Application of oxime cholic acid analogue as TGR5 allosteric agonist

Abstract

The invention belongs to the field of medicinal chemistry, and particularly discloses application of oxime cholic acid analogues as TGR5 allosteric agonists. The preparation method comprises the following steps: by taking cholic acid as a raw material, obtaining cholic acid intermediates of which the 12th site or the 7th site and the 12th site are oximes through a series of reactions, and then carrying out a series of modification on the 24th site and oximes of the intermediates to obtain the cholic acid analogue as shown in a formula 1 or a formula 2. According to the invention, a GloSensor cAMP accumulation experiment is adopted to test the functional activity and allosteric regulation mechanism of all compounds on TGR5 on a G protein dependent signal channel. Pharmacological results show that all the synthesized target compounds have relatively good agonist activity on TGR5, and can positively allosteric regulate the functional activity of chenodeoxycholic acid CDCA, that is, the target compounds disclosed by the invention are all TGR5 positive allosteric modulators or allosteric agonists. # imgabs0 # imgabs1 #

Description

Technical Field

[0001] The invention belongs to the field of medicinal chemistry and specifically discloses the application of an oxime bile acid analog as a G protein-coupled bile acid receptor 1 (TGR5) allosteric agonist. Background Art

[0002] The bile acid receptor TGR5 (Takeda G protein-coupled receptor 5) is an important member of the class A GPCR. It plays an important role in blood glucose homeostasis, energy consumption and liver protection. It is a highly potential drug target for the treatment of metabolic syndrome and inflammatory diseases. Therefore, the development of drugs for the treatment of metabolic diseases targeting TGR5 has great research value.

[0003] In the preliminary structure-activity relationship study of bile acid CA, a potent TGR5 agonist L2 was discovered (Bioorg.Chem.2022,120,105588). The study showed that the 12-OH-retained analog L1 can positively allosterically regulate the functional activity of the TGR5 endogenous ligand CDCA, while the 12-OH-methylated analog L2 lost its allosteric regulatory effect on TGR5. On this basis, it was found that the 7-OH selectively methylated compound L3 is a highly effective TGR5 allosteric modulator (CN115710298A). In order to further investigate the effect of 12-OH on allosteric regulation, we modified it into oxime, and further modified the oxime to obtain a new bile acid analog to explore its allosteric regulatory functional activity on TGR5.

[0004]

[0005] Study on the structure and activity of TGR5 allosteric agonists Summary of the invention

[0006] The purpose of the present invention is to provide an application of an oxime bile acid analog as a TGR5 allosteric agonist. The present invention uses bile acid CA as a lead compound, keeps 3-OH unchanged, performs amide coupling on 24-carboxyl, and then replaces 12-OH or 7,12-OH of these bile acid intermediates with oxime, and then further modifies the oxime to obtain a series of bile acid analogs with novel structures, and tests the agonist activity and allosteric action mechanism of the new compounds on TGR5.

[0007] The oxime bile acid analogs provided by the present invention have structures as shown in formula (1) and formula (2):

[0008]

[0009] Wherein R1 is hydrogen or R 1， R3 is independently one of the following groups:

[0010]

[0011] R2 is one of the following groups:

[0012]

[0013] Further, the bile acid analog, R1 is One of the following groups: R3 is One of them.

[0014] Furthermore, the structure of the bile acid analog is as follows:

[0015]

[0016] The present invention provides a method for synthesizing bile acid analogs of formula (1) and formula (2). The specific synthesis routes of bile acid analogs of formula (1) and formula (2) are as follows:

[0017] 1. Synthesis route and specific synthesis method of TGR5 bile acid analogue formula (1):

[0018]

[0019] Synthesis route of target compound formula (1)

[0020] Among them, the synthesis of compounds 9a, A1, and C1 referred to the previous work of our research group (CN 115710298 A; WO 2023 / 168974Al).

[0021] The synthesis method of the compound of formula (1):

[0022] (1) The intermediate 9a was dissolved in DCM, and the coupling agent 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added at 0°C, and triethylamine (TEA) was slowly added dropwise. After 30 minutes, tetrahydropyrrole was added, and the reaction was carried out at room temperature overnight for 20 hours to obtain compound A1.

[0023] (2) Compound A1 was dissolved in methanol solution, and hydroxylamine hydrochloride (NH2OH·HCl) and sodium acetate trihydrate (NaOAc·3H2O) were added in sequence, wherein the molar ratio was compound A1:NH2OH·HCl:NaOAc·3H2O=1:4:7. The mixture was then condensed and refluxed at 70°C overnight to obtain compound C1.

[0024] (3) Compound C1 was dissolved in redistilled tetrahydrofuran (THF) solvent, solid sodium hydroxide powder and catalyst potassium iodide were added, and the reaction was continued for 30 minutes. A halogenated hydrocarbon was added in an ice bath, and then the reaction was carried out at 70° C. in the dark to obtain the final product.

[0025] 2. Synthesis route and specific synthesis method of TGR5 bile acid analogue formula (2):

[0026]

[0027] Synthesis route of target compound formula (2)

[0028] The synthesis method of the compound of formula (2):

[0029] (1) Compound 1 was dissolved in DCM, and a catalyst of p-dimethylaminopyridine (DMAP), an acid-binding agent of triethylamine (TEA), and an acetylation agent of acetic anhydride (Ac2O) were added thereto. The molar ratio was compound 1:DMAP:TEA:Ac2O=1:0.1:3:0.5. The reaction was carried out at room temperature for 12 hours to obtain compound 2.

[0030] (2) Compound 2 was dissolved in acetone, and an excess of Jones reagent was added dropwise at 0°C to turn the solution into reddish brown. After stirring for 5 minutes, TLC was performed. After the reaction was complete, isopropanol was added to quench the reaction, and the solution was stirred until it turned dark green. The isopropanol and acetone were removed by rotary evaporation, and compound 3 was obtained after post-treatment.

[0031] (3) Compound 3 was dissolved in methanol solvent, and an excess of sodium hydroxide aqueous solution was added. The reaction was hydrolyzed at room temperature for 14-20 hours to obtain intermediate 4.

[0032] (4) Intermediate 4 was dissolved in DCM, and coupling agent 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added at 0°C, and triethylamine (TEA) was slowly added dropwise. After 30 minutes, tetrahydropyrrole was added, and the reaction was allowed to react at room temperature overnight for 20 hours to obtain compound 5.

[0033] (5) Compound 5 was dissolved in methanol solution, and hydroxylamine hydrochloride (NH2OH·HCl) and sodium acetate trihydrate (NaOAc·3H2O) were added in sequence, wherein the molar ratio was compound 5: NH2OH·HCl: NaOAc·3H2O = 1:8:14. The mixture was then condensed and refluxed at 70°C overnight for 12 hours to obtain compound 6.

[0034] (6) Compound 6 was dissolved in redistilled tetrahydrofuran (THF) solvent, solid sodium hydroxide powder and catalyst potassium iodide were added, and the reaction was continued for 30 minutes. Then, a halogenated hydrocarbon was added in an ice bath, and then the reaction was carried out at 70° C. in the dark to obtain the final product.

[0035] The present invention uses the GloSensor cAMP accumulation experiment to test the functional activity and allosteric regulatory mechanism of the synthesized new compounds on TGR5 in the G protein-dependent signaling pathway. Pharmacological results show that all the synthesized target compounds have good agonist activity on TGR5 and can positively allosterically regulate the functional activity of chenodeoxycholic acid CDCA, that is, the target compounds of the present invention are all TGR5 positive allosteric modulators or allosteric agonists.

[0036] The present invention provides the use of the above-mentioned oxime bile acid analogs in the preparation of bile acid receptor TGR5 allosteric agonists.

[0037] The present invention also provides the use of the above-mentioned oxime bile acid analogs in the preparation of drugs for preventing and treating bile acid receptor TGR5-related diseases. The TGR5-related diseases include metabolic diseases, tumors, and inflammatory bowel diseases. Among them, 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 disorders, hyperglycemia, hyperlipidemia, cholelithiasis, cirrhosis, and cholestasis.

[0038] The present invention also provides a pharmaceutical composition comprising one or more of the above-mentioned oxime bile acid analogs.

[0039] The present invention has the following beneficial effects:

[0040] The present invention selects bile acid, an endogenous ligand of TGR5, as a lead compound, firstly obtains bile acid analog intermediates with oxime at positions 12 or 7 and 12 through a series of reactions, and then performs a series of modifications on positions 24 and oxime of these intermediates to obtain the desired novel bile acid analogs. The functional activity of the target compound on TGR5 is tested by the accumulation experiment of GloSensor cAMP. The results show that the synthesized new 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

[0041] Figure 1 This is the allosteric regulatory mechanism of compound D2.

[0042] Figure 2 This is the allosteric regulatory mechanism of compound E2. DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045]

[0046] Synthesis of (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 13R, 14S, 17R, E)-3-hydroxy-12-(hydroxyimino)-7-methoxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoic acid (D1):

[0047] Step 1: Dissolve the intermediate 9a (500 mg, 1.2 mmol) in methanol (15 ml), add hydroxylamine hydrochloride (330 mg, 4.76 mmol) and sodium acetate trihydrate (1.14 g, 8.4 mmol) in turn, and react at 70°C under condensation reflux overnight for 12 hours. After the reaction is completed, extract with DCM (50 mL × 3), wash the organic phase with distilled water, and remove the solvent to obtain a crude product. Column chromatography (V DCM :V MeOH =10:1), and concentrated to obtain 400 mg of compound D1 as a white solid with a reaction yield of 76.6%. 1 H NMR (400MHz, CDCl3) δ3.44 (s, 1H), 3.34 (dd, J1 = 13.0Hz, J2 = 4.8Hz, 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=13.0Hz,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).

[0048] Example 2

[0049]

[0050] Synthesis of (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 13R, 14S, 17R, E)-3-hydroxy-12-(hydroxyimino)-7-methoxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (D2):

[0051] Compound C1 (50 mg, 0.1 mmol) was dissolved in tetrahydrofuran (5 ml) solution from which water had been removed, solid sodium hydroxide powder (12 mg, 0.3 mmol) and catalyst potassium iodide (3.32 mg, 0.002 mmol) were added in sequence, stirred at room temperature for 30 min, and then methyl iodide (31 μL, 0.5 mmol) was added. The mixture was condensed and refluxed at 70°C overnight under light-proof conditions for 24 h. After the reaction was completed, it was extracted with DCM (30 mL × 3), the organic phase was washed with distilled water, and the solvent was removed to obtain a crude product. Column chromatography (V DCM :V MeOH =30:1), and concentrated to obtain 40 mg of compound D2 as a white solid. The reaction yield was 79.6%. 1 H NMR (400MHz, CDCl3) δ3.75 (s, 3H), 3.47-3.40 (m, 5H), 3.22 (s, 4H), 3.13 (dd, J1=1 3.4Hz,J2=5.1Hz,1H),2.39-2.31(m,1H),2.24-2.14(m,2H),2.09(d,J=12.3Hz,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 =13.1Hz,1H),1.48-1.34(m,7H),0.98(d,J=6.5Hz,3H),0.95(s,3H),0.85(s,3H). 13 C NMR (100MHz, CDCl3) δ172.6,164.0,77.3,71.9,60.9,56.1,53.0,49.2,47.1,46.8,45.8,41.8,39.6, 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.

[0052] Example 3

[0053]

[0054] Synthesis of (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 13R, 14S, 17R, E)-12-(ethoxyimino)-3-hydroxy-7-methoxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (D3):

[0055] The other conditions were the same as those for D2, except that methyl iodide was replaced with ethyl bromide to obtain 38 mg of white solid D3 with a yield of 73.6%. 1 H NMR (400MHz, CDCl3) δ3.97 (q, J = 7.0Hz, 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=12.2Hz,1H),2.00-1.90(m,4H),1.83(t,J=7 .1Hz,5H),1.75(s,1H),1.71-1.63(m,5H),1.60-1.54(m,2H),1.49(d,J=13.2Hz,1H),1.45-1. 33(m,5H),1.23(s,1H),1.21(t,J=7.0Hz,4H),0.97(d,J=6.6Hz,4H),0.93(s,3H),0.84(s,3H). 13 C NMR (100MHz, CDCl3) δ172.6,163.7,77.3,71.9,68.4,56.1,53.5,53.1,49.2,47.1,46.7,45.7,41.8,39 .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.

[0056] Example 4

[0057]

[0058] Synthesis of (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 13R, 14S, 17R, E)-3-hydroxy-7-methoxy-10,13-dimethyl-12-(propoxyimino)hexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (D4):

[0059] The other conditions were the same as those for D2, except that methyl iodide was replaced with propyl bromide to obtain 41 mg of white solid D4 with a yield of 77.3%. 1H NMR (400MHz, CDCl3) δ3.87-3.81 (m, J=5.5Hz, 2H), 3.42-3.35 (m, 5H), 3.16 (s, 3H) ,3.14(d,J=5.7Hz,1H),2.33-2.25(m,1H),2.19-2.09(m,2H),2.04(d,J=12.2Hz,1 H),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=6.5Hz,3H),0.90(s,3H),0.85(t,J=7.4Hz,3H),0.81(s,3H). 13 C NMR (100MHz, CDCl3) δ172.5,163.6,74.4,71.7,55.9,53.0,49.1,47.0,46.6,45.6,41.7,39.4,38.3,35.9, 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.

[0060] Example 5

[0061]

[0062] Synthesis of (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 13R, 14S, 17R, E)-3-hydroxy-12-(isopropoxyimino)-7-methoxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (D5):

[0063] The other conditions were the same as those for the synthesis of D2, except that methyl iodide was replaced with isopropyl bromide to obtain 42 mg of a white solid with a yield of 79.2%. 1 H NMR (400MHz, CDCl3) δ3.41-3.34(m,5H),3.16(s,1H),3.12(d,J=5.0Hz,1H),2.32-2.24(m,1H),2.19-1.98(m,3H),1.95-1.85(m,4H ),1.81-1.74(m,5H),1.67-1.48(m,7H),1.45-1.19(m,8H),1.11(t,J=4.9Hz,7H),0.93(d,J=4.2Hz,3H),0.88(s,3H),0.80(s,3H).13 C NMR (100MHz, CDCl3) δ172.6,163.3,77.3,73.9,72.0,56.1,53.3,49.3,47.1,46.7,45.7,41.8,39.5,38 .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.

[0064] Example 6

[0065]

[0066] Synthesis of (R)-4-((3R, 5R, 7Z, 8R, 9S, 10S, 12E, 13R, 14S, 17R)-3-hydroxy-7,12-bis(hydroxyimino)-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentane-1-one (E1):

[0067] Step 1: Dissolve compound 1 (2.5 g, 5.92 mmol) in anhydrous dichloromethane (40 mL), and add triethylamine (2.45 mL, 17.76 mmol), DMAP (72 mg, 0.592 mmol) and acetic anhydride (281 μL, 2.96 mmol) in sequence. After overnight reaction for 20 h, TLC detection was performed. After the reaction was completed, the solvent was removed, extracted with DCM (80 mL × 3), the organic phase was washed with water (100 mL), and the solvent was concentrated to obtain a crude product. Column chromatography (V DCM :V EA =10:1), concentrated to obtain 1.4 g of compound 2, which was a white powdery solid, with a reaction yield of 51%.

[0068] Step 2: Dissolve compound 2 (1 g, 2.2 mmol) in acetone (20 mL), add an appropriate amount of Jones reagent at 0 ° C to turn the solution into reddish brown, stir for 5 minutes and then perform TLC detection. After the reaction is completed, add 20 mL of isopropanol and stir until the solution turns dark green. Remove isopropanol and acetone by rotary evaporation. Extract with EA (50 mL × 3) and water (30 mL) to remove the solvent. Concentrate to obtain 800 mg of crude product 3, which is a white foamy solid with a reaction yield of 79%.

[0069] Step 3: Dissolve compound 3 (500 mg, 1.09 mmol) in methanol (30 mL), add excess 6M / L NaOH aqueous solution, and stir at room temperature for 14 h. After the reaction is completed, add hydrochloric acid solution to adjust the pH value to 2, and remove methanol by rotary evaporation. Wash the solid repeatedly with water and dichloromethane to remove impurities, dry to obtain a crude product, and column chromatography (V DCM :V MeOH =15:1), concentrated to obtain 330 mg of compound 4 as a white solid, with a reaction yield of 74.9%.

[0070] Step 4: Compound 4 (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 slowly added dropwise, tetrahydropyrrole (100 μL, 1.224 mmol) was added after 30 minutes, and the mixture was stirred at room temperature for 20 h. After the reaction was completed, the mixture was extracted with DCM (30 mL × 3), the organic phase was washed with distilled water, and the solvent was removed to obtain a crude product. Column chromatography (V DCM :V MeOH =20:1), concentrated to obtain 360 mg of compound 5 as a white solid, with a reaction yield of 93.6%.

[0071] Step 5: Dissolve compound 5 (360 mg, 0.764 mmol) in methanol (30 ml), add hydroxylamine hydrochloride (424.5 mg, 6.108 mmol) and sodium acetate trihydrate (1.45 g, 10.696 mmol) in turn, and react at 70°C under condensation reflux overnight for 12 hours. After the reaction is completed, extract with DCM (40 mL × 3), wash the organic phase with distilled water, and remove the solvent to obtain a crude product. Column chromatography (V DCM :V MeOH =20:1), and concentrated to obtain 310 mg of compound E1 in the form of a white solid. The reaction yield was 83.2%. 1H NMR (400MHz, DMSO) δ10.15(s,1H),10.08(s,1H),4.44(d,J=4.8Hz,1H),3.34(s,1H),3.19(t,J =6.8Hz,2H),3.07(d,J=9.4Hz,1H),2.85(d,J=12.9Hz,1H),2.45(s,1H),2.38(t,J=10.6Hz,1H ),2.14-2.00(m,3H),1.93(dd,J1=13.0Hz,J2=5.2Hz,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=6.6Hz,3H),0.82(s,3H). 13 C NMR (100MHz, DMSO) δ170.8,162.2,157.2,79.2,78.9,78.6,69.0,54.7,53.0,48.7,46.0,45.2,43.5,4 1.2,36.9,35.5,34.4,31.7,30.4,29.7,27.4,25.7,25.0,23.9,22.2,19.5,14.0,12.0.HRMS(ESI):m / z calcd C 28 H 45 N3O4[M+Na] + 510.3308, found:510.3303.

[0072] Example 7

[0073]

[0074] Synthesis of (R)-4-((3R, 5R, 7Z, 8R, 9S, 10S, 12E, 13R, 14S, 17R)-3-hydroxy-7,12-bis(methoxyimino)-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (E2):

[0075] Compound E1 (30 mg, 0.0615 mmol) was dissolved in tetrahydrofuran (5 ml) solution from which water had been removed, solid sodium hydroxide powder (12 mg, 0.3077 mmol) and catalyst potassium iodide (2 mg, 0.012 mmol) were added in sequence, stirred at room temperature for 30 min, and then methyl iodide (38 μL, 0.625 mmol) was added. The mixture was condensed and refluxed at 70°C overnight under light-proof conditions for 24 h. 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 DCM :V MeOH =30:1), and concentrated to obtain 24 mg of compound E2 in the form of a white solid. The reaction yield was 75.7%. 1 H NMR (400MHz, CDCl3) δ3.74 (d, J = 3.8Hz, 6H), 3.62-3.54 (m, 1H), 3.46-3.40 (m, 4H),3.09(dd,J1=13.1Hz,J2=4.8Hz,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=13.0Hz, 3H),1.48-1.23(m,6H),1.14-1.03(m,5H),0.97(d,J=6.4Hz,3H),0.87(s,3H). 13 C NMR (100MHz, CDCl3) δ172.5,163.2,158.3,70.8,61.1,60.9,52.6,49.2,46.6,46.1,45.6,44.0,43.9,42.0,3 6.7,35.9,34.6,32.2,30.6,29.9,28.2,27.7,26.2,25.3,24.4,22.6,20.5,19.8,12.1,1.01.HRMS(ESI):m / z calcd C 30 H 49 N3O4[M+Na] + 538.3621, found:538.3615.

[0076] Example 8

[0077]

[0078] Synthesis of (R)-4-((3R, 5R, 7Z, 8R, 9S, 10S, 12E, 13R, 14S, 17R)-7,12-bis(ethoxyimino)-3-hydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (E3):

[0079] Other conditions were the same as those for the synthesis of E2, except that iodomethane was replaced by ethyl bromide to obtain 14 mg of a white solid with a yield of 69.2%. 1 H NMR (400MHz, CDCl3) δ3.57(q,J=7.2Hz,1H),3.48(s,8H),3.10(q,J=7.3Hz,1H),2.21(t,J=7.7Hz,2H),2.00(d,J=7.0Hz,3H),1.6 9-1.59(m,6H),1.46(t,J=7.2Hz,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).

[0080] Example 9

[0081]

[0082] Synthesis of (R)-4-((3R, 5R, 7Z, 8R, 9S, 10S, 12E, 13R, 14S, 17R)-3-hydroxy-10,13-dimethyl-7,12-bis(propoxyimino)hexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-1-(pyrrolidin-1-yl)pentan-1-one (E4):

[0083] Other conditions were the same as those for the synthesis of E2, except that methyl iodide was replaced by propyl bromide to obtain 25 mg of a white solid with a yield of 68.2%. 1 H NMR (400MHz, CDCl3) δ3.89 (t, J=6.6Hz, 4H), 3.57 (s, 1H), 3.45-3.39 (m, 4H), 3 .15(dd,J1=13.1Hz,J2=4.7Hz,1H),3.00(d,J=13.2Hz,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=6.4Hz,3H),0.90(s,1H),0.88(s,4H). 13C NMR (100MHz, CDCl3) δ172.6,163.1,158.0,74.9,74.6,71.0,52.9,49.4,46.7,46.1,45.7,44.2,44.1,42.1,36.9 ,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.

[0084] Biological activity test

[0085] Referring to the biological activity test method of CN 115710298A, a cAMP accumulation test was used to test whether the new compounds could allosterically regulate the functional activity of TGR5 endogenous ligand chenodeoxycholic acid (CDCA) to determine whether they were TGR5 positive allosteric modulators (PAMs).

[0086] The results of the target compound's agonist activity test on human TGR5 (hTGR5) and mouse TGR5 (mTGR5):

[0087]

[0088] Table 1. EC of the target compounds of formula (1) and formula (2) for activating TGR5 50 value

[0089]

[0090] The glosensor cAMP accumulation test was used to test the agonist activity of the 9 new analogs synthesized on hTGR5 and mTGR5. The experimental results are shown in Table 1 above. The target compounds of the present invention all have agonist activity on TGR5. The new compounds modified by oxime alkylation at positions 7 and 12 have obvious differences in agonist activity on hTGR5 and mTGR5. Among the D-type compounds, D2, D3, D4 and D5 have a significant difference in EC of hTGR5. 50 The activity of E2 in mTGR5 was 25.58nM, 0.15μM, 0.30μM and 0.16μM, respectively, which was 18, 16, 64 and 82 times higher than that in mTGR5. 50 The activity of hTGR5 was 1.61 μM, which was 19-fold lower than that of hTGR5.

[0091] For hTGR5, the target compound obtained by alkylation of both oximes at positions 7 and 12 is less active than the target compound obtained by alkylation of only oxime at position 12. That is, the target compound obtained by alkylation of monooxime has better agonist activity against TGR5 than the target compound obtained by alkylation of dioxime. For example, among the target compounds, the EC value of D2 for hTGR5 is 50 The activity of D3 was 13 times higher than that of E3; the activity of D4 was 6 times higher than that of E4.

[0092] The 7α-OH of CA was methylated, the 3α-OH and 24-carboxyl groups remained unchanged, and the 12α-OH was modified to oxime to obtain the monooxime compound D1. The EC of D1 for hTGR5 was 50 It was 0.86 μM, which was 45 times higher than that of CA. Then, the carboxyl group at position 24 of D1 was subjected to amide coupling, and the oxime at position 12 was methylated to obtain the target compound D2 with better activity. The activity of D2 was nearly 1500 times higher than that of CA. The 12th position of D2 was extended by one C and two Cs respectively to obtain compounds D3 and D4, whose biological activities decreased by 6 times and 12 times respectively compared with that of D2. In addition, with the extension of the carbon chain of the alkylation modification or the increase of the molecular size, the biological activity of the compound against TGR5 showed a downward trend.

[0093] This rule also applies to the target compound modified by bis-oxime alkylation. We kept the 3α-OH of CA unchanged, performed amide coupling on the 24-position carboxyl group, and modified the 7α-OH and 12α-OH into oximes to obtain the bis-oxime target compound E1. The EC value of E1 for hTGR5 was 50 The EC of E2 for hTGR5 was 1.69 μM, which was 23 times higher than that of CA. Then, the oximes at positions 7 and 12 of E1 were methylated to obtain the target compound E2. 50 The activity of the compound E3 and E4 decreased by nearly 20 times compared with E2 after extending the 7th and 12th positions of E2 by one C and two Cs, respectively. This regular feature is the same as the previous alkylation modification of 7α-OH.

[0094] 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 1As shown in Figure 2, when the concentration of compound D2 increased by several times, the concentration-dependent curve of CDCA mediated by it showed a limited upward shift, indicating that compound D2 can effectively positively allosterically regulate the functional activity of TGR5 endogenous ligand CDCA, that is, D2 is a TGR5 allosteric agonist. The concentration-dependent curve of compound E2 allosterically regulating CDCA (as shown in Figure 2) Figure 2 The results of D2 are consistent with those of D2, that is, the target compounds in the present invention are all positive allosteric modulators (PAM) or allosteric agonists of TGR5.

[0095] 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. An oxime bile acid analogue, characterized in that: The structure of the oxime bile acid analog is shown in formula (1) or formula (2): ， Wherein, R1 is hydrogen or R1 and R3 are independently one of the following groups: ； R2 is one of the following groups: 。 2. The oxime bile acid analogue according to claim 1, characterized in that: R1 is One of: R2 is or ; R3 is One of them.

3. The oxime bile acid analogue according to claim 1 or 2, characterized in that: The structural formula of the oxime bile acid analogue is as follows: 。 4. The use of the oxime bile acid analogue according to any one of claims 1 to 3, characterized in that: The oxime bile acid analogue is used in the preparation of a bile acid receptor TGR5 allosteric agonist.

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

6. The use 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 use of the oxime bile acid analogue according to claim 6, characterized in that: 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 disorders, hyperglycemia, hyperlipidemia, cholelithiasis, cirrhosis, and cholestasis.

8. A pharmaceutical composition, characterized in that: The invention comprises the oxime 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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