A method for synthesizing deoxycholic acid

Abstract

The application discloses a deoxycholic acid synthesis method, and the main process route is as follows: cholic acid is dissolved in DMSO, IBX is added to carry out an oxidation reaction, then DMSO is removed by vacuum distillation, and residual IBX is reduced; hydrazine hydrate, potassium hydroxide, diethylene glycol and a magnetic carbon supported phase transfer catalyst are continuously added to carry out a deoxygenation reduction reaction at 140-160 DEG C, so that deoxycholic acid is obtained. The application adopts a "one-pot" series process, does not need to separate and purify intermediates, directly carries out subsequent reactions in the same reactor, greatly simplifies operation steps, and the total yield of two steps can reach more than 85%, which is significantly improved compared with a traditional step-by-step process. The application reduces the reaction temperature from the conventional 180-200 DEG C to 140-160 DEG C, and significantly reduces energy consumption and safety risks. The series process of the application significantly reduces three waste emissions, and is a more environment-friendly and greener deoxycholic acid synthesis process.

Description

Technical Field

[0001] This invention relates to the field of bile acid compound synthesis technology, specifically to a method for synthesizing deoxycholic acid. Background Technology

[0002] Deoxycholic acid (DCA), chemically named 3α,12α-dihydroxy-5β-cholan-24-acid, is an important class of bile acid compounds with significant application value in pharmaceuticals, medical aesthetics, pharmaceutical excipients, and bile acid derivatives research.

[0003] Cholic acid, chemically known as 3α,7α,12α-trihydroxy-5β-cholan-24-acid, is an important component of natural bile acids and a crucial raw material for the preparation of deoxycholic acid and other bile acid derivatives. The main structural difference between cholic acid and deoxycholic acid lies in the presence of a hydroxyl group at the C7 position in cholic acid, while a methylene group is present at that position in deoxycholic acid. Therefore, the key to preparing deoxycholic acid from cholic acid lies in the targeted removal of the hydroxyl group at the C7 position while retaining the hydroxyl groups at the C3 and C12 positions and the carboxylic acid structure at the C24 position.

[0004] Existing processes for converting cholic acid to deoxycholic acid typically involve selective oxidation, carbonyl reduction deoxygenation, introduction and removal of protecting groups, or other complex intermediate conversion steps. Some methods use chromium-containing oxidants or other highly polluting oxidation systems, resulting in problems such as heavy metal residues, complex post-treatment, and high toxicity. Other processes improve selectivity through multiple protection and deprotection steps, but these are complex, cumbersome, and unfavorable for rapid preparation and subsequent process optimization.

[0005] In addition, existing processes generally suffer from low production efficiency, low yield, and significant safety and environmental risks. Specifically, these issues include: 1) Material loss during intermediate separation and purification, resulting in lower overall yield; 2) Low equipment utilization under step-by-step operation mode, making it difficult to adapt to industrial scale-up requirements; and 3) Easy exposure to air during intermediate transfer, leading to oxidation impurities. Furthermore, step-by-step operation generates a large amount of process waste, resulting in excessive emissions of waste gas, wastewater, and solid waste, and significant environmental pressure.

[0006] Therefore, it is essential to develop a simple, mild, high-yield, and environmentally friendly method for the preparation of deoxycholic acid. Summary of the Invention

[0007] To address the above problems, the present invention aims to provide a green synthesis process for deoxycholic acid that is simple in steps, mild in conditions, easy to operate, and has a high yield.

[0008] The specific technical solution of this invention is as follows:

[0009] A method for synthesizing deoxycholic acid, characterized by comprising the following steps:

[0010] 1) Dissolve cholic acid in dimethyl sulfoxide (DMSO), add 2-iodobenzoic acid (IBX), and react at 20-50°C for 2-6 hours;

[0011] 2) Remove DMSO from the reaction system by vacuum distillation, and then add an appropriate amount of sodium bisulfite to reduce the residual IBX;

[0012] 3) In the same reactor, hydrazine hydrate, potassium hydroxide, diethylene glycol and magnetic carbon supported phase transfer catalyst are added. The reaction is carried out at 80-130℃ for 0.5-4 h, and then the temperature is raised to 140-160℃ and the reaction is continued for 2-8 h to obtain deoxycholic acid. The magnetic carbon supported phase transfer catalyst is a quaternary ammonium salt supported on graphite carbon core-shell Fe3O4@C.

[0013] 4) Cool the reaction system, dilute with water, and adjust the pH to 1-4 to precipitate deoxycholic acid; filter and collect the solid, and obtain pure deoxycholic acid after washing, drying and recrystallization.

[0014] In step 1 above, the molar ratio of bile acid to IBX is 1:1.2 to 1.5; in step 2 above, the molar ratio of sodium bisulfite is 1.1 to 1.2 times that of residual IBX; the reaction temperature for sodium bisulfite to reduce residual IBX is 40 to 60°C, and the reaction time is 20 to 60 min.

[0015] Based on the amount of cholic acid fed in step 1 above, in step 3 above: the molar ratio of hydrazine hydrate to cholic acid is 4-6:1; the molar ratio of potassium hydroxide to cholic acid is 3-6:1; the amount of diethylene glycol used is: 4-6 mL of diethylene glycol is added for every 1 g of cholic acid; the amount of magnetic carbon supported phase transfer catalyst, based on the quaternary ammonium salt active component it supports, is 5-15% of the molar amount of cholic acid.

[0016] In step 3 above, the graphite carbon core-shell Fe3O4@C in the magnetic carbon supported phase transfer catalyst has a core-shell structure, with the core being Fe3O4 nanoparticles and the shell being a carbon layer treated with high temperature graphitization.

[0017] Furthermore, the magnetic carbon-supported phase transfer catalyst is prepared by the following steps:

[0018] a. Fe3O4 nanoparticles were obtained by dissolving FeCl3·6H2O, sodium acetate and polyethylene glycol in ethylene glycol using a solvothermal method and reacting at 180-220℃ for 8-12 h.

[0019] b. Disperse Fe3O4 nanoparticles in 0.5–1.0 M glucose solution, ultrasonically disperse, transfer to a hydrothermal reactor, and react at 180–200 °C for 10–16 h to obtain Fe3O4@C precursor;

[0020] c. Place the Fe3O4@C precursor in a tube furnace, heat it to 800-1000℃ at 5-10℃ / min under an inert atmosphere, hold it at that temperature for 3-5 h, and then cool it naturally to room temperature to obtain graphite carbon core-shell Fe3O4@C;

[0021] d. Graphite carbon core-shell Fe3O4@C was successively modified by amination, acylhalogenation and quaternization to obtain a magnetic carbon supported phase transfer catalyst.

[0022] Preferably, the quaternary ammonium salt supported on the magnetic carbon-supported phase transfer catalyst is benzyltriethylammonium chloride (BTEAC), hexadecyltrimethylammonium chloride (CTAC), or benzyltributylammonium chloride (BTBAC).

[0023] Alternatively, the magnetic carbon-supported phase transfer catalyst is benzyltributylammonium chloride supported on graphite carbon core-shell Fe3O4@C (hereinafter referred to as Fe3O4@C-BTBAC), with a benzyltributylammonium chloride loading of 0.8–1.5 mmol / g.

[0024] In step 1 above, the amount of DMSO used is: 6-20 mL of DMSO for every 1 g of cholic acid; in step 2 above, the vacuum distillation conditions for removing DMSO are: temperature 40-60℃, vacuum degree 650-2500 Pa, and distillation time 20-60 min.

[0025] The beneficial effects of this invention are as follows:

[0026] 1) This invention uniquely adopts a series process, which eliminates the need to separate and purify intermediates after the oxidation reaction. Subsequent reactions can be carried out directly in the same reactor, greatly simplifying the operation steps. The total yield of the two steps can reach more than 85%, which is a significant improvement over the traditional stepwise process.

[0027] 2) This invention reduces the reaction temperature from the conventional 180-200℃ to 140-160℃, significantly reducing energy consumption and safety risks. In addition, the magnetic carbon-supported phase transfer catalyst of this invention is easy to separate and recover after the reaction, and can be recycled more than 5 times without activation, while the total yield is still maintained at more than 85%, thereby greatly reducing production costs.

[0028] 3) The tandem process of this invention reduces the intermediate separation and purification steps, is easy to operate, has high equipment utilization, and significantly reduces the amount of waste emissions compared to the traditional stepwise process. It is a more environmentally friendly and greener deoxycholic acid synthesis process. Attached Figure Description

[0029] Figure 1 HPLC chromatogram of the standard 7-keto intermediate (3α,12α-dihydroxy-7-oxo-5β-cholan-24-acid).

[0030] Figure 2 Example 1: HPLC spectrum of the product after 4 h of reaction of cholic acid with IBX.

[0031] Figure 3 HPLC chromatogram of deoxycholic acid standard.

[0032] Figure 4 Example 1: HPLC chromatogram of the deoxycholic acid product. Detailed Implementation Plan

[0033] The invention's technical solution will be further explained below with reference to specific embodiments.

[0034] Example 1

[0035] 1) Add bile acid to the reaction vessel, then add DMSO and stir thoroughly to dissolve the bile acid in the DMSO. In this step, the amount of DMSO added is: 8 mL of DMSO for every 1 g of bile acid.

[0036] 2) Add IBX in batches at 30℃ to achieve a molar ratio of IBX to cholic acid of 1.3:1 in the reactor. Control the system temperature during the addition process to avoid local overheating. After the addition is completed, react at 35℃ for 4 hours. Take samples every 1 hour during the reaction, dilute with methanol and perform HPLC analysis to monitor the decrease of the cholic acid raw material peak and the formation of the 7-ketone intermediate (3α,12α-dihydroxy-7-oxo-5β-cholan-24-acid) peak. Figure 1 The HPLC chromatogram of the 7-one intermediate standard is shown below, with a main peak retention time of 5.586 min. The HPLC chromatogram of a sample taken after 4 h of reaction is shown below. Figure 2 As shown, the retention time of the main peak was 5.494 min, which was highly consistent with the standard, confirming the formation of the 7-ketone intermediate. The impurity peaks from 4.2 to 6.4 min were trace amounts of over-oxidation byproducts and incompletely separated bile acid raw materials. Calculated by peak area normalization, the oxidation selectivity at the C7 position was 91.2%, the bile acid conversion rate reached 95.5%, and there were no obvious over-oxidation impurities at the C3 and C12 positions, so the reaction was stopped.

[0037] 3) The reaction mixture was subjected to vacuum distillation at 50°C and 1500 Pa for 30 min to remove DMSO. Then, appropriate amounts of deionized water and sodium bisulfite were added to the reactor, and the mixture was stirred at 50°C for 30 min to reduce residual IBX. TLC chromatographic monitoring confirmed the complete disappearance of IBX spots. In this step, the amount of sodium bisulfite added was 1.2 times the amount of residual IBX by molar ratio. The amount of residual IBX was determined based on the amount of IBX added in step 2 and the conversion rate of the reaction.

[0038] 4) Continue adding hydrazine hydrate, potassium hydroxide, diethylene glycol, and Fe3O4@C-BTBAC to the reactor. With mechanical stirring, first maintain the temperature at 100℃ for 2 h, then slowly raise the temperature to 150℃ and continue the reaction for 4 h to obtain deoxycholic acid. This reaction can also be carried out under N2 protection. After the reaction is complete, samples are taken for HPLC analysis, and the results are as follows: Figure 4 As shown, the product peak retention time was 24.992 min, which is similar to that of deoxycholic acid standard ( Figure 3 The retention time (24.945) was highly consistent, confirming the formation of deoxycholic acid product. Water and low-boiling substances generated during the reaction naturally evaporated as the temperature increased, and were collected and removed via a top-of-boiler condenser. In this step, the molar ratio of hydrazine hydrate and potassium hydroxide was 5 times that of cholic acid, with hydrazine hydrate added as an 80% aqueous solution; the amount of diethylene glycol added was 5 mL for every 1 g of cholic acid; the BTBAC loading in the Fe3O4@C-BTBAC was 1.2 mmol / g, the catalyst addition was 1 g, and the active component of BTBAC was 9.8% of the molar weight of cholic acid.

[0039] The preparation steps of the above Fe3O4@C-BTBAC are as follows:

[0040] a. Add FeCl3·6H2O, anhydrous sodium acetate and polyethylene glycol to ethylene glycol and stir until completely dissolved. Transfer the mixed solution to a polytetrafluoroethylene-lined stainless steel hydrothermal reactor and react at 200℃ for 10 h. After naturally cooling to room temperature, collect the product by magnetic separation, wash three times with deionized water and ethanol respectively, and dry under vacuum at 60℃ for 8 h to obtain Fe3O4 nanoparticles.

[0041] b. Fe3O4 nanoparticles were dispersed in a 0.5M glucose aqueous solution, ultrasonically dispersed, and then transferred to a hydrothermal reactor. After reacting at 180℃ for 12 h, the product was collected by magnetic separation. The product was washed with deionized water and ethanol and dried under vacuum at 60℃ to obtain the Fe3O4@C precursor.

[0042] c. The Fe3O4@C precursor was placed in a tube furnace and heated to 800℃ at 5℃ / min under an Ar atmosphere. After holding at that temperature for 5 h, it was naturally cooled to room temperature to obtain graphite carbon core-shell Fe3O4@C.

[0043] d. Graphite core-shell Fe3O4@C was dispersed in anhydrous toluene, and a small amount of 3-aminopropyltriethoxysilane was added. The mixture was refluxed under nitrogen protection for 12 h. After magnetic separation and washing, it was dispersed in anhydrous DMF, and a small amount of p-bromomethylbenzoyl bromide and triethylamine were added. The mixture was reacted at 60 °C for 8 h. After magnetic separation and washing, it was dispersed again in acetonitrile, and an appropriate amount of tributylamine was added. The mixture was refluxed at 80 °C for 24 h. After magnetic separation, it was washed successively with acetonitrile, ethanol, and deionized water, and dried under vacuum at 60 °C to obtain the Fe3O4@C-BTBAC catalyst.

[0044] 5) After cooling the reaction system to below 60℃, Fe3O4@C-BTBAC was separated and recovered using an external magnetic field (magnetic field strength 0.5 T) applied to the bottom of the reactor. The remaining reaction solution was diluted with an appropriate amount of deionized water, and the pH was adjusted to 3 with hydrochloric acid to precipitate deoxycholic acid (white crystals). The filter cake was washed twice with a small amount of pre-cooled ethanol / water mixture (1:1 volume ratio), dried under vacuum at 50℃ to constant weight, yielding pure deoxycholic acid. HPLC analysis showed that the product purity was 98.3% and the overall yield was 86.2%.

[0045] Example 2

[0046] 1) Add cholic acid and DMSO to the reaction vessel and stir thoroughly to dissolve the cholic acid; the ratio of cholic acid to DMSO added is as described in Example 1.

[0047] 2) Add IBX in batches at 30℃ (refer to Example 1 for the addition ratio). Control the system temperature during the addition process to avoid excessive temperature. After the addition is completed, react at 35℃ for 4 hours. Take samples for HPLC analysis during the reaction. Stop the reaction when the bile acid conversion rate is ≥95%.

[0048] 3) After diluting and quenching with an appropriate amount of deionized water, the pH was adjusted to 2 with concentrated hydrochloric acid to precipitate the product. The solid was collected by filtration, and the filter cake was washed with deionized water until the pH of the filtrate was close to neutral. The product was then dried under vacuum at 60°C to obtain crude 7-ketone intermediate (3α,12α-dihydroxy-7-oxo-5β-cholan-24-acid). The crude product was purified by pulping and drying to obtain a purified product with a purity of 86% as determined by HPLC, and the yield of the oxidation step was 75.4%.

[0049] 4) The purified 7-one intermediate, diethylene glycol, potassium hydroxide and 80 wt% hydrazine hydrate were added to the reactor (the proportion of each raw material added is the same as in Example 1). Under mechanical stirring, the mixture was first kept at 100°C for 3 h, and then the temperature was raised to 180°C and kept at 180°C for 6 h to obtain deoxycholic acid.

[0050] 5) Cool the reaction system to below 60℃, dilute with an appropriate amount of deionized water, and adjust the pH to 2 with hydrochloric acid to precipitate the product. Filter and collect the solid. Wash the filter cake with deionized water until nearly neutral, and dry to obtain crude deoxycholic acid. Recrystallize the crude product twice with a 1:1 volume ratio of ethanol / water mixed solvent. After drying, the deoxycholic acid product is obtained. The purity was 94.2% as determined by HPLC, and the overall yield of the two steps was 61.5%.

[0051] Example 3

[0052] Alkali resistance test of catalyst support: 2.0 g of the graphite carbon core-shell Fe3O4@C prepared in Example 1 was placed in 30 mL of diethylene glycol, and 8.40 g of KOH was added to achieve a KOH molar concentration of approximately 5 M. Simulating the high alkali concentration conditions after water distillation in the later stages of the reaction, the reaction was carried out under N2 protection with mechanical stirring at 300 rpm at 160 °C for 6 h. After the reaction, the mixture was cooled to 60 °C, and the solid was collected by magnetic separation. It was washed with deionized water until neutral, dried under vacuum at 60 °C to constant weight, and accurately weighed. The mass loss rate after alkali treatment was calculated. The supernatant after magnetic separation was digested, and the iron ion concentration was determined by ICP-MS. The amount of iron dissolved per unit mass of support was calculated based on the iron ion concentration.

[0053] The results showed that after long-term treatment in a high-concentration alkaline environment, the mass loss rate of the graphite carbon core-shell carrier was only 0.5%, and the iron ion leaching amount was 0.2 μg / g carrier.

[0054] Example 4

[0055] Catalyst cycle stability test: The Fe3O4@GC-BTBAC catalyst recovered in Example 1 was directly fed into the next batch of deoxycholic acid synthesis reaction without any activation (the raw material ratio and reaction conditions were the same as in Example 1), and a total of 10 batches were carried out (including Example 1). The total recovery rate of each batch of reaction and the magnetic separation recovery time of the catalyst after the reaction were determined.

[0056] Table 1. Total recovery rate of deoxycholic acid and magnetic separation recovery time of catalyst after reaction for each batch of reaction.

[0057]

[0058] The results are shown in Table 1. After 10 cycles, the magnetic separation recovery time of the catalyst slightly increased from 8 s to 9 s, while the total yield of deoxycholic acid remained close to 75%, demonstrating that the graphite carbon core-shell Fe3O4@C supported quaternary ammonium salt catalyst used in this invention has good stability and is valuable for industrial recycling. The slow decrease in yield is mainly attributed to the gradual loss of the surface quaternary ammonium salt active components, rather than the destruction of the shell. The total recovery rate of deoxycholic acid in the second and third batches was higher than that in the first batch, which is a normal phenomenon in the early stage of catalyst cycling. In the freshly prepared catalyst, some active sites are covered by residual coating layers and impurities that have not been completely removed. After being washed and rinsed by the solvothermal environment of the first reaction, the quaternary ammonium salt active sites are fully exposed, resulting in a slight increase in the total yield.

Claims

1. A method for synthesizing deoxycholic acid, characterized in that... Includes the following steps: 1) Dissolve cholic acid in dimethyl sulfoxide, add 2-iodobenzoic acid, and react at 20-50°C for 2-6 h; 2) Remove dimethyl sulfoxide from the reaction system by vacuum distillation, and then add an appropriate amount of sodium bisulfite to reduce the residual 2-iodobenzoic acid; 3) In the same reactor, hydrazine hydrate, potassium hydroxide, diethylene glycol and magnetic carbon supported phase transfer catalyst are added. The reaction is carried out at 80-130℃ for 0.5-4 h, and then the temperature is raised to 140-160℃ and the reaction is continued for 2-8 h to obtain deoxycholic acid. The magnetic carbon supported phase transfer catalyst is a quaternary ammonium salt supported on graphite carbon core-shell Fe3O4@C. 4) Cool the reaction system, dilute with water, and adjust the pH to 1-4 to precipitate deoxycholic acid; The solid was collected by filtration, and after washing, drying, and recrystallization, pure deoxycholic acid was obtained.

2. The method for synthesizing deoxycholic acid according to claim 1, characterized in that: In step 1, the molar ratio of cholic acid to 2-iodobenzoic acid is 1:1.2 to 1.5; in step 2, the molar ratio of sodium bisulfite is 1.1 to 1.2 times that of residual 2-iodobenzoic acid; the reaction temperature for sodium bisulfite to reduce residual 2-iodobenzoic acid is 40 to 60°C, and the reaction time is 20 to 60 min.

3. The method for synthesizing deoxycholic acid according to claim 1, characterized in that: Based on the amount of cholic acid fed in step 1, in step 3: the molar ratio of hydrazine hydrate to cholic acid is 4-6:1; the molar ratio of potassium hydroxide to cholic acid is 3-6:1; the amount of diethylene glycol used is: 4-6 mL of diethylene glycol for every 1 g of cholic acid; the amount of magnetic carbon supported phase transfer catalyst used is 5-15% of the molar amount of cholic acid, based on the quaternary ammonium salt active component it supports.

4. The method for synthesizing deoxycholic acid according to claim 1, characterized in that: In step 3, the graphite carbon core-shell Fe3O4@C in the magnetic carbon supported phase transfer catalyst has a core-shell structure, with the core being Fe3O4 nanoparticles and the shell being a carbon layer treated with high temperature graphitization.

5. The method for synthesizing deoxycholic acid according to claim 4, characterized in that: The magnetic carbon-supported phase transfer catalyst was prepared by the following steps: a. Fe3O4 nanoparticles were obtained by dissolving FeCl3·6H2O, sodium acetate and polyethylene glycol in ethylene glycol using a solvothermal method and reacting at 180-220℃ for 8-12 h. b. Disperse Fe3O4 nanoparticles in 0.5–1.0 M glucose solution, ultrasonically disperse, transfer to a hydrothermal reactor, and react at 180–200 °C for 10–16 h to obtain Fe3O4@C precursor; c. Place the Fe3O4@C precursor in a tube furnace, heat it to 800-1000℃ at 5-10℃ / min under an inert atmosphere, hold it at that temperature for 3-5 h, and then cool it naturally to room temperature to obtain graphite carbon core-shell Fe3O4@C; d. Graphite carbon core-shell Fe3O4@C was successively modified by amination, acyl halide and quaternization reactions to obtain a magnetic carbon supported phase transfer catalyst.

6. The method for synthesizing deoxycholic acid according to claim 4 or 5, characterized in that: The quaternary ammonium salt supported on the magnetic carbon-supported phase transfer catalyst is benzyltriethylammonium chloride, hexadecyltrimethylammonium chloride, or benzyltributylammonium chloride.

7. The method for synthesizing deoxycholic acid according to any one of claims 1, 4, and 5, characterized in that: The magnetic carbon-supported phase transfer catalyst is benzyltributylammonium chloride supported on a graphite carbon core-shell Fe3O4@C substrate, with a benzyltributylammonium chloride loading of 0.8–1.5 mmol / g.

8. The method for synthesizing deoxycholic acid according to claim 1, characterized in that: In step 1, the amount of dimethyl sulfoxide used is: 6-20 mL of dimethyl sulfoxide is added for every 1 g of cholic acid; in step 2, the vacuum distillation conditions for removing dimethyl sulfoxide are: temperature 40-60℃, vacuum degree 650-2500 Pa, and distillation time 20-60 min.

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