CN110157764B - Preparation method of dexamethasone intermediate - Google Patents

Preparation method of dexamethasone intermediate Download PDF

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CN110157764B
CN110157764B CN201910454561.1A CN201910454561A CN110157764B CN 110157764 B CN110157764 B CN 110157764B CN 201910454561 A CN201910454561 A CN 201910454561A CN 110157764 B CN110157764 B CN 110157764B
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刘建
蒋随新
王小飞
张小强
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Zhejiang Xianju Pharmaceutical Co Ltd
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Abstract

The preparation method for obtaining the dexamethasone epoxy hydrolysate 8DM by utilizing the Arthrobacter Simple CPCC140451 through one-step fermentation has the advantages of Simple operation mode, stable process and suitability for industrial mass production.

Description

Preparation method of dexamethasone intermediate
Technical Field
The invention belongs to the technical field of steroid biology, and particularly relates to a method for synthesizing dexamethasone intermediate 8DM through biological fermentation.
Background
Dexamethasone is widely used for treating various diseases clinically, such as active rheumatism, rheumatoid arthritis, severe bronchial asthma, severe dermatitis, acute leukemia and the like, and the clinical dosage of dexamethasone is increased year by year. 8DM, also known as dexamethasone epoxy hydrolysate, is one of the important intermediates in steroid pharmacochemistry, whose CAS #: 24916-90-3, the structural formula is as follows:
Figure BDA0002075854700000011
the 8-DM is mainly used for synthesizing glucocorticoid medicaments such as dexamethasone, pitavastatin clocortolone, mometasone furoate and the like.
Initially, dexamethasone was prepared starting from a diene derivative, 8DM being a key intermediate. The diene derivative is derived from dioscin extracted from Curcuma rhizome, and the dioscin is subjected to ring-opening cracking to generate dehydropregnenolone acetate; performing epoxidation, hydrolysis and oxidation by aspergillus ochraceus, and dehydrogenation by arthrobacter to obtain a mold dehydrogenation substance; then, the dexamethasone intermediate is obtained through methylation at the C16 position and iodine replacement on a side chain; then 8DM is obtained through the reactions of hydroxyl bromide and the like. The production line is long, the operation is complex, the pollution is serious, and with the upgrading of the environmental protection requirement and the increasing exhaustion of the diene derivative saponin resource, the price of obtaining 8DM by the diene derivative line is increased, so that the steroid industrial structure is greatly adjusted.
At this time, the dexamethasone synthesis route using phytosterol as a raw material rapidly rises. The phytosterol is a byproduct of soybean oil production, and has low price and rich source. At present, phytosterol is directly fermented to produce 4-AD, 9 alpha at home-OH-AD has already been industrially produced. Russian patent RU2532902C1 discloses a process route for preparing 8DM by adopting 9 alpha-OH-AD, and Chinese patent CN 105440094A discloses a dexamethasone intermediateThe preparation method of (1); also, for example, the chinese patent application with publication No. CN 107417754a discloses a method for preparing a key intermediate of dexamethasone and betamethasone; the three patents specifically research the process route from 9 alpha-OH-AD to dexamethasone, have cost advantage compared with the original route of diene derivatives, and accord with the concept of environmental protection.
Figure BDA0002075854700000021
Thus, 8DM is an important intermediate in the existing synthetic method of dexamethasone. In the prior report, there are two ways to prepare 8DM from the starting material I, 16 alpha-methyl-17 alpha, 21-dihydroxypregn-4-ene-9, 11-epoxy-3, 20-dione-21 acetate: the first is biological method combined with chemical method, i.e. firstly utilizing microbial fermentation to dehydrogenate 1 and 2 positions of the ring A of the starting material I, then utilizing chemical method to hydrolyze 21-position acetate of the ring D, and obtaining 8 DM. For example, chinese patent CN 105440094a discloses a method for preparing dexamethasone intermediate, which combines two steps into one step, wherein chemical hydrolysis generates impurities, resulting in unstable quality of refined product, low yield, and primary yield of weight of about 60%; the second is a biological fermentation method, namely, only the 8DM is prepared by utilizing microbial fermentation and transformation; for example, in the chinese patent CN 106520896a, the reaction of two steps of dehydrogenation and hydrolysis is performed simultaneously by the co-fermentation of Arthrobacter simplex (ATCC 6946) and Bacillus megaterium, and the synergistic effect, so as to directly obtain the product dexamethasone epoxy hydrolysate 8DM, the method has the advantages of mild reaction, environmental protection, high yield, about 70% of primary yield by weight, and about 76% of total yield. Although the result is better than the 8DM obtained by the first mode, the mixed strains have the problems of mutual inhibition of strains, difficult regulation and control of the fermentation process, material degradation, reduced yield and the like; in addition, because the two strains need to be cultured independently and then mixed, the equipment investment is large, the operation is complicated, the risk of system contamination is increased, and the method is not suitable for industrial production.
In order to improve the product quality of 8DM, the industry is always searching and establishing a conversion mode with green and environment-friendly production process, high yield, simple operation and low cost and commercial competitiveness.
Disclosure of Invention
Aiming at the defect of synthesizing the important intermediate 8DM of dexamethasone in the prior art, the invention aims to provide a method for synthesizing 8DM by microbial fermentation, which comprises the following steps: the starter I is subjected to one-step fermentation by utilizing microbial strains to obtain 8DM, wherein the microbes refer to Arthrobacter simplex CPCC 140451; the starting material I is 16 alpha-methyl-17 a, 21-dihydroxypregn-4 ene-9, 11-epoxy-3, 20-diketone-21 acetate, and the conversion formula is shown as follows. After the fermentation process is optimized, the yield of the synthesized 8DM is higher, and the quality is more stable.
Figure BDA0002075854700000031
Although the microorganisms are widely distributed with strains with dehydrogenation activity and hydrolysis activity, but some Arthrobacter and nocardia with dehydrogenation activity do not have strong hydrolysis effect on the initiator I, the strains listed in the following table can not well realize the simultaneous dehydrogenation and hydrolysis of the initiator I to obtain 8DM, while the simple Arthrobacter simplex CPCC140451 is a single bacterial colony with strong hydrolysis and dehydrogenation activity on the initiator I, and the screening results are as follows:
Figure BDA0002075854700000032
as can be seen from the above table, the Arthrobacter simplex CPCC140451 provided by the present invention has unique advantages in the fermentation process of preparing 8DM from the starting material I.
The microorganisms used were: the Arthrobacter simplex CPCC140451 used in the invention is purchased from the China pharmaceutical microorganism strain preservation management center in 2014 3 months, the original preservation time of the strain is 19 days in 1989 in 2 months, the original source of the strain is Anyang second pharmaceutical factory in Henan province in China, and the original number of the strain is A-1-3.
Specifically, the inventors provide the following technical solutions:
(1) strain culture
Eluting a simple arthrobacter slope of a 100mL eggplant-shaped bottle by using 50mL sterile water, inoculating the eluted simple arthrobacter slope into 100mL culture medium (the volume of a culture shake flask is 500mL), and performing primary culture in a shaking table at the culture temperature of 29-35 ℃ and the shaking table rotation speed of 180rpm for about 18-24 h; arthrobacter simplex herein refers to Arthrobacter simplex CPCC 140451.
The first-stage culture medium adopts: 2.0% of glucose, 3.2% of corn steep liquor, 1.5% of yeast extract, 0.3% of monopotassium phosphate, 0.04% of natural killer and 30% of sodium hydroxide for adjusting the pH value to 7.0-7.5.
(2) Fermentation transformation
Transferring the primary seeds into a secondary culture medium under the aseptic condition, culturing at the culture temperature of 29-34 ℃ for about 10-13 h, micronizing an initiator I or dissolving a solvent, feeding, adding coenzyme A, adjusting the fermentation conversion temperature to 30-36 ℃ for conversion, performing 85 ℃ sterilization for 30min when the HPLC content of 8DM is more than or equal to 95.0% (HPLC area normalization), and cooling to 25 ℃ for extraction.
The secondary fermentation culture medium comprises: 2.0% of glucose, 3.2% of corn steep liquor, 1.5% of yeast extract, 0.3% of monopotassium phosphate, 0.2-0.5% of initiator I, 0.04% of natural killer and 30% of sodium hydroxide for adjusting the pH value to 7.5-8.5;
0.2-0.5% of an initiator I is added into the secondary fermentation medium to be used as a fermentation inducer.
Adjusting the pH value of the secondary fermentation medium to 7.5-8.5 by using 30% sodium hydroxide so as to be beneficial to hydrolysis of the initiator I;
the starting material I adopts a solvent dissolving and feeding mode, and the solvent is selected from the following components: one of methanol, ethanol, dioxane, DMF and propylene glycol; the solvent is further preferably: methanol.
The feeding concentration of the starting material I is 30-60 g/L.
The starting material I can also be micronized and fed, and the crushing particle size D90 is less than or equal to 100 mu m;
after the feeding is finished, adding coenzyme A to increase the dehydrogenation capacity of the thalli; when 60g/L of micronized feed is fed, the coenzyme A is added, so that the conversion rate of 8DM is improved by nearly 10%, a good effect of improving the dehydrogenation capacity of thalli is shown, and further, the addition amount of the coenzyme A is 0-0.2%;
(3) extracting and refining
Filtering the fermentation liquor to obtain a filter cake, adding 20-30V of a dichloromethane/methanol mixed solvent and activated carbon accounting for 5% of the weight of the materials into the filter cake, performing reflux extraction, filtering, performing reduced pressure concentration on the filtrate, carrying out water-drying extraction on the extraction solvent by using a certain amount, cooling to 25 ℃, performing suction filtration to obtain an 8DM wet crude product, drying to obtain an 8DM crude product, and refining;
adding the 8DM crude product into a refining bottle, heating by adopting dichloromethane/acetone of 20-30V, refluxing for 30min, concentrating under reduced pressure to acetone of 1-3V, cooling to below 20 ℃, performing suction filtration to obtain a wet refined product, and drying the wet refined product to obtain the 8DM refined product.
The extraction solvent is a mixed solvent of dichloromethane/methanol, wherein the volume mixing ratio of dichloromethane to methanol is 6: 1-2: 1;
the refining solvent is a mixed solvent of dichloromethane and acetone, wherein the mixing ratio of dichloromethane to acetone is 4: 1-4: 3;
the inventors have conducted intensive studies on the kind of microorganism used for transformation, the concentration and mode of feed, the culture of the strain, the transformation conditions, and the like, and have obtained a fermentation process for efficiently producing 8 DM.
To further illustrate the inventive scheme, in example 1, the micronization feeding mode, the starting material I feeding concentration is 30g/L, and the conversion rate is 96.32%;
in comparative example 1, a micronization feeding mode is adopted, coenzyme A is not added, the feeding concentration is 60g/L, and the conversion rate is 78.21%;
in example 2, a micronized feeding mode is adopted, coenzyme A is added, the feeding concentration is 60g/L, and the conversion rate is 87.56%;
in examples 3-7, a solvent dissolving and feeding manner is adopted, coenzyme a is added, the feeding concentration is 60g/L, the dissolving solvents are respectively methanol, ethanol, dioxane, DMF and propylene glycol, the amounts of the coenzyme A are respectively 0.2%, 0.1%, 0.15%, 0.10% and 0.20%, and the conversion rates are respectively 97.32%, 95.21%, 93.21%, 96.99% and 96.70%;
compared with the prior art, the invention has the following beneficial effects:
(1) the 8DM is obtained by simultaneously carrying out dehydrogenation on 1, 2-position of I A ring of an initiator, hydrolysis of 21-position acetate of D ring and one-step fermentation by using Arthrobacter simplex CPCC 140451. Through optimization, the feeding concentration can reach 60g/L, the conversion rate reaches 93-97%, and the one-time yield of 8DM can reach 70-75% while the production process is simplified.
(2) Compared with the fermentation process for producing 8DM by using the mixed strain of the simple arthrobacter for dehydrogenation of the 1-position and 2-position of the A ring and the 21-position hydrolysis of the D ring of the bacillus megaterium, which is reported by the Chinese patent CN 106520896A in the prior art, the method has the advantages of simple operation mode, stable process and reduced risk of bacterial contamination of the system, and is suitable for industrial mass production.
Drawings
FIG. 1 HPLC detection profile of starting material I.
FIG. 2 HPLC transformation profile of example 3.
FIG. 3 HPLC transformation profile of example 6.
FIG. 4 HPLC transformation profile of example 7.
Detailed Description
The present invention will be described in more detail with reference to examples.
In the invention, all the equipment, raw materials and the like can be purchased from the market or commonly used in the industry; the methods in the following examples are conventional in the art unless otherwise specified.
Description of main raw materials and detection method:
the invention relates to a microbial biotransformation starting material I: 8DM is prepared by 16 alpha-methyl-17 alpha, 21-dihydroxypregn-4-ene-9, 11-epoxy-3, 20-dione-21 acetate. The strain used in the examples was Arthrobacter simplex CPCC140451, purchased from the China pharmaceutical culture Collection.
The conversion rate of the product is determined by HPLC, and the separation column is C18The detection wavelength of the column is 254nm, the temperature of the column is 40 ℃, the mobile phase is acetonitrile and pure water which are 26: 74, and the flow rate is 2.3 mL/min. The product conversion was calculated using area normalization.
Note: herein, W represents weight and V represents volume. When W is in units of g, units of V are mL; when W is in units of kg, V is in units of L.
Example 1
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.5 by using 30 percent of sodium hydroxide, sterilizing for 30min at 121 ℃, cooling to room temperature, eluting the inclined plane of Arthrobacter simpliciens CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 30 ℃, at the rotating speed of 180rpm of the shaking table for 19h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.2 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 8.0 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 32 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 32 ℃, and culturing for 12 h. ② microbial transformation stage: the feed concentration was 30g/L, i.e. micronised starter I (D)90Less than or equal to 100 mu m) is added into the reactor, the conversion temperature is adjusted to 34 ℃, and the conversion time is adjusted to 48 h. HPLC analysis (area normalization) was carried out, and the contents of 8DM, 8DM acetate, starting material I, and intermediate II were 96.32%, 2.52%, 0.05%, and 0.65%, respectively.
(4) Extraction and refining: sterilizing the fermentation liquor at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, adding the fermentation filter cake into 90mL of dichloromethane/methanol (3: 1) mixed solvent slowly, adding 1.5g of activated carbon, heating to 40 ℃, keeping the temperature of clear solution for 30min, filtering while hot, performing reduced pressure concentration on the filtrate to be dry, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, and drying. After the 8DM crude product is dissolved by 70mL of dichloromethane/acetone (4: 1) mixed solvent, the mixture is concentrated under normal pressure until no dichloromethane exists, the mixture is continuously concentrated until the volume of the material is 1V acetone, the mixture is cooled to below 20 ℃, the mixture is filtered and dried to obtain 2.18g of fine product (the HPLC purity is more than or equal to 99.0 percent, and single impurity is less than 0.5 percent), the filtrate is concentrated to obtain 0.23g of mother liquor, the primary yield is 72.66 percent, and the total yield is 78.70 percent.
Comparative example 1
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.2 by using 30 percent of sodium hydroxide, sterilizing for 30min, cooling to room temperature, eluting the inclined plane of Arthrobacter simpliciens CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 30 ℃, at the rotating speed of 180rpm of the shaking table for 18h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.2 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 7.5 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 33 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 32 ℃, and culturing for 10 h. ② microbial transformation stage: the feed concentration was 60g/L, i.e. micronised starting material I (D)90Less than or equal to 100 mu m) is added into the reactor, the conversion temperature is 34 ℃, and the conversion time is 76 h. Sampling and HPLC analysis (area normalization method), the 8DM content is 78.21%, the 8DM acetate content is 1.92%, the content of the starting material I is 19.11%,the content of the intermediate II is 0.65%; the conversion time was continued for 12h and samples were taken for HPLC analysis (area normalization) and the 8DM content was 79.89%.
(4) Extraction and refining: sterilizing the fermentation liquor at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, slowly adding 180mL of dichloromethane/methanol (6: 1) mixed solvent into the fermentation filter cake, simultaneously adding 3g of activated carbon, heating to 40 ℃, keeping the temperature of the solution clear for 30min, filtering while hot, performing reduced pressure concentration on the filtrate to obtain a filtrate, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, and drying the wet crude product; after the 8DM crude product is dissolved by 150mL of dichloromethane/acetone (4: 3) mixed solvent, the mixture is concentrated under normal pressure until no dichloromethane exists, the mixture is continuously concentrated until the volume of the material is 3V acetone, the mixture is cooled to be below 20 ℃, the filtration is carried out, the refining step is repeated once, 2.98g of refined product is obtained by drying (the HPLC purity is more than or equal to 99.0 percent, and single impurity is less than 0.5 percent), 1.92g of mother liquor is obtained by concentrating the filtrate, the primary yield is 49.67 percent, and the total yield is 73.03 percent.
Example 2
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.2 by using 30 percent of sodium hydroxide, sterilizing for 30min, cooling to room temperature, eluting the inclined plane of Arthrobacter simplex CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 30 ℃, rotating the shaking table at 180rpm, culturing for 18h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.2 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 7.5 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 33 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain under aseptic conditionCulturing in secondary culture medium at 32 deg.C for 10 hr. ② microbial transformation stage: the feed concentration was 60g/L, i.e. micronised starting material I (D)90Less than or equal to 100 mu m) is added into the reactor, simultaneously 0.2 percent of coenzyme A is added, the conversion temperature is 34 ℃, and the conversion time is 76 h. HPLC analysis (area normalization) was carried out for samples with 87.56% 8DM content, 2.12% 8DM acetate content, 10.61% starting material I content and 0.55% intermediate II content.
(4) Extraction and refining: sterilizing the fermentation liquor at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, slowly adding 180mL of dichloromethane/methanol (3: 1) mixed solvent into the fermentation filter cake, simultaneously adding 3g of activated carbon, heating to 40 ℃, keeping the temperature of the solution clear for 30min, filtering while hot, performing reduced pressure concentration on the filtrate to obtain a filtrate, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, and drying the wet crude product; after the 8DM crude product is dissolved by 150mL of dichloromethane/acetone (4: 3) mixed solvent, the mixture is concentrated under normal pressure until no dichloromethane exists, the mixture is continuously concentrated until the volume of the material is 3V acetone, the mixture is cooled to be below 20 ℃, the filtration is carried out, the refining step is repeated once, the refined product is dried to obtain 4.03g (the HPLC purity is more than or equal to 99.0 percent, and single impurity is less than 0.5 percent), the filtrate is concentrated to obtain 0.92g of mother liquor, the primary yield is 67.16 percent, and the total yield is 79.8 percent.
Example 3
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.5 by using 30 percent of sodium hydroxide, sterilizing for 30min at 121 ℃, cooling to room temperature, eluting the inclined plane of Arthrobacter simpliciens CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 30 ℃, at the rotating speed of 180rpm of the shaking table for 19h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.2 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 8.0 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 32 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 32 ℃, and culturing for 12 h. ② microbial transformation stage: the feeding concentration is 60g/L, namely 6g of dry powder is fed, 4mL of methanol solvent is added at the same time, the coenzyme A content is 0.2 percent, the conversion temperature is 34 ℃, and the conversion time is 72 hours. HPLC analysis (area normalization) was carried out, with 97.32% 8DM content, 1.54% 8DM acetate content, 0.08% starting material I content and 0.76% intermediate II content.
(4) Extraction and refining: sterilizing a fermentation liquid at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, slowly adding 120mL of dichloromethane/methanol (5: 1) mixed solvent into the fermentation filter cake, simultaneously adding 3g of activated carbon, jointly heating to 40 ℃, keeping the temperature of clear solution for 30min, filtering while hot, performing reduced pressure concentration on a filtrate to obtain the dry filtrate, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, drying the crude product, dissolving 8DM crude product with 100mL of dichloromethane/acetone (4: 1) mixed solvent to obtain the clear solution, performing normal pressure concentration to obtain no dichloromethane, continuously concentrating to a material volume of 1V acetone, cooling to below 20 ℃, filtering, drying to obtain a fine product of 4.52g (the HPLC purity is more than or equal to 99.0%, and single impurity is less than 0.5%), concentrating the filtrate to obtain 0.38g of a mother liquid, wherein the primary yield is 75.33%, and the total yield is 80.42.
Example 4
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.0 by using 30 percent of sodium hydroxide, sterilizing for 30min, cooling to room temperature, eluting the inclined plane of Arthrobacter simpliciens CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 31 ℃, rotating the shaking table at 180rpm, culturing for 21h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.5 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 8.5 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 32 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 33 ℃, and culturing for 13 h. ② microbial transformation stage: the feeding concentration is 60g/L, namely 6g of dry powder is fed, 4mL of ethanol solvent is added at the same time, the coenzyme A content is 0.10 percent, the conversion temperature is 33 ℃, and the conversion time is 76 h. HPLC analysis (area normalization) was carried out, with 95.21% 8DM content, 1.82% 8DM acetate content, 0.05% starting material I content and 1.75% intermediate II content.
(4) Extraction and refining: sterilizing the fermentation liquor at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, adding the fermentation filter cake into 160mL of dichloromethane/methanol (2: 1) mixed solvent slowly, adding 3g of activated carbon, heating to 40 ℃, keeping the temperature of the solution clear for 30min, filtering while hot, performing reduced pressure concentration on the filtrate to be dry, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, and drying the crude product. After the 8DM crude product is dissolved by 130mL of dichloromethane/acetone (4: 2) mixed solvent, the mixture is concentrated under normal pressure until no dichloromethane exists, the mixture is continuously concentrated until the volume of the material is 2V acetone, the mixture is cooled to below 20 ℃, the mixture is filtered and dried to obtain 4.30g of fine product (the HPLC purity is more than or equal to 99.0 percent, and single impurity is less than 0.5 percent), the filtrate is concentrated to obtain 0.58g of mother liquor, the primary yield is 71.67 percent, and the total yield is 79.33 percent.
Example 5
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.2 by using 30 percent of sodium hydroxide, sterilizing for 30min, cooling to room temperature, eluting the inclined plane of Arthrobacter simplex CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating the inclined plane into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 35 ℃, at the rotating speed of 180rpm of the shaking table for 24h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.5 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 8.5 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 32 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 33 ℃, and culturing for 12 h. ② microbial transformation stage: the feeding concentration is 60g/L, namely 6g of dry powder is added, 3mL of dioxane solvent is added at the same time, the amount of coenzyme A is 0.15 percent, the conversion temperature is 35 ℃, and the conversion time is 76 h. HPLC analysis (area normalization) was carried out for samples with 93.21% 8DM content, 2.82% 8DM acetate content, 0.08% starting material I content and 2.51% intermediate II content.
(4) Extraction and refining: sterilizing a fermentation liquid at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, slowly adding 180mL of dichloromethane/methanol (4: 1) mixed solvent into the fermentation filter cake, simultaneously adding 3g of activated carbon, jointly heating to 40 ℃, keeping the temperature of clear solution for 30min, filtering while hot, performing reduced pressure concentration on filtrate until the filtrate is completely dried, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, drying the crude product, dissolving 8DM crude product with 140mL of dichloromethane/acetone (4: 3) mixed solvent, concentrating under normal pressure until no dichloromethane exists, continuously concentrating until the volume of the material is 3V acetone, cooling to below 20 ℃, filtering, drying to obtain a fine product of 4.25g (the HPLC purity is more than or equal to 99.0%, and the single impurity is less than 0.5%), concentrating the filtrate to obtain 0.63g of a mother liquid, wherein the primary yield is 70.83%, and the total yield is 79.29%.
Example 6
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.5 by using 30 percent of sodium hydroxide, sterilizing for 30min, cooling to room temperature, eluting the inclined plane of Arthrobacter simpliciens CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 34 ℃, at the rotating speed of 180rpm of the shaking table for 22h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.4 percent of initiator I and 0.04 percent of natural killer, adjusting the pH value to 8.0 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 32 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 33 ℃, and culturing for 12 h. ② microbial transformation stage: the feeding concentration is 60g/L, namely 6g of dry powder is fed, 3mL of DMF solvent is added at the same time, the coenzyme A content is 0.10 percent, the conversion temperature is 33 ℃, and the conversion time is 76 h. Sampling and HPLC analysis (area normalization method), the content of 8DM is 96.99%, the content of 8DM acetate is 1.57%, the content of the starting material I is 0.05%, and the content of the intermediate II is 0.94%.
(4) Extraction and refining: sterilizing the fermentation liquor at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, slowly adding 180mL of dichloromethane/methanol (4: 1) mixed solvent into the fermentation filter cake, simultaneously adding 3g of activated carbon, heating to 40 ℃, keeping the temperature of the solution clear for 30min, filtering while the solution is hot, performing reduced pressure concentration on the filtrate to be dry, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, and drying; after the 8DM crude product is dissolved by 140mL of dichloromethane/acetone (4: 3) mixed solvent, the mixture is concentrated under normal pressure until no dichloromethane exists, the mixture is continuously concentrated until the volume of the material is 3V acetone, the mixture is cooled to below 20 ℃, the mixture is filtered and dried to obtain 4.38g of fine product (the HPLC purity is more than or equal to 99.0 percent, and single impurity is less than 0.5 percent), the filtrate is concentrated to obtain 0.50g of mother liquor, the primary yield is 73.00 percent, and the total yield is 79.78 percent.
Example 7
A preparation method of a dexamethasone intermediate is disclosed, wherein the dexamethasone intermediate is 8-DM, and the preparation method comprises the following steps:
(1) and (3) strain culture: preparing a first-stage culture medium according to the proportion, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.04 percent of foam killer, adjusting the pH value to 7.5 by using 30 percent of sodium hydroxide, sterilizing for 30min, cooling to room temperature, eluting the inclined plane of Arthrobacter simpliciens CPCC140451 cultured in a 100mL eggplant-shaped bottle by using 50mL of sterile water, inoculating into 20 percent to 100mL of culture medium (the volume of a culture shake flask is 500mL), performing first-stage culture by using a shaking table, culturing at the temperature of 33 ℃, rotating the shaking table at 180rpm, culturing for 21h, and performing second-stage seed transfer;
(2) fermentation and transformation: preparing a fermentation culture medium according to a mixture ratio, wherein 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate, 0.3 percent of initiator I and 0.04 percent of foam killer, adjusting the pH value to 8.0 by using 30 percent of sodium hydroxide, sterilizing at 121 ℃ for 30min, cooling to 32 ℃, and waiting for inoculation; the fermentation is divided into two stages: firstly, a thallus growth stage: inoculating 30% of the first-stage culture strain into a second-stage culture medium under aseptic conditions, controlling the temperature at 33 ℃, and culturing for 12 h. ② microbial transformation stage: the feeding concentration is 60g/L, namely 6g of dry powder is fed, 4mL of propylene glycol solvent is added, the amount of coenzyme A is 0.20 percent, the conversion temperature is 33 ℃, and the conversion time is 76 h. Sampling and HPLC analysis (area normalization method), the content of 8DM is 96.70%, the content of 8DM acetate is 1.09%, the content of the starting material I is 0.05%, and the content of the intermediate II is 1.72%.
(4) Extraction and refining: sterilizing a fermentation liquid at 85 ℃ for 30min, cooling to 25 ℃, performing suction filtration to obtain a fermentation filter cake, slowly adding 170mL of dichloromethane/methanol (4: 1) mixed solvent into the fermentation filter cake, simultaneously adding 3g of activated carbon, jointly heating to 40 ℃, keeping the temperature of clear solution for 30min, filtering while hot, performing reduced pressure concentration on filtrate until the filtrate is completely dried, adding a proper amount of water, cooling to 25 ℃, performing suction filtration to obtain a wet crude product, drying the crude product, dissolving 8DM crude product in 130mL of dichloromethane/acetone (4: 2) mixed solvent to obtain the solution, performing normal pressure concentration until no dichloromethane exists, continuously concentrating until the volume of the material is 1.5v acetone, cooling to below 20 ℃, filtering, drying to obtain a fine product of 4.47g (the HPLC purity is more than or equal to 99.0%, and single impurity is less than 0.5%), concentrating the filtrate to obtain 0.46g of mother liquor, achieving the primary yield of 74.50%, and the total yield of 80.68.

Claims (10)

1. A preparation method of dexamethasone intermediate 8DM is characterized in that the preparation method comprises the step of converting a starting material I into 8DM in one step by utilizing Arthrobacter simplex, wherein the Arthrobacter simplex refers toArthrobacter SimplexAccession number CPCC140451, the transformation process being of the formula:
Figure DEST_PATH_IMAGE002
。
2. the method for preparing the dexamethasone intermediate 8DM according to claim 1, wherein the starting material I is fed at a concentration of 30-60 g/L.
3. The method for preparing the dexamethasone intermediate 8DM as claimed in claim 2, wherein the starting material I is micronized and the particle size D90 is less than or equal to 100 μm.
4. The preparation method of the dexamethasone intermediate 8DM as claimed in claim 3, wherein after the feeding is completed, coenzyme A with a concentration of 0-0.2% is added, the fermentation conversion temperature is adjusted to 30-36 ℃, and the fermentation conversion time is adjusted to 48-76 h.
5. The process for preparing dexamethasone intermediate 8DM as claimed in claim 2 wherein starting material I is a solvated charge, the solvent being selected from the group consisting of: one of methanol, ethanol, dioxane, DMF and propylene glycol.
6. The preparation method of the dexamethasone intermediate 8DM as claimed in claim 5, wherein after the feeding is completed, coenzyme A with a concentration of 0-0.2% is added, the fermentation conversion temperature is adjusted to 30-36 ℃, and the fermentation conversion time is adjusted to 48-76 h.
7. The method for preparing the dexamethasone intermediate 8DM according to claim 1, wherein the first-order seed culture medium adopted by the method is prepared according to the following ratio: 2.0 percent of glucose, 3.2 percent of corn steep liquor, 1.5 percent of yeast extract, 0.3 percent of monopotassium phosphate and 0.04 percent of natural killer; and (3) adjusting the pH value to 7.0-7.5 by using 30% sodium hydroxide, and using the mixture for seed culture, wherein the temperature of a shaking table is 29-35 ℃, the rotating speed of the shaking table is 180rpm, and the culture time is 18-24 hours.
8. The method for preparing dexamethasone intermediate 8DM according to claim 7, wherein the transformation medium used in the method is formulated as follows: 2.0% of glucose, 3.2% of corn steep liquor, 1.5% of yeast extract, 0.3% of monopotassium phosphate, 0.2-0.5% of initiator I and 0.04% of natural killer, and adjusting the pH value to 7.5-8.5 by using 30% of sodium hydroxide.
9. The preparation method of the dexamethasone intermediate 8DM as claimed in claim 8, wherein a filter cake generated by fermentation post-treatment is dissolved by a mixed solvent of dichloromethane and methanol, 5% of activated carbon is added for extraction, and an extract is subjected to reduced pressure concentration to obtain a crude fermentation product, wherein the volume mixing ratio of dichloromethane and methanol is 6: 1-2: 1.
10. The preparation method of the dexamethasone intermediate 8DM as claimed in claim 9, wherein the method comprises a refining method of 8DM, and the refining method comprises the steps of dissolving a crude 8DM in a mixed solvent of dichloromethane and acetone, concentrating under reduced pressure to a volume of 1-3V, cooling and filtering to obtain a refined product, wherein the volume mixing ratio of dichloromethane to acetone is 4: 1-4: 3.
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