Method for preparing Aramchol by enzymatic synthesis
Through the enzymatic synthesis method, 3α,7α,12α-trihydroxy-5β-cholic acid is gradually converted into Aramchol by using specific enzyme catalysts, solving the problems of low yield, poor safety and limited industrial applications in the prior art, and achieving efficient, safe and suitable for industrial production.
Patent Information
- Application Number
- CN202411902482.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-14
AI Technical Summary
The existing Aramchol preparation method has problems such as low yield, highly toxic and explosive, high cost and complex side reactions, which limits its industrial application.
By using the enzymatic synthesis method, 3α,7α,12α-trihydroxy-5β-cholic acid was gradually converted into Aramchol through the effects of oxidative Coenzyme I, 3α-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase, the defects such as many hydroxyl bare reaction sites, large consumption of sodium azide and arachidyl chloride were overcome.
It has achieved high yield (process yield up to more than 91%), simple operation, high safety and low energy consumption, and is suitable for industrial production.
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Figure CN119614659A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for synthesizing a steroid compound, in particular to a method for preparing Aramchol by using an enzyme method, and belongs to the technical field of organic synthesis. Background Art
[0002] Aramchol (arachidyl amido cholanoic acid), chemically named [3β-arachidyl amido-7α,12α-dihydroxy-5β-cholestane-24-acid], is a FABACs fatty liver treatment drug that has entered Phase II clinical studies and has the potential to treat cholesterol gallstones and atherosclerosis. Aramchol is a compound in which bile acid is linked to arachidonic acid via an amide bond. The results of the Aramchol II b clinical trial showed that the liver fat rate of patients taking Aramchol was significantly improved compared with the placebo group, and it had significant therapeutic effects in relieving NASH fatty liver symptoms, improving liver fibrosis, and reducing the progression of cirrhosis.
[0003] The Aramchol preparation method of the prior art is mainly based on chemical synthesis. Its technical routes mainly include sodium azide conversion hydroxyl method, DPPA conversion hydroxyl method and Mitsunobu reaction, namely Mitsunobu amination conversion hydroxyl method. However, each preparation method has many disadvantages: sodium azide conversion hydroxyl method is an early idea for synthesizing Aramchol, which is of great significance for the development of chemical synthesis of Aramchol, but its yield is too low and sodium azide is highly toxic and explosive, which limits its industrial application. DPPA conversion hydroxyl method, although DPPA is a substitute for sodium azide and has higher safety, but the cost is too high, and the overall yield is not improved. Mitsunobu amination conversion hydroxyl method, although the reagent used is the commonly used reagent phthalimide, but there are too many reaction auxiliary materials, such as triphenylphosphine, diethyl azodicarboxylate, etc., and there are too many post-treatment impurities, which limits its industrial production application.
[0004] Enzymes are highly efficient biocatalysts and are widely used in the biofermentation synthesis of some organic compounds. The biofermentation of organic compounds simulates the action principle of enzymes in organisms and utilizes the active centers and catalytic mechanisms of enzymes to promote chemical reactions. Enzymatic preparation is usually carried out under milder conditions, such as room temperature and pressure, which makes the enzymatic preparation process more environmentally friendly and efficient. Enzymes have high selectivity and can efficiently catalyze specific substrates to generate products with specific structures. Enzymatic preparation is widely used in food processing, pharmaceutical synthesis and other fields, but there are currently no reports on the preparation method that combines the preparation of Aramchol with enzymatic synthesis technology.
[0005] Therefore, it is very necessary to design a new method for preparing Aramchol by enzymatic synthesis with simple operation, high safety, low energy consumption and high yield to broaden its application. Summary of the invention
[0006] In order to solve the problems existing in the background technology, the object of the present invention is to provide a new method for preparing Aramchol by enzyme synthesis with simple operation, high safety, low energy consumption and high yield. In order to achieve the above object, the technical scheme of the present invention is as follows:
[0007] A method for preparing Aramchol by enzymatic synthesis comprises the following steps: using 3α,7α,12α-trihydroxy-5β-cholic acid as a raw material, converting 3α,7α,12α-trihydroxy-5β-cholic acid into 3β,7α,12α-trihydroxy-5β-cholic acid under the action of oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase (3α HSDH) and 3β-hydroxysteroid dehydrogenase (3βHSDH), reacting 3β,7α,12α-trihydroxy-5β-cholic acid with ammonia water to generate 3β-amino-7α,12α-dihydroxy-5β-cholic acid, and esterifying 3β-amino-7α,12α-dihydroxy-5β-cholic acid with methanol to generate 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl Ester, 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester and condensation with arachidic acid to generate 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester, 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is then hydrolyzed to obtain 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid, i.e. Aramchol. The synthetic route is shown in the attached Figure 1 .
[0008] The method for preparing Aramchol by enzymatic synthesis comprises the following specific steps:
[0009] S1: Preparation of 3β,7α,12α-trihydroxy-5β-cholic acid
[0010] Mix the proportion of 3α,7α,12α-trihydroxy-5β-cholic acid and water, add a 1M sodium hydroxide solution until the cholic acid is completely dissolved, and adjust the pH to 7.7-8.0 with hydrochloric acid. Then add oxidized coenzyme I (NAD+), 3α-hydroxysteroid dehydrogenase (3αHSDH), and 3β-hydroxysteroid dehydrogenase (3βHSDH) in sequence, and stir the reaction in a 30°C water bath for 4-6 hours. After the reaction is completed, filter and dry to obtain 3β,7α,12α-trihydroxy-5β-cholic acid for later use.
[0011] S2: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholic acid
[0012] The 3β,7α,12α-trihydroxy-5β-cholic acid prepared in step S1 was mixed with ammonia water, heated to 90° C. for reaction for 2 h, and after the reaction was completed, cooled to room temperature, filtered, and the filter cake was air-dried to obtain 3β-amino-7α,12α-dihydroxy-5β-cholic acid for later use.
[0013] S3: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0014] Add 3β-amino-7α,12α-dihydroxy-5β-cholic acid and methanol prepared in step S2 into a reaction bottle, slowly add sulfuric acid dropwise at room temperature, react at room temperature for 4 hours, cool the reaction solution to -20°C for crystallization for 12 hours, filter with suction, and dry the filter cake to obtain 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester for later use.
[0015] S4: Preparation of 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0016] The 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester prepared in step S3, arachidic acid, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N'-diisopropylethylamine (DIPEA) and dichloromethane were added to a reaction bottle, reacted at room temperature for 8-15 hours, the reaction was stopped, filtered, the mother liquor was acid washed, washed with water, washed with saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester for later use.
[0017] S5: Preparation of Aramchol
[0018] The 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester prepared in step S4, 10% NaOH solution and methanol were added to a reaction bottle, the temperature was raised and refluxed for reaction for 2 hours, the reaction solution was added with hydrochloric acid to adjust the pH to 2-3, the reaction solution was filtered, the solution was rinsed with water, and the solid was dried to obtain a crude product of 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid, and the crude product was purified with ethanol to obtain Aramchol.
[0019] Preferably, the mass ratio of 3α,7α,12α-trihydroxy-5β-cholic acid, oxidized coenzyme I, 3αHSDH enzyme solution and 3βHSDH enzyme solution in step S1 is 3α,7α,12α-trihydroxy-5β-cholic acid: oxidized coenzyme I: 3αHSDH enzyme solution: 3βHSDH enzyme solution = 1: 0.001~0.02: 0.1~2.0: 0.1~2.0.
[0020] Preferably, the mass ratio of 3α,7α,12α-trihydroxy-5β-cholic acid and water in step S1 is 3α,7α,12α-trihydroxy-5β-cholic acid: water = 1:10-50, and the water is deionized water.
[0021] Preferably, the molar ratio of ammonia water and 3β,7α,12α-trihydroxy-5β-cholic acid in step S2 is ammonia water:3β,7α,12α-trihydroxy-5β-cholic acid=5~20eq.
[0022] Preferably, the molar ratio of 3β-amino-7α,12α-dihydroxy-5β-cholic acid to sulfuric acid in step S3 is 1:0.05-0.5. The mass volume ratio of 3β-amino-7α,12α-dihydroxy-5β-cholic acid to methanol in step S3 is 1:10-50.
[0023] Preferably, the molar ratio of arachidic acid to 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester in step S4 is arachidic acid: 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester = 0.5 to 2.0 eq; the molar ratio of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate: 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester = 1.0~5.0eq; the molar ratio of N,N'-diisopropylethylamine and 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is N,N'-diisopropylethylamine:3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester = 1.0~5.0eq; the volume mass ratio of dichloromethane and 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is 10~50:1; preferably, the reaction time described in step S4 is 12h;
[0024] Preferably, the mass ratio of 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester and 10% sodium hydroxide in step S5 is 1:4-15; the mass volume ratio of 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester and methanol is 1:10-40.
[0025] Compared with the existing detection technology, the beneficial effects of the present invention are embodied in:
[0026] 1. The method for preparing Aramchol by enzymatic synthesis of the present invention comprises the following steps: firstly preparing 3β,7α,12α-trihydroxy-5β-cholic acid by using a specific and selective enzyme catalyst, then reacting 3β,7α,12α-trihydroxy-5β-cholic acid with ammonia water to generate 3β-amino-7α,12α-dihydroxy-5β-cholic acid, and then esterifying 3β-amino-7α,12α-dihydroxy-5β-cholic acid with methanol to generate 3β-amino The 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester is condensed with arachidic acid to generate 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester, and finally 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester is hydrolyzed to obtain Aramchol. This overcomes the defects of the existing chemical synthesis method, such as the large number of exposed reaction sites of the three hydroxyl groups of 3α,7α,12α-trihydroxy-5β-cholic acid, the large consumption of sodium azide and arachidonyl chloride, the complex side reactions and the poor reproducibility.
[0027] 2. The method for preparing Aramchol by enzymatic synthesis of the present invention is simple to operate and convenient for post-processing. The process yield of the method is as high as more than 91%, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 A synthetic route diagram of the method for preparing Aramchol by enzymatic synthesis of the present invention;
[0029] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of Aramchol prepared in Example 1 of the present invention;
[0030] Figure 3 The mass spectrum of Aramchol prepared in Example 1 of the present invention; DETAILED DESCRIPTION
[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0032] Example 1
[0033] S1: Preparation of 3β,7α,12α-trihydroxy-5β-cholic acid
[0034] 50g 3α,7α,12α-trihydroxy-5β-cholic acid (122mmol, Zhongshan Bailing Biotechnology Co., Ltd.) and 500ml water were added to the reaction bottle, and 1M sodium hydroxide solution was added dropwise under stirring until the cholic acid was completely dissolved, and the pH was adjusted to 7.7-8.0 with hydrochloric acid. Then 0.4g oxidized coenzyme I (NAD+, Zhongshan Bailing Biotechnology Co., Ltd.), 20ml 3α-hydroxysteroid dehydrogenase (3αHSDH, Zhongshan Bailing Biotechnology Co., Ltd.), and 30ml 3β-hydroxysteroid dehydrogenase (3βHSDH, Zhongshan Bailing Biotechnology Co., Ltd.) were added in sequence, and the volume was made up to 1250ml with water, and the reaction was stirred in a 30℃ water bath for 4-6h. After the reaction was completed, the mother liquor was filtered, and the pH value of the mother liquor was adjusted to 3.0-4.0 with hydrochloric acid, and then filtered. The filter cake was dried by air to obtain 47.6 g of A1, a white powder, namely 3β,7α,12α-trihydroxy-5β-cholic acid, with a yield of 95.2%.
[0035] S2: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholic acid
[0036] The 3β,7α,12α-trihydroxy-5β-cholic acid (47 g, 115 mmol) prepared in step S1 and 25% ammonia water (80.6 g, 1185 mmol) were added to the reaction bottle, the temperature was raised to 90°C and the reaction was carried out for 2 h. After the reaction was completed, the temperature was lowered to room temperature. A large amount of solid precipitated, which was filtered and washed with water. The filter cake was dried by air to obtain 43.6 g of 3β-amino-7α,12α-dihydroxy-5β-cholic acid white powder with a yield of 93.0%.
[0037] S3: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0038] The 3β-amino-7α,12α-dihydroxy-5β-cholic acid (42 g, 103 mmol) prepared in step S2 and methanol (800 ml) were added to a reaction flask, and sulfuric acid (1.0 g, 10.3 mmol) was slowly added dropwise at room temperature. The reaction was allowed to react for 4 h at room temperature. The reaction solution was cooled to -20°C for crystallization for 12 h, filtered, and the filter cake was dried with air to obtain 39.8 g of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester as a white powder with a yield of 91.6%.
[0039] S4: Preparation of 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0040] The 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester (38 g, 90 mmol), arachidic acid (30.9 g, 99 mol), HATU (51.3 g, 135 mmol), DIPEA (17.4 g, 135 mmol) and dichloromethane (800 ml) prepared in step S3 were added to a reaction flask, reacted at room temperature for 12 h, the reaction was stopped, filtered, the mother liquor was washed with dilute hydrochloric acid, water, and saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, and the mother liquor was evaporated under reduced pressure to dry the solvent to obtain 62.5 g of yellow liquid 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester, with a yield of 96.8%.
[0041] S5: Preparation of Aramchol
[0042] 3β-Arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester (50 g, 70 mmol) prepared in step S4, 10% NaOH solution (300 g) and methanol (1 L) were added to the reaction bottle, heated and refluxed for 2 h, the reaction solution was cooled to room temperature, hydrochloric acid was added to adjust the pH of the reaction solution to about 2-3, a yellow solid was precipitated, filtered, rinsed with water, and the solid was air-dried to obtain a crude product. The crude product was dissolved by reflux with ethanol, slowly cooled to -20°C at room temperature for crystallization for 12 h, filtered, and the filter cake was air-dried to obtain 44.8 g of white powder, i.e. Aramchol (3β-Arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-acid), with a yield of 91.4%.
[0043] The results of the NMR test are attached. Figure 2 The mass spectrometry results are shown in the attached Figure 3 .
[0044] 1 HNMR(DMSO),δ: 12.02 (s, 1H), 7.56 (s, 1H), 4.14 (d, J = 30.4 Hz,2H), 3.86 (d, J = 21.8 Hz, 2H), 3.67 (s, 1H), 2.29 – 1.19 (m, 57H), 0.98 (s, 3H), 0.91 (s, 6H), 0.64 (s, 3H). MS(m / s):703.60.
[0045] Example 2
[0046] S1: Preparation of 3β,7α,12α-trihydroxy-5β-cholic acid
[0047] 60g 3α,7α,12α-trihydroxy-5β-cholic acid (147mmol, Zhongshan Bailing Biotechnology Co., Ltd.) and 600ml water were added to the reaction flask, 1M sodium hydroxide solution was added dropwise under stirring until the cholic acid was completely dissolved, and the pH was adjusted to 7.7-8.0 with hydrochloric acid. Then 0.3g oxidized coenzyme I (NAD+, Zhongshan Bailing Biotechnology Co., Ltd.), 12ml 3α-hydroxysteroid dehydrogenase (3αHSDH, Zhongshan Bailing Biotechnology Co., Ltd.), and 24ml 3β-hydroxysteroid dehydrogenase (3βHSDH, Zhongshan Bailing Biotechnology Co., Ltd.) were added in sequence, and the volume was made up to 1.2L with water, and the reaction was stirred in a 30℃ water bath for 4-6h. After the reaction was completed, the mother liquor was filtered, and the pH value of the mother liquor was adjusted to 3.0-4.0 with hydrochloric acid, and then filtered. The filter cake was dried by air to obtain 57.7 g of A1, a white powder, namely 3β,7α,12α-trihydroxy-5β-cholic acid, with a yield of 96.2%.
[0048] S2: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholic acid
[0049] The 3β,7α,12α-trihydroxy-5β-cholic acid (56.4 g, 138 mmol) prepared in step S1 and 25% ammonia water (48.4 g, 712 mmol) were added to a reaction flask, the temperature was raised to 90°C and the reaction was carried out for 2 h. After the reaction was completed, the temperature was lowered to room temperature. A large amount of solid precipitated, which was filtered off with suction, rinsed with water, and the filter cake was dried by air to obtain 53.1 g of 3β-amino-7α,12α-dihydroxy-5β-cholic acid white powder with a yield of 94.4%.
[0050] S3: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0051] The 3β-amino-7α,12α-dihydroxy-5β-cholic acid (50.4 g, 123.7 mmol) prepared in step S2 and methanol (800 ml) were added to a reaction flask, and sulfuric acid (1.2 g, 6.2 mmol) was slowly added dropwise at room temperature. The reaction was allowed to react for 4 h at room temperature. The reaction solution was cooled to -20°C for crystallization for 12 h, filtered, and the filter cake was dried with air to obtain 50.5 g of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester as a white powder with a yield of 96.9%.
[0052] S4: Preparation of 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0053] The 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester (54.7 g, 129.7 mmol), arachidic acid (32.4 g, 103.7 mol), HATU (49.2 g, 129.6 mmol), DIPEA (16.7 g, 129.6 mmol) and dichloromethane (550 ml) prepared in step S3 were added to a reaction flask, reacted at room temperature for 12 h, the reaction was stopped, filtered, the mother liquor was washed with dilute hydrochloric acid, water, and saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, and the mother liquor was evaporated under reduced pressure to dry the solvent to obtain 89.4 g of yellow liquid 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester, with a yield of 96.2%.
[0054] S5: Preparation of Aramchol
[0055] The 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester (70 g, 98 mmol) prepared in step S4, 10% NaOH solution (280 g) and methanol (700 ml) were added to the reaction bottle, heated and refluxed for 2 h, the reaction solution was cooled to room temperature, hydrochloric acid was added to adjust the pH of the reaction solution to about 2-3, and a yellow solid was precipitated, filtered, rinsed with water, and the solid was air-dried to obtain a crude product. The crude product was dissolved in ethanol by reflux, slowly cooled to -20°C at room temperature for crystallization for 12 h, filtered, and the filter cake was air-dried to obtain 63.6 g of white powder, namely Aramchol (3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-acid), with a yield of 92.7%.
[0056] Embodiment 3:
[0057] S1: Preparation of 3β,7α,12α-trihydroxy-5β-cholic acid
[0058] 100g 3α,7α,12α-trihydroxy-5β-cholic acid (245mmol, Zhongshan Bailing Biotechnology Co., Ltd.) and 1000ml water were added to the reaction bottle, 1M sodium hydroxide solution was added dropwise under stirring until the cholic acid was completely dissolved, and the pH was adjusted to 7.7-8.0 with hydrochloric acid. Then 1.5g oxidized coenzyme I (NAD+, Zhongshan Bailing Biotechnology Co., Ltd.), 100ml 3α-hydroxysteroid dehydrogenase (3αHSDH, Zhongshan Bailing Biotechnology Co., Ltd.), and 100ml 3β-hydroxysteroid dehydrogenase (3βHSDH, Zhongshan Bailing Biotechnology Co., Ltd.) were added in sequence, and the volume was filled to 3L with water. The reaction was stirred in a 30℃ water bath for 4-6h. After the reaction was completed, the mother liquor was filtered, and the pH value of the mother liquor was adjusted to 3.0-4.0 with hydrochloric acid, and the mother liquor was filtered, and the filter cake was dried by air to obtain 95.8 g of A1, a white powder, namely 3β,7α,12α-trihydroxy-5β-cholic acid, with a yield of 95.8%.
[0059] S2: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholic acid
[0060] The 3β,7α,12α-trihydroxy-5β-cholic acid (94 g, 230 mmol) prepared in step S1 and 25% ammonia water (241.6 g, 3552 mmol) were added to a reaction bottle, the temperature was raised to 90°C and the reaction was carried out for 2 h. After the reaction was completed, the temperature was lowered to room temperature. A large amount of solid precipitated, which was filtered off with suction, rinsed with water, and the filter cake was dried by air to obtain 87.8 g of 3β-amino-7α,12α-dihydroxy-5β-cholic acid as a white powder with a yield of 93.6%.
[0061] S3: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0062] The 3β-amino-7α,12α-dihydroxy-5β-cholic acid (84 g, 206 mmol) prepared in step S2 and methanol (2500 ml) were added to a reaction flask, and sulfuric acid (4.0 g, 41.2 mmol) was slowly added dropwise at room temperature. The reaction was allowed to react at room temperature for 4 h. The reaction solution was cooled to -20°C for crystallization for 12 h, filtered, and the filter cake was dried with air to obtain 83.2 g of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester as a white powder with a yield of 95.8%.
[0063] S4: Preparation of 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester
[0064] The 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester (76 g, 180 mmol), arachidic acid (84.3 g, 270 mol), HATU (205.2 g, 540 mmol), DIPEA (69.6 g, 540 mmol) and dichloromethane (2200 ml) prepared in step S3 were added to a reaction flask, reacted at room temperature for 12 h, the reaction was stopped, filtered, the mother liquor was washed with dilute hydrochloric acid, water, and saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, filtered, and the mother liquor was evaporated under reduced pressure to dry the solvent to obtain 125.8 g of yellow liquid 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester, with a yield of 97.5%.
[0065] S5: Preparation of Aramchol
[0066] 3β-Arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester (120 g, 168 mmol) prepared in step S4, 10% NaOH solution (1200 g) and methanol (3.6 L) were added to the reaction bottle, heated and refluxed for 2 h, the reaction solution was cooled to room temperature, hydrochloric acid was added to adjust the pH of the reaction solution to about 2-3, a yellow solid was precipitated, filtered, rinsed with water, and the solid was air-dried to obtain a crude product. The crude product was dissolved by reflux with ethanol, slowly cooled to -20°C at room temperature for crystallization for 12 h, filtered, and the filter cake was air-dried to obtain 109.2 g of white powder, namely Aramchol (3β-Arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-acid), with a yield of 92.8%.
[0067] In summary, the method for preparing Aramchol by enzymatic synthesis of the present invention prepares Aramchol by using a specific and selective enzyme catalyst. It overcomes the defects of the prior art chemical synthesis method, such as the large number of exposed reaction sites of the three hydroxyl groups of 3α, 7α, 12α-trihydroxy-5β-cholic acid, the large consumption of sodium azide and arachidyl chloride, the complex side reactions and the poor reproducibility. The preparation method is simple to operate and convenient for post-processing. The process yield is as high as 91% or more, and is suitable for industrial production.
[0068] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for preparing Aramchol by enzymatic synthesis, characterized in that The following steps are involved: With 3α,7α,12α-trihydroxy-5β-cholic acid as raw material, 3α,7α,12α-trihydroxy-5β-cholic acid is converted into 3β,7α,12α-trihydroxy-5β-cholic acid under the action of oxidative coenzyme I, 3α-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase; 3β,7α,12α-trihydroxy-5β-cholic acid reacts with ammonia water to generate 3β-amino-7α,12α-dihydroxy-5β-cholic acid, and 3β-amino-7α,12α-dihydroxy-5β-cholic acid is then esterified with methanol to generate 3β-amino-7α,12α-dihydroxy- 5β-cholestane-24-acid methyl ester; 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester is then condensed with arachidic acid to generate 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester; 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-acid methyl ester is then hydrolyzed to obtain 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-acid; its synthetic route is as follows: 。 2. A method for preparing Aramchol by enzymatic synthesis according to claim 1, characterized in that The steps include: S1: Preparation of 3β,7α,12α-trihydroxy-5β-cholic acid: Mix a proportion of 3α,7α,12α-trihydroxy-5β-cholic acid and water, add a 1M sodium hydroxide solution dropwise until the cholic acid is completely dissolved, and adjust the pH to 7.7-8.0 with hydrochloric acid; then add oxidized coenzyme I, 3α-hydroxysteroid dehydrogenase, and 3β-hydroxysteroid dehydrogenase in sequence, and stir in a 30°C water bath to react for 4-6 hours; after the reaction is completed, filter and dry to obtain 3β,7α,12α-trihydroxy-5β-cholic acid for later use; S2: preparing 3β-amino-7α,12α-dihydroxy-5β-cholic acid: mixing the 3β,7α,12α-trihydroxy-5β-cholic acid prepared in step S1 with ammonia water, heating to 90° C. for reaction for 2 h, and cooling to room temperature after the reaction is completed. The filter cake is filtered with suction, and air-dried to obtain 3β-amino-7α,12α-dihydroxy-5β-cholic acid for standby use; S3: Preparation of 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester: Add 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid and methanol prepared in step S2 into a reaction flask, slowly add sulfuric acid dropwise at room temperature, react at room temperature for 4 hours, cool the reaction solution to -20°C for crystallization for 12 hours, filter with suction, and dry the filter cake to obtain 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester for later use; S4: Preparation of 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester: Add 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester prepared in step S3, arachidic acid, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N'-diisopropylethylamine and dichloromethane to a reaction flask, react at room temperature for 8-15 hours, and stop the reaction; filter, wash the mother liquor with acid, water, and saturated sodium chloride, dry the organic phase with anhydrous sodium sulfate, filter, and evaporate the solvent under reduced pressure to obtain 3β-arachidylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester for later use; S5: Preparation of Aramchol: 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester prepared in step S4, 10% NaOH solution and methanol were added to a reaction bottle, the temperature was raised and refluxed for reaction for 2 hours, the reaction solution was added with hydrochloric acid to adjust the pH to 2-3, the reaction solution was filtered, the solution was rinsed with water, and the solid was dried to obtain a crude product of 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid, and the crude product was purified with ethanol to obtain Aramchol.
3. A method for preparing Aramchol by enzymatic synthesis according to claim 2, characterized in that: The mass ratio of 3α,7α,12α-trihydroxy-5β-cholic acid, oxidized coenzyme I, 3αHSDH enzyme solution and 3βHSDH enzyme solution described in step S1 is 3α,7α,12α-trihydroxy-5β-cholic acid: oxidized coenzyme I: 3αHSDH enzyme solution: 3βHSDH enzyme solution = 1: 0.001~0.02: 0.1~2.0: 0.1~2.
0.
4. A method for preparing Aramchol by enzymatic synthesis according to claim 2, characterized in that: The mass ratio of 3α,7α,12α-trihydroxy-5β-cholic acid and water in step S1 is 3α,7α,12α-trihydroxy-5β-cholic acid: water = 1:10-50, and the water is deionized water.
5. A method for preparing Aramchol by enzymatic synthesis according to claim 2, characterized in that: The molar ratio of ammonia water and 3β,7α,12α-trihydroxy-5β-cholic acid in step S2 is ammonia water:3β,7α,12α-trihydroxy-5β-cholic acid=5~20eq.
6. A method for preparing Aramchol by enzymatic synthesis according to claim 2, characterized in that: The molar ratio of 3β-amino-7α,12α-dihydroxy-5β-cholic acid and sulfuric acid described in step S3 is 3β-amino-7α,12α-dihydroxy-5β-cholic acid: sulfuric acid = 1:0.05~0.5; the mass volume ratio of 3β-amino-7α,12α-dihydroxy-5β-cholic acid and methanol described in step S3 is 1:10~50.
7. A method for preparing Aramchol by enzymatic synthesis according to claim 2, characterized in that: The molar ratio of arachidic acid to 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester in step S4 is arachidic acid:3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester= 0.5~2.0eq; the molar ratio of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is 1.0~5.0eq; the molar ratio of N,N'-diisopropylethylamine and 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is 1.0~5.0eq; the volume mass ratio of dichloromethane and 3β-amino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester is 10~50:
1.
8. A method for preparing Aramchol by enzymatic synthesis according to any one of claims 2 or 7, characterized in that: The reaction time in step S4 is 12 h.
9. A method for preparing Aramchol by enzymatic synthesis according to claim 2, characterized in that: The mass ratio of 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester and 10% sodium hydroxide in step S5 is 1:4-15; the mass volume ratio of 3β-arachidonylamino-7α,12α-dihydroxy-5β-cholestane-24-oic acid methyl ester and methanol is 1:10-40.