12-O-fatty acid ester oleanolic acid compounds and preparation method and use thereof

Abstract

The present invention belongs to the field of medical technology. It relates to a 12-O-fatty acid ester oleanolic acid compound acting on farnesoid X receptor (FXR) and its preparation method and use. Specifically, it provides a compound as shown in formula (I) and its pharmaceutically acceptable salt, diastereoisomer, the preparation method of these compounds, and the use of these compounds to regulate FXR activity for the treatment and / or prevention of FXR-mediated metabolic diseases such as diabetes, hyperlipidemia, non-alcoholic fatty hepatitis. #imgabs0# R in formula (I) is as defined in the specification.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology. Specifically, the present invention relates to an oleanolic acid derivative acting on farnesoid X receptor (FXR) and a preparation method and use thereof. More specifically, the present invention relates to 12-O-fatty acid ester oleanolic acid compounds and a preparation method and use thereof, as well as their pharmaceutically acceptable salts, diastereoisomers, preparation methods of these compounds, and the use of these compounds in drugs for regulating FXR activity and for treating and / or preventing FXR-mediated diseases. These FXR-mediated diseases include, but are not limited to, metabolic diseases such as diabetes, hyperlipidemia, and atherosclerosis, and hepatobiliary diseases such as cholestasis and non-alcoholic steatohepatitis. Background Art

[0002] The farnesoid X receptor (FXR) was discovered as an orphan nuclear receptor in 1995. In 1999, it was found that bile acids can activate FXR at physiological concentrations. As a receptor for bile acids, FXR maintains the balance of bile acids in the body by regulating the expression of genes involved in bile acid metabolism. In addition, FXR is also involved in the metabolism of lipids and carbohydrates. As a drug target, FXR is expected to provide new treatment options for diabetes, hyperlipidemia, non-alcoholic steatohepatitis, and even tumors and infectious diseases.

[0003] A variety of primary and secondary bile acids at physiological concentrations can activate FXR, among which chenodexycholic acid (CDCA) has the strongest agonist effect. However, the direct use of CDCA for related biological regulation and disease treatment is limited by its poor solubility and high toxicity. Based on the steroid structure, the FXR agonist 6-ethylchenodeoxycholic acid (6-ECDCA) was discovered. 6-ECDCA, also known as obeticholic acid (OCA), was approved by the FDA in 2016 for the treatment of primary biliary cirrhosis and is the first approved new drug targeting FXR. In addition, representative compounds of non-steroidal FXR agonists include GW4064 discovered earlier and Tropifexor and Nidufexor in clinical trials. Since potent FXR agonists in clinical trials can lead to side effects such as increased high-density lipoprotein and itching, there has been an increase in research on partial FXR agonists and even antagonists recently.

[0004] Existing studies have shown that since there are many downstream genes of FXR, selectively regulating some of the genes that are closely related to a certain disease can treat the disease and reduce side effects. Therefore, small molecule compounds that act on FXR and have a selective regulatory effect, also known as selective bile acid receptor modulators (SBARMs), can be used to develop molecules for the treatment of metabolic diseases and hepatobiliary diseases.

[0005] Plants containing oleanolic acid (OA) can be used to treat liver diseases in traditional medicine, and OA has also been approved in China for liver protection and auxiliary treatment of viral hepatitis (through mechanisms such as reducing alanine aminotransferase). In recent years, OA has also been found to have a selective regulatory effect on FXR [Liu W, Wong C. Phytotherapy Research 2010, 24: 369-373.], and can alleviate the symptoms of cholestasis in model animals by antagonizing FXR. However, OA is not very active, so we developed 12-substituted OA analogs based on the structure of this type of pentacyclic triterpenes after modification, which significantly enhanced the effect on FXR and had a selective regulatory effect on downstream genes. Disclosed in the present invention are 12-O-fatty acid ester oleanolic acid compounds that act on FXR, and their preparation methods and applications to FXR-related diseases. Summary of the invention

[0006] The present invention provides a class of compounds represented by formula (I), as well as their pharmaceutically acceptable salts and diastereoisomers, their preparation methods and their use in treating or preventing various FXR-related diseases.

[0007]

[0008] The present invention is achieved through the following technical solutions.

[0009] First, the present invention provides a 12-O-fatty acid ester oleanolic acid compound represented by formula (I), characterized in that the substituent R is selected from hydrogen, unsubstituted or substituted C1-7 alkyl,

[0010] The C1-7 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-n-butyl, n-pentyl, isopentyl, n-hexyl and n-heptyl.

[0011] The substituents of the C1-7 alkyl group are also selected from C1-5 alkanoyl, C1-5 alkanoyloxy, carboxyl, and C1-5 alkoxycarbonyl. Further, C1-5 alkanoyl is selected from acetyl, propionyl, butyryl, valeryl, and hexanoyl; C1-5 alkanoyloxy is selected from formyloxy, acetoxy, propionyloxy, butyryloxy, and valerylalkoxy; C1-5 alkoxycarbonyl is selected from methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, and pentaneoxycarbonyl;

[0012] The compound having the structure of formula (I) is further preferably 12β-O-hydroxy-13H-oleanane-3β-hydroxy-28-carboxylic acid substituted with n-valeryl, n-hexanoyl, acetylpropionyl, acetylbutyryl, (3-carboxy)propionyl, (3-carboxy)butyryl, methoxycarbonylacetyl, methoxycarbonylpropionyl, or methoxycarbonylbutyryl.

[0013] Secondly, the 12-O-fatty acid ester oleanolic acid compounds of the present invention can be synthesized by the method described in the following process and / or other techniques known to those of ordinary skill in the art, but are not limited to the following methods.

[0014]

[0015] The reaction steps are:

[0016] 1. The raw material OA is dissolved in an organic solvent (such as tetrahydrofuran, diethyl ether), and reacted with a benzyl halide (benzyl bromide, benzyl chloride) in the presence of an aqueous solution of a base (potassium carbonate, cesium carbonate, triethylamine, tributylamine, pyridine) to form the compound shown in Intermediate 1.

[0017] 2. Intermediate 1 is dissolved in an organic solvent (dichloromethane, N,N-dimethylformamide), and in the presence of a base (triethylamine, imidazole) and a silylating agent (tert-butyldimethylsilyl trifluoromethanesulfonate, tert-butyldimethylchlorosilane) to form the compound shown in Intermediate 2.

[0018] 3. Intermediate 2 is dissolved in an organic solvent (chloroform), an oxidant (m-chloroperbenzoic acid, tert-butyl hydroperoxide) is added, and the compound shown in Intermediate 3 is formed after post-treatment.

[0019] 4. Dissolve intermediate 3 in an organic solvent (tetrahydrofuran, diethyl ether), add a reducing agent (sodium borohydride, lithium borohydride) to form the compound shown in intermediate 4.

[0020] 5. Dissolve intermediate 4 in an organic solvent (dichloromethane, toluene, tetrahydrofuran, diethyl ether), and react with an appropriate acylating agent, wherein the acylating agent and the base are used, wherein the acylating agent is the corresponding carboxylic acid and the activating agent, the activating agent (N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the base is selected from pyridine, triethylamine, 4-dimethylaminopyridine), to form the compound shown in intermediate 5.

[0021] 6. Intermediate 5 is dissolved in an organic solvent (dichloromethane, acetonitrile) in the presence of a fluorine-containing reagent (hydrofluoric acid-pyridine, tetrabutylammonium fluoride) or an acid (hydrochloric acid, boron trifluoride-ether) to form a compound as shown in Intermediate 6.

[0022] 7. The intermediate 6 is dissolved in a mixed solvent of organic solvents (methanol, ethanol, tetrahydrofuran, diethyl ether), and a catalyst (Pd / C) and a hydrogen donor (hydrogen, 1,4-cyclohexadiene) are added to form a compound as shown in formula (I).

[0023] In the above reaction formula, R is as defined above.

[0024] The present invention provides a compound of formula (I) and a pharmaceutically acceptable salt thereof, which is used for preventing or treating one or more symptoms of a disease mediated by FXR by regulating the effect of FXR, wherein the effect of regulating FXR includes but is not limited to the antagonism of FXR agonists.

[0025] The FXR-mediated diseases involved in the present invention include, but are not limited to, metabolic diseases such as hypercholesterolemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipidemia, hyperglycemia, diabetes, insulin resistance, atherosclerosis, and hepatobiliary diseases such as non-alcoholic fatty hepatitis, liver fibrosis, hepatocellular carcinoma, cholestasis, and cholelithiasis.

[0026] Compared with the prior art, the present invention has the following significant effects:

[0027] The present invention has discovered a series of novel oleanolic acid analogs that act on FXR. As shown in the examples, some of the compounds can strongly antagonize the agonistic effect of FXR agonist CDCA, IC 50 The value reached 100nM, which had a selective regulatory effect on FXR downstream genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Example 8 improves glucose balance in KKay mice. KKay mice were fed a high-fat diet (HFD) for 6 weeks with or without OCA (10 mg / kg, qd) or Example 8 (100 mg / kg and 200 mg / kg, qd). (A) Mouse body weight during the 5-week treatment period. (B) Food intake curve. (C) Fasting blood glucose level. (D) Random blood glucose level. (E) Glycated hemoglobin HbA1c level. (F) 2-week OGTT results. (G) 4-week OGTT results. (H) Oral glucose tolerance-AUC. (I) 3-week ITT results. (J) 5-week ITT results. (k) Insulin tolerance-AUC. Data results are expressed as mean ± SD (n = 9) (*P < 0.05, **P < 0.01 and ***P < 0.001). DETAILED DESCRIPTION

[0029] The present invention is further illustrated by the following examples. These examples are not intended to limit the scope of the invention as defined or claimed above.

[0030] Example 1

[0031] Preparation of Oleanane-28-Benzyl Ester

[0032]

[0033] 21.6 mg of oleanolic acid was dissolved in 0.35 ml of tetrahydrofuran, and 12.9 mg of potassium carbonate, 13 μl of benzyl bromide and 8 μl of water were added at room temperature. The reaction was stopped after 4 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated and chromatographed on a silica gel column with petroleum ether: acetone = 9:1 as eluent to obtain 25.7 mg of the product with a yield of 99%. 1 HNMR (400MHz, CDCl3): δ7.56-7.26(m,5H),5.32(brs,1H),5.12(ABq,2H),3.24(m,1H),2.93(d,J=13 Hz,1H),1.18(s,1H),1.03(s,3H),0.97(s,3H),0.94(s,6H),0.92(s,3H),0.80(s,3H),0.66(s,3H).

[0034] Example 2

[0035] Preparation of 3β-tert-Butyldimethylsilyloleanane-28-benzyl ester

[0036]

[0037] Oleanolic acid-28-benzyl ester (12.5 g) was dissolved in CH2Cl2 (241 mL) and imidazole (8.50 g) was added. Tert-butyldimethylsilyl trifluoromethanesulfonate (16.6 g) was slowly added at room temperature and stirred overnight. The reaction mixture was washed with water (240 mL). The aqueous phase was extracted with CH2Cl2 (2×240 mL). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (2% ethyl acetate / petroleum ether) to give the product 3β-tert-butyldimethylsilyl oleanolic acid-28-benzyl ester in a yield of 81%. 1 H NMR (400MHz, CDCl3): 7.29-7.30(m,5H),5.24(s,1H),5.04(d,J=12.4Hz,1H),4.99(d,J=12.4Hz,1H),3.11-3.14(m,1H),2. 84-2.87(m,1H),1.07(s,3H),0.87(s,6H),0.85(s,3H),0.84(s,9H),0.82(s,3H),0.69(s,3H),0.50(s,3H),-0.02(s,6H).

[0038] Example 3

[0039] Preparation of 3β-tert-butyldimethylsilyl-12-oxo-oleanane-28-benzyl ester

[0040]

[0041] 3β-tert-Butyldimethylsilyloleanane-28-benzyl ester (12.5 g) was dissolved in dichloromethane (248 mL), and 85% meta-chloroperbenzoic acid (11.5 g) was added. The reaction was allowed to react at room temperature for about 9 h. After the reaction was complete, the mixture was quenched with a saturated aqueous sodium bisulfite solution (248 mL). The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (2×248 mL). The organic phases were combined and concentrated under reduced pressure. The concentrate was washed with a saturated sodium carbonate solution and a saturated saline solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The product 3β-tert-butyldimethylsilyl-12-oxo-oleanane-28-benzyl ester was purified by silica gel column chromatography (5% ethyl acetate / petroleum ether) to obtain the product 3β-tert-butyldimethylsilyl-12-oxo-oleanane-28-benzyl ester with a yield of 63%. 1 H NMR(400MHz, CDCl3):7.31-7.33(m,5H),5.05-5.21(m,2H),3.12-3.15(m,1H),2.77-2.80(m,1H),2.42-2.45(m,1H),2.1 7-2.19(m,1H),1.12(s,3H),0.92(s,3H),0.90(s,3H),0.88(s,3H),0.87(s,12H),0.77(s,3H),0.61(s,3H),0.03(s,6H).

[0042] Example 4

[0043] Preparation of 3β-tert-butyldimethylsilyl-12-hydroxy-oleanane-28-benzyl ester

[0044]

[0045] 24.3 mg of 3β-tert-butyldimethylsilyl-12-oxo-oleanane-28-benzyl ester was dissolved in 0.54 ml of tetrahydrofuran, 4.1 mg of sodium borohydride and 54 μl of water were added, and the reaction was terminated after 11 hours of reaction at room temperature. The reaction solution was adjusted to a neutral pH value with a saturated aqueous solution of ammonium chloride, and the organic layer was washed with 3×10 ml of water and 10 ml of a saturated aqueous solution of sodium chloride in turn, and the aqueous layer was extracted with 3×10 ml of dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate. After the organic layer was concentrated, it was chromatographed on a silica gel column, and eluted with petroleum ether: acetone = 50:1 to obtain 8.6 mg of 3β-tert-butyldimethylsilyl-12α-hydroxy-oleanane-28-benzyl ester, with a yield of 35%; 15.7 mg of 3β-tert-butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester, with a yield of 64%.

[0046] 3β-tert-Butyldimethylsilyl-12α-hydroxy-oleanane-28-benzyl ester:1 HNMR (400MHz, CDCl3): δ7.24-7.39(m,5H),5.13(ABq,2H),3.99(brs,1H),3.17(dd,J=6.4Hz,1H),2.49(d,J=12H z,1H),0.97(s,3H),0.96(s,3H),0.88(s,12H),0.83(s,3H),0.79(s,3H),0.71(s,3H),0.60(s,3H),0.02(s,6H).

[0047] 3β-tert-Butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester: 1 HNMR (400MHz, CDCl3): δ7.29-7.42(m,5H),5.15(ABq,2H),3.67(m,1H),3.14(dd,J=6.8Hz,1H),2.74 (m,1H),0.92(s,6H),0.89(s,12H),0.85(s,3H),0.77(s,3H),0.70(s,3H),0.60(s,3H),0.02(s,6H).

[0048] Example 5

[0049] Preparation of 3β-Hydroxy-12β-n-Valeryloxy-Oleanane-28-Carboxylic Acid (7a)

[0050]

[0051] (1) 100 mg of 3β-tert-butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester was dissolved in 3 mL of anhydrous dichloromethane, followed by the addition of 183 mg of 4-dimethylaminopyridine, 76 μL of n-pentanoic acid and 115 μL of N,N'-diisopropylcarbodiimide. The reaction mixture was reacted at room temperature for 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was concentrated under reduced pressure and then diluted with 30 mL of ethyl acetate, washed with 2×10 mL of water and 10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the product 3β-tert-butyldimethylsilyloxy-12β-pentanoyloxy-oleanane-28-benzyl ester was obtained by silica gel column chromatography (dichloromethane: methanol = 200: 1). (2) The product (191 mg) was dissolved in 5.6 mL of anhydrous dichloromethane. Under argon protection at 0°C, 1.4 mL of boron trifluoride ether was slowly added. The reaction was carried out at 0°C for about 2.5 h and monitored by TLC. After the reaction was complete, the reaction mixture was diluted with 25 mL of dichloromethane, washed with 10 mL of water-saturated 2×10 mL of brine, and the organic phase was dried over anhydrous sodium sulfate. The organic solvent was removed by concentration under reduced pressure and the product 3β-hydroxy-12β-n-pentanoyloxy-oleanane-28-benzyl ester was obtained by purification by silica gel column chromatography (petroleum ether:acetone=5:1). (3) The product (80 mg) was dissolved in 1 mL of tetrahydrofuran and 1 mL of methanol, and 45 mg of 10% palladium on carbon was added to the reaction mixture. The reaction was allowed to react for about 12 h at room temperature in the presence of hydrogen and monitored by TLC. After the reaction was complete, the reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 3×12 mL of 6% methanol / dichloromethane. The organic layer was concentrated under reduced pressure and purified by flash silica gel column chromatography (petroleum ether: acetone = 3:1) to obtain the product 3β-hydroxy-12β-n-pentanoyloxy-oleanane-28-carboxylic acid (7a). The total yield of the three steps was 71%.

[0052] 1 H NMR (400MHz, CDCl3): δ4.95-5.00(m,1H),3.48(s,4H),3.19(dd,J=4.8Hz,11.2Hz,1H),2.51-2.54(m,1H),2.27-2.31(m,2H),2.07(dd, J=4.8Hz,11.6Hz,1H),1.06-1.92(m,25H),1.01(s,3H),0.96(s,3H),0.91(s,3H),0.89(s,3H),0.82(s,3H),0.81(s,3H),0.74(s,3H); 13C NMR (100MHz, CDCl3): δ183.8,173.7,78.8,77.3,71.4,55.2,48.8,47.3,41.5,40.7,40.0,38.9,38.6,37.1,36.0,34.3,33.3, 33.2,32.5,31.4,30.5,28.8,28.1,27.4,27.2,27.1,23.3,22.9,22.5,18.3,17.8,16.5,15.9,15.4,13.8.HRMS(ESI-TOF)m / z calcd.for C 35 H 58 O5[M+Na] + :581.4176,found 581.4174.

[0053] Example 6

[0054] Preparation of 3β-Hydroxy-12β-n-hexanoyloxy-oleanane-28-carboxylic acid (7b)

[0055]

[0056] (1) 3β-tert-Butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester (300 mg) and hexanoic acid (137 mg) were dissolved in toluene (8.90 ml), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (677 mg) and 4-dimethylaminopyridine (108 mg). The reaction mixture was stirred at 60° C. for 3 h under argon. The reaction mixture was concentrated in vacuo and then diluted with dichloromethane (18.0 mL), washed with H2O (2×9.00 mL) and brine (9.00 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (2) The crude product was diluted with CH2Cl2 (5.65 mL), and BF3Et2O (1.97 mL) was slowly added at 0°C under argon. The mixture was stirred at 0°C for 3 h. The reaction mixture was diluted with CH2Cl2 (12.0 mL), washed with H2O (6.00 mL) and brine (2×16.0 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (3) The crude product was dissolved in THF (10.8 mL) and ethanol (10.8 mL), and 19 mg of 10% Pd / C was added to the reaction mixture. The mixture was stirred at 30°C under hydrogen for 5 h. The reaction mixture was filtered with diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 6% methanol / CH2Cl2 (3×12.0 mL). The organic layer was concentrated in vacuo, and the residue was purified by flash column chromatography (6% MeOH / CH2Cl2) to obtain the product (7b). The total yield of the three-step reaction was 65%. 1 H NMR (500MHz, CD3OD): δ5.00(td,J=10.8,5.6Hz,1H),3.15(dd,J=10.6,5.4Hz,1H),2.57(d,J=13.7Hz,1H),2.31(hept,J=7.9,7.4Hz,2H),2.1 5(dd,J=11.8,4.8Hz,1H),1.06(s,3H),0.97(s,3H),0.96(s,3H),0.91 (s,3H),0.86(s,3H),0.84(s,3H),0.76(s,3H),0.74(d,J=11.3Hz,1H); 13C NMR (125MHz, CD3OD): δ181.90,175.34,79.43,72.97,56.60,50.08,48.21,4 2.67,41.82,41.20,39.92,39.82,38.16,37.38,35.45,35.38,34.47,33.87 ,33.68,32.81,32.60,31.47,29.93,28.65,28.52,27.91,25.75,24.08,23. 96,23.40,19.40,18.30,16.94,16.42,16.12,14.30; HRMS(ESI)calculated for C 36 H 60 O5[M+Na] + :595.4333, found 595.4324.

[0057] Example 7

[0058] Preparation of 3β-Hydroxy-12β-levulinyloxy-oleanane-28-carboxylic acid (7c)

[0059]

[0060] (1) 100 mg of 3β-tert-butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester was dissolved in 3 mL of anhydrous dichloromethane, followed by the addition of 183 mg of 4-dimethylaminopyridine, 76 μL of 4-methyl-4-oxobutyric acid and 115 μL of N,N'-diisopropylcarbodiimide. The reaction mixture was reacted at room temperature for 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was concentrated under reduced pressure and then diluted with 30 mL of ethyl acetate, washed with 2×10 mL of water and 10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the product 3β-tert-butyldimethylsilyloxy-12β-levulinyloxy-oleanane-28-benzyl ester was obtained by silica gel column chromatography (dichloromethane: methanol = 200: 1). The product (191 mg) was dissolved in 5.6 mL of anhydrous dichloromethane. Under argon protection at 0°C, 1.4 mL of boron trifluoride ether was slowly added. The reaction was carried out at 0°C for about 2.5 h and monitored by TLC. After the reaction was complete, the reaction mixture was diluted with 25 mL of dichloromethane, washed with 10 mL of water and 2×10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The organic solvent was removed by concentration under reduced pressure and purified by silica gel column chromatography (petroleum ether: acetone = 5: 1) to obtain the product 3β-hydroxy-12β-levulinyloxy-oleanane-28-benzyl ester. (3) The product (80 mg) was dissolved in 1 mL of tetrahydrofuran and 1 mL of methanol, and 45 mg of 10% palladium on carbon was added to the reaction mixture. The reaction was allowed to react for about 12 h at room temperature in the presence of hydrogen and monitored by TLC. After the reaction was complete, the reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 3×12 mL of 6% methanol / dichloromethane. The organic layer was concentrated under reduced pressure and purified by flash silica gel column chromatography (petroleum ether: acetone = 3:1) to obtain the product 3β-hydroxy-12β-levulinyloxy-oleanane-28-carboxylic acid (7c). The total yield of the three steps was 65%.

[0061] 1 H-NMR (400MHz, CDCl3): δ4.93-4.98(m,1H),3.17-3.21(m,1H),2.71-2.75(m,2H),2.50-2.59(m,3H),2.19(s,3H),2.08(dd ,J=4.8Hz,11.6Hz,1H),1.05-1.91(m,22H),0.99(s,3H),0.96(s,3H),0.90(s,6H),0.85(s,3H),0.81(s,3H),0.74(s,3H). 13C-NMR (100MHz, CDCl3): δ206.7,184.2,172.5,78.8,72.2,55.2,48.8,47.3,41.4,40.6,39.9,38.8,38.6,37.9,37.1,36.0,34.3,33. 3,33.2,32.4,31.3,30.5,29.9,28.8,28.3,28.0,27.3,27.2,23.2,22.9,18.2,17.7,16.5,15.9,15.4.HRMS(ESI-TOF)m / zcalcd.for C 35 H 56 O6[M+Na] + :595.3969,found 595.3966.

[0062] Example 8

[0063] Preparation of 3β-Hydroxy-12β-acetylbutyryloxy-oleanane-28-carboxylic acid (7d)

[0064]

[0065] (1) 3β-tert-Butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester (300 mg) and 5-methyl-5-oxopentanoic acid (153 mg) were dissolved in toluene (8.90 ml), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (677 mg) and 4-dimethylaminopyridine (108 mg). The reaction mixture was stirred at 60° C. under argon for 3 h. The reaction mixture was concentrated in vacuo and then diluted with dichloromethane (18.0 mL), washed with H2O (2×9.00 mL) and brine (9.00 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (2) The crude product was diluted with CH2Cl2 (5.65 mL), and BF3Et2O (1.97 mL) was slowly added at 0°C under argon. The mixture was stirred at 0°C for 3 h. The reaction mixture was diluted with CH2Cl2 (12.0 mL), washed with H2O (6.00 mL) and brine (2×16.0 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (3) The crude product was dissolved in THF (10.8 mL) and ethanol (10.8 mL), and 19 mg of 10% Pd / C was added to the reaction mixture. The mixture was stirred at 30°C under hydrogen for 5 h. The reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 6% methanol / CH2Cl2 (3×12.0 mL). The organic layer was concentrated in vacuo and the residue was purified by flash column chromatography (6% MeOH / CH2Cl2) or liquid chromatography (5% MeOH / CH2Cl2) to give the product (7d). The total yield of the three steps was 62%.

[0066] 1 H NMR (500MHz, CD3OD): δ5.08–5.00(m,1H),3.17(dt,J=10.9,4.5Hz,1H),2.65–2.53(m,3H),2.37(t,J=6.9Hz,2H),2.16(s ,4H),1.07(s,3H),0.99(s,3H),0.98(s,3H),0.93(s,3H),0.88(s,3H),0.87(s,3H),0.78(s,3H),0.75(d,J=10.6Hz,1H); 13C NMR (125MHz, CD3OD): δ210.88,181.86,174.79,79.46,73.17,56.63,50.1 1,48.23,43.30,42.69,41.83,41.23,39.93,39.81,38.17,37.40,35.35,3 4.48,33.80,33.66,32.87,31.46,29.93,29.84,28.62,28.53,27.93,24.05,23.97,20.03,19.39,18.22,16.92,16.39,16.09; HRMS(ESI)calculated for C 36 H 58 O6[M+Na] + :609.4126,found609.4104.

[0067] Example 9

[0068] Preparation of 3β-Hydroxy-12β-(3-carboxyl)propionyloxy-oleanane-28-carboxylic acid (7e)

[0069]

[0070] (1) 100 mg of 3β-tert-butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester was dissolved in 3 mL of anhydrous dichloromethane, followed by the addition of 183 mg of 4-dimethylaminopyridine, 76 μL of 3-carboxypropionic acid and 115 μL of N,N'-diisopropylcarbodiimide. The reaction mixture was reacted at room temperature for 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was concentrated under reduced pressure and then diluted with 30 mL of ethyl acetate, washed with 2×10 mL of water and 10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the product 3β-tert-butyldimethylsilyloxy-12β-(3-carboxy)propionyloxy-oleanane-28-benzyl ester was obtained by silica gel column chromatography (dichloromethane:methanol=200:1). The product (191 mg) was dissolved in 5.6 mL of anhydrous dichloromethane, and 1.4 mL of boron trifluoride ether was slowly added under argon protection at 0°C. The reaction was carried out at 0°C for about 2.5 h and monitored by TLC. After the reaction was complete, the reaction mixture was diluted with 25 mL of dichloromethane, washed with 10 mL of water and 2×10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic solvent, and purified by silica gel column chromatography (petroleum ether: acetone = 5:1) to obtain the product 3β-hydroxy-12β-(3-carboxyl)propionyloxy-oleanane-28-benzyl ester. (3) The product (80 mg) was dissolved in 1 mL of tetrahydrofuran and 1 mL of methanol, and 45 mg of 10% palladium on carbon was added to the reaction mixture. The reaction was allowed to react for about 12 h at room temperature in the presence of hydrogen and monitored by TLC. After the reaction was complete, the reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 3×12 mL of 6% methanol / dichloromethane. The organic layer was concentrated under reduced pressure and purified by flash silica gel column chromatography (petroleum ether: acetone = 3:1) to obtain the product 3β-hydroxy-12β-(3-carboxyl)propionyloxy-oleanane-28-carboxylic acid (7e). The total yield of the three steps was 65%.

[0071] 1 H-NMR (400MHz, CDCl3): δ8.19(s,1H),4.97-4.98(m,1H),3.22-3.24(m,1H),2.49-2.69(m,5H),2.07-2.11(m,1H) ,1.06-1.96(m,22H),0.99(s,3H),0.96(s,3H),0.91(s,3H),0.88(s,3H),0.83(s,3H),0.81(s,3H),0.75(s,3H). 13C-NMR (100MHz, CDCl3): δ184.3,178.1,171.9,78.8,72.5,55.1,48.7,47.2,41.4,40.6,39.9,38.9,38.6,37.1,34.3,33.3,3 2.4,31.9,31.3,30.5,29.3,28.0,26.9,23.1,22.7,22.6,20.2,19.2,18.3,17.8,16.4,15.9,15.6,14.1.HRMS(ESI-TOF)m / z calcd.for C 34 H 54 O7[M+Na] + :597.3762,found597.3763.

[0072] Example 10

[0073] Preparation of 3β-Hydroxy-12β-(3-carboxyl)butyryloxy-oleanane-28-carboxylic acid (7f)

[0074]

[0075] (1) 100 mg of 3β-tert-butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester was dissolved in 3 mL of anhydrous dichloromethane, followed by the addition of 183 mg of 4-dimethylaminopyridine, 76 μL of 4-carboxybutyric acid and 115 μL of N,N'-diisopropylcarbodiimide. The reaction mixture was reacted at room temperature for 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was concentrated under reduced pressure and then diluted with 30 mL of ethyl acetate, washed with 2×10 mL of water and 10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the product 3β-tert-butyldimethylsilyloxy-12β-(3-carboxy)butyryloxy-oleanane-28-benzyl ester was obtained by silica gel column chromatography (dichloromethane:methanol=200:1). (2) The product (191 mg) was dissolved in 5.6 mL of anhydrous dichloromethane. Under argon protection at 0°C, 1.4 mL of boron trifluoride ether was slowly added. The reaction was carried out at 0°C for about 2.5 h and monitored by TLC. After the reaction was complete, the reaction mixture was diluted with 25 mL of dichloromethane, washed with 10 mL of water and 2×10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The organic solvent was removed by concentration under reduced pressure and the product 3β-hydroxy-12β-(3-carboxyl)butyryloxy-oleanane-28-benzyl ester was obtained by purification by silica gel column chromatography (petroleum ether:acetone=5:1). (3) The product (80 mg) was dissolved in 1 mL of tetrahydrofuran and 1 mL of methanol, and 45 mg of 10% palladium on carbon was added to the reaction mixture. The reaction was allowed to react in the presence of hydrogen at room temperature for about 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 3×12 mL of 6% methanol / dichloromethane. The organic layer was concentrated under reduced pressure and purified by flash silica gel column chromatography (petroleum ether: acetone = 3:1) to obtain the product 3β-hydroxy-12β-(3-carboxyl)butyryloxy-oleanane-28-carboxylic acid (7f). The total yield of the three steps was 57%.

[0076] 1 H-NMR (400MHz, CDCl3): δ4.96-5.01(m,1H),3.19(dd,J=4.4Hz,J=11.2Hz,1H),2.33-2.57(m,5H),2.07(dd,J=4.8Hz,J=11 .6Hz,1H),1.05-1.99(m,24H),1.01(s,3H),0.97(s,3H),0.94(s,3H),0.90(s,3H),0.83(s,3H),0.82(s,3H),0.75(s,3H). 13C-NMR (100MHz, CDCl3): δ185.4,179.9,172.5,78.8,72.1,55.2,48.7,47.4,41.5,40.6,40.3,38.9,38.6,37.1,36.1,34.3,34. 1,33.6,33.3,32.5,31.3,30.5,28.7,28.0,27.4,27.2,26.9,23.4,22.8,20.0,18.3,17.8,16.5,15.9,15.4.HRMS(ESI-TOF)m / z calcd.forC 35 H 56 O7[M+Na] + :611.3918,found 611.3918.

[0077] Embodiment 11

[0078] Preparation of 3β-hydroxy-12β-methoxycarbonylacetyloxy-oleanane-28-carboxylic acid (7 g)

[0079]

[0080] (1) 100 mg of 3β-tert-butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester was dissolved in 3 mL of anhydrous dichloromethane, followed by the addition of 183 mg of 4-dimethylaminopyridine, 76 μL of 3-methoxy-3-oxopropionic acid and 115 μL of N,N'-diisopropylcarbodiimide. The reaction mixture was reacted at room temperature for 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was concentrated under reduced pressure and then diluted with 30 mL of ethyl acetate, washed with 2×10 mL of water and 10 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the product 3β-tert-butyldimethylsilyloxy-12β-methoxycarbonylacetoxy-oleanane-28-benzyl ester was obtained by silica gel column chromatography (dichloromethane:methanol=200:1). (2) The product (191 mg) was dissolved in 5.6 mL of anhydrous dichloromethane. Under argon protection at 0°C, 1.4 mL of boron trifluoride ether was slowly added. The reaction was carried out at 0°C for about 2.5 h and monitored by TLC. After the reaction was complete, the reaction mixture was diluted with 25 mL of dichloromethane, washed with 10 mL of water and 2×10 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the organic solvent. The product 3β-hydroxy-12β-methoxycarbonylacetoxy-oleanane-28-benzyl ester was purified by silica gel column chromatography (petroleum ether:acetone=5:1). (3) The product (80 mg) was dissolved in 1 mL of tetrahydrofuran and 1 mL of methanol, and 45 mg of 10% palladium on carbon was added to the reaction mixture. The reaction was carried out at room temperature in the presence of hydrogen for about 12 h and monitored by TLC. After the reaction was complete, the reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 3×12 mL of 6% methanol / dichloromethane. The organic layer was concentrated under reduced pressure and purified by flash silica gel column chromatography (petroleum ether: acetone = 3:1) to obtain the product 3β-hydroxy-12β-methoxycarbonylacetoxy-oleanane-28-carboxylic acid (7 g). The total yield of the three steps was 25%.

[0081] 1 H-NMR (400MHz, CDCl3): δ5.14(s,1H),3.75(s,3H),3.33-3.35(m,1H),3.21(dd,J=3.6Hz,10.8Hz,1H),2.41-2.44(m, 1H),1.12-2.04(m,24H),1.10(s,3H),0.98(s,3H),0.91(s,3H),0.89(s,3H),0.85(s,3H),0.83(s,3H),0.75(s,3H). 13C-NMR (100MHz, CDCl3): δ186.2,169.0,168.3,78.74,75.44,55.2,52.4,48.7,47.2,41.5,41.4,40.6,39.8,38.8,38.5,37.1, 36.0,34.3,33.2,33.1,32.3,31.2,30.4,28.8,28.0,27.1,27.0,23.1,22.8,18.2,17.7,16.4,15.8,15.3.HRMS(ESI-TOF)m / z calcd.for C 34 H 54 O7[M+Na] + :597.3762,found 597.3765.

[0082] Example 12

[0083] Preparation of 3β-Hydroxy-12β-methoxycarbonylpropionyloxy-oleanane-28-carboxylic acid (7h)

[0084]

[0085] (1) 3β-tert-Butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester (300 mg) and 4-methoxy-4-oxobutyric acid (156 mg) were dissolved in toluene (8.90 ml), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (677 mg) and 4-dimethylaminopyridine (108 mg). The reaction mixture was stirred at 60° C. for 3 h under argon. The reaction mixture was concentrated in vacuo and then diluted with dichloromethane (18.0 mL), washed with H2O (2×9.00 mL) and brine (9.00 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (2) The crude product was diluted with CH2Cl2 (5.65 mL), and BF3Et2O (1.97 mL) was slowly added at 0°C under argon. The mixture was stirred at 0°C for 3 h. The reaction mixture was diluted with CH2Cl2 (12.0 mL), washed with H2O (6.00 mL) and brine (2×16.0 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (3) The crude product was dissolved in THF (10.8 mL) and ethanol (10.8 mL), and 19 mg of 10% Pd / C was added to the reaction mixture. The mixture was stirred at 30°C under hydrogen for 5 h. The reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 6% methanol / CH2Cl2 (3×12.0 mL). The organic layer was concentrated in vacuo and the residue was purified by flash column chromatography (6% MeOH / CH2Cl2) or liquid chromatography (5% MeOH / CH2Cl2) to give the product (7h). The total yield of the three steps was 24%.

[0086] 1 H NMR (500MHz, CD3OD): δ4.99(td,J=10.6,5.5Hz,1H),3.68(s,3H),3.14(dd,J=10.7,5.4Hz,1H),2.72–2.48(m,5H),2 .15(dd,J=11.8,4.9Hz,1H),1.05(s,3H),0.96(d,J=3.5Hz,6H),0.91(s,3H),0.87(s,3H),0.86(s,3H),0.76(s,3H); 13C NMR (125MHz, CD3OD): δ181.90,174.42,173.87,79.44,73.59,56.62,52.2 5,50.13,48.20,42.67,41.84,41.14,39.92,39.81,38.17,37.34,35.41,3 4.47,33.78,33.66,32.80,31.46,30.28,29.97,29.69,28.63,28.43,27.91,24.08,23.68,19.39,18.25,16.93,16.43,16.10.HRMS(TOF)calculated for C 35 H 56 O7[M+Na] + :611.3918, found 611.3896.

[0087] Embodiment 13

[0088] Preparation of 3β-Hydroxy-12β-Methoxycarbonylbutyryloxy-Oleanane-28-Carboxylic Acid (7i)

[0089]

[0090] (1) 3β-tert-Butyldimethylsilyl-12β-hydroxy-oleanane-28-benzyl ester (300 mg) and 5-methoxy-5-oxopentanoic acid (172 mg) were dissolved in toluene (8.90 ml), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (677 mg) and 4-dimethylaminopyridine (108 mg). The reaction mixture was stirred at 60° C. for 3 h under argon. The reaction mixture was concentrated in vacuo and then diluted with dichloromethane (18.0 mL), washed with H2O (2×9.00 mL) and brine (9.00 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (2) The crude product was diluted with CH2Cl2 (5.65 mL), and BF3Et2O (1.97 mL) was slowly added at 0°C under argon. The mixture was stirred at 0°C for 3 h. The reaction mixture was diluted with CH2Cl2 (12.0 mL), washed with H2O (6.00 mL) and brine (2×16.0 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The product was directly used for the next step without chromatographic purification. (3) The crude product was dissolved in THF (10.8 mL) and ethanol (10.8 mL), and 19 mg of 10% Pd / C was added to the reaction mixture. The mixture was stirred at 30°C under hydrogen for 5 h. The reaction mixture was filtered through diatomaceous earth filter aid, and the diatomaceous earth filter aid was washed with 6% methanol / CH2Cl2 (3×12.0 mL). The organic layer was concentrated in vacuo and the residue was purified by flash column chromatography (6% MeOH / CH2Cl2) or liquid chromatography (5% MeOH / CH2Cl2) to give the product (7i). The total yield of the three steps was 32%.

[0091] 1 H NMR (500MHz, CDCl3): δ5.12–4.90(m,1H),3.68(s,3H),3.20(dd,J=11.5,4.4Hz,1H),2.58–2.44(m,1H),2.38(dt,J=21.7 ,7.3Hz,4H),2.08(dd,J=11.7,4.9Hz,1H),1.01(s,3H),0.97(s,3H),0.92(s,3H),0.90(s,3H),0.82(s,6H),0.75(s,3H); 13CNMR (100MHz, CDCl3): δ183.24,173.58,172.80,78.92,71.93,55.30,51.76 ,48.88,47.35,41.57,40.76,40.08,38.98,38.69,37.22,36.19,34.42,33.6 6,33.41,33.38,33.33,32.53,31.50,30.59,28.93,28.15,27.51,27.33,23 .44,23.04,20.26,18.35,17.88,16.59,15.99,15.48; HRMS(TOF)calculated for C 36 H 58 O7[MH] - :601.4110,found601.4114. Pharmacological experiments:

[0092] Experimental Example 1 Testing the effect of FXR at the cellular level

[0093] Take logarithmically growing 293T cells and inoculate them into 96-well plates at 10,000 / well. Use phenol red-free DMEM (70ul / well) + 5% activated carbon-treated FBS to culture for 24h. Use expression vector pGAL4-DBD-binding domain pBIND-FXRα (10ng / well), reporter gene vector pGL4.35[luc2P / 9X GAL4UAS / Hygro] (50ng / well), and high-efficiency transfection reagent FuGENE HD Transfection Reagent (0.3μl / well) for transfection, and perform transient transfection according to the transfection mixture of 10μL / well. 24 hours after transfection, use MEM culture medium containing 0.5% activated carbon-treated FBS with CDCA (50μM), and then add DMSO solution of the test compound. After continuing to culture for 24 hours, detect Luciferase enzyme activity according to the instructions of Dual-Luciferase reporter gene. RL enzyme activity is used as internal reference.

[0094] Table 1 FXR antagonistic activity of compounds in the examples

[0095]

[0096] Experimental Example 2 Pharmacodynamics experiment of the compound of Example 8 on blood glucose in type 2 diabetic KKAy mice

[0097] Spontaneous type 2 diabetic KKAy mice, female (purchased from the Animal Center of the Chinese Academy of Medical Sciences) were used for animal experiments. According to the insulin tolerance test, the mice were randomly divided into two groups according to the percentage of blood glucose decrease in 40 minutes, fasting blood glucose, body weight, plasma triglyceride and cholesterol levels.

[0098] The two groups of KKAy mice were respectively the negative control group (Con group), which was gavaged with water (0.05 ml / 10 g body weight); the compound group, which was gavaged with drugs (0.05 ml / 10 g body weight, dose of 100 mg / kg, 200 mg / kg). The control group, the obeticholic acid (OCA) group, was gavaged with drugs (0.05 ml / 10 g body weight, dose of 10 mg / kg) once a day for 42 consecutive days. During the experiment, the body weight was measured regularly and the amount of food and water intake was recorded every day.

[0099] Fasting blood glucose and random blood glucose levels were measured during the 1st to 5th week of administration.

[0100] Insulin tolerance test (ITT) was performed at 3 and 5 weeks after administration.

[0101] Oral glucose tolerance test (OGTT) was performed at the 2nd and 4th week of administration.

[0102] Blood HbA1C was measured at 4 and 5 weeks after administration.

[0103] The drug was stopped and the animals were processed at the 6th week of administration. The liver tissue was taken and weighed. Various biochemical indicators were tested. The specific results are shown in Figure 1 .

[0104] In summary, Example 8 has a reducing effect on the non-fasting blood glucose, fasting blood glucose and glycosylated hemoglobin levels of KKay mice, and has an improving effect on insulin tolerance and oral glucose tolerance.

Claims

1. A 12-O-fatty acid ester oleanolic acid compound represented by formula (I) or a pharmaceutically acceptable salt thereof: R is selected from unsubstituted or substituted C1-7 alkyl, The substituted substituent is selected from C1-5 alkanoyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, It is characterized in that The C1-7 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-n-butyl, n-pentyl, isopentyl, n-hexyl and n-heptyl.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, Features: The C1-5 alkanoyl group is selected from acetyl, propionyl, butyryl and valeryl.

4. A compound or a pharmaceutically acceptable salt thereof, It is characterized in that The compound is selected from:

5. A method for preparing the compound according to any one of claims 1 to 4, It is characterized in that The method comprises the following preparation steps: Step 1: Oleanolic acid and benzyl halide are reacted with a base to obtain intermediate 1, wherein the benzyl halide is selected from benzyl bromide and benzyl chloride, and the base is selected from potassium carbonate, cesium carbonate, triethylamine, tributylamine, and pyridine; Step 2: Intermediate 1 is reacted with a silylating agent under the action of a base to obtain intermediate 2, wherein the base is selected from triethylamine or imidazole; and the silylating agent is selected from tert-butyldimethylsilyl trifluoromethanesulfonate and tert-butyldimethylchlorosilane; Step 3: The 12,13-double bond of intermediate 2 is oxidized under the action of an oxidant to obtain intermediate 3, wherein the oxidant is selected from m-chloroperbenzoic acid and tert-butyl hydroperoxide; Step 4: Reducing the carbonyl group of intermediate 3 under the action of a reducing agent to obtain intermediate 4, wherein the reducing agent is selected from sodium borohydride and lithium borohydride; Step 5: Intermediate 4 is condensed with an acylating agent to obtain intermediate 5, the reagents used are an acylating agent and a base, wherein the acylating agent is a corresponding carboxylic acid and an activating agent, the activating agent is selected from N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the base is selected from pyridine, triethylamine, 4-dimethylaminopyridine; Step 6: intermediate 5 is subjected to the action of a fluorine-containing reagent or an acid reagent to remove the 28-position protecting group to obtain intermediate 6, wherein the fluorine-containing reagent is selected from hydrofluoric acid-pyridine and tetrabutylammonium fluoride, and the acid reagent is selected from hydrochloric acid and boron trifluoride-ether; Step 7: The intermediate 6 and a hydrogen donor are treated with a catalyst to remove the 3-position protecting group to obtain a compound of formula (I), wherein the catalyst is selected from Pd / C; the hydrogen donor is selected from hydrogen and 1,4-cyclohexadiene.

6. A pharmaceutical composition, It is characterized in that The composition comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing FXR-mediated diseases.

8. The use according to claim 7, It is characterized in that The FXR-mediated disease is selected from hyperlipoproteinemia, diabetes, dyslipidemia or atherosclerosis.

9. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a liver protection drug or a drug for treating hepatobiliary diseases.

10. The use according to claim 9, It is characterized in that The liver protection is selected from the liver protection effect produced by reducing the levels of aspartate aminotransferase and alanine aminotransferase, and the hepatobiliary disease is selected from non-alcoholic fatty hepatitis, liver fibrosis, hepatocellular carcinoma, cholestasis or cholelithiasis.

Images