阅读说明：本技术 一种高质量醋酸***及其中间体的制备方法 (Preparation method of high-quality prednisone acetate and intermediate thereof ) 是由 徐明琴 廖俊 付林 冯蕾 曾建华 田玉林 于 2019-12-06 设计创作，主要内容包括：本发明公开了一种高质量醋酸泼尼松及其中间体的制备方法,包括如下步骤：以表氢化可的松为原料,首先进行微生物脱氢得到11α,17α,21-三羟基-孕甾-1,4-二烯-3,20-二酮中间体,然后经过酯化反应得到11α,17α,21-三羟基-孕甾-1,4二烯-3,20二酮-21-醋酸酯,最后经氧化反应制得醋酸泼尼松,解决了醋酸泼尼松传统发酵生产过程中C1,2位双键引入不理想的工艺难点。该发明所制备的醋酸可的松质量高,杂质含量极低,大大提高了醋酸泼尼松的多方向用途,同时该工艺路线具有成本低,操作简单的特点。(The invention discloses a preparation method of high-quality prednisone acetate and an intermediate thereof, which comprises the following steps of firstly carrying out microbial dehydrogenation on epi-hydrocortisone as a raw material to obtain 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone intermediates, then carrying out esterification reaction to obtain 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone-21-acetate, and finally carrying out oxidation reaction to obtain prednisone acetate.)

1. A preparation method of high-quality prednisone acetate and an intermediate thereof is characterized by comprising the following steps:

s1, preparing an epihydrocortisone compound 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone by using epihydrocortisone as a raw material through microbial dehydrogenation;

s2, carrying out esterification reaction on the fermentation product 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone to prepare 11a, 17a, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone-21-acetate;

s3: esterification reaction products 11a, 17a, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone-21-acetate, and then oxidation reaction is carried out to prepare prednisone acetate;

the specific reaction formula is as follows:

2. the method for preparing high-quality prednisone acetate and the intermediate thereof according to claim 1, wherein in S1, the specific steps of preparing 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-dione from epi-hydrocortisone as a raw material through microbial fermentation and dehydrogenation are as follows:

A. arthrobacter simplex strain culture

Adding a culture medium into a shake flask, inoculating Arthrobacter simplex, culturing at 28-35 ℃, and culturing at 120-200rpm for 20-28 hours for later use;

B. converting the input raw materials to obtain the dehydrogenation product of the hydrogen

Adding hydrocortisone as raw material into the cultured thallus culture medium, adding 7% ethanol, culturing and converting for 1-2 days, detecting by thin layer chromatography until the raw material is completely converted, separating and purifying to obtain the epihydrocodone 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-dione.

3. The method for preparing high-quality prednisone acetate and the intermediate thereof according to claim 2, characterized in that: the shake flask culture medium comprises the following components in parts by weight: 1-3 parts of glucose, 2-4 parts of corn steep liquor, 0.5-2 parts of peptone and 0.5-2 parts of potassium dihydrogen phosphate.

4. The method for preparing high-quality prednisone acetate and the intermediate thereof according to claim 1, characterized in that: the specific steps for carrying out the esterification reaction in S2 are:

putting the epihydric hydride and 3-6 times of acetic acid into a dry and clean three-neck bottle, heating in water bath, keeping the temperature at 40-60 ℃, stirring for 10-30 minutes, then cooling to 20-40 ℃, rapidly adding 0.5-1 times of barium acetate, continuously cooling to 10-20 ℃, and adding 2-3 times of acetic anhydride; heating in water bath, carrying out heat preservation reaction at 20-40 ℃, detecting by TLC, and carrying out heat preservation reaction for 5-6 hours to completely react.

5. The method for preparing high-quality prednisone acetate and the intermediate thereof according to claim 1, characterized in that: the step of performing the oxidation reaction in S3 is:

(1) and preparing an oxidizing solution: adding 1-2 times of manganese chloride into 2 times of water, stirring and dissolving, and then adding 1-2 times of chromic anhydride into the solution, stirring and dissolving for later use;

(2) and oxidation reaction: cooling the esterification reaction liquid to 10-20 ℃, and beginning to dropwise add the standby oxidation liquid; controlling the dropping temperature to be 10-20 ℃, the dropping time to be 30 minutes, keeping the temperature for reaction for 30 minutes at 15-20 ℃ after the dropping is finished, then heating in a water bath to 20-40 ℃ for heat preservation reaction, and carrying out TLC detection to ensure that the reaction is completed after 5-6 hours of heat preservation reaction; pouring the reaction solution into ice water which is pre-cooled to 50-100 times of the temperature below 5 ℃ for precipitation, fully stirring, and standing overnight; and (3) stirring the precipitated liquid, freezing to 0-5 ℃, carrying out suction filtration, draining, washing a filter cake with 100 times of hot water at 50-60 ℃ to neutrality, and drying the filter cake at 50-60 ℃ to obtain a prednisone acetate crude product.

Technical Field

The invention relates to the technical field of preparation of steroid medicines and intermediates thereof, in particular to a preparation method of high-quality prednisone acetate and intermediates thereof.

Background

At present, in various drugs in the world, steroid drugs are second only to antibiotics, and the molecular structures of different steroid drugs are derived from steroid hormone intermediates. The steroid hormone has strong pharmacological actions of resisting infection, virus, allergy and shock, and is an important medicament for treating rheumatism, cardiovascular diseases, lymphatic leukemia, cellular encephalitis, skin diseases, tumors and critical patients, wherein the prednisone acetate is an important steroid medicament, and the structural formula of the prednisone acetate is as follows:

prednisone acetate and corticoids act on glycometabolism to relieve pathological reaction of body tissues to noxious stimulation, and are used for treating adrenal cortex hypofunction, active rheumatism, rheumatoid arthritis, lupus erythematosus and other collagen diseases, severe bronchial asthma, severe dermatitis and other allergic diseases, acute leukemia and the like, and also for the comprehensive treatment of certain infections. It should be used with cautions for hypertension, diabetes, gastroduodenal diseases and heart failure. For long-term administration, the dosage should be gradually decreased when the administration is stopped.

According to the technical assembly of the national raw material medicines, the traditional prednisone acetate production process is to prepare prednisone acetate by fermenting and dehydrogenating cortisone acetate by using Arthrobacter simplex, and the technical route is as follows:

because the introduction of the double bond C1 and 2 in the microbial dehydrogenation process is not thorough enough, the quality of the fermentation product prednisone acetate is difficult to improve, and the prepared prednisone acetate can only reach the quality standard that the content of the prednisone acetate is more than 97 percent, the maximum impurity cortisone acetate is less than 1.5 percent, and the total impurity is less than 2.0 percent according to an external standard method.

Disclosure of Invention

Aiming at the existing problems in the actual production, the invention aims to provide a production method of prednisone acetate, in particular to a production method of high-quality prednisone acetate, which takes epi-hydrocortisone as a raw material and adopts a method of fermentation and synthesis to produce prednisone acetate, thereby solving the technical difficulty that the C1 and 2 double bonds are not ideal in the fermentation production process of prednisone acetate, and simultaneously, the technical process has the characteristics of low cost and simple and convenient operation.

The invention provides a preparation method of high-quality prednisone acetate and an intermediate thereof, which comprises the following steps:

s1, preparing an epihydrocortisone compound 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone by using epihydrocortisone as a raw material through microbial dehydrogenation;

s2, carrying out esterification reaction on the fermentation product 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone to prepare 11a, 17a, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone-21-acetate;

s3: esterification reaction products 11a, 17a, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone-21-acetate, and then oxidation reaction is carried out to prepare prednisone acetate;

the specific reaction formula is as follows:

。

preferably, in S1, the specific steps of preparing 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-dione from epi-hydrocortisone as a raw material through microbial fermentation and dehydrogenation are as follows:

A. arthrobacter simplex strain culture

Adding a culture medium into a shake flask, inoculating Arthrobacter simplex, culturing at 28-35 ℃, and culturing at 120-200rpm for 20-28 hours for later use;

B. converting the input raw materials to obtain the dehydrogenation product of the hydrogen

Adding hydrocortisone as raw material into the cultured thallus culture medium, adding 7% ethanol, culturing and converting for 1-2 days, detecting by thin layer chromatography until the raw material is completely converted, separating and purifying to obtain the epihydrocodone 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-dione.

Preferably, the shake flask culture medium comprises the following components by weight: 1-3 parts of glucose, 2-4 parts of corn steep liquor, 0.5-2 parts of peptone and 0.5-2 parts of potassium dihydrogen phosphate.

Preferably, the specific steps of performing the esterification reaction in S2 are:

putting the epihydric hydride and 3-6 times of acetic acid into a dry and clean three-neck bottle, heating in water bath, keeping the temperature at 40-60 ℃, stirring for 10-30 minutes, then cooling to 20-40 ℃, rapidly adding 0.5-1 times of barium acetate, continuously cooling to 10-20 ℃, and adding 2-3 times of acetic anhydride; heating in water bath, carrying out heat preservation reaction at 20-40 ℃, detecting by TLC, and carrying out heat preservation reaction for 5-6 hours to completely react.

Preferably, the step of performing the oxidation reaction in S3 is:

(1) and preparing an oxidizing solution: adding 1-2 times of manganese chloride into 2 times of water, stirring and dissolving, and then adding 1-2 times of chromic anhydride into the solution, stirring and dissolving for later use;

(2) and oxidation reaction: cooling the esterification reaction liquid to 10-20 ℃, and beginning to dropwise add the standby oxidation liquid; controlling the dropping temperature to be 10-20 ℃, the dropping time to be 30 minutes, keeping the temperature for reaction for 30 minutes at 15-20 ℃ after the dropping is finished, then heating in a water bath to 20-40 ℃ for heat preservation reaction, and carrying out TLC detection to ensure that the reaction is completed after 5-6 hours of heat preservation reaction; pouring the reaction solution into ice water which is pre-cooled to 50-100 times of the temperature below 5 ℃ for precipitation, fully stirring, and standing overnight; and (3) stirring the precipitated liquid, freezing to 0-5 ℃, carrying out suction filtration, draining, washing a filter cake with 100 times of hot water at 50-60 ℃ to neutrality, and drying the filter cake at 50-60 ℃ to obtain a prednisone acetate crude product.

The preparation method of the high-quality prednisone acetate and the intermediate thereof has the beneficial effects that:

1. the preparation process of the intermediate body surface hydrocodone and prednisone acetate is not reported in documents until now, and the fermentation dehydrogenation process in the invention has the characteristics of high feeding concentration and good conversion effect, solves the process difficulty that the C1 and 2 double bond is not ideal in the original fermentation production process of prednisone acetate, and has the characteristics of low cost and simple and convenient operation.

2. The esterification reaction and the oxidation reaction in the steps of the invention are simple, the operation of the used reagent is convenient, the method has low cost and little pollution, and is suitable for industrial production.

3. The product quality is good: the invention improves the selectivity of the final product, reduces the impurity content of the product, and the final product is detected by an HPLC external standard method, and the content of the prednisone acetate is more than 99.5 percent, and the total impurity content is less than 0.5 percent, which is far better than the quality standard of the prednisone acetate in the traditional technique of the prednisone acetate, wherein the content of the prednisone acetate is more than 97 percent, the maximum impurity content is less than 1.5 percent, and the total impurity content is less than 2.0 percent. The prednisone acetate prepared by the method has satisfactory quality and yield.

Detailed Description

The technical solutions and the results of the present invention will be described in detail below with reference to embodiments of the present invention, which, of course, are only a part of the present invention and not all of the present invention. Other embodiments, which can be derived by other persons skilled in the art from the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention.

The invention provides a preparation method of high-quality prednisone acetate, which comprises the following specific steps of firstly carrying out microbial dehydrogenation on epi-hydrocortisone as a raw material to obtain an 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone intermediate, then carrying out esterification reaction to obtain 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone-21-acetate, and finally carrying out oxidation reaction to obtain prednisone acetate, wherein the specific technical route is as follows:

1) the epi-hydrocortisone is used as a raw material, and the epi-hydrocortisone dehydrogenizer is prepared into 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-diketone by dehydrogenation of arthrobacter simplex, and the specific preparation steps are as follows:

A. arthrobacter simplex culture

Adding a culture medium into a shake flask, inoculating Arthrobacter simplex, culturing at 28-35 ℃, and culturing at 120-200rpm for 20-28 hours for later use;

B. the raw materials are added for conversion to obtain the target product

Adding hydrocortisone as raw material into the cultured thallus culture medium, adding 7% ethanol, culturing and converting for 1-2 days, detecting by thin layer chromatography until the raw material is completely converted, separating and purifying to obtain target products of epihydrocortisone 11 α, 17 α, 21-trihydroxy-pregna-1, 4-diene-3, 20-dione.

The shake flask culture medium comprises the following components in parts by weight: 1-3 parts of glucose, 2-4 parts of corn steep liquor, 0.5-2 parts of peptone and 0.5-2 parts of potassium dihydrogen phosphate.

The feeding concentration of the substrate is increased to 40g/L, and the conversion effect is good.

The temperature and the revolution number in the transformation stage are higher than those in the thallus culture stage.

The fermentation conversion period of the invention is shortened to 1-2 days, which is better than the 2-3 days of the prednisone orthoacetate fermentation process.

The purity of the intermediate body surface hydrogen dehydrogenated substance reaches more than 98 percent.

2) The method comprises the following steps of adding the epihydrocodone and 3-6 times of acetic acid into a dry and clean three-necked bottle, heating in a water bath, keeping the temperature at 40-60 ℃, stirring for 10-30min, cooling to 20-40 ℃, rapidly adding 0.5-1 time of barium acetate, continuously cooling to 10-20 ℃, adding 2-3 times of acetic anhydride, heating in the water bath, keeping the temperature at 20-40 ℃ for reaction, detecting by TLC, keeping the temperature for 5-6h, and completely reacting.

3) The oxidation reaction for preparing the prednisone acetate comprises the following specific steps:

① preparing oxidizing solution by adding manganese chloride 1-2 times into water 2 times, stirring, dissolving, adding chromic anhydride 1-2 times into the solution, stirring, and dissolving;

② oxidation reaction, namely cooling the esterification reaction liquid to 10-20 ℃, beginning to dropwise add the standby oxidation liquid, controlling the dropwise adding temperature to 10-20 ℃, dropwise adding for 30min, keeping the temperature for reaction for 30min at 15-20 ℃, then heating in a water bath to 20-40 ℃, keeping the temperature for reaction, detecting by TLC, keeping the temperature for 5-6h to react completely, pouring the reaction liquid into 50-100 times of ice water which is pre-cooled to below 5 ℃ to separate out, fully stirring, standing overnight, stirring the separated liquid, freezing to 0-5 ℃, performing suction filtration, draining, washing a filter cake to be neutral by using 50-60 ℃ hot water which is about 100 times of the temperature, and drying the filter cake at 50-60 ℃ to obtain a prednisone acetate crude product.

In the invention, if not specifically indicated, all the parts by weight and the percentages are weight units, and all the equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following examples are conventional in the art unless otherwise specified.