Non-steroidal cholic acid analogue, preparation method thereof and application of non-steroidal cholic acid analogue as TGR5 allosteric agonist

Abstract

The invention belongs to the field of medicinal chemistry, and discloses application of a non-steroidal cholic acid analogue as a TGR5 allosteric agonist. Cholic acid (CA) is used as a lead compound, 7-OH alkylation is obtained through a series of reactions, an intermediate of seven-membered lactone or lactam is obtained through ring expansion of a B ring or a C ring, and then the intermediate is subjected to a series of modification to obtain the non-steroidal cholic acid analogue. According to the invention, the agonistic activity of all target compounds on TGR5 and whether the functional activity of chenodeoxycholic acid (CDCA) serving as an endogenous ligand of TGR5 can be positively allosterally regulated are tested by virtue of a GloSensor cAMP accumulation experiment. Pharmacological results show that most of the synthesized target compounds have good agonist activity on TGR5, and can positively allosteric regulate the functional activity of CDCA, that is, the target compounds are all TGR5 positive allosteric modulators or allosteric agonists. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry, and specifically discloses a preparation method of a non-steroidal bile acid analog and an application of the non-steroidal bile acid analog as a bile acid receptor TGR5 allosteric agonist. Background Art

[0002] G protein-coupled receptors (GPCRs) are a general term for a large class of membrane protein receptors, and their abnormal expression is closely related to a variety of diseases, such as neurodegenerative diseases, metabolic diseases, cardiovascular diseases, cancer, etc. At present, about 30% of drugs in clinical practice target GPCRs. In recent years, studies have found that GPCRs not only have orthosteric sites, but also allosteric sites that are topologically different from orthosteric sites. Allosteric sites are less conserved than orthosteric sites, so allosteric modulators that bind to this site have higher selectivity for GPCR receptor subtypes and can reduce the side effects of drugs. Bile acid receptor TGR5, also known as G protein-coupled bile acid receptor 1, is one of the important members of the GPCRs family. TGR5 can be activated by endogenous bile acids, such as cholic acid CA, deoxycholic acid DCA, and chenodeoxycholic acid CDCA. In recent years, studies have found that bile acids containing 12-OH, such as CA, can not only activate TGR5, but also have allosteric regulatory functions (Nature, 2020, 587, 499-504.). However, the activity of CA is relatively low. Our research group conducted preliminary structure-activity relationship studies using CA as a lead compound and discovered a potent TGR5 agonist V2 (Bioorg. Chem. 2022, 120, 105588). Mechanistic studies have shown that the 12-OH-retained analog V1 can positively allosterically regulate the functional activity of the TGR5 endogenous ligand CDCA, while the 12-OH-methylated analog V2 loses its allosteric effect on TGR5. Recently, we used structure-based drug design to selectively alkylate 7-OH and discovered a highly efficient TGR5 allosteric agonist V3 (CN 115710298A; WO 2023 / 168974 Al), which significantly improves agonist activity and can allosterically regulate the functional activity of TGR5.

[0003] Summary of the invention

[0004] The purpose of the present invention is a preparation method of a non-steroidal bile acid analog and its application as a bile acid receptor TGR5 allosteric agonist.

[0005] The present invention uses cholic acid CA as a lead compound, keeps 3-OH unchanged, alkylates 7-OH, oxidizes 12-OH to a keto carbonyl, further expands the B ring or C ring to a seven-membered ring of a lactone or lactam, and modifies the 24-carboxyl group to an amide or ester to obtain a series of novel non-steroidal cholic acid analogs, and tests the agonist activity and mechanism of action of the new compounds on TGR5.

[0006] The non-steroidal bile acid analogs provided by the present invention have structures as shown in formula (1) and formula (2):

[0007]

[0008] Wherein, X is an imino group (-NH-) or an oxygen group (O);

[0009] R1 is one of the following groups:

[0010]

[0011] R2 is one of the following groups:

[0012]

[0013] Further, in the non-steroidal bile acid analog, X is O, NH; R1 is R2 is

[0014]

[0015] Furthermore, the structure of the non-steroidal bile acid analog is as follows:

[0016]

[0017] The present invention provides a method for synthesizing non-steroidal bile acid analogs of formula (1) and formula (2). The specific synthesis route of the non-steroidal bile acid analogs of formula (1) is as follows:

[0018]

[0019] The synthesis of compound A refers to patent (CN 115710298 A);

[0020] The specific synthesis steps of A1-A5 are as follows:

[0021] (1) Compound A was dissolved in dichloromethane solvent, and m-chloroperbenzoic acid and p-toluenesulfonic acid hydrate were added in a molar ratio of compound A: oxidant: acid = 1:1.2:0.1, and the mixture was reacted at room temperature for 24 hours to obtain compound A1.

[0022] (2) Compound A1 was dissolved in a solvent, coupling agent HATU was added under ice bath condition, and the mixture was stirred for 30 minutes. A base and an amine compound were slowly added and stirred at room temperature for 2 hours. After post-treatment, A2-A5 was obtained.

[0023] Among them, the solvent is dichloromethane, the coupling agent is HATU or EDC, the base is triethylamine, and the amine compound is specifically tetrahydropyrrole, dimethylamine, diethylamine, morpholine, piperazine, piperidine, methylamine, ethylamine, n-propylamine, isopropylamine, glycine ethyl ester, glycine, tetrahydropyrrole substituted glycine, cyclopropylamine, cyclopentylmethylamine, cyclopentylamine or cyclopentylmethylamine; the molar ratio of compound A1: coupling agent: amine compound: base is 1:(1.1-1.3):1.2:3.

[0024]

[0025] The specific synthesis steps of A6-A10 are as follows:

[0026] (1) Compound A is dissolved in methanol solvent, and hydroxylamine hydrochloride and sodium acetate trihydrate are added in a molar ratio of compound A: hydroxylamine: buffer = 1:4:7, and the mixture is heated under reflux at 80° C. for 12-14 hours to obtain intermediate compound W.

[0027] (2) Compound W was dissolved in anhydrous tetrahydrofuran, and the mixture was evacuated to replace nitrogen. Then, thionyl chloride was added under ice bath condition at a molar ratio of compound W:thionyl chloride = 1:10, and the mixture was reacted at room temperature for 8-12 h to obtain compound A6.

[0028] (3) Compound A6 was dissolved in a solvent, coupling agent HATU was added under ice bath condition, and the mixture was stirred for 30 minutes. A base and an amine compound were slowly added and stirred at room temperature for 20 hours. After post-treatment, A7-A10 was obtained.

[0029] Among them, the solvent is dichloromethane, the coupling agent is HATU or EDC, the base is triethylamine, and the amine compound is specifically tetrahydropyrrole, dimethylamine, diethylamine, morpholine, piperazine, piperidine, methylamine, ethylamine, n-propylamine, isopropylamine, glycine ethyl ester, glycine, tetrahydropyrrole substituted glycine, cyclopropylamine, cyclopentylmethylamine, cyclopentylamine or cyclopentylmethylamine; the molar ratio of compound A6: coupling agent: amine compound: base is 1:1.5:1.5:3.

[0030] The specific synthesis route of the non-steroidal bile acid analogue of formula (2) is as follows:

[0031]

[0032] The specific synthesis steps of B1 are as follows:

[0033] (1) CA was dissolved in DCM, HATU was added under ice bath conditions, a base (such as triethylamine) was slowly added dropwise, and the mixture was stirred for 30 min. An amine compound (such as tetrahydropyrrole) was added, and the mixture was stirred for 20 h at room temperature to obtain intermediate compound 1.

[0034] (2) Compound 1 was dissolved in a solvent of V acetone:V water = 3:1, NBS was added and the mixture was reacted in the dark for 12 h to obtain intermediate compound B.

[0035] (3) Compound B was dissolved in dichloromethane solvent, and m-chloroperbenzoic acid and p-toluenesulfonic acid hydrate were added in a molar ratio of compound B: oxidant: acid = 1:1.2:0.1, and the mixture was reacted at room temperature for 24 hours to obtain compound B1.

[0036] The specific synthesis steps of B2 are as follows:

[0037] (1) Compound B is dissolved in methanol solvent, and hydroxylamine hydrochloride and sodium acetate trihydrate are added in a molar ratio of compound B: hydroxylamine: buffer = 1:4:7, and the mixture is heated under reflux at 80° C. for 12-14 hours to obtain an intermediate compound M.

[0038] (2) Compound M was dissolved in anhydrous tetrahydrofuran, and the mixture was evacuated to replace nitrogen. Then, thionyl chloride was added under ice bath conditions at a molar ratio of compound M to thionyl chloride of 1:10, and the mixture was reacted at room temperature for 8-12 h to obtain compound B2.

[0039] The cumulative experimental results of GloSensor cAMP showed that the synthesized new non-steroidal bile acid analogues have good agonist activity on TGR5 and can allosterically regulate the functional activity of TGR5 endogenous ligands, and can be used to prepare allosteric agonists of bile acid receptor TGR5.

[0040] Further, the use of the non-steroidal bile acid analogs of formula (1) and formula (2) in the preparation of drugs for preventing and treating TGR5-related diseases. The TGR5-related diseases are selected from: TGR5-related atherosclerosis, obesity, non-alcoholic fatty liver disease (NAFLD) (e.g., simple fatty liver or non-alcoholic steatohepatitis (NASH)), cholestatic liver disease, metabolic syndrome, type II diabetes, type I diabetes, insulin resistance, hyperinsulinemia, glucose intolerance, glucose metabolism disorders, hyperglycemia, hyperlipidemia (e.g., hypercholesterolemia), cholelithiasis, cirrhosis, cholestasis (e.g., intrahepatic cholestasis) and other metabolic diseases, as well as tumors and inflammatory bowel disease-related diseases.

[0041] The present invention provides a pharmaceutical composition, comprising a non-steroidal bile acid analog of formula (1) or formula (2) or a pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, solvate, crystal form or metabolite thereof; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0042] The present invention has the following beneficial effects:

[0043] The present invention selects bile acid, an endogenous ligand of TGR5, as a lead compound, keeps 3-OH unchanged through a series of reactions, alkylates 7-OH, further expands the B ring or C ring to a seven-membered ring of a lactone or lactam, and modifies the 24-carboxyl group to an amide to obtain a series of novel non-steroidal bile acid analogs, and tests the functional activity of the target compound on TGR5 through a GloSensor cAMP accumulation experiment. The results show that the synthesized novel non-steroidal bile acid analog has good agonist activity on TGR5 and can allosterically regulate the functional activity of the endogenous ligand of TGR5. The TGR5 allosteric agonist in the present invention will provide a new potential candidate drug for the treatment of metabolic diseases such as type II diabetes, obesity, and non-alcoholic fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the allosteric regulatory mechanism of compound A5.

[0045] Figure 2 This is the allosteric regulatory mechanism of compound A10. DETAILED DESCRIPTION

[0046] The specific implementation of the present invention is described in detail below in conjunction with the embodiments of the specification.

[0047] Example 1

[0048]

[0049] Synthesis of (R)-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxohexadecahydro-2H-cyclopenta[b]naphtho[2,1-d]oxepan-4-yl)pentanoic acid (A1):

[0050] The specific synthesis process of intermediate compound A refers to patent (CN 115710298 A).

[0051] Steps: Compound A (140 mg, 0.3 mmol) was dissolved in DCM (6 mL), and oxidant m-chloroperbenzoic acid (62 mg, 0.36 mmol) and p-toluenesulfonic acid monohydrate (5.7 mg, 0.03 mmol) were added to react at room temperature for 24 h. After the reaction was completed, it was extracted with DCM (20 mL × 3), and the organic phase was washed with distilled water to remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 15:1) was performed and concentrated to obtain 79 mg of white solid A1, with a yield of 60%. 1 H NMR (400MHz, CDCl3) δ3.46 (s, 1H), 3.30-3.26 (m, 2H), 3.22 (s, 3H), 2.65 (d, J = 13.1Hz, 1H), 2.42-2.09 (m, 6H), 1.95-1.84 (m, 4H), 1.75-1.72 (m, 3H) ),1.64-1.60(m,2H),1.45-1.41(m,2H),1.35(s,3H),1.28-1.18(m,6H), 1.06(d,J=6.4Hz,2H),0.99(t,J=7.3Hz,2H),0.86(s,3H).HRMS(ESI):m / z calcd C 25 H 40 O6[M+Na] + 459.2723, found:459.2724.

[0052] Example 2

[0053]

[0054] Synthesis of (R)-N-ethyl-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxohexadecahydro-2H-cyclopenta[b]naphtho[2,1-d]oxazol-4-yl)pentanamide (A2):

[0055] Steps: Compound A1 (100 mg, 0.23 mmol) was dissolved in dichloromethane (5 mL), HATU (119 mg, 0.30 mmol) was added under ice bath conditions, triethylamine (100 μL, 0.69 mmol) was slowly added dropwise, and 2M ethylamine was added in THF (145 μL, 0.28 mmol) after stirring in an ice bath for 30 minutes, and stirred at room temperature for 2 hours, and detected by TCL. After the reaction was completed, it was extracted with dichloromethane (10 mL × 3), the organic phase was washed with saturated brine, and the solvent was concentrated to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 50: 1) was performed and concentrated to obtain 95.7 mg of white solid compound A2 with a yield of 89%. 1H NMR (400MHz, CDCl3) δ5.28(s,3H),3.45(s,1H),3.29-3.22(m,3H),3.20(s,2H),2.63(d,J =13.1Hz,1H),2.37(t,J=12.8Hz,1H),2.31-2.15(m,3H),2.14-2.01(m,2H),1.96-1.77(m, 6H),1.74-1.65(m,3H),1.60(d,J=19.4Hz,1H),1.52(s,1H),1.41(d,J=16.3Hz,1H),1.33 (s,4H),1.21(m,J=17.4Hz,4H),1.11(t,J=7.2Hz,3H),1.03(d,J=6.7Hz,3H),0.85(s,3H). 13 C NMR (101MHz, CDCl3) δ175.11,87.06,71.75,55.82,55.24,50.22,42.69,41.23,38.10 ,36.08,35.65,34.40,33.40,32.93,31.05,27.46,25.22,24.13,22.33,17.60,14.82.

[0056] Example 3

[0057]

[0058] Synthesis of (R)-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxohexadecahydro-2H-cyclopenta[b]naphtho[2,1-d]oxepan-4-yl)-N-isopropylvaleramide (A3):

[0059] The synthesis steps were the same as those of A2, except that ethylamine was replaced with isopropylamine to obtain 86.6 mg of white solid compound A3 with a yield of 79%. 1H NMR (400MHz, CDCl3) δ4.06 (p, J = 7.0Hz, 1H), 3.49-3.40 (m, 1H), 3.20 (s, 3H), 2.63 (d, J = 13.2Hz, 1H), 2.37 (t,J=12.9Hz,1H),2.28(d,J=8.4Hz,1H),2.24-2.12(m,3H),2.11-1.99(m,2H),1.96-1.86(m,4H),1.82(d ,J=2.6Hz,2H),1.72(d,J=10.8Hz,3H),1.60(d,J=18.1Hz,1H),1.51(d,J=9.4Hz,1H),1.42(d,J=12.3Hz,1 H),1.33(s,5H),1.22(dd,J=21.5,11.7Hz,4H),1.12(d,J=6.5Hz,6H),1.03(d,J=6.7Hz,4H),0.85(s,3H). 13 C NMR (101MHz, CDCl3) δ175.40,172.86,87.35,72.08,56.13,55.57,50.52,43.01,41.59,38.41,36 .38,36.19,35.74,34.67,33.84,33.22,31.35,27.76,25.52,24.46,23.22,22.63,17.92,14.94.

[0060] Example 4

[0061]

[0062] Synthesis of (R)-N-cyclopropyl-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxohexadecahydro-2H-cyclopenta[b]naphtho[2,1-d]oxepan-4-yl)pentanamide (A4):

[0063] The synthesis steps were the same as those of A2, except that ethylamine was replaced with cyclopropylamine to obtain 92.8 mg of white solid compound A4 with a yield of 85%. 1H NMR (400MHz, CDCl3) δ3.43(p,J=6.6Hz,1H),3.23(s,1H),3.19(s,3H),2.69-2.58(m,2H),2.36(t,J=12.8Hz,1H),2.26( d,J=8.2Hz,1H),2.17(q,J=12.4Hz,3H),2.05(dd,J=17.6,11.7Hz,2H),1.91(s,2H),1.86(d,J=9.7Hz,2H),1.80(d,J=1 2.9Hz,2H),1.70(d,J=10.7Hz,3H),1.59(d,J=20.5Hz,1H),1.50(d,J=8.8Hz,1H),1.40(d,J=14.9Hz,1H),1.32(d,J=7. 4Hz,5H),1.26-1.14(m,3H),1.08-1.03(m,1H),1.00(d,J=6.7Hz,3H),0.84(s,3H),0.71(d,J=5.5Hz,2H),0.46(s,2H).

[0064] Example 5

[0065]

[0066] Synthesis of (3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-4-((R)-5-oxo-5-(pyrrolidin-1-yl)pentan-2-yl)hexahydro-2H-cyclopenta[b]naphtho[2,1-d]oxepan-2-one (A5):

[0067] The synthesis steps were the same as those of A2, except that ethylamine was replaced with tetrahydropyrrole to obtain 96.9 mg of white solid compound A5 with a yield of 86%. 1 H NMR (400MHz, CDCl3) δ3.46-3.38(m,5H),3.25(s,1H),3.21(s,3H),2.64(d,J=13.2 Hz,1H),2.38(t,J=12.7Hz,1H),2.32-2.24(m,2H),2.21-2.08(m,3H),1.96-1.93( m,4H),1.87-1.80(m,5H),1.74-1.69(m,4H),1.63-1.54(m,3H),1.44-1.41(m,2H) ,1.34(s,3H),1.24-1.19(m,3H),1.05(d,J=6.5Hz,1H),0.88(s,3H),0.84(s,1H). 13C NMR (100MHz, CDCl3) δ175.1,172.5,87.0,72.1,55.7,55.0,53.5,52.9,50.1,46.6,45.7,42.6,41.1,40.4,38 .0,37.2,36.0,35.8,35.3,34.2,32.8,31.0,30.4,30.0,27.4,26.1,25.2,24.4,23.6,20.6,17.6,16.9,14.5.

[0068] Example 6

[0069]

[0070] Synthesis of (R)-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxooctadecylhydrocyclopenta[b]naphtho[2,1-d]azepan-4-yl)pentanoic acid (A6):

[0071] Step 1: Dissolve compound A (200 mg, 0.48 mmol) in methanol (5 mL) solution, add hydroxylamine hydrochloride (132 mg, 1.9 mmol) and sodium acetate trihydrate (457 mg, 3.4 mmol) in sequence, reflux at 80 ° C overnight for 12 hours, extract with DCM (30 mL × 3) after the reaction, wash the organic phase with distilled water, remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 20: 1), concentrate to obtain 171.5 mg of white solid intermediate W, yield 82%. 1 H NMR (400MHz, CDCl3) δ3.44 (s, 1H), 3.34 (dd, J = 4.8Hz, 1H), 3.23 (s, 1H), 3.2 1(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=13.0Hz,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).

[0072] Step 2: Compound W (150 mg, 0.34 mmol) was dissolved in redistilled tetrahydrofuran (5 mL) solvent, and dichlorothionyl (0.25 ml, 3.4 mmol) was added under ice bath conditions, and then reacted at room temperature for 8 h under nitrogen protection. After the reaction, it was extracted with DCM (30 mL × 3), and the organic phase was washed with distilled water to remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 8: 1) was concentrated to obtain 103.7 mg of white solid compound A6, 70%. 1 H NMR (400MHz, DMSO) δ6.72(s,1H),3.38(s,1H),3.25-3.20(m,2H),3.15(s,3H),2.36(t,J=12 .7Hz,1H),2.17-2.10(m,2H),1.93(q,J=13.0Hz,3H),1.82-1.76(m,6H),1.67(d,J=8.2Hz,2H ),1.61-1.57(m,1H),1.50(d,J=9.0Hz,2H),1.45-1.43(m,2H),1.32-1.28(m,2H),1.22-1.1 8(m,3H),1.14(s,3H),0.98(d,J=6.3Hz,2H),0.91-0.85(m,2H),0.81(s,3H).HRMS(ESI):m / z calcd C 25 H 41 NO5[M+Na] + 458.2882, found:458.2892.

[0073] Example 7

[0074]

[0075] Synthesis of (R)-N-ethyl-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxooctadecylhydrocyclopenta[b]naphtho[2,1-d]oxepan-4-yl)pentanamide (A7):

[0076] Steps: A6 (50 mg, 0.11 mmol) was dissolved in DCM (5 mL), and a coupling agent 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (65.4 mg, 0.17 mmol) was added at 0°C, and triethylamine (48 μL, 0.33 mmol) was slowly added dropwise. After 30 minutes, ethylamine (85 μL, 0.17 mmol) was added, and the mixture was reacted overnight at room temperature for 20 hours. After the reaction was completed, the mixture was extracted with DCM (30 mL × 3), and the organic phase was washed with distilled water to remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 20:1) gave 41.1 mg of a white solid product A7 with a yield of 81%. 1 H NMR (400MHz, CDCl3) δ6.66 (s, 1H), 5.93 (s, 1H), 3.44 (p, J = 5.8Hz, 1H), 3.29-3.21 (m, 3H), 3.18 (s, 3H), 2.4 0(d,J=10.8Hz,2H),2.27-2.18(m,1H),2.17-2.06(m,3H),2.03(s,2H),1.95(d,J=15.1Hz,2H),1.83(d,J=1 5.7Hz,2H),1.77(d,J=9.7Hz,1H),1.74-1.68(m,2H),1.64-1.57(m,2H),1.54(d,J=8.9Hz,1H),1.38(t,J=1 1.2Hz,3H),1.24(d,J=12.8Hz,3H),1.18(s,3H),1.11(t,J=7.2Hz,3H),1.03(d,J=6.4Hz,3H),0.84(s,3H). 13 C NMR (101MHz, CDCl3) δ177.38,173.13,71.50,67.48,60.08,55.43,52.56,43.08,40.96,37.72,35.83,35.49 ,35.23,34.67,34.18,32.65,32.26,30.53,29.47,27.06,26.00,25.57,22.14,20.85,17.35,14.58,14.10.

[0077] Example 8

[0078]

[0079] Synthesis of (R)-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxooctadecylhydrocyclopentane[b]naphtho[2,1-d]azepan-4-yl)-N-isopropylvaleramide (A8):

[0080] The synthesis steps were the same as those of A7, except that ethylamine was replaced with isopropylamine to obtain 39.5 mg of white solid product A8 with a yield of 75%. 1 H NMR (400MHz, CDCl3) δ6.04 (s, 1H), 5.38 (d, J = 7.9Hz, 1H), 4.06 (h, J = 6.7Hz, 1H), 3.45 (t, J = 10.0Hz, 1H), 3.20(s,3H),2.39(d,J=6.9Hz,2H),2.22-2.08(m,4H),2.06-1.95(m,3H),1.86(s,1H),1.81(d,J=11.1H z,2H),1.77-1.69(m,3H),1.69-1.57(m,2H),1.53(d,J=6.5Hz,2H),1.39(t,J=9.3Hz,2H),1.33(d,J=5. 1Hz, 1H), 1.26 (d, J = 12.7Hz, 3H), 1.19 (s, 3H), 1.13 (d, J = 6.6Hz, 6H), 1.04 (d, J = 5.1Hz, 3H), 0.86 (s, 4H).

[0081] Example 9

[0082]

[0083] Synthesis of (R)-N-cyclopropyl-4-((3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-2-oxooctahydrocyclopenta[b]naphtho[2,1-d]azepan-4-yl)pentanamide (A9):

[0084] The synthesis steps were the same as those of A7, except that ethylamine was replaced with cyclopropylamine to obtain 45.6 mg of white solid product A9 with a yield of 87%. 1H NMR (400MHz, CDCl3) δ6.06(s,1H),3.47(d,J=12.0Hz,1H),3.20(s,3H),2.69(q,J=3.6Hz,1H),2.40(d,J=7 .5Hz,2H),2.23-2.09(m,4H),2.06-1.95(m,3H),1.85(d,J=15.9Hz,3H),1.77(s,1H),1.73(d,J=13.9Hz,2H ),1.62(d,J=15.9Hz,2H),1.53(d,J=6.4Hz,2H),1.41(d,J=5.4Hz,2H),1.36(s,1H),1.33(d,J=7.7Hz,1H), 1.27(s,2H),1.24(s,4H),1.20(d,J=7.3Hz,3H),1.03(d,J=5.6Hz,3H),0.86(s,4H),0.76(d,J=5.5Hz,1H). 13 C NMR (101MHz, CDCl3) δ176.89,175.96,72.03,60.77,55.87,55.06,52.87,50.75,43.55,41.39,38.02,36.29,3 5.64,35.18,32.66,32.56,30.71,29.92,27.53,26.48,26.00,22.90,22.54,21.15,17.32,14.35,6.71,6.62.

[0085] Example 10

[0086]

[0087] Synthesis of (3aR,4R,6aS,6bR,7R,8aS,10R,12aS,12bS)-10-hydroxy-7-methoxy-3a,12a-dimethyl-4-((R)-5-oxo-5-(pyrrolidin-1-yl)pentan-2-yl)hexahydrocyclopenta[b]naphtho[2,1-d]azepan-2(1H)-one (A10):

[0088] The synthesis steps were the same as those of A7, except that ethylamine was replaced with tetrahydropyrrole to obtain 38.7 mg of white solid product A10 with a yield of 72%. 1H NMR(400MHz, CDCl3)δ6.03(s,1H),3.43-3.35(m,5H),3.21(s,1H),3.17(m,3 H),2.36(d,J=7.0Hz,2H),2.30-2.23(m,1H),2.18-2.06(m,5H),2.00-1.89(m ,4H),1.81(t,J=7.0Hz,4H),1.74-1.61(m,4H),1.56-1.52(m,2H),1.39-1.2 1(m,5H),1.16(s,3H),1.02(d,J=5.4Hz,3H),0.96-0.93(m,1H),0.83(s,3H). 13 C NMR (100MHz, CDCl3) δ176.52,171.54,77.42,71.73,60.11,56.52,55.63,52.93,46.59,45.69,43.32,41.26,38 .05,36.07,35.50,34.71,32.56,31.08,30.93,30.00,27.32,26.23,26.10,25.71,24.38,22.41,18.08,14.51.

[0089] Embodiment 11

[0090]

[0091] Synthesis of (5R,7aS,9S,9aR,10R)-5,9-dihydroxy-7a,9a-dimethyl-10-((R)-5-oxo-5-(pyrrolidin-1-yl)pentan-2-yl)hexahydro-2H-benzo[d]indeno[4,5-b]oxepan-2-one (B1):

[0092] Step 1: CA (300 mg, 0.73 mmol) was dissolved in DCM (15 mL), HATU (418.5 mg, 1.1 mmol) was added at 0 ° C, triethylamine (305 μL, 2.2 mmol) was slowly added dropwise, tetrahydropyrrole (78 μL, 1.1 mmol) was added after 30 minutes, and stirred at room temperature for 20 h. After the reaction, DCM (40 mL × 3) was used for extraction, the organic phase was washed with brine, and the solvent was removed to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 30: 1) was concentrated to obtain 1280 mg of a white solid intermediate with a yield of 83%. The hydrogen and carbon spectra of the compound are consistent with the reference (Bioorg Chem, 2022, 120: 105588.).

[0093] Step 2: Compound 1 (200 mg, 0.43 mmol) and N-bromosuccinimide (169 mg, 0.95 mmol) were dissolved in a mixed solvent of acetone (6 mL) and water (2 mL), the volume ratio of acetone to water was 3:1, and the mixture was reacted at room temperature in the dark for 12 h. After the reaction, it was extracted with DCM (40 mL × 3), the organic phase was washed with distilled water, and the solvent was removed to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 20: 1) was performed and concentrated to obtain 140 mg of white solid intermediate B with a yield of 71%. 1 H NMR (400MHz, CDCl3) δ4.01(s,1H),3.59-3.54(m,1H),3.42(q,J=7.3Hz,4H),3.22(q,J=7.3Hz,1H),2 .83(q,J=6.0Hz,1H),2.51(s,1H),2.39-2.27(m,3H),2.24-2.12(m,4H),1.96-1.91(m,4H),1.85(q, J=6.7Hz,3H),1.80(d,J=5.3Hz,1H),1.76(s,1H),1.69(q,J=9.5Hz,4H),1.57-1.52(m,1H),1.36(q, J=7.4Hz,2H),1.32-1.23(m,3H),1.16(s,3H),1.14-1.10(m,1H),0.89(q,J=6.3Hz,3H),0.67(s,3H). 13 C NMR (100MHz, CDCl3) δ212.5,172.5,71.7,70.5,49.5,46.7,46.4,46.1,46.0,45.7,45.4,40 .4,37.1,35.8,35.5,34.7,34.2,31.8,30.9,29.4,29.1,27.7,26.0,24.3,22.7,17.4,12.8.

[0094] Step 3: Compound B (140 mg, 0.3 mmol) was dissolved in DCM (6 mL), and the oxidant m-chloroperbenzoic acid (62 mg, 0.36 mmol) and p-toluenesulfonic acid monohydrate (5.7 mg, 0.03 mmol) were added to react at room temperature for 24 h. After the reaction was completed, it was extracted with DCM (20 mL × 3), and the organic phase was washed with distilled water to remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 15: 1) was performed and concentrated to obtain 185 mg of a white solid product B with a yield of 60%. 1H NMR (400MHz, CDCl3) δ4.92 (s, 4H), 4.50 (t, J = 9.9Hz, 1H), 3.93 (s, 1H), 3.63-3.56 (m, 1H),3.51(t,J=6.7Hz,2H),3.40(t,J=6.9Hz,2H),3.35-3.24(m,2H),2.43-2.37(m,2 H),2.32-2.21(m,3H),1.98(t,J=6.8Hz,2H),1.93-1.87(m,5H),1.78-1.73(m,4H),1 .68-1.63(m,2H),1.46-1.36(m,5H),1.10(s,3H),1.03(d,J=6.4Hz,3H),0.74(s,3H). 13 C NMR (100MHz, CDCl3) δ175.2,172.4,80.5,71.1,70.5,47.2,46.7,45.7,45.1,41.7,37 .3,35.7,35.2,34.8,31.8,30.8,30.1,29.7,27.3,26.1,24.8,24.4,23.2,17.5,12.5.

[0095] Example 12

[0096]

[0097] Synthesis of (5R,7aS,9S,9aR,10R)-5,9-dihydroxy-7a,9a-dimethyl-10-((R)-5-oxo-5-(pyrrolidin-1-yl)pentan-2-yl)hexahydrobenzo[d]indeno[4,5-b]azacyclopentene-2(1H)-one (B2):

[0098] Step 1: Dissolve compound B (140 mg, 0.3 mmol) in methanol (6 mL), add hydroxylamine hydrochloride (83.4 mg, 1.2 mmol) and sodium acetate trihydrate (285.6 mg, 2.1 mmol) in sequence, reflux at 80 ° C for 12 h, extract with DCM (30 mL × 3) after the reaction, wash the organic phase with distilled water, remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 10: 1), concentrate to obtain 120 mg of white solid intermediate M, with a yield of 83%. 1H NMR(400MHz, CDCl3)δ4.00(s,1H),3.58-3.51(m,1H),3.45–3.39(m,4H),3 .10(d,J=15.2Hz,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=5.8Hz,4H),0.67(s,3H). 13 CNMR (100MHz, CDCl3) δ172.8,161.0,72.4,70.8,46.7,46.6,46.2,45.7,44.8,42.4,40.6,36.4 ,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.

[0099] Step 2: Compound M (120 mg, 0.25 mmol) was dissolved in redistilled tetrahydrofuran (6 mL) solvent, and dichlorothionyl (0.18 ml, 2.5 mmol) was added under ice bath conditions, and then reacted at room temperature for 8 h under nitrogen protection. After the reaction, it was extracted with DCM (20 mL × 3), and the organic phase was washed with distilled water to remove the solvent to obtain a crude product. Column chromatography (V dichloromethane: V methanol = 8: 1) was performed and concentrated to obtain 284 mg of a white solid product B with a yield of 70%. 1 H NMR (400MHz, CDCl3) δ7.43 (s, 1H), 3.92 (s, 1H), 3.64 (s, 1H), 3.41 (dd, J = 7.4Hz, 4H), 3.29 (s ,1H),2.92(d,J=13.8Hz,1H),2.33-2.26(m,1H),2.22-2.13(m,2H),2.10-2.00(m,4H),1.92( q,J=6.8Hz,3H),1.86-1.74(m,6H),1.62(d,J=10.4Hz,4H),1.49(s,1H),1.40(s,2H),1.28(s ,2H),1.19-1.13(m,2H),1.01(s,3H),0.97(d,J=5.8Hz,4H),0.87-0.81(m,1H),0.64(s,3H). 13C NMR (100MHz, CDCl3) δ180.2,172.4,73.8,71.1,53.7,52.1,46.8,46.6,45.8,44.2,42.0,38.6, 36.5,35.5,35.4,34.8,32.0,30.8,29.8,29.5,29.1,27.4,26.3,24.7,24.5,23.1,17.7,12.3.

[0100] Biological activity test

[0101] The GloSensor cAMP accumulation assay (refer to patent CN 115710298 A) was used to test the agonist activity and allosteric regulatory mechanism of the new compounds on human or mouse TGR5.

[0102] The dose-response curve of chenodeoxycholic acid (CDCA) (100 nM-50 μM) was tested. When the bioluminescent signal reached the highest point, a new compound (80 nM-50 μM) with a multiple relationship in concentration was added to test whether the change in bioluminescence at this time showed a concentration-dependent limited upward trend.

[0103] 1. Activity test of target compounds in human TGR5 (hTGR5) and mouse TGR5 (mTGR5):

[0104]

[0105] Table 1. EC of Class A target compounds for activation of TGR5 50 value

[0106]

[0107]

[0108] Table 2. EC of Class B target compounds for activation of TGR5 50 value

[0109]

[0110] From the results in Table 1, it can be seen that the agonist activity of the new compounds A1-A5, which are obtained by expanding the C ring of bile acid to lactone, is significantly improved compared with CA. Among them, the agonist activity of A1 with the best activity on hTGR5 (EC 50 =0.8 μM) was 48 times that of the lead compound CA, and its agonist activity on mTGR5 (EC 50 =0.6 μM) is 49 times that of the lead compound CA; the agonist activity of the new compounds A6-A10 with C ring expansion to lactam is also significantly improved compared with CA, among which A10 has an agonist activity against hTGR5 (EC 50=1.6 μM) was 24 times that of the lead compound CA, and its agonist activity on mTGR5 (EC 50 =1.7μM) is 17 times that of the lead compound CA. A comprehensive comparison of A1-A10 shows that the agonist activity of the new compounds whose corresponding C rings are expanded to lactones is slightly higher than that of the new compounds whose lactams are expanded, that is, the expansion of the new compounds to lactones is beneficial to the improvement of activity. Among them, A1, A5, and A10 have better pharmacological activity, among which A1 has the best activity; followed by A2, A3, A4, and A8. When the 24-position of A5 and A10 is tetrahydropyrrole, they have the highest agonist activity for seven-membered ring lactones or lactams. All new compounds have comparable agonist activity for hTGR5 and mTGR5, with no obvious species differences.

[0111] From the results in Table 2, we can see that the activity of the analog B1 containing a seven-membered lactone ring in the B ring of cholic acid is lower than that of the seven-membered lactam ring B2. Comparison of Table 1 and Table 2 shows that the activity of the new analogs obtained by ring expansion of the C ring of cholic acid is significantly higher than that of the analogs obtained by ring expansion of the B ring.

[0112] 2. Study on the mechanism of allosteric action

[0113] The GloSensor cAMP accumulation assay was used to further investigate whether this class of compounds could allosterically regulate the functional activity of the endogenous ligand CDCA of TGR5, and to further confirm whether this class of compounds is a positive allosteric modulator (PAM) or allosteric agonist of TGR5. Figure 1 As shown), the concentration gradient of compound A5 is set to 80nM, 400nM, 2μM, 10μM, and 50μM, and the concentration increases successively. As the concentration increases, the dose-effect curve of CDCA begins to move upward. When the concentration of A5 is 80nM and 400nM, the upward window is small. When the concentration of A5 reaches 2μM, A5 regulates the hTGR5 endogenous ligand CDCA and a large upward window appears. At this time, when the concentration of A5 continues to increase to 10μM, the dose-effect regulation amplitude of the hTGR5 endogenous ligand CDCA is reduced and almost reaches saturation. At this time, the allosteric effect will no longer increase if the concentration of A5 is continued to increase, and the dose-effect curve of CDCA shows a limited upward shift as the concentration of A5 increases. Compound A10 (as shown) Figure 2 The above results show that the target compounds of the present invention can effectively positively allosterically regulate the functional activity of CDCA, an endogenous ligand of TGR5, that is, the target compounds of the present invention are all positive allosteric modulators (PAMs) or allosteric agonists of TGR5.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A non-steroidal bile acid analogue, characterized in that: The structure of the non-steroidal bile acid analog is shown in formula (1) or formula (2): ； Wherein, X is NH or O; R1 is one of the following groups: ； R2 is one of the following groups: 。 2. The non-steroidal bile acid analog according to claim 1, characterized in that: R1 is One of: R2 is One of them.

3. The non-steroidal bile acid analog according to claim 1, characterized in that: The structure of the non-steroidal bile acid analog is as follows: 。 4. The method for preparing a non-steroidal bile acid analogue according to any one of claims 1 to 3, characterized in that: The steps include: (1) Compound A is dissolved in dichloromethane solvent, an oxidant and an acid are added, the molar ratio of compound A to the oxidant and the acid is 1:1.2:0.1, and the reaction is carried out at room temperature to obtain compound A1 in which the C ring is expanded to a seven-membered lactone ring; Compound A1 is dissolved in dichloromethane, a coupling agent is added under ice bath conditions, and the mixture is stirred for 30 minutes. A base and an amine compound are slowly added and stirred at room temperature for 2 hours, and a compound of formula (1) is obtained after post-treatment, wherein X is O; the molar ratio of compound A1: coupling agent: amine compound: base is 1:(1.1-1.3):1.2:3; Or, (2) Compound A is dissolved in methanol solvent, hydroxylamine and buffer are added, the molar ratio of compound A:hydroxylamine:buffer is 1:4:7, and the reaction is heated under reflux at 80°C for 12-14 hours to obtain intermediate compound W; Compound W is dissolved in anhydrous tetrahydrofuran, thionyl chloride is added under ice bath condition, the molar ratio of compound W:thionyl chloride is 1:10, and the reaction is carried out at room temperature for 8-12 hours under nitrogen protection to obtain a compound of formula (1), wherein X is NH, R2 is OH, and is recorded as compound A6; The structural formula of the compound A is as follows: ; Or, (3) dissolving compound A6 in a solvent, adding a coupling agent under ice bath conditions, stirring for 30 minutes, slowly adding a base and an amine compound, stirring and reacting at room temperature for 20 hours, and post-treating to obtain a compound of formula (1), wherein X is NH, and R2 is not OH; the molar ratio of compound A6: coupling agent: amine compound: base is 1:1.5:1.5:3; Or, (4) dissolving bile acid in dichloromethane, adding coupling agent under ice bath condition, slowly adding base dropwise, stirring and reacting for 30 minutes, adding amine compound, stirring and reacting for 20 hours at room temperature, to obtain intermediate compound 1; dissolving compound 1 in a solvent of V acetone: V water = 3:1, adding NBS and reacting in the dark for 12 hours, to obtain intermediate compound B; dissolving compound B in dichloromethane solvent, reacting at a molar ratio of compound B: oxidant: acid = 1:1.2:0.1 at room temperature for 24 hours, to obtain a compound of formula (2), X = O; Or, (5) Compound B is dissolved in methanol solvent, hydroxylamine and buffer are added, the molar ratio of compound B: reducing agent: buffer is 1:4:7, and the reaction is refluxed at 80°C for 12 hours to obtain intermediate compound M; Compound M is dissolved in anhydrous tetrahydrofuran, thionyl chloride is added under ice bath condition, the molar ratio of compound M: thionyl chloride is 1:10, and the reaction is carried out at room temperature for 8 hours to obtain a compound of formula (2), X=NH.

5. The method for preparing a non-steroidal bile acid analogue according to claim 4, characterized in that: The oxidant is m-chloroperbenzoic acid, the acid is p-toluenesulfonic acid hydrate; and the buffer is sodium acetate trihydrate.

6. The method for preparing a non-steroidal bile acid analogue according to claim 4, characterized in that: The coupling agent is HATU or EDC; the base is triethylamine; the amine compound is one of tetrahydropyrrole, dimethylamine, diethylamine, morpholine, piperazine, piperidine, methylamine, ethylamine, n-propylamine, isopropylamine, glycine ethyl ester, glycine, tetrahydropyrrole substituted glycine, cyclopropylamine, cyclopentylmethylamine, cyclopentylamine or cyclopentylmethylamine.

7. The use of the non-steroidal bile acid analogue according to any one of claims 1 to 3, characterized in that: The non-steroidal bile acid analogue is used in the preparation of drugs for preventing and treating bile acid receptor TGR5 related diseases.

8. The use of the non-steroidal bile acid analogue according to claim 7, characterized in that: The TGR5-related 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 disorders, hyperglycemia, hyperlipidemia, cholelithiasis, cirrhosis, cholestasis and other metabolic diseases, as well as tumors and inflammatory bowel disease.

9. The use of the non-steroidal bile acid analogue according to any one of claims 1 to 3, characterized in that: The non-steroidal bile acid analog is used in the preparation of a bile acid receptor TGR5 allosteric agonist.

10. A pharmaceutical composition, characterized in that The invention comprises the non-steroidal bile acid analogue according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, solvate, crystal form or metabolite thereof, and a pharmaceutically acceptable carrier or excipient.

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