A method for the chemo-enzymatic preparation of dehydroepiandrosterone

Abstract

The present invention provides a method for preparing dehydroepiandrosterone by a chemical-enzymatic method, belonging to the technical field of pharmaceutical compounds. In the present invention, water, glacial acetic acid and sodium D-ascorbate are mixed and then tert-butanol, a strong base catalyst and 4-androstenedione are sequentially added for reaction to obtain 5-androstenedione; a phosphate buffer system is obtained by mixing tert-butanol with a phosphate buffer solution; 5-androstenedione, magnesium chloride, ketoreductase, coenzyme and a coenzyme regeneration system are added to the phosphate buffer system for an enzymatic reaction, and dehydroepiandrosterone is obtained. The target product is synthesized in two steps by the combined chemical-enzymatic method in the present invention, and the yield of the product is as high as 95%, and the purity is above 99%. The synthesis method of the present invention effectively solves the problems of complex chemical process and difficult post-treatment of biological method, not only simplifies the process, but also improves the yield and purity of DHEA, and can realize large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compounds, and particularly to a method for preparing dehydroepiandrosterone by a chemo-enzymatic method. Background Art

[0002] Dehydroepiandrosterone (DHEA) is a key intermediate for synthesizing numerous steroid drugs such as mifepristone, drospirenone, norethisterone, abiraterone acetate, ulipristal acetate, etc. DHEA is also the most abundant steroid substance in human blood circulation and is a prerequisite for the synthesis of more than 40 hormones in the human body. Its content in the human body changes with age. Currently, the methods for obtaining DHEA mainly include chemical synthesis, biosynthesis, and extraction from natural products.

[0003] The chemical synthesis of DHEA has the following route:

[0004]

[0005] This method uses 4-AD as the raw material, acetyl chloride and acetic anhydride as the 3-position enoesterification reagent, pyridine hydrochloride as the highly efficient catalyst to catalyze the protection of the 17-position carbonyl with ethylene glycol ketal, reduction with potassium borohydride, and hydrolysis with dilute hydrochloric acid to remove the 17-position protection to prepare DHEA. The total yield is 70%, and the product purity is greater than 99.0%. This process is a pure chemical method, with a relatively complex process, requiring the use of expensive catalytic reagents, and the product yield is not high.

[0006] The biosynthesis of DHEA has the following route:

[0007]

[0008] This method uses microbial fermentation to produce DHEA, with phytosterol as the raw material. First, the 3-position hydroxyl group is protected, then the 17-position saturated side chain is removed by microbial fermentation, and finally DHEA is obtained through hydrolysis. Biosynthesis has the advantages of wide raw material sources and short reaction steps. However, the reaction liquid composition is complex, the post-treatment separation is cumbersome, and the product yield is reduced.

[0009] Therefore, how to obtain a synthesis method of DHEA with a simple process, high yield, and high purity is a problem that needs to be solved currently. Summary of the Invention

[0010] The purpose of the present invention is to provide a method for preparing dehydroepiandrosterone by a chemo-enzymatic method to solve the technical problems of the complex synthesis method and low product yield of dehydroepiandrosterone currently.

[0011] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0012] The present invention provides a method for preparing dehydroepiandrosterone by a chemical-enzymatic method, comprising the following steps:

[0013] 1) Under a protective atmosphere, water, glacial acetic acid and sodium D-ascorbate are mixed and successively added with tert-butanol, a strong base catalyst and 4-androstenedione for reaction to obtain 5-androstenedione;

[0014] 2) Tert-butanol and a phosphate buffer solution are mixed to obtain a phosphate buffer system;

[0015] 3) 5-Androstenedione, magnesium chloride, ketoreductase, coenzyme and a coenzyme regeneration system are added to the phosphate buffer system for an enzymatic reaction to obtain dehydroepiandrosterone.

[0016] Further, the mass ratio of the water, glacial acetic acid and sodium D-ascorbate is 10-15:0.2-0.5:0.3-0.6.

[0017] Further, the strong base catalyst includes one or more of potassium tert-butoxide, sodium tert-butoxide, sodium ethoxide and butyl lithium.

[0018] Further, the mass ratio of the sodium D-ascorbate, tert-butanol, strong base catalyst and 4-androstenedione is 0.3-0.6:2-5:1-3:1.

[0019] Further, in step 1), the protective atmosphere includes nitrogen and / or argon; the reaction temperature is 25-40 °C, and the reaction time is 1.5-3 h.

[0020] Further, the phosphate buffer solution includes glucose monohydrate, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and water with a mass ratio of 1.0-2.0:0.1-0.2:0.1-0.4:3.0-5.0; the pH of the phosphate buffer system is 6.8-7.8;

[0021] In step 2), the content of the tert-butanol is 0.4-1.0 times the mass of the phosphate buffer solution.

[0022] Further, the ketoreductase includes one or more of KRED-101, KRED-119 and KRED-130.

[0023] Further, the coenzyme includes NAD and NADH, and the mass ratio of NAD and NADH is 1-3:1-3.

[0024] Further, the coenzyme regeneration system comprises glutamic acid and glutamate dehydrogenase with a mass ratio of 0.5 - 0.7:0.001 - 0.003; the mass ratio of 5-androstenedione, magnesium chloride, ketoreductase, coenzyme, and coenzyme regeneration system is 1:0.2 - 0.3:0.003 - 0.005:0.01 - 0.015:0.4 - 0.8.

[0025] Further, the temperature of the enzymatic reaction is 35 - 45 °C, and the time of the enzymatic reaction is 3 - 5 h.

[0026] Advantages of the present invention:

[0027] By using sodium D-ascorbate to control the oxygen content in the system, the present invention avoids the reaction of the intermediate 5-AD with oxygen, enabling 5-AD to exist more stably in the reaction system; by using a specific ratio of ketoreductase, coenzyme, and coenzyme regeneration system, 5-AD can be more stably and rapidly converted into DHEA.

[0028] The process route of the present invention is simple. Compared with the chemical method for synthesizing DHEA, the route is shorter and the required reaction conditions are milder. The yield of the chemical method for synthesizing DHEA is only 70%, while the total product yield obtained by the process method of the present invention is as high as 95%, and the product purity is above 99%, making it of higher industrial application value. Specific embodiments

[0029] The present invention provides a method for preparing dehydroepiandrosterone by a chemo-enzymatic method, comprising the following steps:

[0030] 1) Under a protective atmosphere, water, glacial acetic acid, and sodium D-ascorbate are mixed and successively added with tert-butanol, a strong base catalyst, and 4-androstenedione for reaction to obtain 5-androstenedione;

[0031] 2) Tert-butanol and phosphate buffer solution are mixed to obtain a phosphate buffer system;

[0032] 3) 5-Androstenedione, magnesium chloride, ketoreductase, coenzyme, and coenzyme regeneration system are added to the phosphate buffer system for enzymatic reaction to obtain dehydroepiandrosterone.

[0033] In the present invention, the mass ratio of water, glacial acetic acid, and sodium D-ascorbate is 10 - 15:0.2 - 0.5:0.3 - 0.6, preferably 11 - 14:0.3 - 0.4:0.35 - 0.55, and further preferably 12 - 13:0.35 - 0.38:0.44 - 0.52.

[0034] In the present invention, the strong base catalyst comprises one or more of potassium tert-butoxide, sodium tert-butoxide, sodium ethoxide, and butyllithium, preferably potassium tert-butoxide and / or sodium tert-butoxide.

[0035] In the present invention, the mass ratio of sodium D-ascorbate, tert-butanol, strong base catalyst and 4-androstenedione is 0.3 - 0.6:2 - 5:1 - 3:1, preferably 0.35 - 0.55:3 - 4:1.5 - 2.5:1, and more preferably 0.44 - 0.52:3.5 - 3.8:1.8 - 2.0:1.

[0036] In the present invention, in step 1), the protective atmosphere contains nitrogen and / or argon, preferably nitrogen.

[0037] In the present invention, in step 1), the temperature of the reaction is 25 - 40 °C, and the reaction time is 1.5 - 3 h; preferably, the reaction temperature is 30 - 38 °C, and the reaction time is 2 - 2.5 h; more preferably, the reaction temperature is 32 - 35 °C, and the reaction time is 2.5 h.

[0038] In the present invention, the phosphate buffer solution contains glucose monohydrate, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and water in a mass ratio of 1.0 - 2.0:0.1 - 0.2:0.1 - 0.4:3.0 - 5.0, preferably 1.5 - 1.9:0.14 - 0.18:0.2 - 0.3:4.0 - 4.5, and more preferably 1.82:0.145:0.265:4.4.

[0039] In the present invention, in step 2), the content of tert-butanol is 0.4 - 1.0 times the mass of the phosphate buffer solution, preferably 0.5 - 0.8 times, and more preferably 0.6 - 0.7 times.

[0040] In the present invention, the pH of the phosphate buffer system is 6.8 - 7.8, preferably 7.0 - 7.5, and more preferably 7.2 - 7.4.

[0041] In the present invention, the pH of the phosphate buffer system is adjusted with a sodium hydroxide solution, wherein the concentration of the sodium hydroxide solution is 0.5 - 1.5 mol / L, preferably 1.0 mol / L.

[0042] In the present invention, the ketoreductase comprises one or more of KRED-101, KRED-119 and KRED-130, preferably KRED-101.

[0043] In the present invention, the coenzyme comprises NAD and NADH, and the mass ratio of NAD and NADH is 1 - 3:1 - 3, preferably 1.5 - 2.5:1.5 - 2.5, and more preferably 2:2.

[0044] In the present invention, the coenzyme regeneration system comprises glutamic acid and glutamate dehydrogenase with a mass ratio of 0.5 to 0.7: 0.001 to 0.003, preferably 0.55 to 0.65: 0.0015 to 0.002, and more preferably 0.60: 0.002.

[0045] In the present invention, the mass ratio of the 5-androstenedione, magnesium chloride, ketoreductase, coenzyme and coenzyme regeneration system is 1: 0.2 to 0.3: 0.003 to 0.005: 0.01 to 0.015: 0.4 to 0.8, preferably 1: 0.22 to 0.28: 0.0035 to 0.0045: 0.012 to 0.014: 0.5 to 0.7, and more preferably 1: 0.25: 0.004: 0.013: 0.6 to 0.65.

[0046] In the present invention, the temperature of the enzymatic reaction is 35 to 45 °C, and the time of the enzymatic reaction is 3 to 5 h; preferably, the temperature of the enzymatic reaction is 38 to 42 °C, and the time of the enzymatic reaction is 3.5 to 4.5 h; more preferably, the temperature of the enzymatic reaction is 40 °C, and the time of the enzymatic reaction is 4 h.

[0047] In the present invention, the post-treatment steps of the product obtained from the enzymatic reaction are as follows:

[0048] a. The obtained product is concentrated under reduced pressure to a paste and then centrifuged to obtain a wet material. Acetone, the wet material and activated carbon are mixed and then filtered and concentrated to a paste, and then centrifuged to obtain a dry material;

[0049] b. The dry material is purified with ethyl acetate to obtain dehydroepiandrosterone.

[0050] In the present invention, in the step a, the rotation speed of the centrifugation is independently 200 to 500 rpm, preferably 300 to 400 rpm, and more preferably 350 rpm.

[0051] In the present invention, the mass ratio of acetone, the wet material and activated carbon is 1.5 to 2.0: 1.0 to 2.0: 0.07 to 0.1, preferably 1.57 to 1.82: 1.6 to 1.8: 0.08 to 0.09, and more preferably 1.60: 1.7: 0.07.

[0052] In the present invention, in the step b, the mass-to-volume ratio of the dry material and ethyl acetate is 80 to 100 kg: 150 to 250 L, preferably 88 to 95 kg: 180 to 220 L, and more preferably 88 kg: 200 L.

[0053] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] Preparation of dehydroepiandrosterone:

[0056] 1) Under nitrogen protection, 120 g of water, 3.85 g of glacial acetic acid and 4.4 g of sodium D-ascorbate were pumped into the water separation kettle to dissolve sodium D-ascorbate by stirring and keep the temperature at 25 °C. Then, 38.6 g of tert-butanol, 18.25 g of potassium tert-butoxide and 10 g of 4-androstenedione were successively pumped into the water separation kettle. The temperature in the kettle dropped to 35 °C and the reaction was carried out for 1.5 h to obtain 5-AD with a yield of 98%;

[0057] 2) 35.6 g of tert-butanol, 18.2 g of glucose monohydrate, 1.45 g of disodium hydrogen phosphate dodecahydrate, 2.65 g of sodium dihydrogen phosphate dihydrate and 44 g of water were successively pumped into the enzyme reaction kettle and dissolved by stirring. The pH in the kettle was adjusted to 6.8 with 1.0 mol / L sodium hydroxide solution. Then, 10 g of 5-AD was put into the enzyme reaction kettle, and 2.54 g of magnesium chloride, 0.04 g of ketoreductase KRED-101, 0.1 g of NAD, 0.05 g of NADH and coenzyme regeneration system (where the coenzyme regeneration system contains 5 g of glutamic acid and 0.02 g of glutamate dehydrogenase) were successively added. The temperature was controlled at 40 °C and the reaction was carried out for 4 h to obtain a white solid product with a yield of 97%.

[0058] 3) The obtained product was concentrated under reduced pressure to a paste and then centrifuged at 300 rpm for 20 min to obtain wet material. 15.7 g of acetone, 16 g of wet material and 0.8 g of activated carbon were mixed and then filtered and concentrated to a paste, and then centrifuged at 400 rpm to obtain dry material; 10 g of dry material was purified with 20 mL of ethyl acetate to obtain dehydroepiandrosterone. After HPLC detection, the purity was 99.0%.

[0059] Example 2

[0060] Preparation of dehydroepiandrosterone:

[0061] 1) Under nitrogen protection, 150 g of water, 4.25 g of glacial acetic acid and 5.0 g of sodium D-ascorbate were pumped into the water separation kettle to dissolve sodium D-ascorbate by stirring and keep the temperature at 25 °C. Then, 39.2 g of tert-butanol, 16.35 g of potassium tert-butoxide and 10 g of 4-androstenedione were successively pumped into the water separation kettle. The temperature in the kettle dropped to 35 °C and the reaction was carried out for 2 h to obtain 5-AD with a yield of 97.6%;

[0062] 2) 40.3 g of tert-butanol, 16.8 g of glucose monohydrate, 1.56 g of disodium hydrogen phosphate dodecahydrate, 2.65 g of sodium dihydrogen phosphate dihydrate and 44 g of water were successively pumped into the enzyme reaction kettle, stirred and dissolved, and the pH in the kettle was adjusted to 7.0 with 1.0 mol / L sodium hydroxide solution. Then 10 g of 5-AD was put into the enzyme reaction kettle, and 2.63 g of magnesium chloride, 0.05 g of ketoreductase KRED-119, 0.1 g of NAD, 0.05 g of NADH and the coenzyme regeneration system (where the coenzyme regeneration system contains 6 g of glutamic acid and 0.02 g of glutamate dehydrogenase) were successively added. The temperature was controlled at 35 °C and the reaction was carried out for 4 h to obtain a white solid product with a yield of 97.9%.

[0063] 3) The obtained product was concentrated under reduced pressure to a paste and then centrifuged at 300 rpm for 20 min to obtain a wet material. 18.2 g of acetone, 18 g of the wet material and 0.9 g of activated carbon were mixed and then filtered and concentrated to a paste, and then centrifuged at 400 rpm to obtain a dry material; 10 g of the dry material was purified with 22 mL of ethyl acetate to obtain dehydroepiandrosterone. After HPLC detection, the purity was 99.0%.

[0064] Example 3

[0065] Preparation of dehydroepiandrosterone:

[0066] 1) Under nitrogen protection, 100 g of water, 26.3 g of glacial acetic acid and 6.0 g of sodium D-ascorbate were pumped into the water separation kettle to dissolve the sodium D-ascorbate by stirring and keep it at 25 °C. Then 35.8 g of tert-butanol, 15.3 g of potassium tert-butoxide and 10 g of 4-androstenedione were successively pumped into the water separation kettle. The temperature in the kettle was lowered to 35 °C and the reaction was carried out for 2 h to obtain 5-AD with a yield of 96.9%.

[0067] 2) 39.8 g of tert-butanol, 15.3 g of glucose monohydrate, 1.40 g of disodium hydrogen phosphate dodecahydrate, 2.51 g of sodium dihydrogen phosphate dihydrate and 44 g of water were successively pumped into the enzyme reaction kettle, stirred and dissolved, and the pH in the kettle was adjusted to 7.5 with 1.0 mol / L sodium hydroxide solution. Then 10 g of 5-AD was put into the enzyme reaction kettle, and 2.35 g of magnesium chloride, 0.03 g of ketoreductase KRED-130, 0.1 g of NAD, 0.05 g of NADH and the coenzyme regeneration system (where the coenzyme regeneration system contains 6 g of glutamic acid and 0.03 g of glutamate dehydrogenase) were successively added. The temperature was controlled at 45 °C and the reaction was carried out for 3 h to obtain a white solid product with a yield of 98.1%.

[0068] 3) The obtained product was concentrated under reduced pressure to a paste, centrifuged at 300 rpm for 20 min to obtain wet material. Then, 16.0 g of acetone, 17 g of wet material and 0.8 g of activated carbon were mixed and filtered by suction and concentrated to a paste, and then centrifuged at 400 rpm to obtain dry material. 10 g of the dry material was purified with 20 mL of ethyl acetate to obtain dehydroepiandrosterone. After HPLC detection, the purity was 99.0%.

[0069] Example 4

[0070] Preparation of dehydroepiandrosterone:

[0071] 1) Under nitrogen protection, 145 g of water, 3.62 g of glacial acetic acid and 5.0 g of sodium D-ascorbate were pumped into a water separation kettle to dissolve sodium D-ascorbate by stirring until clear, and maintained at 25 °C. Then, 36.5 g of tert-butanol, 15.34 g of potassium tert-butoxide and 10 g of 4-androstenedione were successively pumped into the water separation kettle. The temperature in the kettle dropped to 35 °C and reacted for 2 h to obtain 5-AD, and the yield was 98%.

[0072] 2) 42.5 g of tert-butanol, 17.5 g of glucose monohydrate, 1.56 g of disodium hydrogen phosphate dodecahydrate, 2.65 g of sodium dihydrogen phosphate dihydrate and 44 g of water were successively pumped into an enzyme reaction kettle and stirred to dissolve. The pH in the kettle was adjusted to 7.0 with 1.0 mol / L sodium hydroxide solution. Then, 10 g of 5-AD was put into the enzyme reaction kettle, and 2.63 g of magnesium chloride, 0.05 g of ketoreductase KRED-119, 0.05 g of NAD, 0.1 g of NADH and a coenzyme regeneration system (where the coenzyme regeneration system contains 7 g of glutamic acid and 0.03 g of glutamate dehydrogenase) were successively added, and the temperature was controlled at 40 °C and reacted for 4 h to obtain a white solid product, and the yield was 96.5%.

[0073] 3) The obtained product was concentrated under reduced pressure to a paste, centrifuged at 300 rpm for 20 min to obtain wet material. Then, 15.7 g of acetone, 16 g of wet material and 0.8 g of activated carbon were mixed and filtered by suction and concentrated to a paste, and then centrifuged at 400 rpm to obtain dry material. 10 g of the dry material was purified with 18 mL of ethyl acetate to obtain dehydroepiandrosterone. After HPLC detection, the purity was 99.0%.

[0074] As can be seen from the above examples, the present invention provides a method for preparing dehydroepiandrosterone by a chemical-enzymatic method. The present invention synthesizes the target product in two steps by combining a chemical-enzymatic method. The yield of the product is as high as 95%, and the purity is above 99%. The synthesis method of the present invention effectively solves the problems of complex chemical process and difficult post-treatment of biological methods, not only simplifies the process, but also improves the yield and purity of DHEA, and can realize large-scale production.

[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing dehydroepiandrosterone by a chemo-enzymatic method, characterized in that, It includes the following steps: 1) Under a protective atmosphere, water, glacial acetic acid and sodium D-ascorbate are mixed and successively added with tert-butanol, a strong base catalyst and 4-androstenedione for reaction to obtain 5-androstenedione; 2) Tert-butanol is mixed with a phosphate buffer solution to obtain a phosphate buffer system; 3) 5-Androstenedione, magnesium chloride, ketoreductase, coenzyme and coenzyme regeneration system are added to the phosphate buffer system for an enzymatic reaction to obtain dehydroepiandrosterone; The coenzyme contains NAD and NADH, and the mass ratio of NAD to NADH is 1-3:1-3; The coenzyme regeneration system contains glutamic acid and glutamate dehydrogenase with a mass ratio of 0.5-0.7:0.001-0.003; the mass ratio of 5-androstenedione, magnesium chloride, ketoreductase, coenzyme and coenzyme regeneration system is 1:0.2-0.3:0.003-0.005:0.01-0.015:0.4-0.8; The phosphate buffer solution contains glucose monohydrate, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and water with a mass ratio of 1.0-2.0:0.1-0.2:0.1-0.4:3.0-5.0; the pH of the phosphate buffer system is 6.8-7.8; In step 2), the content of tert-butanol is 0.4-1.0 times the mass of the phosphate buffer solution; The ketoreductase is one or more of KRED-101, KRED-119 and KRED-130; The strong base catalyst is potassium tert-butoxide.

2. The method for preparing dehydroepiandrosterone by chemo-enzymatic method according to claim 1, wherein The mass ratio of water, glacial acetic acid and sodium D-ascorbate is 10-15:0.2-0.5:0.3-0.

6.

3. The method for preparing dehydroepiandrosterone by chemo-enzymatic method according to claim 1, characterized in that, The mass ratio of sodium D-ascorbate, tert-butanol, strong base catalyst and 4-androstenedione is 0.3-0.6:2-5:1-3:

1.

4. The method for preparing dehydroepiandrosterone by chemo-enzymatic method according to claim 1 or 2 or 3, characterized in that, In step 1), the protective atmosphere contains nitrogen and / or argon; the temperature of the reaction is 25-40°C, and the reaction time is 1.5-3 h.

5. The method for preparing dehydroepiandrosterone by chemo-enzymatic method according to claim 1, characterized in that, The temperature of the enzymatic reaction is 35-45°C, and the enzymatic reaction time is 3-5 h.

Images