture of 3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oates (65:35 ratio) was obtained as a white residue (7.83 g, 18.1 mmol, 78%). First, both compounds led to the same mixture of alkenes in the next step. (Compound A:) [ka] LRMS(ESI + ) m / z: 450.3 [M+NH4] + ,100%. (Compound B:) [ka] LRMS(ESI + ) m / z: 450.4 [M+NH4] + ,100%.

[0181] (C. Methyl-6α-ethyl-7-oxo-5β-cholesterol-2-en-24-oate and Methyl-6α-ethyl- 7-Oxo-5β-chol-3-en-24-oate [ka] Step B: Methyl-3β-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate (6.31 g, 1 To a solution of 4.6 mmol, 1.0 equiv) in dichloromethane (120 mL), dimethylaminopyridine (3.56 g , 29.2 mmol, ∼2.0 equiv.) was added at room temperature. The reaction mixture was cooled to 0°C and trifluoroacetate was added. Fluoromethanesulfonic anhydride (2.57 mL, 15.3 mmol, ~1.05 equiv) was added dropwise over 5 min. After warming to 12° C. for 2 hours, the reaction was complete by TLC and the reaction mixture was diluted with 2M hydrochloric acid. (100 mL). The aqueous phase was separated and extracted with dichloromethane (3×100 mL) and The combined organic fraction was washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. This gave 7.56 g of crude material as an orange oil. NAP KP-Sil 100 g cartridge) with PE 40-60 / acetone (90 / 10) as eluent The mixture was purified to obtain methyl-6α-ethyl-7-oxo-5β-cholesterol-2-en-24-oate and methyl-6α- An inseparable mixture of ethyl 7-oxo-5β-chol-3-en-24-oate (80:20 ratio) was dissolved in water to give a colorless oil. (2.70 g, 6.51 mmol, 45%) [ka] LRMS(ESI + ) m / z: 432.20 [M+NH4] + ,100%.

[0182] (D. Methyl-2β,3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl 3β,4β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate [ka] Step C: Methyl-6α-ethyl-7-oxo-5β-cholesterol-2-en-24-oate and Methyl-6α -Ethyl-7-oxo-5β-chol-3-en-24-oate (80:20 ratio) (5.00 g, 12.1 mmol, ~1.0 equiv.) m-perchlorobenzoic acid (3.12 g, 18.1 mm After 3 hours at room temperature, the reaction was confirmed to be complete by TLC, and the reaction mixture was The mixture was quenched with saturated Na2S2O3 solution (150 mL). After stirring for 10 min, the aqueous phase was separated and Extract with methane (3 x 100 mL) and dry the combined organic fractions over Na2SO4, filter, and evacuate under vacuum. Concentration under reduced pressure gave 5.28 g of crude material as a pale yellow residue. Flash column chromatography The solution was diluted with PE 40-60 / acetone (95 / 5- 90 / 10) as the eluent, and purified to obtain methyl-2β,3β-epoxy-6α-ethyl-7-oxo- 5β-Cholan-24-oate and methyl-3β,4β-epoxy-6α-ethyl-7-oxo-5β-cholane An inseparable mixture of 1,2-dihydro-2,4-trimethylamino-1, ... By flash column chromatography (Biotage SNAP KP-Sil 340 g cartridges) The compound was purified using PE 40-60 / acetone (95 / 5-90 / 10) as the eluent to obtain methyl-2β, 3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate as a colorless oil (3.71 g, 8. 62 mmol, 72%), and the compound methyl-3β,4β-epoxy-6α-ethyl-7-oxo-5β-cholane- The 24-oate was obtained as a colorless oil (1.18 g, 2.74 mmol, 23%). (Compound A:) [ka] LRMS(ESI + ) m / z: 448.30 [M+NH4] + ,100%. (Compound B:) [ka] LRMS(ESI + ) m / z: 448.26 [M+NH4] + ,100%.

[0183] (E. Methyl-2α-fluoro-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-ol (Rate) [ka] Step D: Methyl-2β,3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate ( A dry solution of 1.0 eq. (3.33 g, 7.73 mmol) in dichloromethane (100 mL) was added at 0°C under argon. Add HF.pyridine (70%) complex (100 mL, 3.86 mol, ~500 equiv.) to an open 100 mL bottle under atmospheric pressure. Immediately add the solution to the cooled reaction flask through a glass funnel under a steady stream of argon. After the addition of the reagents, the vessel and funnel were rinsed with dichloromethane (20 mL). After 3 h at 0 °C, The reaction mixture was then diluted with dichloromethane (200 mL) and saturated NaHCO3 solution (500 mL) was slowly added. The mixture was quenched with 5.0 g of NaHCO3 in portions of 100 mg each while stirring at room temperature for 1 h. The aqueous phase was then separated and extracted with dichloromethane (3 × 250 mL) and the combined organic fractions were washed with Na2 Drying over SO4, filtration, and concentration under vacuum reduced pressure gave 3.61 g of the crude material as a colorless oil. P was isolated by rush column chromatography (Biotage SNAP KP-Sil 100 g cartridge). The compound methyl-2α-furan was purified using E 40–60 / acetone (95 / 5–90 / 150) as the eluent. Fluoro-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate was obtained as a white residue. (2.39 g, 5.30 mmol, 69%) [ka] LRMS(ESI + ) m / z: 468.28 [M+NH4] + ,100%.

[0184] (F. Methyl-2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate) [ka] Step E Methyl-2α-fluoro-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholane To a solution of 1.00 g, 2.26 mmol, 1.0 equiv. of 24-aminopropyl ester in dichloromethane (20 mL), In periodinane (1.92 g, 4.52 mmol, ∼2.0 equiv) and H2O (0.25 mL) were added at room temperature. After 3 h at room temperature, the reaction was complete as determined by TLC, and the reaction mixture was diluted with saturated NaHCO3 solution (25 mL ), filtered through Celite and washed with dichloromethane (90 mL). The organic phases were separated and extracted with dichloromethane (2 × 50 mL) and the combined organic phases were washed with saturated Na2S2O3 solution (150 1 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give 1.21 g of the crude material as a pale yellow solid. The product was obtained as a yellow oil by flash column chromatography (Biotage SNAP KP-Sil 25 g column). The product was purified by a column chromatography (LCC) using PE 40-60 / acetone (95 / 5-90 / 10) as the eluent. The compound methyl-2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate was dissolved in water to give a white residue. Obtained as a distillate (568 mg, 1.27 mmol, 56%). [ka] LRMS(ESI + ) m / z: 466.55 [M+NH4] + ,100%.

[0185] (G. 2α-Fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid and 2β-Fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid xo-6α-ethyl-5β-cholanic acid) [ka] Methyl-2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate (Step F) To a solution of the product (878 mg, 1.95 mmol, 1.0 equiv.) in methanol (20 mL) was added sodium hydroxide ( 0.0 g) was added at room temperature. After 19 hours at room temperature, the reaction was complete as determined by TLC, and the reaction mixture was The mixture was acidified to pH 4.0 and concentrated under reduced pressure in vacuo. The residue was dissolved in ethyl acetate (50 mL) and diluted with 1M HCl (50 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give 968 mg of crude hexane. The raw material was obtained as a colorless oil. Flash column chromatography (Biotage SNAP KP-Sil 2 5 g cartridge) and dichloromethane / methanol (98 / 2 to 90 / 10) as the eluent. and purified to obtain 2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid and 2β-fluoro-3, An inseparable mixture of 7-dioxo-6α-ethyl-5β-cholanic acid (40:60 ratio) was obtained as a white residue. (772 mg, 1.77 mmol, 91%). (2α-Fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid:) [ka] LRMS(ESI + ) m / z: 452.51 [M+NH4] + ,100%. (2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid:) [ka] LRMS(ESI + ) m / z: 452.51 [M+NH4] + ,100%.

[0186] (H. Methyl-2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate) [ka] Step G: 2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid and 2β-fluoro-3 ,7-Dioxo-6α-ethyl-5β-cholanic acid (40:60 ratio) (750 mg, 1.72 mmol, ~1.0 equiv.) Cesium carbonate (840 mg, 2.58 mmol, ~1.5 equiv.) was added to a solution of ethylformamide (17 mL) at room temperature. After 20 min at room temperature, iodomethane (0.54 mL, 8.59 mmol, ~5.0 equiv) was added dropwise. After 19 hours at room temperature, the reaction was complete as determined by TLC, and the reaction mixture was concentrated under vacuum. Concentrate. The residue is dissolved in ethyl acetate (25 mL) and H2O (20 mL). The aqueous phase is separated and diluted with acetic acid. The mixture was extracted with ethyl acetate (3×25 mL) and the combined organic phases were washed with brine (100 mL) and dried over Na2SO4. The crude material was extracted with ethyl acetate, filtered and concentrated under vacuum to give 917 mg of a pale yellow oil. PE 40 was purified by column chromatography (Biotage SNAP Ultra KP-Sil 25 g cartridge). The compound methyl-2β-fluorouracil was purified using 50 / 60 / acetone (95 / 5–90 / 10) as the eluent. The 3,7-dioxo-6α-ethyl-5β-cholan-24-oate was obtained as a white residue (416 mg , 0.95 mmol, 54%). The corresponding 2α-fluoro derivative was not isolated. [ka] LRMS(ESI + )m / z:466.59 [M+NH4] + ,100%.

[0187] (I. Methyl-2β-fluoro-3β,7α-dihydroxyl-6α-ethyl-5β-cholan-24-ol and methyl-2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-ol to) [ka] Step H: Methyl-2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate ( 390 mg, 0.87 mmol, 1.0 equiv) in dry methanol (20 mL) at room temperature under argon. At rt, sodium borohydride (164 mg, 4.44 mmol, 5.0 equiv.) was added. After 1 h at rt The reaction mixture was concentrated under reduced pressure in vacuo. The residue was diluted with dichloromethane (20 mL) and H2O (20 mL). The aqueous phase was separated and extracted with dichloromethane (3×20 mL). Dry over Na2SO4, filter, and concentrate under vacuum to give 446 mg of crude material as a pale yellow oil. Flash column chromatography (Biotage SNAP Ultra KP-Sil 25 g cartridge) The compound was purified using PE 40-60 / acetone (95 / 5-90 / 10) as the eluent to obtain methyl 2β-fluoro-3β,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate was dissolved in a colorless oil. (161 mg, 0.36 mmol, 41%) as the compound methyl-2β-fluoro-3α,7α-dihydroxyl -6α-Ethyl-5β-cholan-24-oate was obtained as a colorless oil (146 mg, 0.32 mmol, 37%). (Methyl-2β-fluoro-3β,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate: ) [ka] LRMS(ESI + ) m / z: 470.64 [M+NH4] + ,100%. (Methyl-2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate: ) [ka] LRMS(ESI + ) m / z: 470.64 [M+NH4] + ,100%.

[0188] (J. 2β-Fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholanic acid) [ka] Methyl-2β-fluoroacetamide from step I in a solution of 1,4-dioxane (9.8 mL) and water (3.6 mL) -3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate (119 mg, 0.26 mmol, 1.0 To a solution of 1.2 mL of concentrated (37%) hydrochloric acid (1.2 mL, 9:3:1 ratio) was added at room temperature. After refluxing for 1 h, the reaction The reaction was complete as determined by TLC, and the reaction mixture was neutralized with saturated NaHCO3 solution (20 mL). The extract was extracted with ethyl acetate (3×15 mL) and the combined organic fractions were dried over Na2SO4, filtered and concentrated in vacuo Concentration under reduced pressure gave 141 mg of the crude material as a colorless oil. Dichloromethane / methanol was measured by Biotage SNAP Ultra KP-Sil 10 g cartridge. The compound 2β-fluoro-3α,7α-dihydrazine was purified using methanol (95 / 5-90 / 10) as the eluent. The resulting solution was treated with 5-methyl-6α-ethyl-5β-cholanic acid as a white residue (92 mg, 0.21 mmol, 80%). [ka] LRMS(ESI + ) m / z: 456.60 [M+NH4] + ,100%.

[0189] This compound was synthesized in a similar manner using general procedures 1-4 as described in Examples 1-3 above. It can be converted to the sulfonylurea or sulfonamide analogues.

[0190] Biological Examples For Biological Examples 8 and 9 below, Professor Kim Watson and Dannie All work was done and data kindly provided by Ille Kydd-Sinclair.

[0191] Example 7 EC in FXR receptor 50 and effectiveness measurement) The compounds of the present invention were analyzed for agonist activity at the FXR receptor. EC of the clear example compound 50 The values ​​and effective values ​​are compared with those of the comparative example compound, obeticholic acid, and and the known FXR agonist GW4064, which has the following structure: [ka] The values ​​are shown in comparison with those of . Efficacy was defined as the maximum point on the dose-response curve, and the efficacy value of GW4064 in Table 1 was set as 100%. did. EC in Table 1 50 The EC value for GW4064 was taken as 25 nM. 50 Normalized against .

[0192] Obeticholic acid is disclosed in International Publication WO02 / 072598 or the inventors' application International Publication WO2016 / 079518 , International Publication WO2016 / 079518, International Publication WO2016 / 079519 and International Publication WO2016 / 079520 It can be prepared as follows.

[0193] (FXR's EC 50 / Efficacy Protocol) Dose-response assays were performed using the human farnesoid X receptor (NR1H4, FXR) reporter assay system. Technical manual for the Indigo Biosciences Human Farnesoid X Receptor (NR1H4, FXR) As described in the Reporter Assay System, Technical Manual (version 7.1b, www.indigobiosciences.com) The experiment was carried out as described.

[0194] It consists of an FXR-responsive promoter gene functionally linked to a luciferase gene. FXR reporter cells were thawed and seeded in 96-well plates, and the cells were immediately transfected with the recombinant FXR vector. Test compounds were administered at different concentrations (10-0.05 μM) according to the manufacturer's protocol. After 24 h of incubation in the presence of either the inhibitor or vehicle (DMSO), fluorescence-based live cell multiplexing was performed. Live Cell Multiplex Assay (LCM) (Indigo Biosciences Live Cell Multiplex Assay, Technical Manual (3 We used the Indigobiosciences ELISA Kit (Version 1.1, available at www.indigobiosciences.com) to identify cells from these treated and untreated reporter cells. Cell viability was measured to eliminate false negative results. Fluorescence from live cells was measured using the following formula: [485nmEx|535nmEm] The measurements were made using a plate reader equipped with a combination filter. Induction of luciferase activity, a measure of transactivation activity, was measured using a luminometer according to the manufacturer's protocol. Quantitative analysis was performed using a TECAN meter.

[0195] The positive control was EC 50 The efficacy of GW4064 was set at 100%, and the efficacy of each arm was set at 25 nM. The efficacy and EC 50 was compared with GW4064. The results are shown in Table 1.

[0196] (Table 1) [Table 2] TIFF2024088648000170.tif238170TIFF2024088648000171.tif121170

[0197] All compounds of the present invention have FXR agonist activity. All compounds except compound 11 showed significantly improved EC compared with obeticholic acid. 50 Furthermore, all of the present invention The efficacy values ​​of the compounds are at least as good as those of GW4064, and in most cases better. is.

[0198] Comparison of the results for compound 2 with those for its non-fluorinated analogue, compound A, indicates that fluorination is 50 The results of aromatic sulfonamide compounds 14-20 show that both the efficacy and safety of the compound are improved. , compared with the results of carbocyclic sulfonamide comparative compound B and methylsulfonamide comparative compound C The comparison shows the importance of aromatic substituents on the side chain.

[0199] Example 8 EC in TGR5 receptor 50 and effectiveness measurement) Compounds 2 and 14, TGR5 (control), and the taurine and glycine conjugates of obeticholic acid Agonist models were identified using the HitHunter® cAMP assay available from DiscoverX. In the 1990s, activation of the TGR5 (GPBAR1) receptor was mediated through Gi and Gs second messenger signaling. Activation was monitored to test activity at the TGR5 receptor. Data are compared with the ligand (TGR5) control. Normalized to the maximum and minimum responses observed in the presence and in the presence of vehicle control .

[0200] (Assay Design: GPCR cAMP Regulation) (Cell manipulation) 1. The cAMP Hunter cell line was grown from frozen stocks according to standard procedures. 2. Cells were seeded in a total volume of 20 μL into white-walled 384-well microplates and incubated at 37°C until appropriate incubation. The mixture was incubated for the appropriate time. 3. cAMP modulation was measured using the DiscoverX HitHunter® cAMP XS+ Assay. It was decided.

[0201] (Gs agonist format) 1. For agonist determination, cells were incubated with the sample to induce a response. 2. Media was aspirated from cells and replaced with 15 μL of 2:1 HBSS / 10 mM Hepes:cAMP XS+Ab Reagent. . 3. Intermediate dilutions of sample stocks were made to create 4x samples in assay buffer. Add 4.5 μL of 4× sample to the cells and incubate at 37° C. or room temperature for 30 or 60 minutes. The vehicle concentration was 1%.

[0202] (Signal Detection) 1. After appropriate compound incubation, lyse the cells in 20 μL of cAMP XS+ED / CL lysis cocktail at room temperature. Incubate for 2 hours, followed by 20 μL of cAMP XS+EA reagent for 3 hours. Thus, an assay signal was generated. 2. Following signal generation, the chemiluminescent signal was detected using a PerkinElmer Envision™ instrument. Then, the microplate was subjected to measurement.

[0203] (Data Analysis) 1. Compound activity was analyzed using the CBIS data analysis suite (ChemInnovation, CA). 2. Percent activity is calculated according to the following formula for Gs agonist mode assay:

number

[0204] 5. Percent activity is calculated according to the following formula for Gi agonist mode assay:

number

[0205] (result) The results are shown in Table 2. (Table 2) [Table 3]

[0206] The results showed that, unlike the obetichol acid conjugate, neither compound 2 nor compound 14 inhibited the TGR5 receptor. These compounds therefore have no agonist activity at the receptor. It is a potent FXR agonist.

[0207] Example 9 Quantitative Analysis of Ligand-Induced Gene Expression Compound 2 and obeticholic acid were investigated for their effects on the expression of many FXR target genes. In this example, the effect of the compound of the present invention and obeticholic acid on the cellular level was examined. Cell-based assays and gene expression analysis to observe functional activation of FXR The following was stated.

[0208] Quantitative real-time analysis was performed to assess specific changes in gene expression in response to test compounds. Precise quantification and analysis were performed using real-time PCR (qPCR). Hepatoma cells (Huh7) were placed in a 6-well plate at 1 × 106 Cells were seeded at 1000 cells / well and incubated at 37°C for 24 hours. The cells were incubated and allowed to attach. 50 Or EC 90 Either of the Exposure to test compound or vehicle (DMSO) was for 24 hours.

[0209] Compound 2 and OCA were also tested in the human hepatocellular carcinoma cell line, HepG2. E-commerce 50 or EC 90 The cells were incubated for 24 hours in a medium containing either OCA or compound 2 at a concentration of I did. Tests in Huh7 and HepG2 cells were also performed with compound 14 in the same manner as compound 2.

[0210] (Isolation of total RNA from cultured cells) Total RNA was extracted using the RNAqueous™ Total RNA Isolation kit (Ambion); and All reagents were provided in the kit or prepared according to the manufacturer's instructions. For cultured cells, the medium was removed and the cells were washed with 1x PBS to remove cell debris and residual medium. Total RNA was extracted from fresh cells. 6 For cells, 350 μl of cell lysis buffer Buffer was added directly to the wells and the cells were scraped off with a pipette tip. The solution was mixed with 64% ethanol and thoroughly mixed by pipetting. The mixture was transferred to the column and spun at 12,000 x G for 1 minute, and the flow-through was discarded. The membrane was centrifuged at 12,000 x G for 1 minute after adding Wash Buffer 1 to the column. Then, discard the flow-through and repeat this step twice with Wash Buffer 2. The empty cartridge was spun an additional time to completely dry the ethanol membrane. Finally, the total RNA was eluted in 50 μl of pre-warmed elution buffer (containing a trace of EDTA, nuclease-free). The solution was eluted as two successive aliquots of 0.01% glycerol-free water.

[0211] (Analysis of RNA quantity, purity and integrity) RNA concentration was determined by measuring absorbance at 260 nm using a Nanodrop Lite spectrophotometer (Thermo Scientific). The RNA purity was measured and quantified. 260:280 By analyzing the ratio, A value of 1.8–2.1 indicates that the product is free of protein contamination and can be used in downstream applications. RNA integrity was assessed by running samples on a denaturing formaldehyde agarose gel. To visualize nucleic acids, 1% agarose (Sigma Aldrich), 1× MOPS (S Sigma Aldrich), 6.6% formaldehyde (Fisher Scientific) gels were prepared using 1× SYBR™ Safe DNA stain (Invitrogen) was added. Add 1 μg RNA dye (Ambion) to 1 μg RNA, heat the sample to 70°C for 10 min, and then immediately place on ice for 2–3 min. The gel was run at 90V for 1 hour and 30 minutes using the NuGenius gel doc system (Syngene). The 28S and 18S rRNA bands were visualized by UV light at approximately 5 kb and 1.9 kb, respectively. The upper 28S band corresponds to the lower 18S band of intact RNA. The intensity of the 18S rRNA band appeared to be twice that of the 18S rRNA band. NA, whereas smearing and / or bands above 28S rRNA indicate DNA contamination. Immediately after analysis, the RNA was used for reverse transcription studies.

[0212] (Reverse Transcription) Reverse transcription was performed using the iScript™ Advanced cDNA Synthesis Kit for RT-qPCR (Biorad). 1 μg of DNase-treated RNA was treated with 4 μl of 5× iScript™ Advanced reaction mix and 1 μl of iScript™ Advanced Reverse Transcriptase was added. Nuclease-free water was added to the final volume The reaction was incubated at 46°C for 20 min, then at 95°C for 1 min. The newly synthesized cDNA was diluted 10-fold in TE buffer (10 mM Tris pH 8, 1 mM EDTA). The mixture was diluted, aliquoted, and stored at -20°C until use in qPCR experiments.

[0213] (Qualitative real-time PCR analysis) Reference genes were selected based on data from existing literature. The selected target genes are listed in Table 3. As shown in. Table 3 - Selected target genes for qualitative real-time PCR analysis [Table 4]

[0214] (Optimization of primers for qPCR) KicQStart® SYBR® Green Predesigned Primers for the above target genes - (selected according to highest rating) were purchased from Sigma Life Science. Free water was added to the lyophilized primers for a 100 μM stock concentration. The assay was diluted to a working concentration of 10 μM in enzyme-free water. To test the overall mix range, 10-fold serial dilutions were prepared using Human Reference RNA ( The kit consisted of five concentrations of cDNA (outlined above) generated from a 100% Agilent Following qPCR of these samples, the standard curve was plotted at the threshold cycle (C t ) value (y-axis) versus log cDNA concentration (x-axis) The primer amplification efficiency (E) for one cycle in the exponential phase is calculated by the equation E = 10 ( -1 / 勾配) The precision of these qPCR reactions was determined using a standard curve. R 2 The specificity of each primer is determined by the PCR amplification rate, with values ​​above 0.98 being appropriate. The melting temperature was determined by melting curve analysis at the end of the experiment, which produced a single peak at one melting temperature. When the primers were used, they showed amplification of only one product, indicating that the primers were highly specific. The amplified products were analyzed by agarose gel electrophoresis (2% agarose, 1×TAE, Electrophoresis at 100 V for 30 min was performed to confirm that the size of the amplified products was as expected and that only one product was identified. It was observed that it was approved.

[0215] (Quantitative PCR) iTaq™ Universal SYBR® Green Suspension Master Mix Permix (Biorad) was used for all qPCR reactions. Typical reactions for each gene were 5 μl 2x iTaq™ Universal SYBR® Green Supermix, 500 nm forward primer and 500 nm reverse primer, approximately 15 ng of cDNA, and nuclease-free water. The final volume was 10 μl. Illuminations were performed in triplicate in Optical MicroAmp 96-well plates (Applied Biosystems). The tray was sealed with an optical adhesive seal (Applied biosystems) and briefly placed on a plate shaker. The components were mixed and centrifuged. The reactions were monitored using the Applied Biosystem Step One Plus real-time PCR system. Use a 30-μl PCR system and follow the cycling conditions: initial denaturation step at 95 °C for 15 min; 40 cycles of denaturation at 94°C for 15 s, annealing and extension were combined. Amplification consisted of a 1 min step at 60°C and was performed with a single fluorescence measurement. Solution curve analysis was performed immediately after each run by increasing the temperature from 60°C to 95°C in 0.3°C increments and analyzing the solution by fluorescence. This was carried out by measuring the separation.

[0216] (Data Analysis) To quantify gene expression, baseline-corrected C t The values ​​were calculated using the qPCR system software. Relative changes in gene expression were determined automatically by the Live k or ΔΔC t The method was used to determine the effect of the vehicle / untreated sample on the The C of the target gene was determined for both the control (control) and compound-treated samples (test samples). t Value reference C of the legend t The ΔC value of the test sample was then normalized to t The value is the ΔC t Normalized to value Finally, the expression ratio was calculated using Eq.-ΔΔC t In total, three There were biological replicates and all data are expressed as mean ± SE. Treatments were analyzed by one-way ANOVA. followed by Tukey and Dunnett's post-hoc tests.

[0217] (result) Direct target genes nr0b2 (SHP) and slc51a (OSTα) are involved in bile acid homeostasis , which are known to be positively regulated by FXR (Goodwin et al., 2000; Landrier et al., (2006) and shows a significant increase upon treatment of Huh7 with Compound 2. The expression level of SHP was significantly increased by Compound 2. After treatment with 50 concentration) to 2.5 times (EC 90 In addition, OstαmR NA was also significantly upregulated by treatment with compound 2, and its EC 50 and E.C. 90 In concentration The expression of α-terminal β-lactamase was increased 11-fold and 18-fold, respectively (Figure 2).

[0218] FXR activation suppresses CYP7A1 through both the SHP- and FGF19-mediated pathways. CYP7A1 is downregulated with increasing concentrations of compound 2 (Figure 3). HepG2 cells are characterized by their EC 50 The effect of compound 2 on TGFβ1 expression levels in response to treatment at The results show a significant downregulation of IFN-γ expression (Figure 4). SHP is an EC 90 A moderate increase of more than two-fold in its expression level upon treatment with compound 14 at a concentration Compound 14 induced a highly significant increase in the FXR target Ostα, which Cells treated with IFN-γ achieved 20-40 fold higher mRNA expression levels than vehicle-treated cells. This was achieved (Figure 6).

[0219] EC 50 At the concentrations, compound 14 significantly reduced CYP7A1 expression, less than that seen in vehicle control cells. reduced it to just over half of its level (Figure 7). Similar to compound 2, compound 14 induced a decrease in TGFβ1 expression with increasing compound concentration. (Figure 8).

[0220] The inventors have provided numerous background sections as well as methodology and data for the biological examples. We would like to thank Dannielle Kydd-Sinclair of the University of Reading for providing us with the data, and several David Evans and Simon Holland of the University of Southampton, who synthesized the example compounds, and Lawrence Tam.

[0221] (References) [ka] TIFF2024088648000177.tif248170TIFF2024088648000178.tif247170TIFF2024088648000179.tif163170

Claims

[Claim 1] The novel products, methods and processes substantially as herein described.

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