Use of oxime-based cholic acid analogs as allosteric agonists for TGR5

Abstract

The present invention relates to the field of medicinal chemistry. Specifically disclosed is a use of oxime-based cholic acid (CA) analogs as allosteric agonists for TGR5. In the present invention, CA used as a raw material undergoes a series of reactions to obtain CA intermediates each having an oxime at either or both of positions 7 and 12, and a series of modifications are then carried out on positions 24 and the oximes of these intermediates to obtain CA analogs as represented by formula 1, formula 2, or formula 3. The present invention uses a GloSensor cAMP accumulation assay to test the functional activity and allosteric mechanism of all target compounds on TGR5. Pharmacological results have shown that all synthesized target compounds have relatively good agonistic activity on TGR5 and can positively allosterically regulate the functional activity of chenodeoxycholic acid (CDCA) or lithocholic acid (LCA), that is, the target compounds are all positive allosteric modulators or allosteric agonists for TGR5.

Description

Application of oxime cholic acid analogues as TGR5 allosteric agonists TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and specifically discloses application of cholic acid analogues as allosteric agonists of G protein-coupled bile acid receptor 1 (TGR5). BACKGROUND

[0002] The bile acid receptor TGR5 (Takeda G protein-coupled receptor 5) is an important member of class A GPCRs, plays an important role in blood glucose homeostasis, energy consumption and liver protection, and is a very potential drug target for treating metabolic syndrome and inflammatory diseases, so developing drugs for treating metabolic diseases with TGR5 as a target has great research value.

[0003] In the preliminary structure-activity relationship study of cholic acid CA, a potent TGR5 agonist L2 (Bioorg. Chem. 2022, 120, 105588) was found. Studies have shown that the 12-OH-retained analogue L1 can positively allosterically modulate the functional activity of the TGR5 endogenous ligand CDCA, while the 12-OH-methylated analogue L2 loses the effect of allosteric modulation of TGR5. On this basis, it is found that the compound L3 modified by selectively methylating 7-OH is a high-efficiency TGR5 allosteric modulator (CN 115710298A). In order to further investigate the effect of 12-OH on allosteric modulation, we modified it into an oxime, and further modified the oxime to obtain new cholic acid analogues, and explored their allosteric modulation functional activity on TGR5. SUMMARY

[0004] The purpose of the present application is to provide a preparation method and application as a TGR5 allosteric agonist of oxime cholic acid analogues. In the present application, cholic acid analogues are all based on CA as a lead compound. First, a series of reactions are performed to obtain cholic acid intermediates with oxime at positions 7 and 12 or oxime at both positions 7 and 12. Then, further modifications are made to the oxime and position 24 of these intermediates to obtain a series of novel cholic acid analogues in structure, and the agonistic activity and allosteric mechanism of the new compounds on TGR5 are tested.

[0005] The oxime cholic acid analogue provided by the present application has a structure as shown in formula (1), formula (2) and formula (3):

[0006] wherein R1 is hydrogen or R1, R3 are each independently one of the following groups:

[0007] R2 is one of the following groups:

[0008] Further, the cholic acid analogs, R1 is one of the following groups: R3 is one of the following groups.

[0009] Still further, the cholic acid analogs have the following structure:

[0010] The present application provides a synthesis method of cholic acid analogs of formula (1), formula (2) and formula (3), and the synthesis route of cholic acid analogs of formula (1), formula (2) and formula (3) is as follows:

[0011] I. The synthesis route and specific synthesis steps of TGR5 cholic acid analogs of formula (1):

[0012] Scheme 1. The synthesis route of the target compound of formula (1)

[0013] The specific synthesis steps are as follows:

[0014] (1) Dissolve cholic acid in DCM, add coupling agent N, N, N', N'-tetramethyl-O-(7-azabenzotriazol-1-yl) urea hexafluorophosphate (HATU) at 0°C, slowly drop triethylamine (TEA), after 30 min, add alkyl, aryl or ester group for coupling reaction, and react at room temperature overnight for 20 h to obtain compound 1.

[0015] (2) Dissolve compound 1 and N-bromosuccinimide (NBS) in a mixed solvent of acetone and water, the volume ratio of acetone to water is 3:1, react in the dark at room temperature for 12 h to obtain compound 2.

[0016] (3) Dissolve compound 2 in methanol solution, sequentially add hydroxylamine hydrochloride (NH2OH·HCl) and sodium acetate trihydrate (NaOAc·3H2O), and condense and reflux at 80°C overnight to obtain compound 3.

[0017] (4) Dissolve compound 3 in freshly distilled tetrahydrofuran (THF) solvent, add sodium hydroxide solid powder and react for 30 min, then add halogenated hydrocarbon under ice bath condition, and then react at 70°C in the dark to obtain compound 4.

[0018] II. The synthesis route and specific synthesis steps of TGR5 cholic acid analogs of formula (2):

[0019] Scheme 2. The synthesis route of the target compound of formula (2)

[0020] wherein, the synthesis of compound 5, A1, C1 refers to the previous work of the research group (CN 115710298 A; WO 2023 / 168974 Al).

[0021] The specific synthesis steps are as follows:

[0022] (1) Dissolve intermediate 5 in DCM, add coupling agent 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) at 0°C, slowly add triethylamine (TEA), add tetrahydropyrrole after 30 minutes, and react overnight at room temperature to obtain compound A1.

[0023] (2) Dissolve compound A1 in methanol solution, add hydroxylamine hydrochloride (NH2OH·HCl) and sodium acetate trihydrate (NaOAc·3H2O) in sequence, and the molar ratio is compound A1:NH2OH·HCl:NaOAc·3H2O = 1:4:7. Then condense and reflux at 70°C overnight to obtain compound C1.

[0024] (3) Dissolve compound C1 in freshly distilled tetrahydrofuran (THF) solvent, add sodium hydroxide solid powder and catalyst potassium iodide, and after 30 minutes of reaction, add halogenated hydrocarbon under ice bath conditions, and then react at 70°C in the dark to obtain the final product 6.

[0025] (4) Dissolve compound 5 in methanol solvent, add hydroxylamine hydrochloride, sodium acetate trihydrate according to the molar ratio of compound 5:reducing agent:buffer = 1:4:7, heat and reflux at 70°C for 14h to obtain compound 7.

[0026] Scheme 3. Synthesis route of target compound formula (3)

[0027] The specific synthesis steps are as follows:

[0028] (1) Dissolve compound 8 in DCM, add catalyst p-dimethylaminopyridine (DMAP), acid binder triethylamine (TEA), and acetylation reagent acetic anhydride (Ac2O) to it. The molar ratio is compound 1:DMAP:TEA:Ac2O = 1:0.1:3:0.5. After 12h of reaction at room temperature, compound 9 is obtained.

[0029] (2) Dissolve compound 9 in acetone, and add excess Jones reagent at 0°C to make the solution red-brown. After stirring for 5 minutes, TLC detection is performed. After the reaction is complete, isopropyl alcohol is added to quench, and the solution is stirred until it turns dark green. Isopropyl alcohol and acetone are removed by rotary evaporation, and after post-treatment, compound 10 is obtained.

[0030] (3) Compound 10 was dissolved in methanol solvent, and an excess amount of sodium hydroxide aqueous solution was added, and after hydrolysis reaction at room temperature for 14-20 h, intermediate 11 was obtained.

[0031] (4) Intermediate 11 was dissolved in DCM, and coupling agent 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added at 0°C, and triethylamine (TEA) was slowly added dropwise, and after 30 min, tetrahydropyrrole was added, and after overnight reaction at room temperature for 20 h, compound 12 was obtained.

[0032] (5) Compound 12 was dissolved in methanol solution, and hydroxylamine hydrochloride (NH2OH·HCl) and sodium acetate trihydrate (NaOAc·3H2O) were sequentially added, and the molar ratio was compound 12:NH2OH·HCl:NaOAc·3H2O = 1:8:14. Then, after condensation reflux at 70°C overnight for 12 h, compound 13 was obtained.

[0033] (6) Compound 13 was dissolved in freshly distilled tetrahydrofuran (THF) solvent, and sodium hydroxide solid powder and catalyst potassium iodide were added, and after reaction for 30 min, halogenated hydrocarbon was added under ice bath conditions, and then the final product 14 was obtained under dark conditions at 70°C.

[0034] The present application tests the functional activity of the synthesized new compound on TGR5 in the G protein-dependent signal pathway and the allosteric regulation mechanism through the accumulation experiment of GloSensor cAMP. The pharmacological results show that all the target compounds synthesized have good agonistic activity on TGR5, and can positively allosterically regulate the functional activity of chenodeoxycholic acid CDCA and lithocholic acid LCA, that is, the target compounds of the present application are all positive allosteric modulators or allosteric agonists of TGR5.

[0035] The present application provides the use of the oxime-type cholic acid analogues in the preparation of the allosteric agonists of the bile acid receptor TGR5.

[0036] The present application also provides the use of the oxime-type cholic acid analogues in the preparation of the drugs for preventing and treating the diseases related to the bile acid receptor TGR5. The TGR5-related diseases include metabolic diseases, tumors, and inflammatory bowel diseases. The metabolic diseases include atherosclerosis, obesity, non-alcoholic fatty liver disease, cholestatic liver disease, metabolic syndrome, type II diabetes, type I diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, glucose metabolism disorder, hyperglycemia, hyperlipidemia, gallstones, liver cirrhosis, and cholestasis.

[0037] The present application provides a pharmaceutical composition comprising the cholic acid analog or its pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, solvate, crystal form or metabolite form thereof; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0038] The present application has the following beneficial effects:

[0039] The present application selects the TGR5 endogenous ligand cholic acid CA as a lead compound, obtains cholic acid intermediates with oxime at positions 7 and 12 or oxime at both positions 7 and 12 through a series of reactions, and then modifies the 24 position and oxime of these intermediates to obtain new cholic acid analogs. The functional activity of the target compounds on TGR5 is tested through the accumulation experiment of GloSensor cAMP. The results show that the synthesized new cholic acid analogs have good agonistic activity on TGR5 and can allosterically regulate the functional activity of the TGR5 endogenous ligand. The TGR5 allosteric agonist in the present application will provide a new potential candidate drug for treating metabolic diseases such as type II diabetes, obesity, non-alcoholic fatty liver and the like. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is the mechanism of allosteric regulation of CDCA by compound 4a;

[0041] Figure 2 is the mechanism of allosteric regulation of CDCA by compound 4b;

[0042] Figure 3 is the mechanism of allosteric regulation of CDCA by compound 6a;

[0043] Figure 4 is the mechanism of allosteric regulation of CDCA by compound 14a;

[0044] Figure 5 is the mechanism of allosteric regulation of LCA by compound 4a;

[0045] Figure 6 is the mechanism of allosteric regulation of LCA by compound 6a;

[0046] Figure 7 is the mechanism of allosteric regulation of LCA by compound 14a. DETAILED DESCRIPTION

[0047] The present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0048] Example 1

[0049] Synthesis of (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-3,12-dihydroxy-7- (hydroxyimino)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-1- (pyrrolidin-1-yl)pentan-1-one (3):

[0050] Step one: dissolve CA (600 mg, 1.46 mmol) in DCM (15 mL), add HATU (417.0 mg, 2.2 mmol) at 0 °C, slowly drop triethylamine (610 μL, 4.4 mmol), add tetrahydropyrrole (156 μL, 2.2 mmol) after 30 min, stir at room temperature for 20 h. After the reaction is completed, extract with DCM (50 mL x 3), wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain the crude product. Column chromatography (V DCM :V MeOH = 30: 1), concentrate to obtain 620 mg of white solid 1, with a yield of 91.5%.

[0051] Step two: dissolve compound 1 (600 mg, 1.29 mmol) and N-bromosuccinimide (507 mg, 2.85 mmol) in a mixed solvent of acetone (15 mL) and water (5 mL), with a volume ratio of acetone to water of 3: 1, react in the dark at room temperature for 12 h. After the reaction is completed, extract with DCM (60 mL x 3), wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain the crude product. Column chromatography (V DCM: V MeOH = 20: 1), concentrate to obtain 450 mg of white solid 2, with a yield of 75.2%.

[0052] Step three: dissolve compound 2 (420 mg, 0.9 mmol) in methanol (15 mL), and then add hydroxylamine hydrochloride (250.2 mg, 3.6 mmol) and sodium acetate trihydrate (856.8 mg, 6.3 mmol) in sequence, and condense to reflux at 80 °C for 12 h. After the reaction is completed, extract with DCM (40 mL x 3), wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain the crude product. Column chromatography (V DCM :V MeOH = 10: 1), concentrate to obtain 360 mg of white solid 3, with a yield of 83%. 1H NMR (400 MHz, CDC13) δ 4.00 (s, 1H), 3.58-3.51 (m, 1H), 3.45-3.39 (m, 4H), 3.10 (d, J = 15.2 Hz, 1H), 2.35-2.29 (m, 1H), 2.23-2.13 (m, 3H), 2.11-1.99 (m, 4H), 1.97-1.90 (m, 3H), 1.87-1.80 (m, 3H), 1.78-1.50 (m, 8H), 1.46-1.34 (m, 4H), 1.30-1.23 (m, 2H), 1.02 (s, 4H), 0.99 (d, J = 5.8 Hz, 4H), 0.67 (s, 3H). 13 C NMR (100 MHz, CDC13) δ 172.8, 161.0, 72.4, 70.8, 46.7, 46.6, 46.2, 45.7, 44.8, 42.4, 40.6, 36.4, 35.5, 35.1, 34.6, 31.9, 30.9, 29.5, 28.5, 27.9, 27.6, 26.1, 24.9, 24.4, 22.9, 17.5, 14.2, 12.8.

[0053] Example 2

[0054] Synthesis of (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-3,12-dihydroxy-7- (methoxyimino)-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)- 1-(pyrrolidin-l-yl)pentan-l-one (4a):

[0055] Step one: Compound 3 (60 mg, 0.128 mmol) was dissolved in dehydrated tetrahydrofuran (5 mL) solution, sodium hydroxide powder (15.36 mg, 1.824 mmol) and potassium iodide (21.25 mg, 0.128 mmol) were added in turn, after stirring at room temperature for 30 min, iodomethane (49 μL, 0.632 mmol) was added, and the reaction was carried out at 70 °C under light-avoiding condition for 24 h. After the reaction was completed, the product was extracted with DCM (40 mL x 3), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and then the solvent was removed by rotary evaporation to obtain the crude product. Column chromatography (V DCM :V MeOH = 10: 1) to obtain 44 mg of white solid 4a with a yield of 71%. 1H NMR (400 MHz, CDC13) δ 4.00 (s, 1H), 3.78 (s, 3H), 3.61-3.54 (m, 1H), 3.47-3.40 (m, 4H), 2.95 (dd, J = 13.0, 2.4 Hz, 1H), 2.35-2.28 (m, 2H), 2.20-2.14 (m, 2H), 2.08-2.00 (m, 2H), 1.99-1.91 (m, 3H), 1.87-1.81 (m, 3H), 1.75-1.70 (m, 5H), 1.67-1.63 (m, 4H), 1.55-1.42 (m, 3H), 1.39-1.24 (m, 4H), 1.08-1.03 (m, 4H), 1.00 (d, J = 6.3 Hz, 3H), 0.69 (s, 3H). 13 C NMR (100 MHz, CDC13) δ 172.4, 159.3, 72.4, 71.0, 61.0, 46.7, 46.6, 46.5, 45.7, 44.5, 42.21, 41.1, 36.7, 36.3, 35.2, 35.0, 34.5, 31.6, 30.8, 30.0, 28.6, 28.2, 27.5, 26.1, 24.7, 24.4, 23.0, 17.7, 12.9. HRMS (ESI): m / z calcd for C 29 H 48 NO4[M+Na] + 511.3512, found: 511.3508.

[0056] Example 3

[0057] Synthesis of (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-7-(ethoxyimino)-3,12- dihydroxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)-l- (pyrrolidin-l-yl)pentan-l-one (4b):

[0058] Other conditions are the same as the synthesis of 4a, replace iodomethane with ethyl bromide to obtain white solid 39 mg, yield 74%. 1H NMR (400 MHz, CDC13) δ 4.01 - 3.97 (m, 3H), 3.55 - 3.48 (m, 1H), 3.40 (q, J = 7.4 Hz, 4H), 2.96 (d, J = 12.0 Hz, 3H), 2.33 - 2.22 (m, 2H), 2.18 - 2.08 (m, 2H), 2.05 - 1.98 (m, 2H), 1.96 - 1.89 (m, 3H), 1.85 - 1.78 (m, 3H), 1.70 - 1.59 (m, 6H), 1.52 - 1.48 (m, 1H), 1.42 - 1.30 (m, 4H), 1.28 - 1.22 (m, 2H), 1.18 (t, J = 7.0 Hz, 3H), 1.03 - 0.94 (m, 8H), 0.66 (s, 3H). 13 C NMR (100 MHz, CDC13) δ 172.3, 158.7, 72.4, 71.0, 68.5, 53.5, 46.7, 46.6, 46.5, 45.7, 44.6, 42.2, 41.2, 36.7, 36.3, 35.2, 35.0, 34.5, 31.6, 30.9, 30.0, 28.6, 28.2, 27.6, 26.2, 24.4, 23.0, 17.7, 15.0, 13.0. HRMS (ESI): m / z calcd for C 30 H 50 N2O4[M+Na] + 525.3668, found: 525.3666.

[0059] Example 4

[0060] (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-3,12-dihydroxy-10,13-dimethyl-7- (propoxyimino)hexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)-l- (pyrrolidin-l-yl)pentan-l-one (4c) was synthesized according to the procedure described for 4a, replacing iodomethane with n-bromopropane, to give 41 mg of white solid in 75.4% yield.

[0061] Other conditions are the same as the synthesis of 4a, replacing iodomethane with n-bromopropane to give 41 mg of white solid in 75.4% yield. 1H NMR (400 MHz, CDC13) δ 3.93 (s, 1H), 3.89-3.79 (m, 2H), 3.51-3.43 (m, 1H), 3.36 (q, J = 6.9 Hz, 4H), 2.93 (d, J = 11.0 Hz, 2H), 2.45 (s, 1H), 2.28-2.19 (m, 2H), 2.13-2.07 (m, 2H), 2.04-1.93 (m, 3H), 1.91-1.85 (m, 3H), 1.81-1.72 (m, 4H), 1.66-1.61 (m, 3H), 1.57-1.52 (m, 4H), 1.48-1.44 (m, 1H), 1.38-1.27 (m, 4H), 1.24-1.15 (m, 2H), 0.99-0.88 (m, 8H), 0.83 (t, J = 10.0 Hz, 3H), 0.62 (s, 3H). 13 C NMR (100 MHz, CDC13) δ 172.3, 158.7, 74.7, 72.3, 70.8, 46.7, 46.6, 46.5, 45.7, 42.2, 41.2, 36.7, 36.2, 35.2, 34.9, 34.5, 31.7, 30.9, 29.9, 28.6, 28.2, 27.6, 26.1, 24.8, 24.4, 23.0, 22.6, 17.7, 12.9, 10.5. HRMS (ESI): m / z calcd C 31 H 52 N2O4[M+Na] + 539.3825, found: 539.3821.

[0062] Example 5

[0063] Synthesis of (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-3,12-dihydroxy-7- (isopropoxyimino)-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren- 17-yl)-l- (pyrrolidin-l-yl)pentan-l-one (4d):

[0064] Other conditions are the same as the synthesis of 4a, replace methyl iodide with isopropyl bromide to obtain 38 mg of white solid, yield is 69.9%. 1H NMR (400 MHz, CDC13) δ 4.25 - 4.16 (m, 1H), 3.98 (s, 1H), 3.56 (s, 1H), 3.41 (q, J = 9.9 Hz, 4H), 2.99 (d, J = 17.1 Hz, 1H), 2.34 - 2.27 (m, 2H), 2.15 - 2.12 (m, 2H), 2.07 - 1.97 (m, 3H), 1.93 (d, J = 8.2 Hz, 5H), 1.87 - 1.80 (m, 3H), 1.73 - 1.48 (m, 7H), 1.48 - 1.32 (m, 4H), 1.28 - 1.23 (m, 3H), 1.16 (d, J = 8.2 Hz, 5H), 1.05 - 0.98 (m, 7H), 0.67 (s, 3H). 13 C NMR (100 MHz, CDC13) δ 172.4, 158.3, 74.0, 72.4, 70.9, 46.7, 46.6, 46.4, 45.7, 44.6, 42.2, 41.3, 36.6, 36.2, 35.2, 34.9, 34.5, 31.7, 30.9, 30.0, 28.6, 28.2, 27.6, 26.1, 24.9, 24.4, 23.0, 22.0, 21.8, 17.7, 12.9. HRMS (ESI): m / z calcd for C 31 H 52 N2O4[M+Na] + 539.3825, found: 539.3822.

[0065] Example 6

[0066] Synthesis of (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-7-(butyloxyimino)-3,12- dihydroxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)-l- (pyrrolidin-l-yl)pentan-l-one (4e):

[0067] Other conditions are the same as the synthesis of 4a, replace iodomethane with 1-bromobutane to get 55 mg white solid, yield is 62%. 1H NMR (300 MHz, CDCI3) δ 4.00-3.93 (m, 3H), 3.57 (s, 1H), 3.47-3.39 (m, 4H), 3.00 (dd, J = 13.0, 2.1 Hz, 1H), 2.37-2.28 (m, 2H), 2.21-2.13 (m, 2H), 2.08-1.92 (m, 5H), 1.90-1.79 (m, 5H), 1.70-1.53 (m, 10H), 1.45-1.24 (m, 9H), 1.02-0.99 (m, 6H), 0.91 (t, J = 7.3 Hz, 3H), 0.69 (s, 3H).

[0068] Example 7

[0069] Synthesis of (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R,Z)-7-((benzyloxy)imino)- 3,12-dihydroxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)- 1 -(pyrrolidin- 1 -yl)pentan- 1 -one (4f):

[0070] Other conditions are the same as the synthesis of 4a, replace methyl iodide with benzyl bromide to get 35 mg white solid, yield is 84%. 1 H NMR (300 MHz, CDCI3) δ 7.27-7.23 (m, 4H), 7.20 (s, 1H), 4.99-4.92 (m, 2H), 3.94 (s, 1H), 3.49 (s, 1H), 3.40-3.33 (m, 4H), 2.98 (d, J = 13.0 Hz, 1H), 2.29-2.22 (m, 1H), 2.19-2.08 (m, 3H), 2.02-1.92 (m, 4H), 1.90-1.76 (m, 7H), 1.67-1.53 (m, 7H), 1.47-1.29 (m, 5H), 1.21 (s, 1H), 1.19 (s, 5H), 0.95 (s, 3H), 0.93 (s, 2H), 0.83-0.77 (m, 4H), 0.61 (s, 1H). 13 C NMR (100 MHz, CDCI3) δ 172.4, 159.8, 138.8, 128.3, 127.5, 75.4, 72.5, 71.1, 46.8, 46.7, 45.8, 44.7, 42.4, 41.4, 36.9, 36.5, 35.2, 35.0, 34.5, 32.0, 31.7, 30.9, 30.1, 29.8, 28.8, 28.5, 27.6, 26.3, 24.8, 24.5, 23.1, 17.8, 14.3, 13.1.

[0071] Example 8

[0072] Synthesis of (R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R,E)-3-hydroxy-12- (hydroxyimino)-7-methoxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren- 17-yl)pentanoic acid (7):

[0073] Step one: Intermediate 5 (500 mg, 1.2 mmol) was dissolved in methanol (15 ml) solution, hydroxylamine hydrochloride (330 mg, 4.76 mmol) and sodium acetate trihydrate (1.14 g, 8.4 mmol) were added in turn, and the reaction was condensed at 70 °C and refluxed overnight for 14 h. After the reaction was completed, the organic phase was extracted with DCM (50 mL x 3) and washed with distilled water. The solvent was removed to obtain the crude product. Column chromatography (V DCM :V MeOH = 10:1) to obtain 400 mg of compound 7 as a white solid, with a reaction yield of 76.6%. 1 H NMR (400 MHz, CDC13) δ 3.44 (s, 1H), 3.34 (dd, Ji = 13.0 Hz, J2= 4.8 Hz, 1H), 3.23 (s, 1H), 3.21 (s, 3H), 2.51-2.46 (m, 1H), 2.30-2.23 (m, 1H), 2.10-2.02 (m, 6H), 1.89-1.82 (m, 3H), 1.78-1.75 (m, 2H), 1.66 (t, J = 13.0 Hz, 7H), 1.45-1.35 (m, 4H), 1.25 (s, 1H), 1.07-1.04 (m, 1H), 0.98 (s, 3H), 0.94-0.92 (m, 4H), 0.91 (s, 2H).

[0074] Example 9

[0075] Synthesis of (R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R,E)-3-hydroxy-12- (hydroxyimino)-7-methoxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren- 17-yl)-l-(pyrrolidin-l-yl)pentan-l-one (6a):

[0076] Compound C1 (50 mg, 0.1 mmol) was dissolved in water-free tetrahydrofuran (5 mL), and solid sodium hydroxide powder (12 mg, 0.3 mmol) and catalyst potassium iodide (3.32 mg, 0.002 mmol) were added successively. After stirring at room temperature for 30 min, iodomethane (31 μL, 0.5 mmol) was added, and the reaction was condensed and refluxed at 70 °C in the dark overnight for 24 h. After the reaction was completed, the organic phase was extracted with DCM (30 mL x 3), washed with distilled water, and the solvent was removed to obtain the crude product. Column chromatography (V DCM :V MeOH = 30:1) to obtain 40 mg of compound 6a in the form of a white solid, with a reaction yield of 79.6%. 1 H NMR (400 MHz, CDC13) δ 3.75 (s, 3H), 3.47-3.40 (m, 5H), 3.22 (s, 4H), 3.13 (dd, J1= 13.4 Hz, J2= 5.1 Hz, 1H), 2.39-2.31 (m, 1H), 2.24-2.14 (m, 2H), 2.09 (d, J = 12.3 Hz, 1H), 2.04-1.91 (m, 4H), 1.88-1.81 (m, 4H), 1.77 (s, 1H), 1.75-1.63 (m, 7H), 1.53 (d, J = 13.1 Hz, 1H), 1.48-1.34 (m, 7H), 0.98 (d, J = 6.5 Hz, 3H), 0.95 (s, 3H), 0.85 (s, 3H). 13 C NMR NMR (100 MHz, CDC13) δ 172.6, 164.0, 77.3, 71.9, 60.9, 56.1, 53.0, 49.2, 47.1, 46.8, 45.8, 41.8, 39.6, 38.5, 36.1, 35.6, 35.5, 35.1, 32.3, 30.8, 30.7, 28.0, 27.8, 26.3, 24.6, 23.7, 22.4, 20.2, 20.0, 12.0.

[0077] Example 10

[0078] Synthesis of (R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R,E)-12-(ethoxyimino)-3- hydroxy-7-methoxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren- 17-yl)-l-(pyrrolidin-l-yl)pentan-l-one (6b)

[0079] Other conditions were the same as those for the synthesis of 6a, except that iodomethane was replaced by bromoethane, to obtain 38 mg of white solid 6b, with a yield of 73.6%.1 H NMR (400 MHz, CDC13) δ 3.97 (q, J = 7.0 Hz, 2H), 3.45 - 3.39 (m, 4H), 3.20 (s, 3H), 3.18 - 3.14 (m, 1H), 2.37 - 2.29 (m, 1H), 2.23 - 2.13 (m, 2H), 2.08 (d, J = 12.2 Hz, 1H), 2.00 - 1.90 (m, 4H), 1.83 (t, J = 7.1 Hz, 5H), 1.75 (s, 1H), 1.71 - 1.63 (m, 5H), 1.60 - 1.54 (m, 2H), 1.49 (d, J = 13.2 Hz, 1H), 1.45 - 1.33 (m, 5H), 1.23 (s, 1H), 1.21 (t, J = 7.0 Hz, 4H), 0.97 (d, J = 6.6 Hz, 4H), 0.93 (s, 3H), 0.84 (s, 3H). 13 C NMR NMR (100 MHz, CDC13) δ 172.6, 163.7, 77.3, 71.9, 68.4, 56.1, 53.5, 53.1, 49.2, 47.1, 46.7, 45.7, 41.8, 39.5, 38.4, 36.0, 35.6, 35.5, 35.1, 32.2, 30.7, 27.9, 27.8, 26.3, 24.5, 23.7, 22.4, 20.3, 19.9, 15.0, 12.0.

[0080] Example 11

[0081] Synthesis of (R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R,E)-3-hydroxy-7-methoxy-10,13-dimethyl-12- (propoxyimino)hexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)-l-(pyrrolidin-l-yl)pentan- 1-one (6c):

[0082] Other conditions are the same as the synthesis of 6a, replace iodomethane with n-bromopropane to obtain 41 mg of white solid 6c, yield 77.3%. 1H NMR (400 MHz, CDC13) δ 3.87-3.81 (m, 2H), 3.42-3.35 (m, 5H), 3.16 (s, 3H), 3.14 (d, J = 5.7 Hz, 1H), 2.33-2.25 (m, 1H), 2.19-2.09 (m, 2H), 2.04 (d, J = 12.2 Hz, 1H), 2.00-1.86 (m, 5H), 1.82-1.76 (m, 5H), 1.72 (s, 1H), 1.69-1.50 (s, 9H), 1.47-1.28 (m, 6H), 0.93 (d, J = 6.5 Hz, 3H), 0.90 (s, 3H), 0.85 (t, J = 7.4 Hz, 3H), 0.81 (s, 3H). 13 C NMR NMR (100 MHz, CDC13) δ 172.5, 163.6, 74.4, 71.7, 55.9, 53.0, 49.1, 47.0, 46.6, 45.6, 41.7, 39.4, 38.3, 35.9, 35.5, 35.4, 35.1, 32.1, 30.6, 30.5, 27.8, 27.7, 26.2, 24.4, 23.5, 22.6, 22.3, 20.2, 19.8, 11.9, 10.6, 1.01.

[0083] Example 12

[0084] Synthesis of (R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R,E)-3-hydroxy-12- (isopropoxyimino)-7-methoxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren- 17-yl)-l-(pyrrolidin-l-yl)pentan-l-one (6d):

[0085] Other conditions are the same as the synthesis of 6a, replace iodomethane with isopropyl bromide to obtain 42 mg of white solid, yield 79.2%. 1 H NMR (400 MHz, CDC13) δ 3.87-3.81 (m, 2H), 3.42-3.35 (m, 5H), 3.16 (s, 3H), 3.14 (d, J = 5.7 Hz, 1H), 2.33-2.25 (m, 1H), 2.19-2.09 (m, 2H), 2.04 (d, J = 12.2 Hz, 1H), 2.00-1.86 (m, 5H), 1.82-1.76 (m, 5H), 1.72 (s, 1H), 1.69-1.50 (s, 9H), 1.47-1.28 (m, 6H), 0.93 (d, J = 6.5 Hz, 3H), 0.90 (s, 3H), 0.85 (t, J = 7.4 Hz, 3H), 0.81 (s, 3H). 13C NMR (100 MHz, CDC13) δ 172.6, 163.3, 77.3, 73.9, 72.0, 56.1, 53.3, 49.3, 47.1, 46.7, 45.7, 41.8, 39.5, 38.4, 36.0, 35.5, 35.2, 32.2, 30.7, 30.6, 27.9, 27.8, 26.3, 24.6, 23.6, 22.4, 22.1, 21.8, 20.4, 20.0, 12.0.

[0086] Example 13

[0087] Synthesis of (R)-4-((3R,5R,7Z,8R,9S,10S,12E,13R,14S,17R)-3-hydroxy-7,12- bis(hydroxyimino)-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)- 1 -(pyrrolidin- 1 -yl)pentan- 1 -one (13):

[0088] Step one: Compound 8 (2.5 g, 5.92 mmol) was dissolved in anhydrous dichloromethane (40 mL), and triethylamine (2.45 mL, 17.76 mmol), DMAP (72 mg, 0.592 mmol) and acetic anhydride (281 μL, 2.96 mmol) were added successively. After 20 h of reaction, TLC detection was performed. After the reaction was completed, the solvent was removed, and the organic phase was extracted with DCM (80 mL x 3), washed with water (100 mL), and concentrated to obtain the crude product. Column chromatography (V DCM :V EA = 10: 1) was performed, and 1.4 g of compound 9 was obtained in the form of a white powdery solid at a reaction yield of 51%.

[0089] Step two: Compound 9 (1 g, 2.2 mmol) was dissolved in acetone (20 mL), and an appropriate amount of Jones reagent was added dropwise at 0°C to make the solution red-brown, and TLC detection was performed after stirring for 5 min. After the reaction was completed, 20 mL of isopropanol was added, and the solution was stirred until it became dark green. Isopropanol and acetone were removed by rotary evaporation. Extraction was performed with EA (50 mL x 3) and water (30 mL), and the solvent was removed. Concentration resulted in 800 mg of crude product 10 in the form of a white foamy solid at a reaction yield of 79%.

[0090] Step three: Compound 10 (500 mg, 1.09 mmol) was dissolved in methanol (30 mL), and an excess of 6 M / L aqueous NaOH solution was added, and stirring was performed at room temperature for 14 h. After the reaction was completed, hydrochloric acid solution was added to adjust the pH to 2, and methanol was removed by rotary evaporation. The solid was repeatedly washed with water and dichloromethane to remove impurities, and the crude product was obtained by drying. Column chromatography (V DCM :VMeOH = 15: 1), concentrated to give 330 mg of compound 11 as a white solid, with a reaction yield of 74.9%.

[0091] Step four: Compound 11 (330 mg, 0.816 mmol) was dissolved in DCM (20 mL), HATU (465.12 mg, 1.224 mmol) was added at 0 °C, triethylamine (340 μL, 2.447 mmol) was added dropwise slowly, and tetrahydropyrrole (100 μL, 1.224 mmol) was added after 30 minutes. The mixture was stirred at room temperature for 20 h. After the reaction was completed, the organic phase was extracted with DCM (30 mL x 3), washed with distilled water, and the solvent was removed to obtain the crude product. Column chromatography (V DCM :V MeOH = 20: 1), concentrated to give 360 mg of compound 12 as a white solid, with a reaction yield of 93.6%.

[0092] Step five: Compound 12 (360 mg, 0.764 mmol) was dissolved in methanol (30 ml), and hydroxylamine hydrochloride (424.5 mg, 6.108 mmol) and sodium acetate trihydrate (1.45 g, 10.696 mmol) were added in sequence. The mixture was condensed and refluxed at 70 °C overnight for 12 h. After the reaction was completed, the organic phase was extracted with DCM (40 mL x 3), washed with distilled water, and the solvent was removed to obtain the crude product. Column chromatography (V DCM :V MeOH = 20: 1), concentrated to give 310 mg of compound 13 as a white solid, with a reaction yield of 83.2%. 1 H NMR (400 MHz, DMSO) δ 10.15 (s, 1H), 10.08 (s, 1H), 4.44 (d, J = 4.8 Hz, 1H), 3.34 (s, 1H), 3.19 (t, J = 6.8 Hz, 2H), 3.07 (d, J = 9.4 Hz, 1H), 2.85 (d, J = 12.9 Hz, 1H), 2.45 (s, 1H), 2.38 (t, J = 10.6 Hz, 1H), 2.14 - 2.00 (m, 3H), 1.93 (dd, J 1 = 13.0 Hz, J 2 = 5.2 Hz, 1H), 1.84 - 1.77 (m, 3H), 1.73 - 1.64 (m, 4H), 1.59 - 1.51 (m, 4H), 1.48 - 1.24 (m, 4H), 1.18 - 0.94 (m, 9H), 0.85 (d, J = 6.6 Hz, 3H), 0.82 (s, 3H). 13C NMR (100 MHz, DMSO) δ 170.8, 162.2, 157.2, 79.2, 78.9, 78.6, 69.0, 54.7, 53.0, 48.7, 46.0, 45.2, 43.5, 41.2, 36.9, 35.5, 34.4, 31.7, 30.4, 29.7, 27.4, 25.7, 25.0, 23.9, 22.2, 19.5, 14.0, 12.0. HRMS (ESI): m / z calcd for C 28 H 45 N3O4[M+Na] + 510.3308, found: 510.3303.

[0093] Example 14

[0094] Synthesis of (R)-4-((3R,5R,7Z,8R,9S,10S,12E,13R,14S,17R)-3-hydroxy-7,12- bis(methoxyimino)-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)- 1 -(pyrrolidin- 1 -yl)pentan- 1 -one (14a):

[0095] Compound 13 (30 mg, 0.0615 mmol) was dissolved in water-free tetrahydrofuran (5 mL), and solid sodium hydroxide powder (12 mg, 0.3077 mmol) and a catalyst potassium iodide (2 mg, 0.012 mmol) were added in turn. After stirring at room temperature for 30 min, iodomethane (38 μL, 0.625 mmol) was added, and the reaction was condensed and refluxed at 70 °C in the dark overnight. After the reaction was completed, the organic phase was extracted with DCM (30 mL x 3) and washed with distilled water, and the solvent was removed to obtain the crude product. Column chromatography (V DCM :V MeOH = 30: 1) to obtain 24 mg of compound 14a in the form of a white solid, with a reaction yield of 75.7%. 1H NMR (400 MHz, CDC13) δ 3.74 (d, J = 3.8 Hz, 6H), 3.62 - 3.54 (m, 1H), 3.46 - 3.40 (m, 4H), 3.09 (dd, Ji = 13.1 Hz, J2= 4.8 Hz, 1H), 2.96 - 2.93 (m, 1H), 2.38 - 2.26 (m, 3H), 2.22 - 2.13 (m, 2H), 2.03 - 1.91 (m, 4H), 1.87 - 1.75 (m, 7H), 1.65 (t, J = 13.0 Hz, 3H), 1.48 - 1.23 (m, 6H), 1.14 - 1.03 (m, 5H), 0.97 (d, J = 6.4 Hz, 3H), 0.87 (s, 3H). 13 C NMR (100 MHz, CDC13) δ 172.5, 163.2, 158.3, 70.8, 61.1, 60.9, 52.6, 49.2, 46.6, 46.1, 45.6, 44.0, 43.9, 42.0, 36.7, 35.9, 34.6, 32.2, 30.6, 29.9, 28.2, 27.7, 26.2, 25.3, 24.4, 22.6, 20.5, 19.8, 12.1, 1.01. HRMS (ESI): m / z calcd C 30 H 49 N3O4[M+Na] + 538.3621, found: 538.3615.

[0096] Example 15

[0097] Synthesis of (R)-4-((3R,5R,7Z,8R,9S,10S,12E,13R,14S,17R)-7,12-bis(ethoxyimino)- 3-hydroxy-10,13-dimethylhexadecahydro-lH-cyclopenta[a]phenanthren-17-yl)-l- (pyrrolidin-l-yl)pentan-l-one (14b):

[0098] Other conditions are the same as the synthesis of 14a, replace iodomethane with ethyl bromide, to get 14 mg of white solid, yield 69.2%. 1H NMR (400 MHz, CDC13) δ 3.57 (q, J = 7.2 Hz, 1H), 3.48 (s, 8H), 3.10 (q, J = 7.3 Hz, 1H), 2.21 (t, J = 7.7 Hz, 2H), 2.00 (d, J = 7.0 Hz, 3H), 1.69 - 1.59 (m, 6H), 1.46 (t, J = 7.2 Hz, 2H), 1.42 - 1.36 (m, 5H), 1.32 (s, 3H), 1.28 (s, 4H), 1.24 (s, 11H), 0.87 (s, 3H), 0.85 - 0.82 (m, 4H).

[0099] Example 16

[0100] Synthesis of (R)-4-((3R,5R,7Z,8R,9S,10S,12E,13R,14S,17R)-3-hydroxy-10,13- dimethyl-7,12-bis(propoxyimino)hexadecahydro-lH-cyclopenta[a]phenanthren- 17-yl)-l- (pyrrolidin-l-yl)pentan-l-one (14c):

[0101] Other conditions are the same as the synthesis of 14a, replace iodomethane with bromopropane, to get 25 mg white solid, yield 68.2%. 1 H NMR (400 MHz, CDC13) δ 3.89 (t, J = 6.6 Hz, 4H), 3.57 (s, 1H), 3.45 - 3.39 (m, 4H), 3.15 (dd, Ji = 13.1 Hz, J2= 4.7 Hz, 1H), 3.00 (d, J = 13.2 Hz, 1H), 2.37 - 2.24 (m, 3H), 2.20 - 2.12 (m, 2H), 2.03 - 1.90 (m, 6H), 1.86 - 1.79 (m, 6H), 1.71 - 1.56 (m, 9H), 1.49 - 1.23 (m, 8H), 1.08 (s, 4H), 0.96 (d, J = 6.4 Hz, 3H), 0.90 (s, 1H), 0.88 (s, 4H). 13 C NMR (100 MHz, CDC13) δ 172.6, 163.1, 158.0, 74.9, 74.6, 71.0, 52.9, 49.4, 46.7, 46.1, 45.7, 44.2, 44.1, 42.1, 36.9, 35.9, 34.7, 32.3, 30.7, 30.0, 28.3, 27.9, 26.3, 25.4, 24.5, 22.7, 22.7, 22.6, 20.8, 19.9, 12.3, 10.6, 10.6, 1.1.

[0102] Biological activity test

[0103] 1. GloSensor cAMP Accumulation Assay to test cAMP levels:

[0104] cAMP is a key signaling molecule for many G protein-coupled receptors. The accumulation level of cAMP was tested using GloSensor, a bioluminescence-based biosensor that can directly detect intracellular cAMP (Promega). The principle is that a cAMP binding domain was inserted at the N- and C-terminus of firefly luciferase by genetic engineering, which makes the enzyme in an inactive state. When cAMP binds to the cAMP binding domain, the enzyme is activated, which generates bioluminescence by oxidizing the substrate luciferin. The cAMP accumulation experiment was used to test the functional activity of target compounds on TGR5 and to clarify whether the new compounds are positive allosteric modulators (PAM) of TGR5.

[0105] The cAMP accumulation assay was used to test whether the new compounds can allosterically modulate the functional activity of the endogenous ligands of TGR5, chenodeoxycholic acid (CDCA) and lithocholic acid (LCA), to determine whether they are positive allosteric modulators (PAM) of TGR5. Briefly, the dose-response curves of CDCA (1 nM-100 μΜ) and LCA (1 nM-100 μΜ) were first tested, and when their bioluminescence signals reached the highest, the new compounds (1 nM-100 μΜ) were added in a concentration multiple relationship, and whether the change in bioluminescence at this time showed a concentration-dependent limited upward shift trend was tested. The specific experimental steps are the same as the above GloSensor cAMP accumulation assay. The experimental results showed that the target compounds can positively allosterically modulate the functional activity of the endogenous ligands CDCA and LCA of TGR5.

[0106] 2. The agonistic activity test results of target compounds on human TGR5 (hTGR5) and murine TGR5 (mTGR5):

[0107] Table 1. EC50 values of target compounds of formula (1), formula (2) and formula (3) to activate TGR5 50

[0108] ​The agonistic activities of the 16 synthesized cholic acid analogues on hTGR5 and mTGR5 were tested by using Glosensor cAMP accumulation experiment, and the experimental results are shown in Table 1 above. The target compounds in the present application all have agonistic activities on TGR5, and some of the compounds have obvious differences in agonistic activities on hTGR5 and mTGR5. The compound 4a obtained by alkylating modification of the 7-position oxime has an activity on hTGR5 which is 5 times that on mTGR5. When we only alkylate the 12-position oxime, the EC 50 values of 6a, 6b, 6c and 6d on hTGR5 are 25.58 nM, 0.15 μM, 0.30 μM and 0.16 μM respectively, which are increased by 18, 16, 64 and 82 times respectively compared with the activities on mTGR5. The better active 14a has an EC 50 value of 1.61 μM on mTGR5, which is decreased by 19 times compared with the activity on hTGR5.

[0109] For hTGR5, from the data in Table 1, the 3α-OH and 12α-OH of CA are kept unchanged, the 24-position carboxyl is amide coupled, and the 7α-OH is modified into an oxime to obtain a mono-oxime compound. It is found that the activity is obviously improved after the 7-position oxime is substituted with a methyl group (4a). The EC 50 value of 4a on hTGR5 is only 0.70 μM, which is increased by 55 times compared with the activity of the lead compound CA, and is increased by 29 times compared with the activity of the precursor 3. The compound 4b is obtained by extending the length of the 7-position of 4a by one carbon. It is found that the activity of 4b on hTGR5 starts to decrease, which is decreased by 2.7 times compared with 4a. And with the extension of the carbon chain or the increase of the molecular size of the alkylated modification group, the biological activities of compounds 4c, 4d, 4e and 4f on hTGR5 and mTGR5 all show a decreasing trend.

[0110] The 7α-OH of CA is methylated, the 3α-OH and the 24-position carboxyl are kept unchanged, and the 12α-OH is modified into an oxime to obtain a mono-oxime compound 7. The EC 50 value of 7 on hTGR5 is 0.86 μM, which is increased by 45 times compared with CA. Then the 24-position carboxyl of 7 is amide coupled, and the 12-position oxime is methylated to obtain the better active target compound 6a. The activity of 6a is increased by nearly 1500 times compared with CA. The compounds 6b and 6c are obtained by extending the length of the 12-position of 6a by one carbon atom and two carbon atoms respectively. The biological activities of 6b and 6c are decreased by 6 times and 12 times respectively compared with 6a. And with the extension of the carbon chain or the increase of the molecular size of the alkylated modification group, the biological activities of the compounds on TGR5 show a decreasing trend.

[0111] This rule also applies to the target compound of the bis-oxime alkylated modification, we keep the 3a-OH of CA unchanged, the carboxyl group at 24 position is amide coupled, and the 7a-OH and 12a-OH are both modified into oxime to obtain the target compound 13 of bis-oxime, the EC 50 of 13 to hTGR5 is 1.69 μM, which is 23 times higher than that of CA, then the oxime at 7 position and 12 position of 13 is methylated to obtain the target compound 14a, the EC 50 of 14a to hTGR5 is 86.96 nM, which is nearly 450 times higher than that of CA, and 19 times higher than that of the bis-oxime target compound 13 without methyl modification, but when we extend the length of 7 position and 12 position of 14a by one carbon atom and two carbon atoms respectively to obtain compounds 14b and 14c, the biological activity of 14b and 14c is nearly 20 times lower than that of 14a. This rule is the same as the alkylated modification of 7a-OH.

[0112] 3. Allosteric mechanism research

[0113] Further research on whether the compounds can allosterically regulate the functional activity of the endogenous ligand CDCA and LCA of TGR5 through GloSensor cAMP accumulation experiment, and further confirm whether the compounds are positive allosteric modulators (PAM) or allosteric agonists of TGR5. Take compound 4a as an example (as shown in Figure 1), when the concentration of compound 4a is doubled, the concentration-dependent curve of CDCA mediated by compound 4a shows limited upward shift, indicating that compound 4a can effectively positively allosterically regulate the functional activity of the endogenous ligand CDCA of TGR5, that is, 4a is an allosteric agonist of TGR5. The concentration-dependent curve of CDCA mediated by compound 4b (as shown in Figure 2), 6a (as shown in Figure 3), 14a (as shown in Figure 4) is consistent with the result of 4a, that is, the target compounds in the present application are all positive allosteric modulators (PAM) or allosteric agonists of TGR5. In addition, the research results show that the target compounds 4a, 6a and 14a not only make the LCA dose-effect curve show limited upward shift, but also make the EC 50 of the LCA dose-effect curve obviously left shift. As 4a (Figure 5) left shifts the EC 50 of LCA by 3.3 times, 6a (Figure 6) left shifts the EC 50 of LCA by 1.9 times, and 14a (Figure 7) left shifts the EC 50 of LCA by 3.7 times, which further indicates that the target compounds in the present application are all positive allosteric modulators of TGR5.

[0114] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. An oxime-cholic acid analogue, characterized in that: The structures of the oxime bile acid analogues are shown in formula (1), formula (2) or formula (3): Wherein, R1 is hydrogen or R1 and R3 are each independently one of the following groups: R2 is one of the following groups:

2. The oxime-cholic acid analogue according to claim 1, characterized in that: R1 is One of them; R2 is one of the following groups: R3 is One of them.

3. The oxime-cholic acid analogue according to claim 1 or 2, characterized in that: The structural formula of the oxime-cholic acid analogue is as follows:

4. The application of the oxime bile acid analogue as described in any one of claims 1 to 3, characterized in that: The application of the oxime bile acid analogues in the preparation of bile acid receptor TGR5 allosteric agonists.

5. The application of the oxime bile acid analogue as described in any one of claims 1 to 3, characterized in that: The application of the oxime bile acid analogues in the preparation of drugs for the prevention and treatment of bile acid receptor TGR5-related diseases.

6. The application of the oxime bile acid analogue according to claim 5, characterized in that: The TGR5-related diseases include metabolic diseases, tumors, and inflammatory bowel disease.

7. The application of the oxime bile acid analogue according to claim 6, characterized in that: The metabolic diseases mentioned include: atherosclerosis, obesity, non-alcoholic fatty liver disease, cholestatic liver disease, metabolic syndrome, type II diabetes, type I diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, glucose metabolism disorders, hyperglycemia, hyperlipidemia, gallstones, cirrhosis, and cholestasis.

8. A pharmaceutical composition, characterized in that: Includes the oxime bile acid analogues as described in any one of claims 1 to 3, or pharmaceutically acceptable salts or esters, prodrugs, stereoisomers, hydrates, solvates, crystal forms, or metabolites thereof.

9. The pharmaceutical composition according to claim 8, characterized in that: It also contains pharmaceutically acceptable carriers or excipients.

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