Synthesis method of glycocholic acid dimer

Abstract

The invention belongs to the technical field of drug synthesis, and particularly discloses a synthesis method of glycocholic acid dimer, which comprises the following steps: step 1, under alkaline condition, glycocholic acid and a condensing agent are condensed to obtain an active ester intermediate A; 2, carrying out a substitution reaction on the intermediate A and tert-butyl diphenyl chlorosilane under an alkaline condition to obtain an intermediate B; 3, under the action of a coupling reagent, the intermediate B and glycocholic acid are subjected to a coupling reaction, and an intermediate C is obtained; and 4, under the action of a deprotection agent, removing the tert-butyl diphenyl silyl group from the intermediate C to obtain the target product glycocholic acid dimer. According to the invention, a glycocholic acid dimer reference substance can be provided for research on impurities of glycocholic acid and related preparations thereof, so that research on drug impurities is promoted, and the method has positive significance for improving the quality standard of glycocholic acid drugs and guaranteeing the medication safety of residents.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis technology, and specifically relates to a method for synthesizing a glycocholic acid dimer. Background Art

[0002] To date, 30% of the chemical drugs listed in pharmacopoeias are poorly soluble, and 60% of those under development are also poorly soluble. This poor solubility leads to low bioavailability, necessitating improvements in the solubility of these drugs to enhance their bioavailability. Solubilization of poorly soluble drugs has always been a critical issue, particularly for injectable dosage forms, where it remains a key constraint in product development and R&D. Therefore, the search for new, efficient, and safe solubilizers is a crucial approach to addressing this issue.

[0003] Glycocholic acid (GDCA) is a pharmaceutical excipient, mainly used as a carrier for the injection of poorly soluble drugs. It is widely used in the field of injections. Its structural formula is as follows:

[0004]

[0005] Glycocholic acid will generate corresponding impurities during the preparation and storage process. The content of these impurities may affect the pharmacology and pharmacokinetics of the drug, and may also reduce the activity of the drug and significantly increase toxic side effects.

[0006] Glycocholic acid dimer (GDCA Dimer) is an impurity produced during the preparation of glycocholic acid. During the storage of the product, glycocholic acid itself will also polymerize to form glycocholic acid dimer. The molecular formula of glycocholic acid dimer is: C 52 H 84 N2O 11 ; Molecular weight: 913.23; Specific structural formula is as follows:

[0007] According to ICHQ3 and relevant technical guidelines for drug development in my country, this impurity should be controlled below 0.15%. To improve the quality of glycocholic acid and its related preparations and reduce the risks of clinical use, detailed research and monitoring of glycocholic acid dimer impurities are necessary.

[0008] In the prior art, Shu Bing et al. used obeticholic acid as the starting material, protected the carboxyl group and hydroxyl group with benzyl and TMS groups, respectively, to obtain two key intermediates: benzyl-protected 3α,7α-dihydroxy-6α-ethyl-5-β-cholanoic acid benzyl ester A-1 and TMS-protected 3α-trimethylsilyloxy-7α-hydroxy-6α-ethyl-5-β-cholanoic acid A-3. The two were condensed under DCC / DMAP conditions to obtain an ester, and then the benzyl and TMS protecting groups were removed to finally obtain obeticholic acid dimer A-5 with a total yield of 30% (Shu Bing, Zheng Tao, Xie Xiaoling, et al. Synthesis of a key impurity dimer of obeticholic acid [J]. Guangzhou Chemistry, 2021, 46(03): 42-46.). This method has a low yield and uses a hydrogen palladium carbon reduction system for reduction, which is highly dangerous and not suitable for industrial production.

[0009] Niu Weiping et al. used ursodeoxycholic acid, chenodeoxycholic acid and obeticholic acid as starting materials, and prepared the target compound through electrophilic substitution, dehydration condensation, hydrogenation and other reactions. The specific synthesis process was not disclosed (Niu Weiping, Niu Weixiao, Zhang Guoning, et al. Design, synthesis and inhibition of hepatocyte apoptosis of bile acid dimer compounds [J]. Chinese Journal of Pharmaceutical Biotechnology, 2020, 15(04): 370-377.).

[0010] North China Pharmaceutical Group New Drug Research and Development Co., Ltd. disclosed a method for preparing obeticholic acid dimer in patent document CN 107674106 A. The method involves protecting the carboxyl and hydroxyl groups of obeticholic acid to obtain 3α,7α-dihydroxy-6α-ethyl-5β-cholanoic acid benzyl ester (VI) and 3α,7α-di-(4-methoxytriphenylmethyl)-6α-ethyl-5β-cholanoic acid (IV). Compounds IV and VI are then esterified under alkaline conditions, and the hydroxyl and carboxyl protecting groups are sequentially removed to obtain obeticholic acid dimer. However, this method involves six steps and two Pd / C hydrogenation reduction debenzylation reactions, which is complex. The maximum content of obeticholic acid dimer obtained is 97.88%, and the overall maximum yield is only 6.66%, making it unsuitable for industrial production.

[0011] Currently, no standard or reference substance for the glycocholic acid dimer impurity is available. The Chinese Pharmacopoeia (ChP), United States Pharmacopoeia (USP), and European Pharmacopoeia (EP) do not include this impurity, and there are no reports on the preparation of glycocholic acid dimer in the prior art. Therefore, it is necessary to develop a synthesis method for glycocholic acid dimer to provide a basis for quality research on glycocholic acid and its related preparations, which is of great significance for improving the safety, efficacy, and quality control of glycocholic acid and its preparations. Summary of the Invention

[0012] The present invention provides a method for synthesizing a glycocholic acid dimer for the first time, provides a glycocholic acid dimer impurity reference substance for the research of glycocholic acid and its related preparations, and provides a basis for the quality research of glycocholic acid and its related preparations, thereby promoting the research of drug impurities and improving the quality standards of glycocholic acid drugs, which has positive significance for ensuring the safety of residents' use of drugs.

[0013] The synthetic route of the technical solution of the present invention is as follows:

[0014]

[0015] It is characterized in that it comprises the steps of:

[0016] (1) Under alkaline conditions, glycocholic acid and a condensing agent are condensed to obtain an active ester intermediate A;

[0017]

[0018] (2) Under alkaline conditions, intermediate A reacts with tert-butyldiphenylsilyl chloride to obtain intermediate B;

[0019]

[0020] (3) Under the action of coupling reagent, intermediate B undergoes coupling reaction with glycocholic acid to obtain intermediate C;

[0021]

[0022] (4) Under the action of a deprotecting agent, the tert-butyldiphenylsilyl group of intermediate C is removed to obtain the target product, glycocholic acid dimer;

[0023]

[0024] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the condensing agent in step (1) includes one of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and N-hydroxysuccinimide;

[0025] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the step (1) comprises one of N,N-diisopropylethylamine and triethylamine;

[0026] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction temperature in step (1) is 25-40°C;

[0027] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction solvent in step (1) comprises one of N,N-dimethylformamide and tetrahydrofuran;

[0028] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the molar ratio of glycocholic acid, base and condensing agent in step (1) is 1:2~3:1.5~2.0.

[0029] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the alkaline substance used in the reaction in step (2) includes one of imidazole and potassium carbonate; and the molar ratio of intermediate A, tert-butyldiphenylchlorosilane and alkaline substance in step (2) is 1:2~3:2~3.

[0030] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction temperature of intermediate B in step (2) is 0~5°C.

[0031] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction solvent in step (2) comprises one of N,N-dimethylformamide and tetrahydrofuran.

[0032] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the molar ratio of intermediate B, coupling reagent and glycocholic acid in step (3) is 1:1.5~2.0:1.8~2.5.

[0033] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the coupling reagent in step (3) includes one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0034] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the base in step (3) comprises one of 4-dimethylaminopyridine, triethylamine and 1-hydroxybenzotriazole; and the molar ratio of the intermediate B to the base is 1:0.5~1.0.

[0035] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction temperature in step (3) is 20~30°C.

[0036] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction solvent in step (3) includes N,N-dimethylformamide.

[0037] The above-mentioned method for preparing a glycocholic acid dimer is characterized in that the deprotecting agent in step (4) comprises one of a 1 mol / L tetrabutylammonium fluoride / tetrahydrofuran solution and a 5% volume fraction hydrochloric acid / methanol solution; and the plastochemical ratio of the intermediate C to the deprotecting agent is 1 g: 15-20 ml.

[0038] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the deprotection reaction temperature in step (4) is 20~30°C.

[0039] The above-mentioned method for preparing glycocholic acid dimer is characterized in that the reaction solvent in step (4) comprises one of tetrahydrofuran and methanol.

[0040] Beneficial effects of the present invention:

[0041] 1. The present invention adopts a synthetic route for glycocholic acid dimer developed by retrosynthetic analysis, which avoids the use of highly dangerous reagents and high-value catalysts: the glycocholic acid dimer is split into two molecules of glycocholic acid, and then one of the glycocholic acid molecules is protected at the carboxyl and hydroxyl groups to obtain a highly stable carboxylic acid active ester intermediate, and then the hydroxyl-protected glycocholic acid dimer is obtained through intermolecular condensation. Finally, the target product glycocholic acid dimer is obtained through deprotection.

[0042] 2. The present invention obtains glycocholic acid 3-position condensation dimer with high selectivity, which can effectively avoid the formation of dimers condensed at other positions. By controlling various process parameters in the preparation composition, the yield and purity of glycocholic acid dimer are improved. The yield of the prepared glycocholic acid dimer is as high as 65.21%, and the purity is not less than 98%.

[0043] 3. The reaction conditions of the present invention are mild, the raw materials are readily available, the process is simple, the preparation steps are short, and the reaction process is easy to control. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The present invention provides a synthetic route for preparing glycocholic acid dimer;

[0045] Figure 2 This is the H NMR spectrum of glycocholic acid dimer;

[0046] Figure 3 This is the C-NMR spectrum of glycocholic acid dimer;

[0047] Figure 4 is the mass spectrum of glycocholic acid dimer;

[0048] Figure 5 This is the liquid phase spectrum of the glycocholic acid dimer prepared in Example 2, wherein Figure A is the liquid phase spectrum of the glycocholic acid dimer, and Figure B is the peak result of the liquid phase spectrum of the glycocholic acid dimer. DETAILED DESCRIPTION

[0049] The following examples are intended only to illustrate the methods for implementing the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention is not limited to these examples. Those skilled in the art will appreciate that equivalent substitutions or corresponding improvements made to the contents of the present invention still fall within the scope of protection of the present invention.

[0050] Example 1

[0051] Preparation of intermediate A:

[0052] 10.00 g (21.48 mmol) of glycocholic acid was dissolved in 100 ml of N,N-dimethylformamide. 5.55 g of N,N-diisopropylethylamine (42.95 mmol; 2.0 eq) was added dropwise and stirred until uniform. Then, 9.70 g of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (32.22 mmol; 1.5 eq) was added and allowed to react at 25°C. TLC monitored the reaction for completion. The reaction solution was poured into 500 ml of purified water and stirred for 10 min. Ethyl acetate was then added for extraction three times, 100 ml each time. The ethyl acetate phase was collected and washed three times with saturated sodium chloride solution, 100 ml each time. The ethyl acetate phase was collected and concentrated until no liquid remained. 60 ml of anhydrous ethanol and 10 ml of purified water were added for dissolution. After complete dissolution, 260 ml of purified water was added dropwise for crystallization. The mixture was filtered and dried to obtain 10.88 g of Intermediate A, a yield of 90.0%.

[0053] 2. Preparation of intermediate B:

[0054] Intermediate A (10.88 g; 19.33 mmol) obtained in the previous step was dissolved in 110 ml of N,N-dimethylformamide. The temperature was maintained at 0–5°C, and 2.63 g of imidazole (38.66 mmol; 2 eq) and 10.63 g of tert-butyldiphenylsilyl chloride (38.66 mmol; 2 eq) were added. After addition, the temperature was maintained at 0–5°C, and the reaction was complete by TLC. The mixture was diluted with 550 ml of purified water and extracted with ethyl acetate three times, 110 ml each time. The ethyl acetate phase was collected and washed three times with saturated sodium chloride solution, 110 ml each time. The upper ethyl acetate phase was collected. 330 ml of purified water was then added, and the mixture was heated to reflux, then cooled to 0–5°C for crystallization. The mixture was filtered and dried to obtain 13.78 g of intermediate B, with a yield of 89.0%.

[0055] 3. Preparation of Intermediate C:

[0056] Dissolve Intermediate B (13.78 g; 19.33 mmol) and 14.42 g of glycocholic acid (30.97 mmol; 1.8 eq) obtained in the previous step in 130 ml of N,N-dimethylformamide. Add 1.05 g of 4-dimethylaminopyridine (8.6 mmol; 0.5 eq). Then cool the mixture to 0°C and add 5.32 g of N,N'-dicyclohexylcarbodiimide (25.80 mmol; 1.5 eq). After addition, warm the temperature to 20°C. Monitor the reaction by TLC. Dilute the mixture with 650 ml of purified water and extract with ethyl acetate three times, each time using 130 ml. Collect the ethyl acetate phase and wash the mixture three times with saturated sodium chloride solution, each using 130 ml. The upper ethyl acetate phase was collected, 650 ml of purified water was added, and the mixture was heated to reflux for dissolution. The mixture was then cooled to 0-5°C for crystallization, filtered, and dried to obtain 15.85 g of intermediate C with a yield of 80.0%.

[0057] 4. Preparation of glycocholic acid dimer:

[0058] Intermediate C (15.85 g; 13.76 mmol) obtained in the previous step was dissolved in 238 ml of a 1 mol / L tetrabutylammonium fluoride / tetrahydrofuran solution (the volume ratio of intermediate C to 1 mol / L tetrabutylammonium fluoride / tetrahydrofuran solution was 1 g:15 ml). The reaction was maintained at 20°C for 8 hours, and completion was monitored by TLC. The solution was concentrated until no liquid remained, dissolved in 1 mol / L sodium hydroxide solution, and the pH was adjusted to 10-12. Extraction was then performed three times with 238 ml of ethyl acetate. The lower aqueous phase was collected, and the pH was adjusted to 1-2 with 1 mol / L dilute hydrochloric acid. Crystallization was performed, filtered, and dried to obtain 10.05 g of glycocholic acid dimer, with a yield of 80.0% and a purity of 98.67%.

[0059] Example 2

[0060] 1. Preparation of intermediate A:

[0061] 18.00 g (38.66 mmol) of glycocholic acid was dissolved in 180 ml of tetrahydrofuran, and 8.33 g of N,N-diisopropylethylamine (42.95 mmol; 3.0 eq) was added dropwise with stirring. Then, 12.93 g of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (42.95 mmol; 2.0 eq) was added. The mixture was allowed to react at 40°C and monitored for completion by TLC. The mixture was concentrated until no liquid remained, and then dissolved in 108 ml of anhydrous ethanol and 18 ml of purified water. After complete dissolution, 468 ml of purified water was added dropwise to crystallize the mixture. The mixture was filtered and dried to obtain 20.66 g of Intermediate A (95.0% yield).

[0062] 2. Preparation of intermediate B:

[0063] Intermediate A (20.66 g, 36.73 mmol) obtained in the previous step was dissolved in 200 ml of tetrahydrofuran. The temperature was maintained at 0–5°C. 7.50 g of imidazole (110.18 mmol, 3 eq) and 30.28 g of tert-butyldiphenylsilyl chloride (110.18 mmol, 3 eq) were added. After addition, the temperature was maintained at 0–5°C. TLC confirmed the reaction was complete. The mixture was concentrated to a dry state, and 330 ml of ethyl acetate was added for dissolution. Then, 330 ml of purified water was added. The mixture was heated to reflux, then cooled to 0–5°C for crystallization. The mixture was filtered and dried to obtain 27.95 g of Intermediate B (95.0% yield).

[0064] 3. Preparation of Intermediate C:

[0065] Dissolve Intermediate B (27.95 g; 34.89 mmol) and 40.61 g of glycocholic acid (87.22 mmol; 2.5 eq) obtained in the previous step in 280 ml of N,N-dimethylformamide. Add 3.53 g of triethylamine (34.89 mmol; 1.0 eq). Then cool to 0°C and add 25.92 g of N,N'-dicyclohexylcarbodiimide (125.60 mmol; 2.0 eq). After addition, warm to 30°C. Monitor the reaction by TLC. Dilute with 1400 ml of purified water and extract with ethyl acetate three times, using 280 ml each time. Collect the ethyl acetate phase and wash with saturated sodium chloride solution three times, using 280 ml each time. The upper ethyl acetate phase was collected, 840 ml of purified water was added, and the mixture was heated to reflux for dissolution. The mixture was then cooled to 0-5°C for crystallization, filtered, and dried to obtain 34.15 g of intermediate C with a yield of 85.0%.

[0066] 4. Preparation of glycocholic acid dimer:

[0067] Intermediate C (34.15 g; 29.66 mmol) obtained in the previous step was dissolved in 683 ml of a 1 mol / L tetrabutylammonium fluoride / tetrahydrofuran solution (the volume ratio of intermediate C to 1 mol / L tetrabutylammonium fluoride / tetrahydrofuran solution was 1 g:20 ml). The reaction was maintained at 30°C for 6 hours, and the reaction was monitored by TLC. The solution was concentrated until no liquid remained, dissolved in 1 mol / L sodium hydroxide solution, and the pH was adjusted to 10-12. Extraction was then performed three times with 340 ml of ethyl acetate. The lower aqueous phase was collected and the pH was adjusted to 1-2 with 1 mol / L dilute hydrochloric acid. Crystallization was performed, filtered, and dried to yield 23.02 g of glycocholic acid dimer, with a yield of 85.0% and a purity of 98.70%.

[0068] Example 3

[0069] 1. Preparation of intermediate A:

[0070] 25.00 g (53.69 mmol) of glycocholic acid was dissolved in 250 ml of N,N-dimethylformamide. 10.87 g of triethylamine (107.38 mmol; 2.0 eq) was added dropwise and stirred until uniform. Then, 9.27 g of N-hydroxysuccinimide (80.54 mmol; 1.5 eq) was added and allowed to react at 29°C. TLC monitored the reaction for completion. The reaction solution was poured into 1250 ml of purified water and stirred for 10 min. The solution was then extracted with ethyl acetate three times, 250 ml each time. The ethyl acetate phase was collected and washed three times with saturated sodium chloride solution, 250 ml each time. The ethyl acetate phase was collected and concentrated until no liquid remained. Then, 150 ml of anhydrous ethanol and 25 ml of purified water were added for dissolution. After complete dissolution, 650 ml of purified water was added dropwise for crystallization. The product was filtered and dried to obtain 27.89 g of intermediate A, a yield of 92.3%.

[0071] 2. Preparation of intermediate B:

[0072] Intermediate A (27.89 g, 49.56 mmol) obtained in the previous step was dissolved in 280 ml of N,N-dimethylformamide. 13.70 g of potassium carbonate (99.12 mmol, 2 eq) and 27.24 g of tert-butyldiphenylsilyl chloride (99.12 mmol, 2 eq) were added at a temperature of 0–5°C. After addition, the reaction was allowed to proceed at a temperature of 0–5°C. TLC confirmed the reaction was complete. The mixture was diluted with 1400 ml of purified water and extracted with ethyl acetate three times, 280 ml each time. The ethyl acetate phase was collected and washed three times with saturated sodium chloride solution, 280 ml each time. The upper ethyl acetate phase was collected. 840 ml of purified water was then added, and the mixture was dissolved under reflux. The mixture was then cooled to 0–5°C for crystallization, filtered, and dried to obtain 36.29 g of intermediate B, with a yield of 91.4%.

[0073] 3. Preparation of Intermediate C:

[0074] Dissolve Intermediate B (36.29 g; 45.30 mmol) and 37.96 g of glycocholic acid (37.96 mmol; 1.8 eq) obtained in the previous step in 363 ml of N,N-dimethylformamide. Add 3.06 g of 1-hydroxybenzotriazole (22.65 mmol; 0.5 eq). Then cool the mixture to 0°C and add 8.57 g of N,N'-diisopropylcarbodiimide (67.94 mmol; 1.5 eq). After addition, warm the temperature to 20°C. Monitor the reaction by TLC. Dilute the mixture with 1815 ml of purified water and extract with ethyl acetate three times, each time using 363 ml. Collect the ethyl acetate phase and wash the mixture three times with saturated sodium chloride solution, each using 363 ml. The upper ethyl acetate phase was collected, 1089 ml of purified water was added, and the mixture was heated to reflux for dissolution. The mixture was then cooled to 0-5°C for crystallization, filtered, and dried to obtain 42.57 g of intermediate C with a yield of 81.6%.

[0075] 4. Preparation of glycocholic acid dimer:

[0076] Intermediate C (42.57 g; 36.96 mmol) obtained in the previous step was dissolved in 638 ml of a 5% (volume fraction) hydrochloric acid / methanol solution (the volume ratio of intermediate C to 5% (volume fraction) hydrochloric acid / methanol solution was 1 g:15 ml). The reaction was maintained at 20°C for 7 hours, and the reaction was monitored by TLC. The solution was concentrated until no liquid remained, dissolved in 1 mol / L aqueous sodium hydroxide, and the pH was adjusted to 10-12. Extraction was then performed three times with 425 ml of ethyl acetate. The lower aqueous phase was collected and the pH was adjusted to 1-2 with 1 mol / L dilute hydrochloric acid. Crystallization was performed, filtered, and dried to yield 27.37 g of glycocholic acid dimer, with a yield of 81.1% and a purity of 98.51%.

[0077] Example 4

[0078] 1. Preparation of intermediate A:

[0079] 50.00 g (107.38 mmol) of glycocholic acid was dissolved in 500 ml of N,N-dimethylformamide. 32.60 g of triethylamine (322.15 mmol; 3.0 eq) was added dropwise and stirred until uniform. Then, 24.72 g of N-hydroxysuccinimide (214.77 mmol; 2.0 eq) was added and allowed to react at 40°C. TLC monitored the reaction for completion. The reaction solution was poured into 2500 ml of purified water and stirred for 10 min. The solution was then extracted with ethyl acetate (3 times, 500 ml each). The ethyl acetate phase was collected and washed with saturated sodium chloride (3 times, 500 ml each). The ethyl acetate phase was collected and concentrated until no liquid remained. 300 ml of anhydrous ethanol and 50 ml of purified water were added for dissolution. After complete dissolution, 1300 ml of purified water was added dropwise for crystallization. The solution was filtered and dried to obtain 55.41 g of intermediate A (91.7% yield).

[0080] 2. Preparation of intermediate B:

[0081] Intermediate A (55.41 g; 98.47 mmol) obtained in the previous step was dissolved in 550 ml of N,N-dimethylformamide. The temperature was maintained at 0–5°C. 40.83 g of potassium carbonate (295.41 mmol; 3 eq) and 81.20 g of tert-butyldiphenylsilyl chloride (295.41 mmol; 3 eq) were added. After addition, the temperature was maintained at 0–5°C for reaction. TLC confirmed the reaction was complete. The mixture was diluted with 2750 ml of purified water and extracted with ethyl acetate three times, 550 ml each time. The ethyl acetate phase was collected and washed three times with saturated sodium chloride solution, 550 ml each time. The upper ethyl acetate phase was collected. 1650 ml of purified water was then added, and the mixture was dissolved under reflux. The mixture was then cooled to 0–5°C for crystallization, filtered, and dried to obtain 71.47 g of Intermediate B (90.6% yield).

[0082] 3. Preparation of Intermediate C:

[0083] Intermediate B (71.47 g; 89.21 mmol) and 91.39 g of glycocholic acid (196.27 mmol; 2.2 eq) obtained in the previous step were dissolved in 715 ml of N,N-dimethylformamide. 12.05 g of 1-hydroxybenzotriazole (89.21 mmol; 1.0 eq) was added, and the mixture was cooled to 0°C. 34.20 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (178.43 mmol; 2.0 eq) was added. After addition, the temperature was naturally raised to 30°C and the reaction was allowed to proceed for 5 hours. The reaction was monitored by TLC. The mixture was diluted with 3575 ml of purified water and extracted with ethyl acetate three times, each time using 2145 ml of ethyl acetate. The ethyl acetate phase was collected and washed three times with saturated sodium chloride solution, each using 2145 ml of ethyl acetate. The upper ethyl acetate phase was collected, 6435 ml of purified water was added, and the mixture was heated to reflux for dissolution. The mixture was then cooled to 0-5°C for crystallization, filtered, and dried to obtain 83.84 g of intermediate C with a yield of 82.3%.

[0084] 4. Preparation of glycocholic acid dimer:

[0085] Intermediate C (83.84 g; 72.80 mmol) obtained in the previous step was dissolved in 1677 ml of a 5% (volume fraction) hydrochloric acid / methanol solution (the volume ratio of intermediate C to 5% (volume fraction) hydrochloric acid / methanol solution was 1 g:20 ml). The reaction was maintained at 30°C for 6 hours, and the reaction was monitored by TLC. The solution was concentrated until no liquid remained, dissolved in 1 mol / L aqueous sodium hydroxide, and the pH was adjusted to 10-12. Extraction was then performed three times with 838 ml of ethyl acetate. The lower aqueous phase was collected and the pH was adjusted to 1-2 with 1 mol / L dilute hydrochloric acid. Crystallization was performed, filtered, and dried to yield 54.25 g of glycocholic acid dimer, with a yield of 80.9% and a purity of 98.26%.

Claims

1. A method for synthesizing a glycocholic acid dimer, characterized in that: The glycocholic acid dimer is: ； The synthesis method comprises the following steps: Step 1: In the presence of a base in a reaction solvent A, glycocholic acid and a condensation agent are condensed to obtain an active ester intermediate A; ； The reaction solvent A is selected from one of N,N-dimethylformamide and tetrahydrofuran; The condensing agent is selected from one of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and N-hydroxysuccinimide; The reaction temperature is 25-40°C; Step 2: In a reaction solvent B, in the presence of a base, intermediate A undergoes a substitution reaction with tert-butyldiphenylsilyl chloride to obtain intermediate B; ； The reaction solvent B is selected from one of N,N-dimethylformamide and tetrahydrofuran; The reaction temperature of the intermediate B is 0~5°C; Step 3: In the reaction solvent C, in the presence of a base and a coupling reagent, the intermediate B undergoes a coupling reaction with glycocholic acid to obtain the intermediate C; ； The reaction solvent C is N,N-dimethylformamide; The reaction temperature is 20-30°C; Step 4: Under the action of a deprotecting agent, the intermediate C removes the tert-butyldiphenylsilyl group to obtain the target product, glycocholic acid dimer; ； The deprotecting agent is selected from one of tetrabutylammonium fluoride / tetrahydrofuran solution and hydrochloric acid / methanol solution; The deprotection reaction temperature is 20-30°C.

2. The method according to claim 1, characterized in that The base in step 1 is selected from one of N,N-diisopropylethylamine and triethylamine; The molar ratio of the glycocholic acid, the base and the condensing agent is 1:2-3:1.5-2.

0.

3. The method according to claim 1, characterized in that The base in step 2 is one of imidazole and potassium carbonate; The molar ratio of the intermediate A, tert-butyldiphenylchlorosilane and the base is 1:2-3:2-3.

4. The method according to claim 1, characterized in that In step 3, the coupling reagent is selected from one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and the base is selected from one of 4-dimethylaminopyridine, triethylamine and 1-hydroxybenzotriazole.

5. The method according to claim 1, characterized in that The molar ratio of intermediate B, coupling reagent and glycocholic acid in step 3 is 1:1.5~2.0:1.8~2.5; The molar ratio of the intermediate B to the base is 1: 0.5~1.

0.

6. The method according to claim 1, wherein the deprotecting agent in step 4 is selected from tetrabutylammonium fluoride / tetrahydrofuran solution and hydrochloric acid / methanol solution, characterized in that: The tetrabutylammonium fluoride concentration of the tetrabutylammonium fluoride / tetrahydrofuran solution is 1 mol / L; the hydrochloric acid volume fraction of the hydrochloric acid / methanol solution is 5%.

7. The method according to claim 1, characterized in that The plastochemical ratio of the intermediate C to the deprotecting agent in step 4 is 1: 15-20.

8. The method according to any one of claims 1 to 7, characterized in that: The yield of the prepared glycocholic acid dimer is greater than or equal to 50%.

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