Fluorinated bile acid derivatives

文档序号：620842 发布日期：2021-05-07 浏览：26次中文

阅读说明：本技术 氟化胆汁酸衍生物 (Fluorinated bile acid derivatives ) 是由 A·C·韦茅斯-威尔逊 G·帕克 B·J·P·林克劳 D·基德-辛克莱 K·A·沃森于 2019-07-30 设计创作，主要内容包括：通式(I)的化合物：其中R～(2a)、R～(2b)、R～(3a)、R～(3b)、R～5、Y和R～7如本文所定义,该化合物为FXR受体的选择性激动剂,并且用于治疗或预防疾病和病症,包括非酒精性脂肪性肝炎(NASH)；原发性胆汁性肝硬化；原发性硬化性胆管炎；胆道闭锁；胆汁郁积性肝病；丙型肝炎感染；酒精性肝病；纤维化；或因纤维化导致的肝损伤。(A compound of the general formula (I): wherein R is 2a 、R 2b 、R 3a 、R 3b 、R 5 Y and R 7 As herein describedAs defined, which are selective agonists of the FXR receptor and are useful for the treatment or prevention of diseases and disorders, including nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; closing the biliary tract; cholestatic liver disease; hepatitis c infection; alcoholic liver disease; fiberizing; or liver damage due to fibrosis.)

1. A compound of the general formula (I):

wherein

R^2a、R^2b、R^3aAnd R^3bEach independently is H or F, with the proviso that R^2bAnd R^3bAt least one of (a) is F;

R⁵is CR^6aR^6bR⁸、OR⁸、SR⁸Or NR^6aR⁸；

R^6a、R^6bAnd R⁸Each independently is H or methyl;

y is a bond or C_1-4Alkylene or C_2-4Alkenylene linking groups, one of which is optionally substituted with one or more R¹⁰Substitution;

wherein R is¹⁰Each independently is halogen or OH;

R⁷selected from C (O) NR¹⁷S(O)₂R¹⁵、NR¹⁷C(O)NR¹⁸S(O)₂R¹⁵、NR¹⁷C(S)NR¹⁸S(O)₂R¹⁵And NR¹⁷C(NH²⁰)NR¹⁸S(O)₂R¹⁵；

R¹⁵Is a 5 to 10 membered aryl or heteroaryl ring, optionally substituted with one or more substituents selected from C_1-6Alkyl radical, C_1-6Haloalkyl, halogen, O (C)_1-6Alkyl) and O (C)_1-6Haloalkyl);

R¹⁷and R¹⁸Each independently is H or methyl;

R²⁰is H, methyl or CN;

or a salt or isotopic variation thereof.

2. A compound according to claim 1, wherein R^3bIs F, and R^3a、R^2aAnd R^2bAre each H.

3. A compound according to claim 1, wherein R^2bIn the case of F, the content of the compound,and R is^2a、R^3aAnd R^3bAre each H.

4. A compound according to claim 1, wherein:

R^3bis F, R^3aIs H, R^2aAnd R^2bOne is F, and R^2aAnd R^2bIs H; or R^3aAnd R^3bAre both F, and R^2aAnd R^2bAre all H.

5. A compound according to any one of claims 1-4, wherein R⁵Is ethyl.

6. A compound according to any one of claims 1 to 5, wherein Y is a bond or C_1-3An alkylene linker optionally substituted with one or more OH groups.

7. A compound according to any one of claims 1 to 6, wherein R⁷Is C (O) NR¹⁷S(O)₂R¹⁵Or NR¹⁷C(O)NR¹⁸S(O)₂R¹⁵Wherein R is¹⁵、R¹⁷And R¹⁸As defined in claim 1.

8. The compound according to claim 7, wherein independently or in any combination:

R¹⁷and R¹⁸Each (if present) of (a) is H; and/or

R¹⁵Selected from phenyl and 5-or 6-membered heteroaryl, which may optionally be unsubstituted or substituted by one or more substituents as defined in claim 1.

9. A compound according to claim 8, wherein R¹⁵Is phenyl, unsubstituted or substituted by a mono-substituent selected from fluoro, C_1-4Alkyl radical, C_1-4Fluoroalkyl group, O (C)_1-4Alkyl) and O (C)_1-4Fluoroalkyl groups).

10. A compound according to claim 1, selected from:

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-toluenesulfonylurea (compound 1);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -benzenesulfonylurea (compound 2);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (tert-butyl) benzenesulfonylurea (compound 3);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -m-toluenesulfonylurea (compound 4);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -o-toluenesulfonylurea (compound 5);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-fluorobenzenesulfonylurea (compound 6);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -m-fluorobenzenesulfonylurea (compound 7);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -o-fluorobenzenesulfonylurea (compound 8);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p- (trifluoromethyl) benzenesulfonylurea (compound 9);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5B-cholan-23-yl) -m- (trifluoromethyl) benzenesulfonylurea (compound 10);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -o- (trifluoromethyl) benzenesulfonylurea (compound 11);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (trifluoromethoxy) benzenesulfonylurea (compound 12);

n, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-methoxybenzenesulfonylurea (compound 13);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -p-trifluoromethoxybenzenesulfonamide (compound 14);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -p-fluorobenzenesulfonamide (compound 15);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -m-fluorophenyl sulfonamide (compound 16);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -o-fluorophenyl sulfonamide (compound 17);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -4-trifluoromethylphenylsulfonamide (compound 18);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -3-trifluoromethylphenylsulfonamide (compound 19);

n- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -2-trifluoromethylphenylsulfonamide (compound 20);

n, N' - (3 α, 7 α -dihydroxy-4, 4-difluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -benzenesulfonylurea (Compound 21)

N- (3 α, 7 α -dihydroxy-4, 4-difluoro-6 α -ethyl-5 β -cholan-24-yl) -benzenesulfonamide (compound 22);

and salts and isotopic variations thereof.

11. A compound according to any one of claims 1 to 10 for use in medicine.

12. A compound according to any one of claims 1 to 10 for use in the treatment or prevention of nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; closing the biliary tract; cholestatic liver disease; hepatitis c infection; alcoholic liver disease; fiberizing; or liver damage due to fibrosis.

13. Use of a compound according to any one of claims 1-10 for the manufacture of a medicament for the treatment or prophylaxis of nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; closing the biliary tract; cholestatic liver disease; hepatitis c infection; alcoholic liver disease; fiberizing; or liver damage caused by fibrosis.

14. Treating or preventing nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; closing the biliary tract; cholestatic liver disease; hepatitis c infection; alcoholic liver disease; fiberizing; or liver damage due to fibrosis, which method comprises administering to a patient in need of such treatment an effective amount of a compound according to any one of claims 1-10.

15. The compound for use according to claim 12, the use according to claim 13 or the method according to claim 14, wherein the fibrosis is selected from the group consisting of liver, kidney and intestinal fibrosis.

16. The compound for use according to claim 15, the use or the method according to claim 15, wherein liver fibrosis is associated with NASH, alcoholic or non-alcoholic fatty liver disease, or with an infection such as hepatitis, especially hepatitis b or c or parasitic liver disease, or is caused by damage induced by congenital diseases, such as Wilson's disease, gaucher's disease, glycogen storage disorders, hemochromatosis, Zellweger's syndrome and congenital liver fibrosis, or by drugs, such as chlorpromazine, tolbutamide, methotrexate, isoniazid and methyldopa; and/or

Renal fibrosis associated with diseases such as diabetic nephropathy, hypertensive nephrosclerosis, glomerulonephritis, interstitial nephritis, transplantation-related glomerulopathy and polycystic kidney disease; and/or

Intestinal fibrosis associated with intestinal diseases.

17. A pharmaceutical composition comprising a compound according to any one of claims 1-10 and a pharmaceutically acceptable excipient or carrier.

18. The pharmaceutical composition according to claim 17, further comprising one or more additional active agents suitable for treating or preventing metabolic syndrome, including non-alcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; closing the biliary tract; cholestatic liver disease; hepatitis c infection; alcoholic liver disease; fiberizing; or liver damage due to fibrosis.

19. A process for the preparation of a compound according to any one of claims 1 to 10, which process comprises:

A. for the compounds of the general formula (I), wherein R⁷Is NHC (O) N (R)¹⁸)S(O)₂R¹⁵：

Reacting a compound of formula (III):

y, R therein^2a、R^2b、R^3a、R^3bAnd R⁵As defined in claim 1, and R⁴⁰Is a protected OH group;

with a sulfonamide of formula (IV) in the presence of a catalyst such as 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU):

wherein R is¹⁵And R¹⁸As defined in claim 1;

to form a compound of formula (II):

y, R therein^2a、R^2b、R^3a、R^3bAnd R⁵As defined in claim 1, and R⁴⁰Is a protected OH group; and is

Deprotecting a compound of formula (II); or

B. For the compounds of the general formula (I), wherein R⁷Is NHC (O) N (R)¹⁸)S(O)₂R¹⁵：

Reacting a compound of formula (XIII):

y, R therein^2a、R^2b、R^3a、R^3bAnd R⁵As defined in claim 1, and R⁴⁵And R⁴⁶Each independently is a protected OH group;

with a sulfonamide of formula (IV) as defined above in the presence of a catalyst such as 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) to form a compound of formula (XII):

y, R therein^2a、R^2b、R^3a、R^3bAnd R⁵As defined in claim 1, and R⁴⁵And R⁴⁶Each independently is a protected OH group; and is

Deprotecting a compound of formula (XII); or

C. For the compounds of the general formula (I), wherein R⁷Is C (O) N (R)¹⁷)S(O)₂R¹⁵：

Reacting a compound of formula (XXIII):

y, R therein^2a、R^2b、R^3a、R^3bAnd R⁵As defined in claim 1, and R⁴⁶Is a protected OH group;

with sulfonamides of the formula (XXIV)

Wherein R is¹⁵And R¹⁷As defined in claim 1;

in the presence of a coupling agent such as 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDCI) and a base such as dimethylaminopyridine:

to give a compound of formula (XXII):

y, R therein^2a、R^2b、R^3a、R^3b、R⁵、R¹⁵And R¹⁷As defined in claim 1, and

R⁴⁶as defined for the compounds of the general formula (XII); and is

Deprotecting a compound of formula (XXII).

Technical Field

The present invention relates to compounds which are bile acid derivatives and which may be used for the treatment of liver diseases. In particular, the present invention relates to compounds that are selective agonists of the Farnesoid X receptor (Farnesoid X receptor) and are therefore useful in the treatment of diseases such as non-alcoholic steatohepatitis (NASH) and primary cholangitis. The invention also relates to pharmaceutical compositions comprising the compounds of the invention.

Background

Nonalcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver disease worldwide and is rapidly becoming a major indicator of liver transplantation (Bellentani, 2017). NAFLD describes a range of physiological conditions, ranging from simple lipid accumulation in the liver (sebaceous gland disease) to non-alcoholic steatohepatitis (NASH), characterized by lobular inflammation and hepatocellular injury (Haas, frame and stals, 2016). In the west, NAFLD is considered a huge public health burden, estimated to affect 30% of the population in the uk (Dyson, anste and McPherson, 2014). Increased prevalence of NAFLD reflects increased prevalence of obesity and type 2diabetes, and NAFLD is considered a hepatic manifestation of metabolic syndrome (Cave et al, 2016).

In patients with NAFLD, at least 10-20% continue to develop NASH, which is accompanied by a patient's predisposition to hepatic and extrahepatic complications such as fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and cardiovascular disease. Although little is known, the pathological progression from steatosis to NASH is thought to consist of multiple "hits" with lipotoxicity, oxidative stress and endoplasmic reticulum stress, sensitizing the liver to other damaging systems mediated by the innate immune defense system and causing cytokine-induced cell damage (Pacana and Sanyal, 2015). One "hit" involves liver neoadipogenesis, which is activated by hyperinsulinemia and carbohydrate-rich diet. Furthermore, as a result of insulin resistance, lipolysis in dysfunctional adipocytes is not inactivated, leading to the penetration of Free Fatty Acids (FFA) into the circulatory system. These FFAs accumulate in ectopic tissues (e.g. liver) and are stored as triglycerides. However, due to triglyceride-derived toxic metabolites, the excessive accumulation of FFA exceeds the triglyceride storage threshold, eventually leading to lipotoxicity. In addition, a decrease in triglyceride clearance and a decrease in Very Low Density Lipoprotein (VLDL) output also contribute to the accumulation of fat in the liver. These events are offset by mitochondria and peroxisomes, which attempt to oxidize fatty acids, but ultimately result in damage to these organelles, resulting in overproduction of Reactive Oxygen Species (ROS) and destruction of associated molecular pattern molecules (DAMPs). In the second "hit", lipid peroxidation and FFA and ROS activate inflammatory cytokines to trigger inflammation and apoptosis and can activate the natural immune defense system through Toll-like receptors, further prolonging NASH progression by exacerbating insulin resistance and initiating fibrogenesis (cui, 2012). Furthermore, recent evidence suggests that the liver-gut axis is involved in the development of disease. In NAFLD patients, the composition of the intestinal flora is altered and intestinal permeability is increased, and inflammatory body (inflamesome) -mediated dysbiosis is also thought to contribute to the development of NASH (Henao-mejia et al, 2012; Mouzaki et al, 2013). The complex, multifactorial, trans-systemic nature of this metabolic abnormality is highlighted by the lipotoxic hepatic events in combination with the indirect effects of inflammatory mediators of the adipose tissue, gut and immune system, and importantly reflects a number of potential therapeutic targets for NASH (Haas, Francque and stals, 2016).

Currently, there is no effective medical therapy for the treatment or prevention of NASH. Most commonly, weight loss and improved insulin sensitivity through changes in diet and lifestyle are recommended, but since many patients cannot initiate or sustain these changes, long-term pharmacological solutions are needed (Neuschwander-Tetri et al, 2015). Research has focused on potential molecular targets for the treatment of NASH, including several nuclear hormone receptors of the NR1 subfamily. These receptors, in particular the Farnesoid X Receptor (FXR), are attractive targets due to their potential role in several pathways contributing to the etiology of the disease.

Like all nuclear receptors, Farnesoid X Receptors (FXRs) act as ligand-activated transcription factors that can modulate the cellular mechanisms responsible for controlling epigenetic changes. There are two genes encoding FXR: FXR α (NR1H4), which is highly conserved in many species, and FXR β (NR1H5), which exists as a pseudogene in humans. The FXR α gene encodes four different isoforms that arise from different promoter applications and alternative mRNA splicing combinations. Although all four isoforms have highly conserved ligand binding domains, suggesting that FXR ligand will bind to any isoform in a non-selective manner, there is clearly differential expression where 2 isoforms are predominantly expressed in hepatocytes and cells with active steroid metabolism, and others are predominantly expressed in the colon, intestine and other cells involved in the enterohepatic cycle (Huber et al, 2002; Vaquero et al, 2013). Furthermore, certain FXR target genes are more responsive to certain isoforms than others, and the overall pattern of isoform expression is believed to have a significant impact on the sensitivity of specific tissues to FXR ligands and transcriptional response (Zhang, Kast-woelbern and Edwards, 2003; Vaquero et al, 2013).

FXR stands for its structural organization and activationTypical nuclear receptors. Briefly, FXR consists of an N-terminal DNA Binding Domain (DBD) composed of two Zn's responsible for recognition and binding of a common hormonal response element²⁺Finger components, which are connected by a variable hinge region to the C-terminal Ligand Binding Domain (LBD), which is the hydrophobic pocket necessary for the identification and precipitation of small molecule ligands (Chiang, 2013). Similar to other nuclear receptors, FXR binds to DNA to a heterodimeric Retinoid X Receptor (RXR) with an obligatory partner. FXR is inactive in the absence of ligand. Typically, FXR/RXR heterodimers are pre-associated with AGGTCA reverse heavy response elements of their target genes in the form of complexes with co-repressor peptides (Neuschwander-Tetri, 2012). Upon activation by ligand binding, the receptor undergoes a conformational change, releasing the co-repressor complex, exposing the binding site of the LXXLL co-activation motif to the hydrophobic groove of the ligand binding pocket (Copple and Li, 2016). Hydrogen bonding between the LBD surface and the two ends of coactivators will form "charge clamps" and coactivators are recruited to the site. Eventually, this results in a change in the chromatin structure of the target gene, bringing the universal transcription factor and RNA polymerase close to its promoter and thus initiating its transcription (anathanarayanan et al, 2004).

A typical endogenous ligand for FXR is bile acid. Bile acids are steroid acids found in mammalian bile and include compounds such as cholic acid, chenodeoxycholic acid, lithocholic acid, and deoxycholic acid, all found in humans.

The general numbering system for steroids and the numbering of the carbon atoms in chenodeoxycholic acid are shown below.

FXR acts as a major regulator of bile acid metabolism. The main role of FXR is to promote nutrient and energy transfer along the entero-hepatic-fatty axis in fed and fasted states (Evans and magelsdorf, 2014). After postprandial stimulation, bile acids cause lipid absorption and activate FXR-mediated signal transduction pathways. This promotes nutrient absorption by the gut and stimulates energy metabolism in the liver by the action of FXR target gene fibroblast growth factor 19(FGF 19). In addition to intestinal FGF19, expression of FXR transcription targets (short heterodimer partners (SHPs)) in the liver also leads to down-regulation of de novo bile acid synthesis and tight control of enterohepatic bile acid pool according to metabolic needs. Studies in NASH patients indicate that decreased expression of FXR and bile acid biosynthetic enzymes, cholesterol 7 alpha hydroxylase (CYP7a1), and sterol 27 hydroxylase (CYP27a1) are all directly proportional to the severity of the disease; NAFLD patients also showed impaired response to hepatic FGF19 (Yang, Shen and Sun, 2010; Min et al, 2013; Cave et al, 2016).

Recently, FXR has become a major participant in lipid, glucose and cholesterol homeostasis, regulating genes involved in liver lipogenesis, VLDL synthesis, insulin sensitivity, and partly due to interactions with other nuclear receptors, gluconeogenesis and glycogenesis (Kast et al, 2001; Watanabe et al, 2004; Ma et al, 2006; Zhang et al, 2006). As a result of acting through the SHP signaling cascade, FXR down-regulates the sterol regulatory element binding protein (SREBP1) to reduce fatty acid synthesis, while up-regulating peroxisome proliferator-activated receptor alpha (PPAR α) to increase fatty acid catabolism by mitochondrial β -oxidation, thereby reducing liver fatty acid accumulation. FXR also increases expression levels of apolipoprotein C2(APOC2) and VLDL receptors, which are responsible for VLDL hydrolysis and clearance. Studies using FXR deficient mice show phenotypic similarities to human NASH patients, including significantly elevated hepatic triglyceride levels, elevated circulating FFA, and hepatic steatosis (Maloney et al, 2000; Zhang et al, 2004). Furthermore, FXR activation by natural and synthetic agonists has been able to improve plasma triglyceride levels in rodents (Kast et al, 2001). By reducing the levels of triglycerides and FFA, it is believed that FXR activation may also increase insulin sensitivity in the liver and surrounding tissues, as observed in FXR null mice, which exhibit mild glucose intolerance and insulin signaling oscillations in both liver and muscle (Ma et al, 2006). Consistent with this finding, semi-synthetic FXR agonist obeticholic acid (OCA) may improve insulin sensitivity in human subjects and animal obesity models of NASH (Cipriani et al, 2010; Mudaliar et al, 2013). Also, FXR has been proposed to be primarily mediated in glucose homeostasis by FGF19 signaling; lowering plasma glucose concentrations, lowering the expression and activity of three key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1, 6-bisphosphatase (FBP1) and glucose-6-phosphatase (G6Pase), and inhibiting cAMP regulatory element binding protein (CREB) and downstream PPAR γ coactivator 1- α (PGC1 α) to promote storage of glucose as glycogen play an important role (Zhang et al, 2006). However, contradictory results from animal models suggest that FXR involvement may be only part of the complex network of receptors and pathways (Watanabe et al, 2011).

Notably, FXR is involved in the inhibition of hepatitis, where complex pathways are involved that lead to the negative regulation of specific nuclear factor κ B (NF- κ B) target genes and proinflammatory cytokines (Wang et al, 2008). Furthermore, although the underlying mechanisms are still poorly understood, FXR is thought to play an important role in gut protection and maintenance of the gut barrier of the gut flora (Inagaki et al, 2006). FXR knockout mice fed a high-fat diet exhibit excessive intestinal bacterial growth and elevated levels of proinflammatory and profibrogenic mediators, such as tumor necrosis factor alpha (TNF α), Tissue Inhibitors of Metalloproteinases (TIMP), and transforming growth factor beta (TGF β -1) (Kong et al, 2009). Furthermore, preclinical evidence suggests that the involvement of FXR agonists inhibits NF κ B expression in primary hepatocytes and in mouse models of NASH, thereby improving the inflammatory microenvironment and fibrosis (Kong et al, 2009; Ma et al, 2013).

To further support its role in NASH (and HCC in particular), FXR has been shown to modulate the expression of tumor suppressor genes, and FXR agonists were shown to reduce tumor growth and metastasis in mouse liver xenograft tumor models (Deuschle et al, 2012; Jiang et al, 2013). Thus, this evidence, in addition to playing a central role in the gut-liver-fat axis, supports the concept that FXR dysregulation leads to the development of NASH in terms of maintaining intestinal barrier integrity, inhibiting inflammation, and regulating bile acid, glucose, and lipid metabolism, and thus confirms that FXR is an ideal target for a therapeutic agent for NASH.

Many FXR agonists are known, including various non-steroidal compounds. More recently, bile acid analogs having FXR agonist activity have been developed. They include obeticholic acid (OCA; INT-747) described in WO 02/072598 and EP 1568706. Analogs and medical uses of OCAs and methods of making OCAs and analogs are described in the following references: WO 2005/092925, WO 2005/089316, WO 2006/122977, WO 2007095174, WO 2008/002573, WO 2008/091540, WO 2010/014836, WO 2010/059853, WO 2010/059859, WO 2013/192097, WO 2014/066819, WO 2015/085474, WO 2014/184271, WO 2016/127019, WO 2016/144946, WO 2016/164413, WO 2016/176208, WO 2016/205475, WO 2017/019524, WO 2017/027396, WO 2017/053428, WO 2017/053826, WO 2017/062763, WO 2017/079062, WO 2017/111979 and WO 2017/156024 (all inclusive Pharmaceuticals).

Additional 6-alkylcholytic acid analogs with modified side chains are described in WO 2016/073767, WO 2016/086115, WO 2016/086134, WO 2016/086169, WO 2016/086218, WO 2016/130809, WO 2016/161003, WO2017/147137, WO 2017/147159 and WO 2017/147174 (all from Enanta Pharmaceuticals, Inc).

Other references relating to similar compounds include CN105646634, WO 2016/173524, WO 2016/173397, CN105348365, US 2014/0206657.

Our earlier applications WO 2016/079518, WO 2016/079518, WO 2016/079519, WO 2016/079520, WO 2017/199036, WO 2017/199039 and WO 2017/199033 all relate to methods of preparing these bile acid analogues and intermediates in their synthesis.

One of the problems with the bile acid analogues described in the prior art is that, in addition to their activity as FXR agonists, they are also modulators of the G protein-coupled receptor TGR 5. It is a member of the rhodopsin-like superfamily of G protein-coupled receptors and plays an important role in the bile acid signaling network. For example, one of the adverse effects associated with the use of OCA is pruritus and is believed to result from OCA activation of off-target receptors such as TGR5 (Alemi et al, 2013).

Xiao et al, 2017, relates to the synthesis and biological evaluation of OCA and its series of OCA derivatives as FXR agonists. In the derivatives, the carboxylic acid groups are replaced by various substitutes. The authors indicate that all tested compounds showed low to moderate TGR5 potency and the best selectivity obtained was 30 times the selectivity towards FXR, which was achieved by the tetrazole derivative. With the highest liver: compound 18, having CH, at a plasma concentration ratio₂CH₂C(O)NH-S(O)₂CH₃Side chains.

The present invention relates to novel compounds which maintain FXR agonist activity and have enhanced selectivity for FXR over TGR 5.

Furthermore, bile acid derivatives fluorinated at the 2 and/or 4 position and having arylsulfonamide or sulfonylurea side chains have increased agonist activity towards the FXR receptor compared to known bile acid derivatives. The inventors postulate that this increased agonist activity is due to the binding of the molecule into the canonical and allosteric pocket of the FXR ligand binding domain. However, the effectiveness of the compounds of the present invention is not affected by the correctness of this presumption or otherwise.

Summary of The Invention

Accordingly, the present invention provides a compound of general formula (I):

wherein

R^2a、R^2b、R^3aAnd R^3bAre each independently H or F, with the proviso that R^2bAnd R^3bAt least one of (a) is F;

R⁵is CR^6aR^6bR⁸、OR⁸、SR⁸Or NR^6aR⁸；

R^6a、R^6bAnd R⁸Each independently is H or methyl

Y is a bond or C_1-4Alkylene or C_2-4Alkenylene linking group, among themOptionally substituted by one or more R¹⁰Substitution;

wherein R is¹⁰Each independently is halogen or OH;

R⁷selected from C (O) NR¹⁷S(O)₂R¹⁵、NR¹⁷C(O)NR¹⁸S(O)₂R¹⁵、NR¹⁷C(S)NR¹⁸S(O)₂R¹⁵And NR¹⁷C(NR²⁰)NR¹⁸S(O)₂R¹⁵；

R¹⁵Is a 5 to 10 membered aryl or heteroaryl ring, optionally substituted with one or more substituents selected from C_1-6Alkyl radical, C_i-6Haloalkyl, halogen, O (C)_1-6Alkyl) and O (C)_1-6Haloalkyl);

R¹⁷and R¹⁸Each independently is H or methyl;

R²⁰is H, methyl or CN;

or a salt or isotopic variation thereof.

Some fluorinated bile acid derivatives are known, for example, Roda et al 1995, Honorio et al 2006, US 5,175,320 and WO 97/44043 all relate to 6-analogues of bile acid analogues, while WO 2014/160441 describes 6, 6-biliac acid analogues. Sato et al 2008 relates to 7-fluorolithocholic acid derivatives, and sieven et al 2008 and Cushman et al 1995 disclose 3, 3-difluorocholane-24-acid and its methyl ester, respectively. EP 3290429 discloses bile acid derivatives, which are believed to be cited for the treatment of FXR-mediated diseases. The disclosed compounds include some 4-fluoro bile acid derivatives. Both Clerici et al 2006 and Macchiarulo et al 2008 relate to 3 α -6 α -dihydroxy-7 α -fluoro-5 β -cholanic acid ester, which is believed to be cited for treatment of liver disease. WO2016/154216 relates to 3-and 7-fluorinated derivatives of UDCA, which are cited for the treatment of neurodegenerative diseases. WO2016/173493 relates to bile acid derivatives with modified side chains and which are modulators of FXR and/or TGR 5. US 2018/0148470 relates to 4 β -fluorinated bile acid derivatives which are believed to be useful in the treatment of FXR-mediated diseases. However, no compounds of the general formula (I) are taught in the prior art.

The compounds of formula (I) are selective FXR agonists and are therefore useful in the treatment of diseases and disorders such as nonalcoholic steatohepatitis (NASH); primary Biliary Cirrhosis (PBC); primary sclerosing cholangitis; closing the biliary tract; cholestatic liver disease; hepatitis c infection; alcoholic liver disease; fiberizing; and liver damage due to fibrosis.

While not wishing to be bound by this theory, the present inventors have speculated that the presence of fluorine in the ring alters the hydrogen bonding capability of the 3 α hydroxyl group, thereby affecting the receptor activity and selectivity of the compound towards FXR and TGR 5. Furthermore, it is clear that compounds having at position 2 or 4 of the steroid ring system may have increased metabolic stability.

The present inventors have also found that, as shown in the following examples, a side chain group (-Y-R)⁷) Has a significant effect on FXR agonist activity.

In this specification, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

In the present specification, reference to "pharmaceutical use" is to be taken to a human or animal, in particular a human or a mammal, such as a domestic or domestic animal mammal, for the purpose of treating or preventing a disease or medical condition. The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, and "pharmaceutically acceptable" refers to an active agent suitable for use in a pharmaceutical composition. Other similar terms should be construed accordingly.

In the context of this specification, the term "plurality" means two or more.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

Drawings

In the figure, p is < 0.05, p is < 0.01, and p is < 0.001.

FIG. 1 shows the human hepatoma cell line Huh7 in comparison with the control, OCA (in EC)₅₀And EC₉₀) And Compound 2 (in EC)₅₀And EC₉₀) Change in SHP expression after 24 hours incubation together.

FIG. 2 shows the human hepatoma cell line Huh7 in comparison with the control, OCA (in EC)₅₀And EC₉₀) And Compound 2 (in EC)₅₀And EC₉₀) Change in the expression of OST α after 24 hours incubation together.

FIG. 3 shows the human hepatoma cell line HepG2 together with the control, OCA (in EC)₅₀And EC₉₀) And Compound 2 (in EC)₅₀And EC₉₀) Change in CYP7a1 expression after 24 hours incubation together.

FIG. 4 shows the human hepatocellular carcinoma cell line HepG2 and control, OCA (in EC)₅₀And EC₉₀) And Compound 2 (in EC)₅₀And EC₉₀) Change in TGF-beta 1 expression after 24 hours incubation together.

FIG. 5 shows human hepatocytesTumor cell line Huh7 and control, OCA (in EC)₅₀And EC₉₀) And compound 14 (in EC)₅₀And EC₉₀) Change in SHP expression after 24 hours incubation together.

FIG. 6 shows the human hepatoma cell line Huh7 in comparison with control, OCA (in EC)₅₀And EC₉₀) And compound 14 (in EC)₅₀And EC₉₀) Change in the expression of OST α after 24 hours incubation together.

FIG. 7 shows the human hepatocellular carcinoma cell line HepG2 and control, OCA (in EC)₅₀And EC₉₀) And compound 14 (in EC)₅₀And EC₉₀) Change in CYP7a1 expression after 24 hours incubation together.

FIG. 8 shows the human hepatocellular carcinoma cell line HepG2 and control, OCA (in EC)₅₀And EC₉₀) And compound 14 (in EC)₅₀And EC₉₀) Change in TGF-beta 1 expression after 24 hours incubation together.

Examples

In the examples, the following abbreviations are used.

Ac₂O acetic anhydride

DBU 1, 8-diazabicyclo [5.4.0] undec-7-ene

DMAP dimethylaminopyridine

EDCI 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

Equiv equivalent

Et₃N-Triethylamine

EtOAc ethyl acetate

IPA isopropyl alcohol

h hours

HDCA hyodeoxycholic acid

HPLC high performance liquid chromatography

LDA lithium diisopropylamide

MeOH methanol

n-BuLi n-butyl lithium

OCA obeticholic acid

PE Petroleum Ether

PTFE Polytetraene

pTSA p-toluenesulfonic acid

RT Room temperature

sat saturation

TBAF tetrabutylammonium fluoride

TBDMS-OTf triflic acid tert-butyldimethylsilyl ester

TEMPO (2, 2, 6, 6-tetramethylpiperidin-1-yl) oxidinyl

THF tetrahydrofuran

TMS-Cl trimethylsilyl chloride

TMS-OTf Trimethylsilyl triflate

TLC thin layer chromatography

Example 1-3 α -hydroxy-4 β -fluoro-6 α -ethyl-7 α -hydroxy-5 β -cholane with sulfonylurea-substituted side chain Synthesis of acid analogs

A.6 alpha-ethyl-3 alpha, 7 alpha-dihydroxy-5 beta-cholane-24-oic acid methyl ester

To a solution of OCA (23.5g, 55.87mmol) in MeOH (540mL) at RT was added p-toluenesulfonic acid (1.02mg, 5.59mmol,. about.0.1 equiv.) and sonicated at 30 ℃ for 3 h. Upon completion, the reaction mixture was concentrated in vacuo. The residue was dissolved in chloroform (500mL) and saturated NaHCO was used₃(500mL)、H₂O (500mL) and brine (500mL), MgSO₄Drying, filtration and concentration in vacuo gave the title compound as a white solid in quantitative yield. The resulting solid was used without further purification.

¹H NMR(400MHz，CDCl₃)：δ3.70(1H，s)，3.67(3H，s，)，3.44-3.37(1H，m)，2.40-2.32(1H，m)，2.26-2.18(1H，m)，1.96(1H，dt，J＝12.0，2.6Hz)，1.92-1.76(6H，m)，1.69-1.59(3H，m)，1.58-1.12(14H，m)，1.00(1H，td，14.2，3.3Hz)，0.93(3H，d，J＝6.3Hz)，0.90(3H，s)，0.90(3H，t，J＝7.4Hz)，0.66(3H，s)ppm。

LRMS(ESI⁺)m/z：452.4[M+NH4]⁺，100％。

B.6 alpha-Ethyl-7 alpha-hydroxy-3-oxo-5 beta-cholan-24-oic acid methyl ester

Stirred methyl 6 α -ethyl-3 α, 7 α -dihydroxy-5 β -cholan-24-oate (9.53g, 21.9mmol) from step A at RT in H₂To a solution of O (22mL) and t-butanol (88mL) was added KBr (5.22g, 43.9mmol,. about.2.0 equiv.), KHCO₃(22.0g, 219mmol,. about.10 equivalents) and TEMPO (4.45g, 28.5mmol,. about.1.3 equivalents). The reaction mixture was cooled to 0 deg.C and received dropwise addition of NaClO (28mL, 32.9mmol,. about.1.5 equiv.) at a rate of 4 mL/hr over a period of 7 hours. On completion, by slow addition of 1: 1 saturated Na₂S₂O₃The reaction was quenched (250mL) and diluted with EtOAc (200 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3X150 mL). The organic phases were combined and MgSO₄Drying, filtration and concentration in vacuo gave 14.2g of crude material as an orange oil. The resulting oil was purified by column chromatography (acetone 40-60, 0-20% gradient in PE) to give the title compound as a white solid (8.48g, 89%).

¹H NMR(400MHz，CDCl₃)：δ3.78(1H，d，J＝2.2Hz)，3.67(3H，s)，3.07(1H，dd，J＝15.2，13.5Hz)，2.46-2.33(2H，m)，2.29-1.91(7H，m)，1.84-1.77(1H，m)。1.74-1.15(18H，m)，1.00(3H，s)，0.94(3H，d，J＝6.5Hz)，0.91(3H，t，J＝7.4Hz)，0.70(3H，s)ppm。

LRMS(ESI⁺)m/z：450.3[M+NH₄]⁺，100％。

C.6 alpha-Ethyl-4 beta-fluoro-7 alpha-hydroxy-3-oxo-5 beta-cholan-24-oic acid methyl ester

To a stirred, pre-cooled solution of diisopropylamine (0.78mL, 5.54mmol,. about.12 equivalents) in dry THF (6.9mL) was added dropwise a solution of n-BuLi in hexane (1.44mL, 2.31mmol,. about.5.0 equivalents) at-78 deg.C under an argon atmosphere over 0.25 h. After addition, trimethylsilyl chloride (0.29mL, 2.31mmol,. about.5.0 equiv.) was added and stirred for 1 h. A solution of methyl 6 α -ethyl-7 α -hydroxy-3-oxo-5 β -cholan-24-oate (200mg, 0.46mmol) from step B in dry THF (3mL) and triethylamine (1.16mL, 8.32mmol, 18 equivalents) was then added. After the addition was complete, the reaction was gradually warmed to-20 ℃ and stirred for 2 h. Upon completion, saturated NaHCO was added dropwise₃The reaction was quenched (5mL) and warmed to RT for 2 h. The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3X 10 mL). The combined organic phases were washed with brine (30mL) and MgSO₄Drying, filtration and concentration in vacuo gave 271mg of crude material as a yellow residue.

To a stirred solution of the crude material obtained in MeCN (13mL) was addedThe mixture was stirred for 16 h. Upon completion, the reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAC (20mL) and acidified with 2M HCl (30 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3 × 15 mL). The combined organic phases were washed with brine (100mL) and MgSO₄Drying, filtration and concentration in vacuo gave 196mg of crude material as a green solid. Purification by HPLC using hexane/acetone (90/10) as eluent gave the title compound and a mixture of methyl-2 β -3-oxo-6 α -ethyl-7 α -hydroxy-5 β -cholan-24-oate which could not be separated as a colorless oil (79mg, 0.18mmol, 37% title compound, accounting for 1% methyl-2 β -fluoro-3-oxo-6 α -ethyl-7 α -hydroxy-5 β -cholan-24-oate contamination by¹H NMR)。

¹H NMR(400MHz，CDCl₃)：δ5.94(1H，dd，J＝46.5，10.9Hz)，3.88(1H，s)，3.65(3H，s)，2.49(1H，td，J＝14.6，5.0Hz)，2.38-2.09(4H，m)，2.01-1.30(18H，m)，1.25-1.14(3H，m)，1.04(3H，s)，0.93-0.89(6H，m)，0.68(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：-194.3(1F，dd，J＝46.8，13.9Hz)ppm。

LRMS(ESI⁺)m/z：468.4[M+NH₄]⁺，100％。

D.6 alpha-ethyl-4 beta-fluoro- (3 alpha, 7 alpha) -dihydroxy-5 beta-cholan-24-oic acid methyl ester

To a stirred solution of methyl 6 α -ethyl-4 β -fluoro-7 α -hydroxy-3-oxo-5 β -cholan-24-oate (75mg, 0.17mmol) from step C in dry THF (6.7mL) at RT under argon was added NaBH₄(19mg, 0.50mmol,. about.3.0 equiv.) and stirred for 16 h. On completion, by dropwise addition of H₂The reaction was quenched with O (8mL) 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 are combined with H₂O (100mL) wash, MgSO₄Drying, filtration and concentration in vacuo gave 77mg of crude material as a white residue. Purification by HPLC using hexane/acetone (90/10) as eluent gave the title compound as a colorless oil (55mg, 0.12mmol, 74%).

¹H NMR(400MHz，CDCl₃)：δ5.31(1H，ddd，J＝50.0，10.4，8.9Hz)，3.82(1H，s)，3.67(3H，s)，3.57-3.50(1H，m)，2.36(1H，ddd，J＝15.4，10.1，5.7Hz)，2.27-2.18(1H，m)，1.96-1.92(2H，m)，1.83-1.07(23H，m)，0.97(3H，s)，0.94-0.92(6H，m)，0.66(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-189.0(1F，d，J＝50.3Hz)ppm。

LRMS(ESI⁺)m/z：470.4[M+NH₄]⁺，100％。

E.3 alpha, 7 alpha-dihydroxy-4 beta-fluoro-6 alpha-ethyl-5 beta-cholanic acid

To a stirred solution of methyl 6 α -ethyl-4 β -fluoro- (3 α, 7 α) -dihydroxy-5 β -cholan-24-oate (58mg, 0.13mmol) in MeOH (5mL) was added NaOH (250mg, 5% solution) at RT and stirred for 18 h. Upon completion, the reaction mixture was concentrated in vacuo, and the residue was acidified to pH 2 with 1M HCl and diluted with EtOAc (20 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine (100mL) and MgSO₄Drying, filtration and concentration in vacuo gave 76mg of crude material as a yellow oil. Activation by HPLC using hexane/acetone (70/30) as eluent gave the title compound as a colorless oil (41mg, 0.09mmol, 72%).

¹H NMR(400MHz，CDCl₃)：δ5.31(1H，dt，J＝49.9，9.5Hz)，3.83(1H，s)，3.60-3.50(1H，m)，2.36(1H，ddd，J＝15.5，10.4，5.3Hz)，2.26(1H，ddd，J＝15.8，9.5，6.6Hz)，1.97-1.91(2H，m)，1.85-1.08(21H，m)，0.97(3H，s)，0.95-0.91(6H，m)，0.67(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-188.7(1F，d，J＝48.6Hz)ppm。

LRMS(ESI⁺)m/z：456.2，[M+NH₄]⁺，100％。

Synthesis of Compounds with sulfonylurea-substituted side chains

The following method exemplifies 4 β -fluoro derivatives, but may also be used for 2 β -fluorinated, 4-difluoroor 2, 4-difluoro compounds.

F.3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-5 beta-cholanic acid

Stirring of 3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -chola from step E under argon atmosphere at RTA solution of alkanoic acid (2.08g, 4.74mmol) in dry THF (160mL) was added dropwise with NaHCO₃(2.04g, 23.7mmol,. about.5.0 equivalents) and Ac₂O (2.29mL, 23.7mmol,. about.5.0 equiv.) over 5 min. After addition, the reaction mixture was heated at 70 ℃ for 16 h. On completion, the reaction mixture was cooled to RT and H was added dropwise₂O (100mL) was quenched, acidified with 1M HCl (20mL), and diluted with EtOAc (100 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3 × 150 mL). The combined organic phases were washed with brine (400mL) and MgSO₄Dried, filtered and concentrated in vacuo to give a yellow oil. The resulting oil (MeOH CH) was purified by column chromatography₂Cl₂Gradient elution of the solution 0-3%) gave the title compound as a white solid (660mg, 29%).

¹H NMR(400MHz，CDCl₃)：δ5.47(1H，dt，J＝49.4，9.4Hz)，4.78(1H，dddd，J＝14.1，11.9，9.3，5.0Hz)，3.84(1H，s)，2.41(1H，ddd，J＝15.5，10.2，5.3Hz)，2.27(1H，ddd，J＝15.8，9.7，6.6Hz)，2.06(3H，s)，1.97-1.89(2H，m)，1.86-1.80(3H，m)，1.70-1.14(19H，m)，0.99(3H，s)，0.94(3H，d，J＝6.2Hz)，0.92(3H，t，J＝7.1Hz)，0.67(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-188.6(1F，dt，J＝50.3，12.1Hz)ppm。

LRMS(ESI⁺)m/z：498.2，[M+NH₄]⁺，100％。

G.3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-5 beta-cholane-24-acyl azide

To a solution of 3 α -acetoxy-4 β -fluoro-6 α -ethyl-7 α -hydroxy-5 β -cholanic acid (200mg, 0.42mmol) from step F in dry THF (4mL) was added Et3N (0.12mL, 0.83mmol,. about.2.0 equiv) dropwise at RT under an argon atmosphere. After addition, the reaction mixture was cooled to 0 ℃ and diphenylphosphoryl azide was added dropwiseSubstance (0.13mL, 0.62mmol,. about.1.5 equiv). After addition, the reaction mixture was stirred for 3h after a strong gas flow hood. Upon completion, the reaction was quenched with brine (5mL) and CH₂Cl₂(5mL) dilution. Removing the organic phase with CH₂Cl₂Back extract the aqueous phase (3 × 5 mL). The organic phases were combined and MgSO₄Drying, filtration and concentration in vacuo at 0 ℃ gave a yellow oil. The resulting oil was used without further purification.

¹H NMR-characteristic peaks (400MHz, CDCl)₃)：δ5.47(1H，ddd，J＝49.4，10.4，9.2Hz)，4.82-4.74(1H，m)，3.83(1H，s)，2.38(1H，ddd，J＝15.8，10.0，5.3Hz)，2.29-2.23(1H，m)，2.06(3H，s)，0.98(3H，s)，0.920(3H，d，J＝6.5Hz)，0.919(3H，t，J＝7.2Hz)，0.67(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-186.8(1F，dt，J＝50.3Hz)ppm。

H. 3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-24-nor-5 beta-cholan-23-yl isocyanate

A stirred solution of the crude oily 3 α -acetoxy-4 β -fluoro-6 α -ethyl-7 α -hydroxy-5 β -cholane-24-acyl azide from step G (105mg putative, 0.42mmol) in dry toluene (3.1mL) was heated to 125 ℃ under an argon atmosphere. After 5h, the reaction was cooled to RT. The resulting solution was used without further purification.

1H NMR-characteristic peaks (400MHz, CDCl)₃)：δ5.47(1H，ddd，J＝49.4，10.4，9.2Hz)，4.81-4.73(1H，m)，3.83(1H，s)，3.35(1H，ddd，J＝12.9，7.8，4.5Hz)，3.30-3.24(1H，m)，2.05(3H，s)，0.98(3H，s)，0.94(3H，d，J＝6.6Hz)，0.92(3H，t，J＝7.2Hz)，0.68(3H，s)ppm。

¹⁹F NMR(¹H non-uncoupled, 376MHz, CDCl 3): delta-186.5 (1F, dt, J ═ 49.3, 12.7)Hz)ppm。

General procedure 1 for the conversion of isocyanates into sulfonylureas

To a stirred crude solution of 3 α -acetoxy-4 β -fluoro-6 α -ethyl-7 α -hydroxy-24-nor-5 β -cholan-23-yl isocyanate from step H in toluene was added sulfonamide (-1.5 equivalents) and DBU (-1.5 equivalents) and stirred for 16H. Upon completion, the reaction was quenched by dropwise addition of 1M HCl (2mL) and diluted with EtOAc (5 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3X5 mL). The organic phases were combined and MgSO₄Drying, filtering and vacuum concentrating. The resulting product was purified by column chromatography (acetone gradient elution in PE, 40-60, 5-20%) to give the desired sulfonylurea.

N, N' - (3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-24-nor-5 beta-cholan-23-yl) -p-toluenesulfonylurea (intermediate 1)

Prepared according to general procedure 1 using 53.4mg p-toluenesulfonamide to give intermediate 1 as a yellow oil (51.7mg, 38%).

¹H NMR(400MHz，CDCl₃)：δ7.78(2H，d，J＝8.2Hz)，7.32(2H，d，J＝8.3Hz)，6.50(1H，t，J＝4.8Hz)，5.47(1H，dt，J＝49.4，9.8Hz)，4.83-4.73(1H，m)，3.83(1H，s)，3.33-3.25(1H，m)，3.19-3.11(1H，m)，2.44(3H，s)，2.06(3H，s)，1.96-1.80(5H，m)，1.72-1.38(15H，m)，1.23-1.14(5H，m)，0.99(3H，s)，0.93(3H，d，J＝6.6Hz)，0.92(3H，t，J＝7.5Hz)，0.65(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-186.6(1F，dt，J＝49.0，12.8Hz)ppm。

LRMS(ESI⁺)m/z：666.4，[M+NH₄]⁺，100％。

N, N' - (3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-24-nor-5 beta-cholan-23-yl) -benzenesulfonylurea (intermediate 2)

Prepared according to general procedure 1 using 49.0mg benzenesulfonamide to give intermediate 2 as a yellow oil (49.9mg, 38%).

¹H NMR(400MHz，CDCl₃)：δ7.90(2H，d，J＝7.6Hz)，7.63(1H，t，J＝7.0Hz)，7.50(2H，t，J＝7.7Hz)，6.60(1H，s)，5.48(1H，dt，J＝49.2，9.8Hz)，4.84-4.74(1H，m)，3.83(1H，s)，3.31-3.25(1H，m)，3.18-3.10(1H，m)，2.06(3H，s)，1.95-1.80(5H，m)，1.69-1.38(13H，m)，1.29-1.12(7H，m)，0.99(3H，s)，0.93(3H，d，J＝6.7Hz)，0.92(3H，t，J＝7.0Hz)，0.65(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-186.5(1F，dt，J＝48.6，12.1Hz)ppm。

LRMS(ESI⁺)m/z：652.3，[M+NH4]+，100％。

N, N' - (3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-24-nor-5 beta-cholan-23-yl) -4- (tert-butyl) benzenesulfonylurea (intermediate 3)

Prepared according to general procedure 1 using 53.2mg 4- (tert-butyl) benzenesulfonamide to give intermediate 3 as a colorless oil (81.6mg, 71%).

¹H NMR(400MHz，CDCl₃)：δ7.81(2H，d，J＝8.7Hz)，7.54(2H，d，J＝8.7Hz)，6.56(1H，s)，5.48(1H，ddd，J＝49.4，10.4，9.3Hz)，4.83-4.73(1H)，3.84(1H，s)，3.33-3.29(1H，m)，3.21-3.14(1H，m)，2.07(3H，s)，1.97-1.80(4H，m)，1.74-1.44(13H，m)，1.36(9H，s)，1.29-1.16(7H，m)，0.99(3H，s)，0.95(3H，d，J＝6.6Hz)，0.93(3H，t，J＝7.0Hz)，0.67(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-186.6(1F，dt，J＝49.9，13.2Hz)ppm。

LRMS(ESI⁺)m/z：708.4，[M+NH₄]⁺，100％。

N, N' - (3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-24-nor-5 beta-cholan-23-yl) -m-toluenesulfonylurea (intermediate 4)

Prepared according to general procedure 1 using 42.6mg of m-toluenesulfonamide to give intermediate 4 as a colorless oil (85.7mg, 80%).

¹H NMR(400MHz，CDCl₃)：δ7.71-7.68(2H，m)，7.45-7.38(2H，m)，6.54(1H，s)，5.47(1H，ddd，J＝49.4，10.3，9.4Hz)，4.83-4.73(1H，m)，3.83(1H，s)，3.33-3.26(1H，m)，3.19-3.12(1H，m)，2.42(3H，s)，2.06(3H，s)，1.96-1.80(4H，m)，1.72-1.11(21H，m)，0.99(3H，s)，0.94(3H，d，J＝6.6Hz)，0.93(3H，t，J＝7.3Hz)，0.65(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-186.6(1F，dt，J＝49.4，13.4Hz)ppm。

LRMS(ESI⁺)m/z：666.3，[M+NH₄]⁺，100％。

N, N' - (3 alpha-acetoxy-4 beta-fluoro-6 alpha-ethyl-7 alpha-hydroxy-24-nor-5 beta-cholan-23-yl) -o-toluenesulfonylurea (intermediate 5)

Prepared according to general procedure 1 using 42.6mg o-toluenesulfonamide to give intermediate 5 as a colorless oil (55.4mg, 51%).

¹H NMR(400MHz，CDCl₃)：δ7.94(1H，dd，J＝8.3，1.0Hz)，7.51(1H，td，J＝7.6，1.2Hz)，7.34(2H，d，J＝7.3Hz)，6.47(1H，s)，5.47(1H，dt，J＝49.3，9.5Hz)，4.83-4.73(1H，m)，3.83(1H，s)，3.28-3.22(1H，m)，3.15-3.08(1H，m)，2.65(3H，s)，2.21-2.17(1H，m)，2.06(3H，s)，1.94-1.80(4H，m)，1.68-1.10(20H，m)，0.98(3H，s)，0.92(3H，d，J＝6.7Hz)，0.90(3H，t，J＝7.3Hz)，0.63(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-186.6(1F，dt，J＝49.9，12.4Hz)ppm。

LRMS(ESI⁺)m/z：666.3，[M+NH₄]⁺，100％。

General procedure for deprotection of 3 alpha-acetate sulfonylureas 2

To the flask to which the protected sulfonylurea was added, NaOH in MeOH (5% solution, 10mL) was added and stirred for 16 h. Upon completion, the reaction was acidified to pH 7.0 with 1M HCl and diluted with EtOAc (10 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3X 10 mL). The organic phases are combined and washed with NaHCO₃The solution (50mL) was washed with MgSO₄Drying, filtering and vacuum concentrating. The resulting product (in CH) was purified by column chromatography₂Cl₂MeOH in gradient elution, 0-5%) to give the deprotected sulfonylurea.

N, N' - (3 alpha, 7 alpha-dihydroxy-4 beta-fluoro-6 alpha-ethyl-24-nor-5 beta-cholan-23-yl) -p-toluenesulfonylurea (Compound 1)

Prepared according to general method 2 using 49.7mg of intermediate 1 to give compound 1 as a colorless residue (18.6mg, 40%).

¹H NMR(400MHz，CDCl₃)：δ7.78(2H，d，J＝8.3Hz)，7.34(2H，d，J＝8.1Hz)，6.50(1H，t，J＝4.8Hz)，5.32(1H，ddd，J＝50.0，10.0，9.2Hz)，3.83(1H，s)，3.59-3.51(1H，m)，3.33-3.26(1H，m)，3.21-3.12(1H，m)，2.46(3H，s)，1.96-1.06(26H，m)，0.98(3H，s)，0.94(3H，t，J＝6.2Hz)，0.93(3H，t，J＝6.4Hz)，0.66(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-188.8(1F，dt，J＝50.3，10.4Hz)ppm。

LRMS(ESI⁺)m/z：624.4，[M+NH₄]⁺，100％。

N, N' - (3 alpha, 7 alpha-dihydroxy-4 beta-fluoro-6 alpha-ethyl-24-nor-5 beta-cholan-23-yl) -benzenesulfonylurea (Compound 2)

Prepared according to general procedure 2 using 44.8mg of intermediate 2 to give compound 2 as a colorless residue (28.5mg, 64%).

¹H NMR(400MHz，CDCl₃)：δ7.91(2H，d，J＝7.5Hz)，7.65(1H，t，J＝7.3Hz)，7.54(2H，t，J＝7.8Hz)，6.51(1H，s)，5.31(1H，ddd，J＝50.1，10.3，9.1Hz)，3.82(1H，s)，3.60-3.50(1H，m)，3.35-3.26(1H，m)，3.20-3.12(1H，m)，1.95-1.36(17H，m)，1.27-1.11(9H，m)，0.97(3H，s)，0.94(3H，d，J＝6.2Hz)，0.93(3H，t，J＝6.7Hz)，0.65(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-189.0(1F，dt，J＝50.3，12.1Hz)ppm。

LRMS(ESI⁺)m/z：610.2，[M+NH₄]⁺，100％。

N, N' - (3 alpha, 7 alpha-dihydroxy-4 beta-fluoro-6 alpha-ethyl-24-nor-5 beta-cholan-23-yl) -4- (tert-butyl) benzenesulfonylurea (Compound 3)

Prepared according to general procedure 2 using 79.6mg of intermediate 3 to give compound 3 as a colorless residue (50.7mg, 65%).

¹H NMR(400MHz，CDCl₃)：δ7.82(2H，d，J＝8.6Hz)，7.53(2H，t，J＝8.4Hz)，6.54(1H，s)，5.32(1H，ddd，J＝49.9，10.3，9.1Hz)，3.82(1H，s)，3.60-3.50(1H，m)，3.35-3.25(1H，m)，3.19-3.11(1H，m)，1.95-1.41(16H，m)，1.34(9H，s)，1.28-1.08(10H，m)，0.97(3H，s)，0.930(3H，t，J＝6.9Hz)，0.927(3H，d，J＝6.2Hz)，0.65(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-188.8(1F，dt，J＝50.3，12.1Hz)ppm。

LRMS(ESI⁺)m/z：666.4，[M+NH₄]⁺，100％。

N, N' - (3 alpha, 7 alpha-dihydroxy-4 beta-fluoro-6 alpha-ethyl-24-nor-5 beta-cholan-23-yl) -m-toluenesulfonylurea (Compound 4)

Prepared according to general method 2 using 83.7mg of intermediate 4 to give compound 4 as a colorless residue (29.0mg, 37%).

¹H NMR(400MHz，CDCl₃)：δ7.71-7.69(2H，m)，7.45-7.38(2H，m)，6.52(1H，s)，5.32(1H，ddd，J＝49.9，10.4，8.9Hz)，3.82(1H，s)，3.60-3.50(1H，m)，3.35-3.26(1H，m)，3.20-3.10(1H，m)，2.43(3H，s)，1.95-1.39(17H，m)，1.28-1.11(10H，m)，0.97(3H，s)，0.94(3H，d，J＝6.2Hz)，0.93(3H，t，J＝6.5Hz)，0.65(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-188.9(1F，dt，J＝48.6，10.4Hz)ppm。

LRMS(ESI⁺)m/z：624.3，[M+NH₄]⁺，100％。

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -o-toluenesulfonylurea (Compound 5)

Prepared according to general procedure 2 using 53.4mg of intermediate 5 to give compound 5 as a colorless residue (24.2mg, 48%).

¹H NMR(400MHz，CDCl₃)：δ7.94(1H，d，J＝8.1Hz)，7.52(1H，td，J＝7.6，1.1Hz)，7.35(2H，d，J＝7.7Hz)，6.47(1H，t，J＝4.6Hz)，5.31(1H，ddd，J＝49.9，10.4，8.9Hz)，3.82(1H，s)，3.60-3.50(1H，m)，3.30-3.22(1H，m)，3.17-3.08(1H，m)，2.67(3H，s)，1.94-1.06(25H，m)，0.97(3H，s)，0.91(3H，t，J＝7.5Hz)，0.90(3H，d，J＝6.6Hz)，0.63(3H，s)ppm。

¹⁹F NMR(¹H non-decoupled, 376MHz, CDCl₃)：δ-189.0(1F，dt，J＝48.6，12.1Hz)ppm。

LRMS(ESI⁺)m/z：624.3，[M+NH₄]⁺，100％。

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-fluorobenzenesulfonylurea (compound 6)

This compound was prepared by a method similar to that described above for compounds 1-5.

¹H NMR(400MHz，MeOD)：δ7.93-7.(2H，m)，7.20-7.17(2H，m)，5.19(1H，dq，J＝49.3，10.5，8.9Hz)，3.65(1H，s)，3.31(1H，m)，3.06(1H，m)，2.95(1H，m)，1.94-1.06(21H，m)，0.84-0.76(9H，m)，0.63(3H，s)ppm。

¹⁹F NMR(¹H non-uncoupled, 376MHz, MeOD): delta-107.29 (1F, m), -186.6(1F, m) ppm.

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -m-fluorobenzenesulfonyl urea (compound 7)

This compound was prepared by a method similar to that described above for compounds 1-5.

¹H NMR(400MHz，CDCl₃)δ7.71(ddd，J＝7.8，1.6，1.0Hz，1H)，7.61(br dt，J＝8.0，2.1Hz，1H)，7.54(td，J＝8.1，5.3Hz，1H)，7.35(tdd，J＝8.3，2.6，0.7Hz，1H)，6.50(br t，J＝5.1Hz，1H)，5.32(ddd，J＝49.9，10.5，8.8Hz，1H)，3.83(s，1H)，3.55(dddd，J＝14.2，12.0，8.8，5.1Hz，1H)，3.32(ddt，J＝13.1，9.5，5.1Hz，1H)，3.18(dtd，J＝13.0，8.0，6.1Hz，1H)，1.96-1.86(m，2H)，1.85-1.74(m，2H)，1.71-1.58(m，6H)，1.54-1.41(m，7H)，1.31-1.08(m，9H)，0.98(s，3H)，0.95(d，J＝6.5Hz，3H)，0.93(t，J＝7.0Hz，3H)，0.66(s，3H)ppm。

¹⁹F NMR(376MHz，CDCl₃)δ-108.9(br s，1F)，-188.9(br d，J＝50.3Hz，1F)ppm。

¹⁹F{1H}NMR(376MHz，CDCl₃)δ-108.9(s，1F)，-188.9(s，1F)ppm。

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -o-fluorobenzenesulfonyl urea (compound 8)

This compound was prepared by a method similar to that described above for compounds 1-5.

¹H NMR(400MHz，CDCl₃)δ7.91(ddd，J＝7.8，7.2，1.7Hz，1H)，7.67(dddd，J＝8.3，7.5，5.0，1.7Hz，1H)，7.33(td，J＝7.7，1.1Hz，1H)，7.28(ddd，J＝10.0，8.4，0.9Hz，1H)，6.45(br t，J＝5.1Hz，1H)，5.31(ddd，J＝49.9，10.5，8.7Hz，1H)，3.83(br s，1H)，3.55(dddd，J＝13.8，11.9，8.9，5.4Hz，1H)，3.29(ddt，J＝12.8，9.4，5.3Hz，1H)，3.16(dtd，J＝13.5，7.8，5.6Hz，1H)，1.96-1.74(m，4H)，1.71-1.39(m，14H)，1.25-1.09(m，8H)，0.98(s，3H)，0.94(t，J＝7.3Hz，3H)，0.93(d，J＝6.6Hz，3H)，0.65(s，3H)ppm。

¹⁹F NMR(376MHz，CDCl₃)δ-109.0(ddd，J＝10.4，6.9，5.2Hz，1F)，-189.2(dt，J＝50.3，11.3Hz，1F)ppm。

¹⁹F{1H}NMR(376MHz，CDCl₃)δ-109.0(s，1F)，-189.2(s，1F)ppm。

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p- (trifluoromethyl) benzenesulfonylurea (Compound 9)

This compound was prepared by a method similar to that described above for compounds 1-5.

¹H NMR(400MHz，CDCl₃)δ8.07(d，J＝8.2Hz，2H)，7.80(d，J＝8.1Hz，2H)，6.46(br t，J＝4.8Hz，1H)，5.32(ddd，J＝49.8，10.4，8.8Hz，1H)，3.82(s，1H)，3.55(dddd，J＝14.2，11.9，8.8，5.4Hz，1H)，3.28(ddt，J＝13.6，7.7，4.9Hz，1H)，3.15(dtd，J＝13.5，7.6，6.0Hz，1H)，1.96-1.75(m，4H)，1.72-1.55(m，7H)，1.53-1.37(m，7H)，1.25-1.06(m，8H)，0.97(s，3H)，0.933(d，J＝6.1Hz，3H)，0.927(t，J＝6.5Hz，3H)，0.63(s，3H)ppm。

¹⁹F NMR(376MHz，CDCl₃)δ-63.5(s，3F)，-188.5(br d，J＝48.6Hz，1F)ppm。

¹⁹F{1H}NMR(376MHz，CDCl₃)δ-63.4(s，3F)，-188.6(br s，1F)ppm。

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -m- (trifluoromethyl) benzenesulfonylurea (Compound 10)

This compound was prepared by a method similar to that described above for compounds 1-5.

¹H NMR(400MHz，CDCl₃)δ8.17(s，1H)，8.11(d，J＝8.0Hz，1H)，7.91(br d，J＝7.7Hz，1H)，7.71(t，J＝7.9Hz，1H)，6.49(br t，J＝4.7Hz，1H)，5.32(ddd，J＝49.9，10.2，9.1Hz，1H)，3.83(s，1H)，3.56(dddd，J＝14.1，11.7，8.7，5.1Hz，1H)，3.32(ddt，J＝13.5，9.4，5.3Hz，1H)，3.17(dtd，J＝12.6，7.8，6.2Hz，1H)，1.96-1.87(m，2H)，1.84-1.74(m，2H)，1.72-1.58(m，6H)，1.53-1.41(m，7H)，1.29-1.09(m，9H)，0.98(s，3H)，0.95(d，J＝6.5Hz，3H)，0.93(t，J＝6.9Hz，3H)，0.66(s，3H)ppm。

¹⁹F NMR(376MHz，CDCl₃)δ-63.1(s，3F)，-189.0(br s，1F)ppm。

¹⁹F{1H}NMR(376MHz，CDCl₃)δ-63.1(s，3F)，-189.0(br s，1F)ppm。

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -o- (trifluoromethyl) benzenesulfonylurea (Compound 11)

This compound was prepared by a method similar to that described above for compounds 1-5.

¹H NMR(400MHz，CDCl₃)δ8.26(dd，J＝6.3，2.3Hz，1H)，7.92(dd，J＝6.6，2，3Hz，1H)，7.77(m，2H)，6.35(br t，J＝4.8Hz，1H)，5.31(ddd，J＝49.9，10.4，8.9Hz，1H)，3.82(s，1H)，3.56(dddd，J＝14.2，12.1，8.7，5.1Hz，1H)，3.29(ddt，J＝13.5，9.1，5.1Hz，1H)，3.13(dtd，J＝13.5，7.7，6.2Hz，1H)，1.96-1.72(m，5H)，1.70-1.35(m，14H)，1.25-1.06(m，7H)，0.97(s，3H)，0.93(d，J＝7.0Hz，3H)，0.92(t，J＝6.5Hz，3H)，0.63(s，3H)ppm。

¹⁹F NMR(376MHz，CDCl₃)δ-58.0(s，3F)，-188.9(br d，J＝48.6Hz，1F)ppm。

¹⁹F{1H}NMR(376MHz，CDCl₃)δ-58.0(s，3F)，-188.9(s，1F)ppm。

N, N' - (3 α, 7 α -dihydroxy-6 α -ethyl-24-nor-5 β -cholan-23-yl) -benzenesulfonylurea (comparative compound A)

This compound was prepared by a similar method as described for compounds 5-9 above.

¹H NMR(400MHz，MeOD)：δ7.86-7.81(2H，m)，7.53(1H，m)，7.47-7.41(2H，m)，3.52(1H，br.s)，3.22(1H，m)，3.04(1H，m)，2.93(1H，m)，1.87-0.83(25H，m)，0.81-0.76(9H，m)，0.52(3H，s)ppm。

Example 2 alternative Synthesis of Compounds with sulfonylurea-substituted side chains

The following methods are exemplified for 4 β -fluoro derivatives, but may also be used for 2 β -fluorinated, 4-difluorinated or 2, 4-difluorinated compounds.

A.6 alpha-ethyl-4 beta-fluoro-7 alpha-trimethylsilyloxy-3-oxo-5 beta-cholan-24-oic acid methyl ester

To a stirred pre-cooled diisopropylamine (0.78mL,5.54mmol,. about.12 equivalents) in dry THF (6.9mL) was added n-BuLi in hexane (1.44mL, 2.31mmol,. about.5.0 equivalents) dropwise over 0.25 h. After addition, trimethylsilyl chloride (0.29mL, 2.31mmol,. about.5.0 equiv.) was added and stirred for 1 h. A solution of methyl 6 α -ethyl-7 α -hydroxy-3-oxo-5 β -cholane-24-oate (200mg, 0.46mmol) from example 1, step B, in dry THF (3mL) and triethylamine (1.16mL, 8.32mmol, 18 equiv.) was then added. After the addition, the reaction was gradually warmed to-20 ℃ and stirred for 2 h. Upon completion, saturated NaHCO was added dropwise₃The reaction was quenched (5mL) and warmed to RT for 2 h. The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3X 10 mL). The combined organic phases were washed with brine (30mL) and MgSO₄Drying, filtration and concentration in vacuo gave 271mg of crude material as a yellow residue.

To a solution of the crude material (1.16g, 2.3mmol) in dry MeCN (55mL) was added(1.23g, 3.47 mmol). After stirring at RT for 14.5h, the mixture was diluted with ethyl acetate (100mL) and 5% NaHCO₃(100mL) and 10% NaCl (50 mL). The aqueous phase was extracted with ethyl acetate (3X 100mL) and MgSO₄The combined organic phases were dried, filtered and concentrated in vacuo to give an orange/yellow oil. Purification of the crude material (SiO) by column chromatography₂0-40% EtOAc in heptane) to give the title compound as a colorless oil (319.5 mg).

B.6 alpha-Ethyl-4 beta-fluoro-7 alpha-trimethylsilyloxy-3 alpha-hydroxy-5 beta-cholan-24-oic acid methyl ester

Crude methyl 6 α -ethyl-4 β -fluoro-7 α -trimethylsiloxy-3-oxo-5 β -cholan-24-oate (319.5mg, 0.71mmol) from step a was dissolved in THF (28mL) under an argon atmosphere while stirring. Adding NaBH₄(80.5mg, 2.13mmol), the reaction was stirred at RT for 16.5h, after which time NaBH was added₄(0.24g, 6.38 mmol). The mixture was stirred for a further 4.5h, then water (20 μ L) was added and the mixture was stirred for-60 h. After this time, the reaction was quenched by addition of water (15mL) and diluted with EtOAc (50 mL). The phases were separated and the aqueous phase was extracted with EtOAc (3 × 50 mL). With MgSO₄The combined extracts were dried, filtered and concentrated in vacuo to give a clear syrup (0.34 g). Purification of the crude material (SiO) by column chromatography₂0-40% EtOAc in heptane) to give the title compound as a clear oil (162.3 mg).

C.6 alpha-Ethyl-4 beta-fluoro-7 alpha-trimethylsilyloxy-3 alpha-o-tert-butyldimethylsilyl-5 beta-cholan-24-oic acid methyl ester

Methyl 6 α -ethyl-4 β -fluoro-7 α -trimethylsilyloxy-3 α -hydroxy-5 β -cholan-24-oate (0.48g, 0.92mmol) from step B was dissolved in dry DCM (12mL) and cooled to 0 ℃ under an argon atmosphere while stirring. 2, 6-lutidine (1.1mL, 9.17mmol) was added followed by TBMDS-OTf (0.32mL, 1.38mmol) dropwise. The reaction was warmed to RT, stirred for 24h, then cooled to 0 ℃ and quenched by the dropwise addition of 10% citric acid (5 mL). The phases were separated and the aqueous phase was extracted with DCM (3 × 5 mL). With 10% citric acid (5mL), NaHCO₃The combined extracts were washed with aqueous (5mL) and water (5mL) and MgSO₄Dried, filtered and concentrated in vacuo to give a yellow oil (0.69 g). Purification of the crude material (SiO) by column chromatography₂0-20% EtOAc in heptane) to give the title compound as a clear oil (0.58 g).

6 α -Ethyl-4 β -fluoro-7 α -trimethylsiloxy-3 α -o-tert-butyldimethylsilyl-5 β -cholanic acid

Stirring while stirring 6 alpha-ethyl-4 beta-fluoro-7 alpha-trimethylsiloxy-3 alpha-o-tert-butyl ether from step CButyldimethylsilyl-5 β -cholan-24-oic acid methyl ester (0.58g) was dissolved in IPA (5.8 mL). 0.5M NaOH (5.8mL) was added and the reaction was stirred at RT for 15 h. The reaction mixture was concentrated under reduced pressure to-half volume, then water (5mL) was added by the addition of 2M H₂SO₄The solution was neutralized and diluted with EtOAc (10 mL). With 2M H₂SO₄The mixture was acidified to pH1, the phases were separated and the aqueous phase was extracted with EtOAc (10 mL). The combined extracts were washed with water (5mL) and brine (5mL), MgSO₄Drying, filtration and concentration in vacuo gave a white foam (0.52 g). Purification of the crude material (SiO) by column chromatography₂0-50% acetone in toluene) to give the title compound as a white solid (0.41g, 72%).

E.6 alpha-Ethyl-4 beta-fluoro-7 alpha-trimethylsiloxy-3 alpha-o-tert-butyldimethylsilyl-5 beta-cholan-24-acyl azide

To a stirred solution of 6 α -ethyl-4 β -fluoro-7 α -trimethylsiloxy-3 α -o-tert-butyldimethylsilyl-5 β -cholanic acid from step D (197mg, 0.32mmol) in dry THF (3.2mL) was added dropwise Et under argon atmosphere at RT₃N (0.09mL, 0.64mmol,. about.2.0 equiv). After addition, the reaction mixture was cooled to 0 ℃ and diphenylphosphoryl azide (0.1mL, 0.48mmol,. about.1.5 equivalents) was added dropwise. After addition, the reaction mixture was stirred for 2.5h behind a strong gas flow hood. Upon completion, the reaction was quenched with brine (3mL) and extracted with DCM (3 × 5 mL). With MgSO₄The combined organic phases were dried, filtered and concentrated in vacuo at 0 ℃. The resulting oil was used without further purification.

General procedure 3 for the formation of sulfonylureas

Stirring in an argon atmosphere from example 2A solution of the crude 6 α -ethyl-4 β -fluoro-7 α -trimethylsilyloxy-3 α -o-tert-butyldimethylsilyl-5 β -cholan-24-acyl azide from step E (69mg) in dry toluene (2.1mL) was heated to 125 ℃. After 5h, the reaction was cooled to RT. The resulting solution was used without further purification. The solution was stirred under an argon atmosphere and sulfonamide (1.5 equiv.) and DBU (1.5 equiv.) were added. Upon completion, the reaction was quenched by dropwise addition of 1M HCl (1mL) and diluted with EtOAc (5 mL). The organic phase was removed and the aqueous phase was back-extracted with EtOAc (3X5 mL). The combined organic phases were washed with brine (3mL) and MgSO₄Dried, filtered and concentrated in vacuo (231.7 mg). The resulting product is purified by column chromatography to give the desired sulfonylurea as a crude inseparable mixture.

N, N '- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (trimethoxy) benzenesulfonylurea (Compound 12) and N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-methoxybenzenesulfonylurea (Compound 13)

Compounds 12 and 13 were prepared according to general procedure 3 above by reacting the crude isocyanate product with 4- (trifluoromethoxy) benzenesulfonamide and 4- (methoxy) benzenesulfonamide, respectively.

To obtain pure product, crude compounds 12 and 13 were converted to protected species (intermediates 12 and 13), purified, and then deprotected to regenerate compounds 12 and 13. The method is as follows.

N, N' - (3 α, 7 α -di-o-tert-butyldimethylsilyl-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (trifluoromethoxy) benzenesulfonylurea (intermediate 12)

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (trifluoromethoxy) benzenesulfonylurea (24.3mg, 0.036mmol) was dissolved in dry DCM (1mL) and cooled to 0 ℃ under an argon atmosphere while stirring. Adding 2, 6-lutidine (0.04mL, 0.36mmol), and adding TBMDS-OTf (0.02 mmol) dropwisemL, 0.108 mmol). The reaction was warmed to RT, stirred for 1.5h, then cooled to 0 ℃ and quenched by the dropwise addition of 10% citric acid (1 mL). The phases were separated and the aqueous phase was extracted with DCM (3 × 1 mL). With 10% citric acid (1mL), NaHCO₃The combined extracts were washed with aqueous (1mL) and water (1mL) over MgSO₄Dried, filtered and concentrated in vacuo to give a yellow oil. Purification of the crude material (SiO) by column chromatography₂0-50% EtOAc in heptane) to give the title compound as a clear oil (9.4mg, 33%).

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (trifluoromethoxy) benzenesulfonylurea (Compound 12)

N, N' - (3 α, 7 α -di-o-tert-butyldimethylsilyl-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -4- (trifluoromethoxy) benzenesulfonylurea (9.4mg) was dissolved in dry THF (1mL) under an argon atmosphere while stirring. A1M solution of TBAF in THF (31. mu.L, 0.03mmol) was added and the reaction stirred at RT for 6 days. The crude solution was dried on silica gel and purified by column chromatography (SiO)₂50-100% EtOAc in heptane) to give the title compound (1 mg).

N, N' - (3 α, 7 α -di-o-tert-butyldimethylsilyl-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-methoxybenzenesulfonylurea (intermediate 13)

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-methoxybenzenesulfonylurea (26.1mg, 0.042mmol) was dissolved in dry DCM (1mL) and cooled to 0 ℃ under an argon atmosphere while stirring. 2, 6-lutidine (0.05mL, 0.419mmol) was added followed by TBMDS-OTf (0.03mL, 0.126mmol) dropwise. The reaction was warmed to RT, stirred for 16h, then cooled to 0 ℃ by dropwise addition of 10% citric acid (1 mL). The phases were separated and the aqueous phase was extracted with DCM (3 × 1 mL). With 10% citric acid (1mL), NaHCO₃The combined extracts were washed with aqueous (1mL) and water (1mL) over MgSO₄Dried, filtered and concentrated in vacuo to give a yellow oil (28.1 mg). Purification of the crude material (SiO) by column chromatography₂0-80% MeOH in DCM) and then repurified by column chromatography (SiO)₂0-50% acetone in toluene) to give the title compound (7.8 mg).

N, N' - (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-methoxy and sulfonylurea (Compound 13)

N, N' - (3 α, 7 α -di-o-tert-butyldimethylsilyl-4 β -fluoro-6 α -ethyl-24-nor-5 β -cholan-23-yl) -p-methoxybenzenesulfonylurea (7.8mg) was dissolved in dry THF (1mL) under an argon atmosphere while stirring. A1M THF solution of TBAF (28. mu.L, 0.03mm0L) was added and the reaction was stirred at RT for 17 h. The crude solution was dried on silica gel and purified by column chromatography (SiO)₂0-80% acetone in toluene) to give the title compound (3.4mg, 59.6%).

¹H NMR(400MHz，CDCl₃)：δ7.97-7.81(2H，m)，7.11-7.02(2H，m)，5.30(1H，ddd，J＝49.6，10.3，8.9Hz)，3.88(3H，s)，3.76(1H，br.s)，3.42(1H，m)，3.15(1H，m)，3.06(1H，m)，1.99-0.96(26H，m)，0.95-0.80(6H，m)，0.64(3H，s)ppm。

EXAMPLE 3 Synthesis of Compounds with sulfonamide-substituted side chains

The following method exemplifies 4 β -fluoro derivatives, but may also be used for 2 β -fluorinated, 4-difluorinated or 2, 4-difluorinated compounds. Steps A and B are as described, for example, in FIG. 2.

C.6 alpha-ethyl-4 beta-fluoro-7 alpha-trimethylsiloxy-3 alpha-hydroxy-5 beta-cholanic acid

Methyl 6 α -ethyl-4 β -fluoro-7 α -trimethylsilyloxy-3 α -hydroxy-5 β -cholan-24-oate (162.3mg) from step B was dissolved in IPA (1.6mL) with stirring. 0.5M NaOH (1.6mL) was added and the reaction was stirred at RT for 16 h. The reaction mixture was concentrated under reduced pressure to-half volume, then water (5mL) was added by the addition of 2M H₂SO₄The solution was neutralized and diluted with EtOAc (10 mL). With 2M H₂SO₄The mixture was acidified to pH1, the phases were separated and the aqueous phase was extracted with EtOAc (10 mL). The combined extracts were washed with water (3mL) and brine (5mL), MgSO₄Dried, filtered and concentrated in vacuo to give a white foam (151.1 mg). Purification of the crude material (SiO) by column chromatography₂0-80% EtOAc in heptane) to give the title compound as a clear oil (164.1 mg).

General procedure for acyl sulfonamide side chain formation 4

6 α -Ethyl-4 β -fluoro-7 α -trimethylsiloxy-3 α -hydroxy-5 β -cholanic acid (50mg, 0.11mmol) was dissolved in dry DCM (2 mL). EDCI (43.7mg, 0.23mmol) and DMAP (27.8mg, 0.23mmol) were added followed by the appropriate sulfonamide (3 equiv.). After stirring overnight at RT, water (5mL) was added, the phases separated and the aqueous phase extracted with DCM (2 × 5 mL). The combined extracts were washed with 1M HCl (2mL) and brine (2mL), MgSO₄Drying, filtering, and vacuum concentrating to obtain crude material as yellow-white solid.

N- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -p-trifluoromethoxybenzenesulfonamide (compound 14)

Stirring while under argonCrude N- (6 α -ethyl-4 β -fluoro-7 α -trimethylsilyloxy-3 α -hydroxy-5 β -cholan-24-yl) -trifluoromethoxy benzenesulfonamide (81.6mg), obtained using trimethoxybenzenesulfonamide according to general procedure 4, was dissolved in dry THF (5mL) under an atmosphere. A1M solution of TBAF in THF (0.48mL, 0.48mmol) was added and the reaction stirred at RT for 17.5 h. The crude solution was dried on silica gel and purified by column chromatography (SiO)₂0-100% EtOAc in heptane). Fractions containing the desired product were combined, concentrated in vacuo, dissolved in EtOAc (5mL) and washed with 2M HCl (5 mL). The aqueous phase was extracted with EtOAc (2X 5mL) over MgSO₄The combined extracts were dried, filtered and concentrated in vacuo to give a white solid which was purified by column chromatography (SiO)₂0-25% acetone in toluene) to give the title compound as a clear residue (4.9 mg).

¹H NMR(400MHz，CDCl₃)：δ8.21-8.10(2H，m)，7.38-7.36(2H，dd，J＝8.9，0.8Hz)，5.31(1H，ddd，J＝49.8，10.4，9.0Hz)，3.82(1H，br.s)，3.56(1H，m)，2.31(1H，ddd，J＝15.6，10.1，5.0Hz)，2.17(1H，m)，1.92-1.07(23H，m)，0.97(3H，s)，0.93(3H，t，J＝6.9Hz)，0.86(3H，d，J＝6.4Hz)，0.62(3H，s)ppm。

N- (3 α, 7 α -dihydroxy-4 β -fluoro-6 α -ethyl-5 β -cholan-24-yl) -p-fluorobenzenesulfonamide (compound 15)