阅读说明：本技术 一种新型制备去氢表雄酮的方法 (Novel method for preparing dehydroepiandrosterone ) 是由 王友富 邵振平 王荣 罗敏 王洪福 黄橙橙 雷灵芝 于 2021-07-30 设计创作，主要内容包括：一种新型制备去氢表雄酮的方法。本发明公开了一种去氢表雄酮的制备方法,属于化合物的制备加工技术领域。该方法以4-雄烯二酮(Ⅰ)为起始原料,经过乙酰化反应得到3-乙酰氧基-Δ3,5-雄甾二烯-17-酮(Ⅱ),再将得到的中间化合物(Ⅱ)经生物酶法反应得到产物去氢表雄酮。本发明中的化合物Ⅱ作为关键中间体,其性质较5-AD更加稳定,且纯度高,从而有效的确保进入下一步生物酶法的稳定性；本发明反应路线的生物酶法采用了特定配比的复合生物酶,该特定配比的复合生物酶作用于化合物Ⅱ具备更好更稳定的转化效果；本发明路线的单批次收率＞90％,且产品化合物Ⅲ纯度高,纯度＞99％,工业化应用价值更高。(A novel method for preparing dehydroepiandrosterone is provided. The invention discloses a preparation method of dehydroepiandrosterone, and belongs to the technical field of preparation and processing of compounds. The method takes 4-androstenedione (I) as an initial raw material, obtains 3-acetoxyl group-delta 3, 5-androstadiene-17-ketone (II) through acetylation reaction, and obtains a product dehydroepiandrosterone through the biological enzyme method reaction of an obtained intermediate compound (II). The compound II is used as a key intermediate, has more stable property than 5-AD and high purity, thereby effectively ensuring the stability of the next biological enzyme method; the biological enzyme method of the reaction route adopts the compound biological enzyme with a specific proportion, and the compound biological enzyme with the specific proportion has better and more stable conversion effect when acting on the compound II; the single-batch yield of the route of the invention is more than 90 percent, and the product compound III has high purity which is more than 99 percent, and has higher industrial application value.)

1. A novel method for preparing dehydroepiandrosterone is characterized in that the synthetic route of the method is as follows:

the method specifically comprises the following steps:

1) under the protection of nitrogen, mixing 4-androstenedione (I), dichloromethane, acetic anhydride and an acidic catalyst or mixing 4-androstenedione (I), acetic anhydride and an acidic catalyst, adding into a reactor, controlling the temperature to be 0-40 ℃, stirring and reacting until the thin layer chromatography detects that raw materials are completely reacted, concentrating to remove dichloromethane, carrying out water precipitation, filtering and drying to obtain 3-acetoxyl group-delta 3, 5-androstadiene-17-ketone (II);

2) taking the compound (II) obtained in the step 1) as an intermediate, forming an enzyme reaction system with a cosolvent and an enzyme buffer solution, adding a biological enzyme, coenzyme and coenzyme regeneration system, carrying out biological enzymatic reaction at the temperature of 26-32 ℃, controlling the pH value in the whole reaction process to be 6.8-8.0, detecting the reaction process of the biological enzyme method by HPLC, finishing the reaction when the conversion rate reaches 98%, and carrying out subsequent treatment to obtain the dehydroepiandrosterone (III).

2. The method for preparing dehydroepiandrosterone according to claim 1, wherein the volume of dichloromethane used in step 1) is 0-5 times that of compound (I); the volume dosage of the acetic anhydride is 2-5 times of that of the compound (I); the acidic catalyst is one of p-toluenesulfonic acid, methanesulfonic acid or trifluoroacetic acid, and the mass amount of the acidic catalyst is 0.1-0.5 times of that of the compound (I).

3. The method for preparing dehydroepiandrosterone according to claim 1, wherein the cosolvent in step 2) is tert-butanol, the bio-enzyme is a complex enzyme obtained by mixing a ketone reductase and an ester hydrolase in a certain ratio, the coenzyme is nicotinamide adenine dinucleotide, the coenzyme regeneration system is a mixture of glucose and glucose dehydrogenase in a certain ratio, and the compound (II): the feeding weight volume ratio of the tertiary butanol is 1: 3; said compound (II) is ketoreductase: the weight ratio of the ester hydrolase to the raw materials is 1: 0.01-0.02: 0.005-0.01; the weight ratio of the compound (II) to the coenzyme is 1: 0.002-0.005; the weight ratio of the glucose to the glucose dehydrogenase of the compound (II) is 1:0.4-0.8: 0.002-0.005;

the enzyme buffer solution is phosphate buffer solution with pH 6.5;

the ketoreductase is the product of expression of the biological enzyme amino acid sequence 1 in a non-pathogenic microorganism;

the ester hydrolase is a product expressed by a biological enzyme amino acid sequence 2 in a non-pathogenic microorganism.

4. The method of claim 3, wherein the non-pathogenic microorganism is Escherichia coli.

5. The method for preparing dehydroepiandrosterone according to claim 1, wherein the post-treatment in step 2) is specifically: cooling to 8-10 ℃ after the reaction is finished, filtering and collecting solid, drying the solid, adding methanol for extraction for three times, extracting for 50-60 minutes each time, combining the extract liquor, concentrating under reduced pressure until a large amount of crystals are separated out, performing water precipitation, filtering at 8-10 ℃ for 2 hours, and drying to obtain the compound (III).

Technical Field

The invention relates to the technical field of preparation and processing of compounds, in particular to a preparation method of dehydroepiandrosterone.

Background

Dehydroepiandrosterone (dehydroepiandrosterone, DHEA for short), chemically (3 β) -3-hydroxy-5-en-17-sterone, molecular formula C19H22O2, molecular weight 288.42, structural formula as follows:

dehydroepiandrosterone, a precursor substance of adrenal hormone secreted by adrenal cortex reticular layer, has physiological activities of regulating obesity, preventing diabetes, resisting carcinogenesis and virus infection, improving memory, immune response and stress response, and relieving stress, and is used for preparing steroid hormone intermediate and participating in the synthesis of adrenal gland to secrete various hormones.

The CN102603839A publication discloses a method for preparing dehydroepiandrosterone by using dehydropregnenolone acetate as starting material and through ketoxime, beckmann rearrangement and hydrolysis reaction, which needs to use environmentally unfriendly phosphorus pentoxide and highly carcinogenic solvent benzene, and is not suitable for industrial production, and the process route is as follows:

CN102603841B discloses a method for preparing dehydroepiandrosterone by acetylation, ketal protection, reduction, and hydrolysis using 4-AD as starting material, which has many reaction steps, generates large isomer impurities difficult to refine and remove during the reduction process, and is not suitable for industrial production, and the synthetic route is as follows:

CN106086148A, CN109312382A, CN105483198A and CN105339382A all disclose a method for preparing dehydroepiandrosterone (hereinafter referred to as 5-AD route) by a chemical-enzymatic method, wherein 4-AD is used as a starting raw material, an intermediate 5-androstenedione (referred to as 5-AD) is synthesized by a chemical method, and the dehydroepiandrosterone is prepared by a biological enzymatic method.

The chemically synthesized intermediate 5-AD in the method has poor stability, according to the reports of org. Process Res. Dev.2016,20,1520-1528, the intermediate state of potassium alcoholate obtained after the 4-AD is treated by tert-butyl alcohol and potassium tert-butylate is easy to react with oxygen, so an antioxidant is required to be added during the glacial acetic acid water precipitation, and the yield of 5-AD obtained from 4-AD is only 32% and is very low according to the patent CN105483198A, while the potassium tert-butylate is used during the synthesis of 5-AD in the methods described in CN105339382A and CN106086148A, sodium ascorbate is also required to inhibit the oxidation of 5-AD, the operation is complex and difficult to control, and the purity of 5-AD is a main problem affecting the next reaction, wherein the impurities bring about the quality and yield of dehydroepiandrosterone, which brings great difficulty to the industrial production.

According to documents org, Process Res, Dev, 2016,20,1520, 1528 and CN105483198A, the 5-AD method is reported to have poor yield and quality, and according to CN105339382A and CN106086148A, even if sodium ascorbate is added to inhibit the oxidation of 5-AD, stability experiment researches show that 5-AD is easy to be converted into other impurities under closed conditions, and the influence on the purity of 5-AD is large, so that the quality and the yield of dehydroepiandrosterone obtained by the next reaction are influenced.

[ summary of the invention ]

In order to solve the problems of the prior art, the invention provides the preparation method of dehydroepiandrosterone, which has the advantages of simple operation, short reaction route, stable intermediate, high yield, low cost and little pollution.

The purpose of the invention is realized by the following steps:

a novel method for preparing dehydroepiandrosterone is characterized in that the synthetic route of the method is as follows:

the method specifically comprises the following steps:

1) under the protection of nitrogen, mixing 4-androstenedione (I), dichloromethane, acetic anhydride and an acidic catalyst or mixing 4-androstenedione (I), acetic anhydride and an acidic catalyst, adding into a reactor, controlling the temperature to be 0-40 ℃, stirring and reacting until the thin layer chromatography detects that raw materials are completely reacted, concentrating to remove dichloromethane, carrying out water precipitation, filtering and drying to obtain 3-acetoxyl group-delta 3, 5-androstadiene-17-ketone (II);

2) taking the compound (II) obtained in the step 1) as an intermediate, forming an enzyme reaction system with a cosolvent and an enzyme buffer solution, adding a biological enzyme, coenzyme and coenzyme regeneration system, carrying out biological enzymatic reaction at the temperature of 26-32 ℃, controlling the pH value in the whole reaction process to be 6.8-8.0, detecting the reaction process of the biological enzyme method by HPLC, finishing the reaction when the conversion rate reaches 98%, and carrying out subsequent treatment to obtain the dehydroepiandrosterone (III).

Further, the volume usage amount of the dichloromethane in the step 1) is 0-5 times of that of the compound (I); the volume dosage of the acetic anhydride is 2-5 times of that of the compound (I); the acidic catalyst is one of p-toluenesulfonic acid, methanesulfonic acid or trifluoroacetic acid, and the mass amount of the acidic catalyst is 0.1-0.5 times of that of the compound (I).

Further, in the step 2), the cosolvent is tert-butyl alcohol, the bio-enzyme is a complex enzyme obtained by mixing ketoreductase and ester hydrolase according to a certain proportion, the coenzyme is nicotinamide adenine dinucleotide, the coenzyme regeneration system is a mixture of glucose and glucose dehydrogenase according to a certain proportion, and the compound (II): the feeding weight volume ratio of the tertiary butanol is 1: 3; said compound (II) is ketoreductase: the weight ratio of the ester hydrolase to the raw materials is 1: 0.01-0.02: 0.005-0.01; the weight ratio of the compound (II) to the nicotinamide adenine dinucleotide is 1: 0.002-0.005; the weight ratio of the glucose to the glucose dehydrogenase of the compound (II) is 1:0.4-0.8: 0.002-0.005;

further, the enzyme buffer solution is phosphate buffer solution with pH 6.5;

further, the ketoreductase is a product expressed by the biological enzyme amino acid sequence 1 in a non-pathogenic microorganism;

further, the ester hydrolase is a product expressed by the biological enzyme amino acid sequence 2 in a non-pathogenic microorganism;

further, the non-pathogenic microorganism is Escherichia coli;

further, the glucose dehydrogenase is obtained by commercial purchase;

preferably, the subsequent treatment in the step 2) is specifically to cool to 8-10 ℃ after the reaction is finished, filter and collect the solid, add methanol for extraction for three times after the solid is dried, extract for 50-60 minutes each time, combine the extracts, concentrate under reduced pressure until a large amount of crystals are separated out, precipitate by water, filter and dry at 8-10 ℃ for 2 hours to obtain the compound (III).

Compared with the prior art, the invention has the beneficial effects that:

1. the compound II is used as a key intermediate, has more stable property than 5-AD, and has high purity, thereby effectively ensuring the stability of the next biological enzyme method.

2. Compared with the strict anhydrous reaction condition of potassium tert-butoxide used in the existing 5-AD route, sodium ascorbate is also needed to control the oxygen content in the system and avoid the reaction of the product 5-AD and oxygen, the acetylation is adopted to prepare the compound II in the invention, so that the reaction condition requirement is low, the product quality is stable, the production operation is easy, the method is suitable for the effect of industrial production, and the market prospect is better.

3. The biological enzyme method of the route of the invention adopts the compound biological enzyme with specific proportion, and the compound biological enzyme with specific proportion has better and more stable conversion effect when acting on the compound II.

4. The subsequent treatment process of the route of the invention has simple operation, single organic solvent, less consumption, less energy consumption and low pollution, and better meets the requirements of industrial production and green chemical industry.

5. The process method provided by the invention can ensure that the yield of the product compound III is higher, compared with the 5AD route, the recovery rate for recycling is 85% for many times, the single-batch yield of the route is more than 90%, the purity of the product compound III is high and more than 99%, and the industrial application value is higher.

[ description of the drawings ]

FIG. 1 is a spectrum of the content of compound II in example 1 of the present invention.

FIG. 2 is a chart showing the stability content of compound II in example 1 of the present invention.

FIG. 3 is a spectrum of the content of compound III in example 2 of the present invention.

FIG. 4 is a graph showing a 5-AD content spectrum in comparative example 3 of the present invention.

FIG. 5 is a content spectrum of 5-AD stability test in comparative example 3 of the present invention.

FIG. 6 is a graph showing the dehydroepiandrosterone content in comparative example 4 of the present invention.

[ detailed description ] embodiments

The invention is further illustrated with reference to the following examples, which are not intended to limit the invention.

The specific experimental procedures or conditions are not shown in the examples, and the procedures can be performed according to the conventional experimental methods described in the publications in the field, and the reagents or equipment used are not indicated by manufacturers, and are all conventional products which can be obtained commercially.

Example 1 preparation and stability testing of compound II, the following are specified:

1) under the protection of nitrogen, 50g of compound I, 250 ml of acetic anhydride and 10g of p-toluenesulfonic acid are mixed and added into a reactor, the temperature is controlled to be 20 ℃, the mixture is stirred and reacted until the thin layer chromatography detects that raw materials are completely reacted, and the mixture is separated out by water, filtered and dried to obtain 55.6g of compound II with the HPLC content of 97.3%, as shown in figure 1.

After the compound II was placed at 20-30 ℃ in a sealed and light-shielded state for 6 hours, the HPLC content stabilized at 97.3%, as shown in FIG. 2.

Embodiment 2a method for preparing dehydroepiandrosterone, comprising the steps of:

1) under the protection of nitrogen, 50g of compound I, 250 ml of acetic anhydride and 10g of p-toluenesulfonic acid are mixed and added into a reactor, the temperature is controlled to be 20 ℃, stirring reaction is carried out until the thin layer chromatography detects that raw materials completely react, water separation, filtration and drying are carried out, 55.5g of compound II is obtained, and the HPLC content is 97.5%.

2) Dissolving 55.5g of the compound II in 167ml of tert-butyl alcohol, adding 390ml of 100mM phosphatase buffer solution with pH6.5, starting stirring, sequentially adding 555mg of ketoreductase, 277.5mg of ester hydrolase, 220mg of nicotinamide adenine dinucleotide, 30g of glucose and 220mg of glucose dehydrogenase, carrying out a biological enzyme reaction at the temperature of 26-28 ℃, and controlling the pH value to be 6.8-7.2 by using a 10% sodium carbonate aqueous solution. Sampling and detecting after 5 hours, wherein the conversion rate is 99.3%, cooling to 8-10 ℃, filtering and collecting solids, drying the solids, adding 180ml of methanol for extraction for three times, extracting for 50-60 minutes each time, combining the extract liquor, concentrating under reduced pressure until a large amount of crystals are separated out, performing elutriation, performing 2 hours at 8-10 ℃, filtering and drying to obtain 45.3g of a compound III, wherein the content is 99.8%, and the yield is 90.6%, as shown in figure 3.

Embodiment 3 a method for preparing dehydroepiandrosterone comprising the steps of:

1) under the protection of nitrogen, 50g of compound I, 100 ml of dichloromethane, 150 ml of acetic anhydride and 25g of methanesulfonic acid are mixed and added into a reactor, the temperature is controlled at 0 ℃ and the mixture is stirred to react until the thin layer chromatography detects that the raw materials are completely reacted, the dichloromethane is concentrated and removed, water is separated out, and the mixture is filtered and dried to obtain 55.6g of compound II with the HPLC content of 97.6%.

2) Dissolving 55.6g of the compound II in 167ml of tert-butyl alcohol, adding 390ml of 100mM phosphate buffer solution with pH6.5, starting stirring, adding 834mg of ketoreductase, 417mg of ester hydrolase, 220mg of nicotinamide adenine dinucleotide, 30g of glucose and 220mg of glucose dehydrogenase in sequence, carrying out the biological enzyme reaction at the temperature of 28-30 ℃, and controlling the pH value to be 7.2-7.6 by using 10% sodium carbonate aqueous solution. Sampling and detecting after 5 hours, wherein the conversion rate is 99.2%, cooling to 8-10 ℃, filtering and collecting solids, drying the solids, adding 180ml of methanol for extraction for three times, extracting for 50-60 minutes each time, combining the extract liquor, concentrating under reduced pressure until a large amount of crystals are separated out, performing elutriation, performing 2 hours at 8-10 ℃, filtering and drying to obtain 45.2g of a compound III, wherein the content is 99.7%, and the yield is 90.4%.

Embodiment 4 a method for preparing dehydroepiandrosterone comprising the steps of:

1) under the protection of nitrogen, 50g of compound I, 250 ml of dichloromethane, 100 ml of acetic anhydride and 5g of trifluoroacetic acid are mixed and added into a reactor, the temperature is controlled to be 40 ℃, the mixture is stirred and reacts until the thin layer chromatography detects that raw materials are completely reacted, dichloroethane is concentrated and removed, the mixture is separated out by water, filtered and dried to obtain 55.6g of compound II, and the HPLC content is 97.3%.

2) Dissolving 55.6g of the compound II in 167ml of tert-butanol, adding 390ml of 100mM phosphate buffer solution with pH6.5, starting stirring, adding 1112mg of ketoreductase, 556mg of ester hydrolase, 220mg of nicotinamide adenine dinucleotide, 30g of glucose and 220mg of glucose dehydrogenase in sequence, carrying out biological enzymatic reaction at the temperature of 30-32 ℃, and controlling the pH value to be 7.6-8.0 by using 10% sodium carbonate aqueous solution. Sampling and detecting after 5 hours, wherein the conversion rate is 99.3%, cooling to 8-10 ℃, filtering and collecting solids, drying the solids, adding 180ml of methanol for extraction for three times, extracting for 50-60 minutes each time, combining the extract liquor, concentrating under reduced pressure until a large amount of crystals are separated out, performing elutriation, performing 2 hours at 8-10 ℃, filtering and drying to obtain 45.3g of a compound III, wherein the content is 99.7%, and the yield is 90.6%.

Comparative example 15 AD preparation

Introducing nitrogen into 330 ml of tert-butyl alcohol at the temperature of 30-35 ℃ for 90 minutes, adding 39.2g of potassium tert-butoxide under the condition of aeration, continuing to introduce nitrogen for 90 minutes, adding 50g of 4-AD, and introducing nitrogen at the temperature of 30-35 ℃ for reaction for 1 hour. Adding 25ml of glacial acetic acid into 1000 ml of water treated by introducing nitrogen for 24 hours, cooling to 20 ℃ under the protection of nitrogen, stirring for 15 minutes, slowly adding the reaction solution under the protection of nitrogen, cooling to 12 ℃ after the addition is finished, stirring for 1 hour, filtering, washing a filter cake with water, and drying the filter cake in vacuum at 40 ℃ to obtain a white solid 5-AD 47.8g with the HPLC content of 75.9%.

Comparative example 25 AD preparation

Putting 50g of 4-AD and 195g of potassium tert-butoxide into a dry three-necked bottle, introducing nitrogen for 25 minutes, adding 1500ml of tert-butanol under the protection of nitrogen, stirring at 30-35 ℃ for 3 hours, quickly pouring the reaction solution into 520ml of 10% acetic acid aqueous solution, stirring for 10 minutes, neutralizing with sodium bicarbonate solution, extracting with 900ml of ethyl acetate in three times, drying with anhydrous sodium sulfate, concentrating under reduced pressure to remove the solvent, and drying to obtain 5-AD 45.6g of yellow solid with HPLC content of 64.3%.

Comparative example 35-AD preparation and stability testing, the following are specified:

introducing nitrogen into 800ml of tert-butyl alcohol at the temperature of 20 ℃ for 15 minutes, adding 60g of potassium tert-butoxide under the protection of nitrogen, stirring for half an hour under the protection of nitrogen, adding 50g of 4-AD, and stirring for 5 hours at the temperature of 35 ℃ under the protection of nitrogen. 35g of sodium ascorbate and 57g of 2000ml of acetic acid water are taken and stirred for 15 minutes, the solution is poured into the reactor and stirred for half an hour under the protection of nitrogen, and the white solid 5-AD 47.3g, HPLC content 82.8% and yield 94.6% are obtained after filtration, water washing and drying, as shown in figure 4.

The product 5-AD was stored in a sealed and light-shielded state at 20-30 ℃ and after standing for 6 hours, the HPLC content decreased from 82.3% to 68.4%, as shown in FIG. 5.

Comparative example 45 route to AD

Introducing nitrogen into 800ml of tert-butyl alcohol at the temperature of 20 ℃ for 15 minutes, adding 60g of potassium tert-butoxide under the protection of nitrogen, stirring for half an hour under the protection of nitrogen, adding 50g of 4-AD, and stirring for 5 hours at the temperature of 35 ℃ under the protection of nitrogen. 35g of sodium ascorbate and 57g of 2000ml of acetic acid water are taken and stirred for 15 minutes, the solution is poured into the reactor and stirred for half an hour under the protection of nitrogen, and the mixture is filtered, washed and dried to obtain 5-AD 47.2g of white solid, the HPLC content is 82.5 percent and the yield is 94.4 percent.

Dissolving 47.2g of the product in 246ml of 2-methyltetrahydrofuran, adding 246ml of 100mM phosphate buffer solution with pH6.5, starting stirring, sequentially adding 49.2g of glucose, 787mg of magnesium chloride hexahydrate, 590mg of ketoreductase (using the ketoreductase described in the application of the invention), 295mg of glucose dehydrogenase, 197mg of NAD, starting reaction at 25 ℃, controlling pH6.5 with 40% sodium hydroxide, detecting for 6 hours, adjusting the conversion rate to 89.3%, adjusting pH to less than 2 with hydrochloric acid, adding 295ml of ethyl acetate and 19.7g of active carbon, stirring for 1 hour, filtering, washing a filter cake with 148ml of ethyl acetate for three times, combining a filtrate and a washing solution, removing an organic solvent by rotary evaporation, adding 394ml of ethyl acetate into an aqueous phase for three times, combining the ethyl acetate, concentrating to 197ml of volume, slowly adding 394ml of n-heptane at room temperature, crystallizing, filtering, drying to obtain 32.3g dehydroepiandrosterone product with 93.7% content and 68.4% yield, as shown in FIG. 6.

And (4) comparing and concluding: from the above examples 1-4 and comparative examples 1-4, it can be seen that the compound I of the present invention can be used as a starting material, and only by using the intermediate compound II of the present invention and a bio-enzyme (complex enzyme of ketoreductase and ester hydrolase), the effects of the product compound III yield > 90% and the product compound III purity > 99% can be achieved.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

<110> Zhejiang Shenzhou pharmaceutical Co., Ltd

<120> novel method for preparing dehydroepiandrosterone

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