A preparation method of prednisone derivatives

Abstract

The present application discloses a method for preparing a prednisone derivative, comprising: reacting an acetate compound shown in Formula 1 under the catalysis of Nocardia to obtain a prednisone derivative shown in Formula 2; wherein R1 is a hydrogen atom, R2 is a hydrogen atom or a carbonyl group; or R1 and R2 form a double bond; R4 is a hydrogen atom, R3 is a hydroxyl group; or R3 and R4 form a double bond. The present application uses an optimized seed culture medium and a fermentation culture medium to increase the fermentation feed concentration to 10 g / L to 80 g / L, and the dehydrogenation and hydrolysis conversion rate reaches more than 90%; using Nocardia, catalysis is performed after fermentation culture, so that the hydrolysis and dehydrogenation processes of the compound of Formula I are carried out in the same system, and a one-step fermentation conversion is achieved to obtain a prednisone derivative, which is simple in process and suitable for industrial production.

Description

Technical Field

[0001] The present application relates to the field of steroid biopharmaceuticals, and in particular to a method for preparing a prednisone derivative. Background Art

[0002] Prednisone is a widely used glucocorticoid in clinical practice. It has multiple pharmacological effects such as anti-inflammatory, anti-allergic and antiviral, and can be used to treat rheumatism, systemic lupus erythematosus, etc. CAS: 53-03-2, chemical name: 17α,21-dihydroxy-1,4-pregnadiene-3,11,20-trione, white or almost white crystalline powder, odorless, bitter taste. It is easily soluble in chloroform, slightly soluble in acetone, slightly soluble in ethanol or ethyl acetate, and insoluble in water.

[0003] The existing reported synthetic prednisone technology process routes are mainly divided into two categories: one is the chemical synthesis method. Patent CN103087135A uses prednisone acetate as the starting material, and the reaction formula is as follows: the process (1) uses methanol-chloroform mixed solvent as solvent, potassium hydroxide-methanol solution as catalyst, and reacts under low temperature conditions. After the reaction is completed, glacial acetic acid is added to neutralize to a pH value of 6-6.5; (2) drinking water is added, concentrated, filtered, and dried to obtain the product prednisone, with a weight yield of 86% to 86.5% and a liquid content of more than 97%. Although the method reports a high yield, the hydrolysis reaction is relatively harsh and needs to be reacted under low temperature conditions. In addition, strong base potassium hydroxide is used for hydrolysis during the reaction process. Impurities are easily generated if not properly controlled, and the discharge of wastewater is likely to cause environmental pollution.

[0004] The other is a bio-fermentation synthesis method. Patent CN106893754A uses cortisone acetate as a raw material and directly produces prednisone through a one-step bio-fermentation process using Arthrobacter Simple ATCC 21032. However, the conversion rate of this method is relatively low, less than 93%, and the concentration of cortisone acetate fed does not exceed 40 g / L.

[0005] Although certain research results have been achieved in the preparation of prednisone and its analogs, the chemical synthesis method in the prior art has the problem of serious environmental pollution; the transformation using Arthrobacter is prone to bacterial contamination, the conversion rate and feed amount are not high, and the material is easily degraded.

[0006] Therefore, it is an urgent problem to be solved by those skilled in the art to develop a preparation method with simple reaction conditions and capable of improving the conversion rate and feed amount of prednisone derivatives. Summary of the invention

[0007] In view of this, the present application provides a method for preparing prednisone derivatives, which can significantly improve the conversion rate, increase the input amount of raw materials and ensure stable conversion, and has good comprehensive performance.

[0008] The present application provides a method for preparing a prednisone derivative, comprising:

[0009] The acetate compound shown in Formula 1 is reacted under the catalysis of Nocardia to obtain the prednisone derivative shown in Formula 2;

[0010] The deposit number of Nocardia is ACCC 10205;

[0011]

[0012] Wherein R1 is a hydrogen atom, R2 is a hydrogen atom or a carbonyl group; or R1 and R2 form a double bond;

[0013] R4 is a hydrogen atom, R3 is a hydroxyl group; or R3 and R4 form a double bond.

[0014] In some specific implementations, the preparation method specifically includes: culturing Nocardia, adding an acetate compound shown in Formula 1, and reacting to obtain a prednisone derivative shown in Formula 2.

[0015] This application first needs to culture Nocardia in a slant culture medium. In some specific implementations, the Nocardia is purchased from the China Agricultural Microbiological Culture Collection Management Center. In some specific implementations, in the slant culture medium, each liter of slant culture medium includes: 7g to 10g glucose, 14g to 16g yeast extract and 19g to 21g agar, and the remaining tap water preferably includes 8g to 9g glucose, 14.5g to 15.5g yeast extract and 19.5g to 20.5g agar. In some specific implementations, the temperature of the slant culture is 28°C to 35°C, preferably 30°C to 33°C, and the time of the slant culture is 24h to 72h, preferably 24h to 48h. In some specific implementations, the pH value of the slant culture medium is 7 to 8.

[0016] Then the present application scrapes out a ring of bacteria in the slant culture medium and accesses the seed culture medium for seed culture. In some specific implementations, in the seed culture medium, each liter of seed culture medium includes: 9g to 12g glucose, 6g to 8g corn steep liquor, 5g to 10g yeast extract, 9g to 12g peptone, 4g to 6g KH2PO4 and 5g to 12g soybean powder, and the remaining tap water preferably includes 10g to 11g glucose, 5.5g to 8g corn steep liquor, 6g to 10g yeast extract, 10g to 11g peptone, 4.5g to 5.5gKH2PO4 and 6g to 10g soybean powder. In some specific implementations, the temperature of the seed culture is 28°C to 34°C, preferably 30°C to 32°C, and the time of the seed culture is 15h to 24h, preferably 18h to 22h. In some specific implementations, the pH value of the seed culture medium is 7 to 8.

[0017] The present application then transfers the Nocardia seeds into a sterilized fermentation medium for fermentation culture. In some specific implementations, the fermentation medium comprises, per liter, 9 to 12 g glucose, 6 to 8 g corn steep liquor, 5 to 10 g yeast extract, 9 to 12 g peptone, 4 to 6 g KH2PO4 and 5 to 12 g soybean powder, and the remainder is water, preferably 10 to 11 g glucose, 5.5 to 8 g corn steep liquor, 6 to 10 g yeast extract, 10 to 11 g peptone, 4.5 to 5.5 g KH2PO4 and 6 to 10 g soybean powder. In some specific implementations, the fermentation culture temperature is 28°C to 34°C, preferably 30°C to 32°C, the fermentation culture time is 10h to 20h, preferably 12h to 18h; the fermentation culture is induced by the compound of formula 1; in the fermentation culture, the concentration of the compound of formula 1 is 1g / L to 2g / L. In some specific implementations, the pH value of the fermentation medium is 7 to 8. In some specific implementations, the fermentation culture is carried out under stirring, and the stirring rate is 150rpm to 300rpm, preferably 220rpm.

[0018] The present application then puts the compound of formula 1 into the already cultured Nocardia fermentation broth, adds a solubilizing agent and adjusts the pH value to react. In some specific implementations, the concentration of the compound of formula 1 in the reaction is 10g / L to 80g / L, preferably 20g / L to 60g / L; the pH value of the reaction is 9 to 10; the temperature of the reaction is 28°C to 35°C, preferably 30°C to 32°C, and the reaction time is 24h to 72h, preferably 30h to 60h. In some specific implementations, the solubilizing agent includes but is not limited to one or more of Tween-80, Triton X-100, N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), and the present application has no special requirements for the selection of solubilizing agents. In some specific implementations, the mass percentage of the solubilizing agent is 0.01% to 0.3%, preferably 0.1% to 0.2%. In some specific implementations, the seed culture is carried out under stirring, and the stirring rate is 150 rpm to 300 rpm, preferably 220 rpm. In some specific implementations, the acetate compound has the structure of Formula 1-1, Formula 1-2, Formula 1-3, Formula 1-4;

[0019]

[0020] The prednisone derivatives have structures of Formula 2-1, Formula 2-2, Formula 2-3, and Formula 2-4;

[0021]

[0022] The present application also requires that the Nocardia transformation liquid after the reaction be inactivated. In some specific implementations, the temperature of the inactivation treatment is 50°C to 80°C, preferably 55°C to 75°C, and the time of the inactivation treatment is 15min to 30min, preferably 20min to 25min.

[0023] In the present application, a solvent is added to the product after the reaction for ultrasonic centrifugation to obtain a prednisone derivative. In some specific implementations, the solvent includes one or more of methanol, acetonitrile or acetone, and the amount of the solvent used is 10 to 50 times the mass of the solution after the reaction, preferably 20 to 40 times. In some specific implementations, the reaction detection method of the present application is HPLC method to track the conversion of the starting material.

[0024] The preparation method optimizes the Nocardia culture process and has the advantages of simple process, high feed amount and conversion rate, small impurities, simple and easy to obtain raw and auxiliary materials, low production cost, etc. It overcomes the defects of the two-step process of chemical hydrolysis and biological dehydrogenation and can be industrialized.

[0025] The present application uses an optimized seed culture medium and a fermentation culture medium, so that the feed concentration of the fermentation culture is increased to 10 g / L to 80 g / L, and the dehydrogenation and hydrolysis conversion rate reaches more than 90%; a single bacterial species Nocardioide simplex, with a preservation number of ACCC 10205, is used, and catalysis is performed after fermentation culture, so that the hydrolysis and dehydrogenation processes of the compound of the formula I structure are carried out in the same system, thereby realizing a one-step fermentation conversion to prednisone derivatives, the process is simple, no intermediate discharging is required, the production cost and environmental pressure are reduced, the method is suitable for industrial production, and has the value of industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the HPLC chart of the prednisone derivative prepared in Example 1 of the present application;

[0027] Figure 2 This is the HPLC chart of the prednisone derivative prepared in Example 2 of the present application;

[0028] Figure 3 This is the HPLC chart of the prednisone derivative prepared in Example 3 of the present application;

[0029] Figure 4 This is the HPLC chart of the prednisone derivative prepared in Example 4 of the present application;

[0030] Figure 5 This is the HPLC chart of the prednisone derivative prepared in Example 5 of the present application;

[0031] Figure 6 This is the HPLC chart of the prednisone derivative prepared in Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0032] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0033] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0034] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0035] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the present application and does not limit the scope of the present application unless the claims are set forth. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the present application.

[0036] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0037] The present application provides a method for preparing a prednisone derivative, comprising:

[0038] The acetate compound shown in Formula 1 is reacted under the catalysis of Nocardia to obtain the prednisone derivative shown in Formula 2;

[0039] The accession number of Nocardia is ACCC 10205;

[0040]

[0041] Wherein R1 is a hydrogen atom, R2 is a hydrogen atom or a carbonyl group; or R1 and R2 form a double bond;

[0042] R4 is a hydrogen atom, R3 is a hydroxyl group; or R3 and R4 form a double bond.

[0043] The present application uses an optimized seed culture medium and a fermentation culture medium, so that the feed concentration of the fermentation culture is increased to 10 g / L to 80 g / L, and the dehydrogenation and hydrolysis conversion rate reaches more than 90%; a single bacterial species Nocardioide simplex, with a preservation number of ACCC 10205, is used, and catalysis is performed after fermentation culture, so that the hydrolysis and dehydrogenation processes of the compound of the formula I structure are carried out in the same system, thereby realizing a one-step fermentation conversion to prednisone derivatives, the process is simple, no intermediate discharging is required, the production cost and environmental pressure are reduced, the method is suitable for industrial production, and has the value of industrial application.

[0044] The present application is further described below in conjunction with the following embodiments. The protection scope of the present application is not limited by the following embodiments.

[0045] Example 1

[0046] Cortisone acetate (17α, 21-dihydroxypregnane-4-ene-3,11,20-trione 21-acetate, CAS: 50-04-4) is used as a raw material, i.e., the structure of formula 1-1;

[0047]

[0048] Strain culture process: Inoculate the strain ACCC 10205 (Nocardioide simplex, deposit number ACCC10205, purchased from China Agricultural Microbiological Culture Collection Management Center in March 2014) on a slant medium (including 8g of glucose, 15g of yeast extract, 20g of agar per 1L of water, pH7.0-8.0), culture at 30°C for 2 days, scrape a loop of bacteria and inoculate into a seed medium (including 10g of glucose, 8g of corn steep liquor, 10g of yeast extract, 11g of peptone, 9.5g of soybean powder, 5g of KH2PO4 per 1L of water, pH7.0-8.0), culture at 200rpm, 32°C for 1 day, and then transfer to a fermentation medium (including 10g of glucose, 8g of corn steep liquor, 10g of yeast extract, 11g of peptone, 9.5g of soybean powder, 5g of KH2PO4 per 1L of water) with a transfer amount of 1%. 5g, pH 7.0-8.0, add 1g / L cortisone acetate before sterilization), 220rpm, 32℃ culture for 10-20h to obtain Nocardia fermentation broth for feeding.

[0049] Weigh 8g of cortisone acetate and add it to 100mL of fermentation liquid adjusted to pH 9.0. Add 0.1% Tween-80 by mass, react at 30℃, 220rpm for 48h, heat to 55℃ to terminate the reaction, keep it for 25min to inactivate, take 2.5mL of conversion liquid, add 50mL of acetone, and then perform ultrasonic centrifugation for HPLC analysis. Figure 1 , Figure 1 This is the HPLC diagram of the prednisone derivative prepared in Example 1 of the present application, Figure 1 It can be seen that the conversion rate is 94.500%, and the hydrolyzate of cortisone acetate shown in the structure of formula 2-1, i.e., cortisone, is 3.819%.

[0050] Example 2

[0051] Anecortave acetate (17α-hydroxypregnane-4,9(11)-diene-3,20-dione-21-acetate, CAS: 7753-60-8, H6) is used as the raw material, i.e., the structure of formula 1-2;

[0052]

[0053] The bacterial cell culture process was the same as in Example 1, wherein 1.5 g / L anecortave acetate was added before the fermentation medium was sterilized.

[0054] Weigh 8 g of the starting material anecortave acetate and add it to 100 mL of fermentation broth adjusted to pH 9.5. Add 0.2% DMSO and shake well. React at 32°C, 220 rpm for 72 h. Heat to 60°C to terminate the reaction and keep it for 20 min for inactivation. Take 2.5 mL of the conversion solution, add 50 mL of acetone and perform ultrasonic centrifugation for HPLC analysis. Figure 2 , Figure 2 This is the HPLC diagram of the prednisone derivative prepared in Example 2 of the present application, Figure 1 It can be seen that the conversion rate is 96.050%, and the hydrolyzate of anecortave acetate shown in the structure of formula 2-2 is 1.426%.

[0055] Example 3

[0056] RSA (17α, 21-dihydroxypregnane-4-ene-3,20-dione-21-acetate) is used as the raw material, i.e., the structure of formula 1-3;

[0057]

[0058] The bacterial culture process was the same as in Example 1, wherein 2.0 g / L RSA was added before the fermentation medium was sterilized.

[0059] Weigh 6 g of RSA and add it to 100 mL of fermentation broth adjusted to pH 10.0. Add 0.1% DMSO and shake well. React at 33°C, 250 rpm for 65 h. Heat to 80°C to terminate the reaction and keep it for 20 min to inactivate. Take 1 mL of the conversion solution, add 30 mL of methanol and perform ultrasonic centrifugation for HPLC analysis. Figure 3 , Figure 3 This is the HPLC chart of the prednisone derivative prepared in Example 3 of the present application, Figure 1 It can be seen that the conversion rate is 95.118%, and the RSA hydrolyzate shown in Formula 2-3, namely RS, is 2.918%.

[0060] Example 4

[0061] Triene (pregnane-4,9,16-triene-3,20-dione 21-acetate) is used as the raw material, i.e., the structure of formula 1-4;

[0062]

[0063] The bacterial cell culture process was the same as in Example 1, wherein 1.0 g / L triene was added before the fermentation medium was sterilized.

[0064] Weigh 3 g of triene and add it to 100 mL of fermentation broth adjusted to pH 9.0. Add 0.2% DMF, shake well and react at 33°C, 250 rpm for 65 h. Heat to 80°C to terminate the reaction and keep it for 20 min for inactivation. Take 1 mL of the conversion solution, add 40 mL of acetone and perform ultrasonic centrifugation for HPLC analysis. Figure 4 , Figure 4 This is the HPLC diagram of the prednisone derivative prepared in Example 4 of the present application, Figure 1 It can be seen that the conversion rate is 95.938%, and the triene hydrolyzate shown in formula 2-4 is 0.431%.

[0065] Example 5

[0066] A 50L tank fermentation pilot was carried out using anecortave acetate as the starting material for the conversion;

[0067] Strain culture process: Inoculate the strain on a slant culture medium (each liter of culture medium includes 8g of glucose, 15g of yeast extract, 20g of agar, pH7.0-8.0, and the balance is tap water), culture at 30°C for 2 days, scrape a loop of bacteria and inoculate it into a seed culture medium (each liter of culture medium includes 10g of glucose, 8g of corn steep liquor, 10g of yeast extract, 11g of peptone, 9.5g of soybean powder, 5g of KH2PO4, pH7.0-8.0, and the balance is tap water), culture at 200rpm, 32°C for 1 day, and then transfer it into a fermentation culture medium with a transfer amount of 5% (each liter of culture medium includes 10g of glucose, 8g of corn steep liquor, 10g of yeast extract, 11g of peptone, 9.5g of soybean powder, 5g of KH2PO4, pH7.0-8.0, and 2g / L of anecortave acetate is added as an induction before sterilization), the liquid volume is 30L, and the ventilation volume is adjusted to 0.6m 3 / h, 250rpm, 32℃ for 20h before feeding.

[0068] Weigh 0.9 kg of the starting material anecortave acetate, add 2700 mL of sterile water (divided into 2 bottles, 1350 mL each), after quenching at 100 ° C for 15 minutes, put it into the fermentation broth whose pH value has been adjusted to 9.0, add Tween-80 with a mass concentration of 0.1%, and transform at 30 ° C, 250 rpm for 72 hours. After the transformation is completed, heat it to 50 ° C to terminate the reaction, take 2.5 mL of the transformation liquid, add 50 mL of acetone, and then perform ultrasonic centrifugation for HPLC analysis, see Figure 5 , Figure 5 This is the HPLC chart of the prednisone derivative prepared in Example 5 of the present application, Figure 5 It can be seen that the conversion rate is 91.062%, and that of anecortave acetate hydrolyzate is 1.166%.

[0069] Comparative Example 1

[0070] Cortisone acetate is used as the starting material for the conversion;

[0071] Strain culture process: Inoculate the strain on a slant culture medium (each liter of culture medium includes 11g of glucose, 17g of yeast extract, 20g of agar, pH7.0-8.0, and the balance is tap water), culture at 30°C for 2 days, scrape a loop of bacteria and inoculate it into a seed culture medium (each liter of culture medium includes 13g of glucose, 5g of corn steep liquor, 1g of yeast extract, 8g of peptone, 3g of KH2PO4, pH7.0-8.0, and the balance is tap water), culture at 200rpm, 32°C for 1 day, and then transfer it into a fermentation culture medium (each liter of culture medium includes 13g of glucose, 5g of corn steep liquor, 1g of yeast extract, 8g of peptone, 3g of KH2PO4, pH7.0-8.0, and the balance is tap water. 1.0g / L of cortisone acetate is added as an induction before sterilization), culture at 220rpm, 32°C for 10-20h, and then use it for feeding.

[0072] 8 g of the starting material cortisone acetate was weighed and added to 100 mL of fermentation broth adjusted to pH 9.5. DMSO with a mass concentration of 0.2% was added and shaken, and the mixture was reacted at 32° C. and 220 rpm for 72 h. The reaction was terminated by heating to 60° C. and maintained for 20 min for inactivation. 2.5 mL of the conversion solution was taken, 50 mL of acetone was added, and then ultrasonic centrifugation was performed for HPLC analysis. The test results are shown in Table 1. The conversion rate was 87.253%, the cortisone was 5.397%, and the impurity was 5.396%. It can be seen that the conversion result was not good and a large amount of impurities were produced.

[0073] Table 1

[0074] Serial number Peak time / RT Area percentage Remark 1 5.194 0.069％ 2 6.880 0.066％ 3 7.456 5.396％ Impurities 4 8.352 0.130％ 5 8.499 0.177％ 6 9.498 0.048％ 7 10.357 0.105％ 8 12.179 0.043％ 9 12.776 87.253％ Formula 2-1 structure 10 14.353 5.397％ Cortisone 11 22.998 0.342％ 12 26.898 0.268％ 13 28.911 0.079％ 14 29.918 0.364％ 15 30.532 0.177％ Cortisone acetate substrate 16 35.402 0.087％

[0075] Comparative Example 2

[0076] The difference between this comparative example and comparative example 1 is that 1.5 g / L anecortave acetate is added as an induction before the fermentation medium is sterilized.

[0077] Weigh 8 g of the starting material anecortave acetate and add it to 100 mL of fermentation broth adjusted to pH 9.5. Add 0.2% DMSO and shake well. React at 32°C, 220 rpm for 72 h. Heat to 60°C to terminate the reaction and keep it for 20 min for inactivation. Take 2.5 mL of the conversion solution, add 50 mL of acetone and perform ultrasonic centrifugation for HPLC analysis. Figure 6 and Table 2, Figure 6 HPLC diagram of the prednisone derivative prepared in Comparative Example 2 of the present application; Table 2 is Figure 6 The statistical diagram of the spectrum shown is given by Figure 1As shown in Table 2, the conversion rate is 74.435%, and the esterified hydrolyzate of cortisone is 1.912%. It can be seen that the conversion result is not good, and the remaining substrate is 20.842% which is not completely converted.

[0078] Table 2

[0079] Serial number Peak time / RT Area percentage Remark 1 5.257 2.123％ 2 7.831 0.218％ 3 8.964 74.435％ Formula 2-2 structure, product 4 12.496 1.912％ 5 20.213 0.063％ 6 22.875 0.407％ 7 24.743 20.842％ (Anecortave acetate) substrate

[0080] Comparative Example 3

[0081] The difference between this comparative example and comparative example 1 is that 2.0 g / L RSA is added as an induction before the fermentation medium is sterilized.

[0082] Weigh 6g of RSA and put it into 100mL of fermentation broth adjusted to pH 10.0. Add DMSO with a mass concentration of 0.1% and shake well, then react at 33°C, 250rpm for 65h. Heat to 80°C to terminate the reaction and keep it for 20min for inactivation. Take 1mL of the conversion solution, add 30mL of methanol and perform ultrasonic centrifugation for HPLC analysis. The test results are shown in Table 3. The conversion rate is 5.45% of Formula 2-3, and the RSA hydrolyzate, i.e. RS, is 93.50%. The results show that the conversion is not good, and 93.50% of the intermediates are not dehydrogenated.

[0083] Table 3

[0084]

[0085]

[0086] Comparative Example 4

[0087] The difference between this comparative example and comparative example 1 is that 1.0 g / L triene is added as an induction before the fermentation medium is sterilized.

[0088] Weigh 3 g of triene and add it to 100 mL of fermentation broth adjusted to pH 9.0, add DMF with a mass concentration of 0.2%, shake well and react at 33 ° C, 250 rpm for 65 h, heat to 80 ° C to terminate the reaction, keep it for 20 min for inactivation, take 1 mL of conversion solution, add 40 mL of acetone and then perform ultrasonic centrifugation for HPLC analysis. The test results are shown in Table 4. The conversion rate is 54.294% of Formula 2-4, the triene hydrolyzate is 5.627%, and the substrate remains 39.240%.

[0089] Table 4

[0090] Serial number Peak time / RT Area percentage Remark 1 5.953 0.527％ Impurities 2 10.625 54.294％ Formula 2-4 structure 3 15.926 5.627％ Triene hydrolyzate 4 23.028 39.552％ Substrate-triene

[0091] Compared with the comparative example, the conversion rate of the prednisone derivative obtained by the preparation method described in the present application is higher, and the culture medium ratio and components are optimized to increase the enzyme production and enzyme activity, thereby increasing the conversion rate.

[0092] The above is only a preferred specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes within the technical scope disclosed in the present application according to the technical solution and application concept of the present application, which should be covered by the protection scope of the present application.

Claims

1. A method for preparing a prednisone derivative, characterized in that: include: The acetate compound shown in Formula 1 is reacted under the catalysis of Nocardia to obtain the prednisone derivative shown in Formula 2; The deposit number of Nocardia is ACCC 10205; Wherein R1 is a hydrogen atom, R2 is a hydrogen atom or a carbonyl group; or R1 and R2 form a double bond; R4 is a hydrogen atom, R3 is a hydroxyl group; or R3 and R4 form a double bond.

2. The preparation method according to claim 1, characterized in that: Specifically include: Nocardia is fermented and cultured in a fermentation medium, and an acetate compound shown in Formula 1 is added to react to obtain a prednisone derivative shown in Formula 2; each liter of the fermentation medium comprises: 9g to 12g of glucose, 6g to 8g of corn steep liquor, 5g to 10g of yeast extract, 9g to 12g of peptone, 4g to 6g of KH2PO4 and 5g to 12g of soybean powder, and the balance is water; and the pH value of the fermentation medium is 7 to 8.

3. The preparation method according to claim 2, characterized in that: The fermentation culture temperature is 28° C. to 34° C., and the fermentation culture time is 10 h to 20 h. The fermentation culture is induced by the compound of formula 1, and the concentration of the compound of formula 1 is 1 g / L to 2 g / L.

4. The preparation method according to claim 2, characterized in that: In the reaction, the concentration of the compound of formula 1 in the fermentation broth is 10 g / L to 80 g / L; the pH value of the reaction is 9 to 10; the temperature of the reaction is 28° C. to 35° C., and the reaction time is 24 h to 72 h.

5. The preparation method according to claim 1, characterized in that: The reaction is carried out in the presence of a solubilizing agent, which includes one or more of Tween-80, Triton X-100, N,N-dimethylformamide or dimethyl sulfoxide; the mass percentage of the solubilizing agent is 0.01% to 0.3%.

6. The preparation method according to claim 2, characterized in that: The method further comprises the following steps before fermenting Nocardia: Nocardia is sequentially subjected to slant culture in a slant culture medium and seed culture in a seed culture medium; the temperature of the slant culture is 28°C to 35°C, and the time of the slant culture is 24h to 72h; the temperature of the seed culture is 28°C to 34°C, and the time of the seed culture is 15h to 24h.

7. The preparation method according to claim 6, characterized in that: In the slant culture medium, each liter of the slant culture medium includes: 7g to 10g of glucose, 14g to 16g of yeast extract and 19g to 21g of agar, and the balance is tap water; the pH value of the slant culture medium is 7 to 8.

8. The preparation method according to claim 6, characterized in that: The seed culture medium comprises, per liter, 9 to 12 g of glucose, 6 to 8 g of corn steep liquor, 5 to 10 g of yeast extract, 9 to 12 g of peptone, 4 to 6 g of KH2PO4 and 5 to 12 g of soybean powder, with the remainder being tap water; the pH value of the seed culture medium is 7 to 8.

9. The preparation method according to claim 1, characterized in that: The reaction further includes an inactivation treatment, the temperature of the inactivation treatment is 50° C. to 80° C., and the time of the inactivation treatment is 15 min to 30 min.

10. The preparation method according to claim 9, characterized in that: After the inactivation treatment, a solvent is added for ultrasonic centrifugation to obtain a prednisone derivative, wherein the solvent comprises one or more of methanol, acetonitrile or acetone, and the amount of the solvent used is 10 to 50 times the mass of the solution after the reaction.

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