A process for the preparation of alfaxalone

Abstract

The application belongs to the technical field of organic synthesis, and particularly relates to a preparation method of alpha soludex. The preparation method takes 11-ketoprogesterone as raw material, firstly acetylates the ketone group at the 3 position, then obtains 3beta-hydroxyl through a composite biological enzyme method, and finally obtains alpha soludex through hydrogenation and translocation reaction. The composite biological enzyme method is used to obtain 3beta-hydroxyl, has good specificity, mild reaction conditions, does not need special equipment, and has high conversion efficiency. The preparation method of the application is easy to purify the reaction products of each step, the total mass yield of the final alpha soludex product is greater than 85%, and the HPLC purity is greater than 99.5%. The preparation method of the application has low cost and is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a method for preparing alfasal. Background Technology

[0002] Alfaxalone, also known as 3α-hydroxy-5α-pregnane-11,20-dione, is a neuroactive steroid anesthetic and a GABAAR agonist. It possesses sedative, anesthetic, anticonvulsant, and neuroprotective effects, and is characterized by rapid onset, quick recovery, and relatively high safety. The structural formula of alfaxalone is shown in Formula I. Formula I.

[0003] Currently, there are three main methods for preparing alpha-salicylate. Route 1 uses 11-ketoprogesterone as a raw material to prepare 5α-H via lithium ammonia solution. This synthesis method has harsh reaction conditions, and the preparation of lithium ammonia is hazardous and does not meet modern safety and environmental protection requirements. The reaction formula is as follows: .

[0004] Route 2 involves a one-step synthesis of 5α-H and 3β-OH via a Pd catalyst and various promoters. The 3β-OH is then esterified with methanesulfonyl chloride, followed by configuration inversion in the presence of DMF and potassium nitrite to generate the 3α-OH product. However, this method, with hydrogenation via the Pd catalyst, yields 5α-H and 5β-H isomers in similar proportions, making product purification difficult (it's hard to obtain a single 5α-H configuration product with high purity). Column separation is required, resulting in low yields. The reaction formula is as follows: - .

[0005] Route 3 involves preparing the 5α-H-dominant product using a Pd catalyst, followed by purification to obtain a pure 5α-H intermediate. This intermediate is then selectively reduced using a rare metal catalyst to prepare the 3α-OH-dominant product. Finally, under specific conditions, a sterically hindered esterification reagent reacts only with the β-OH, allowing solvent purification to remove the β-OH and yield the pure product. However, this synthetic method suffers from poor stereospecificity (the ratio of 5α-H to 5β-H isomers is nearly 1:1), requires multiple purification processes, and has a low overall yield (only 50-60%). The reaction formula is as follows: .

[0006] Therefore, there is an urgent need to develop a method for synthesizing alpha-salt with good stereospecificity and the ability to improve product yield. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a method for preparing alpha-salt. The method for preparing alpha-salt provided by this invention has good stereospecificity and high product yield.

[0008] This invention provides a method for preparing alfasal, comprising the following steps: An acetylation reaction was carried out by mixing 11-ketoprogesterone, acetic anhydride and an acidic catalyst to obtain 3-acetoxypregn-3,5-diene-11,20-dione. The 3-acetoxypregn-3,5-diene-11,20-dione, a solubilizer, a buffer solution, and a biological enzyme system are mixed and subjected to an enzymatic reaction to obtain 3β-hydroxypregn-5-diene-11,20-dione; the biological enzyme system includes a biological enzyme, a coenzyme, and a coenzyme regenerator, and the biological enzyme includes a ketone reductase and a hydrolase. The 3β-hydroxypregn-5-ene-11,20-dione, a hydrogenation catalyst, and an organic solvent were mixed, and hydrogen gas was introduced into the resulting liquid to carry out a hydrogenation reaction to obtain 3β-hydroxy-5α-pregnane-11,20-dione. The 3β-hydroxy-5α-pregnane-11,20-dione, nucleophilic precursor, proton donor, phosphorus reagent, azocarboxylic acid ester compound, and organic solvent were mixed and subjected to a transposition reaction to obtain alfasal.

[0009] Preferably, the mass ratio of the 3-acetoxypregn-3,5-diene-11,20-dione, ketone reductase, and hydrolase is 1:0.2~0.3:0.1~0.2.

[0010] Preferably, the temperature of the enzymatic reaction is 18~25℃.

[0011] Preferably, the coenzyme is nicotinamide adenine dinucleotide; the mass ratio of 3-acetoxypregn-3,5-diene-11,20-dione to the coenzyme is 1:0.002~0.005.

[0012] Preferably, the coenzyme regeneration comprises glucose and glucose dehydrogenase; the mass ratio of 3-acetoxypregn-3,5-diene-11,20-dione, glucose and glucose dehydrogenase is 1:0.4~0.8:0.02~0.04.

[0013] Preferably, the mass ratio of the 11-ketoprogesterone to the volume ratio of acetic anhydride is 1 g: 2~10 mL.

[0014] Preferably, the acidic catalyst includes one or more of p-toluenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid; the mass ratio of 11-ketoprogesterone to the acidic catalyst is 1:0.1~0.5; and the temperature of the acetylation reaction is 20~50°C.

[0015] Preferably, the hydrogenation catalyst is one of palladium / calcium carbonate and palladium / carbon; the mass ratio of the 3β-hydroxypregn-5-ene-11,20-dione to the hydrogenation catalyst is 1:0.03~0.5; and the temperature of the hydrogenation reaction is 20~50℃.

[0016] Preferably, the phosphorus reagent includes one or more of triphenylphosphine, tributylphosphine, trimethoxyphosphine, and triethyl phosphite; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the phosphorus reagent is 1:1~2; the azocarboxylic acid ester compound includes one or more of diethyl azodicarboxylate, di-tert-butyl azodicarboxylate, and dibenzyl azodicarboxylate; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the volume ratio of the azocarboxylic acid ester compound is 1g:0.5~2mL.

[0017] Preferably, the nucleophilic precursor includes one or more of benzoic acid, sodium benzoate, p-nitrobenzoic acid, sodium p-nitrobenzoate, p-fluorobenzoic acid, sodium p-fluorobenzoate, o-fluorobenzoic acid, and sodium o-fluorobenzoate; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the nucleophilic precursor is 1:0.5~2; and the temperature of the transposition reaction is 20~50℃.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing alfasal, comprising the following steps: mixing 11-ketoprogesterone, acetic anhydride, and an acidic catalyst to perform an acetylation reaction to obtain 3-acetoxypregn-3,5-diene-11,20-dione; mixing the 3-acetoxypregn-3,5-diene-11,20-dione, a solubilizer, a buffer solution, and a biological enzyme system to perform an enzymatic reaction to obtain 3β-hydroxypregn-5-diene-11,20-dione; wherein the biological enzyme system comprises a biological enzyme, a coenzyme, and... Coenzyme regeneration, wherein the biological enzymes include ketone reductase and hydrolase; the 3β-hydroxypregn-5-ene-11,20-dione, a hydrogenation catalyst, and an organic solvent are mixed, and hydrogen gas is introduced into the resulting solution to carry out a hydrogenation reaction to obtain 3β-hydroxy-5α-pregnane-11,20-dione; the 3β-hydroxy-5α-pregnane-11,20-dione, a nucleophilic reagent precursor, a proton donor, a phosphorus reagent, an azocarboxylic acid ester compound, and an organic solvent are mixed to carry out a transposition reaction to obtain alfasal.

[0019] This invention uses 11-ketoprogesterone as a starting material. First, the keto group at the 3-position is acetylated. Then, a novel enzymatic method is used to modify the A and B rings of 11-ketoprogesterone, achieving the transfer of the C4,5 double bond to the C5,6 double bond, and directionally constructing a C3 β-hydroxyl group with good specificity. Moreover, compared to traditional, stringent processes, the reaction conditions are mild and do not require special equipment. This invention successfully and directionally constructs the C3 β-hydroxyl group and the C5,6 double bond, providing a favorable spatial configuration for α-hydrogenation of C5. The C5 hydrogenation process does not result in a β-hydrogenation configuration, leading to high product configuration specificity and a high alfasalin yield.

[0020] The preparation method provided by this invention allows for easy purification of the reaction products at each step, resulting in a final product yield of over 85% and an HPLC purity of over 99.5%. The preparation method of this invention has low overall production cost, high operability, and is suitable for large-scale industrial production, offering significant economic benefits. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the HPLC spectrum of the product alfasalin from Example 1. Detailed Implementation

[0023] This invention provides a method for preparing alfasal, comprising the following steps: An acetylation reaction was carried out by mixing 11-ketoprogesterone, acetic anhydride and an acidic catalyst to obtain 3-acetoxypregn-3,5-diene-11,20-dione. The 3-acetoxypregn-3,5-diene-11,20-dione, a solubilizer, a buffer solution, and a biological enzyme system are mixed and subjected to an enzymatic reaction to obtain 3β-hydroxypregn-5-diene-11,20-dione; the biological enzyme system includes a biological enzyme, a coenzyme, and a coenzyme regenerator, and the biological enzyme includes a ketone reductase and a hydrolase. The 3β-hydroxypregn-5-ene-11,20-dione, a hydrogenation catalyst, and an organic solvent were mixed, and hydrogen gas was introduced into the resulting liquid to carry out a hydrogenation reaction to obtain 3β-hydroxy-5α-pregnane-11,20-dione. The 3β-hydroxy-5α-pregnane-11,20-dione, nucleophilic precursor, proton donor, phosphorus reagent, azocarboxylic acid ester compound, and organic solvent were mixed and subjected to a transposition reaction to obtain alfasal.

[0024] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0025] In this invention, 11-ketoprogesterone, acetic anhydride and an acidic catalyst are mixed and subjected to an acetylation reaction to obtain 3-acetoxypregn-3,5-diene-11,20-dione.

[0026] In this invention, the preferred mass ratio of 11-ketoprogesterone to acetic anhydride is 1 g: 2~10 mL, specifically 1 g: 10 mL. The acetic anhydride also serves as a reaction solvent. The CAS number of the 11-ketoprogesterone is 81800-93-3, and its structural formula is shown below: .

[0027] In this invention, the acidic catalyst preferably includes one or more of p-toluenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid; the mass ratio of 11-ketoprogesterone to the acidic catalyst is preferably 1:0.1 to 0.5, specifically 1:0.1.

[0028] In this invention, the acetylation reaction is preferably carried out at a temperature of 20-50°C, specifically 30°C, and the reaction time is preferably until the reaction of the raw materials is complete as detected by thin-layer chromatography. The acetylation reaction is preferably carried out under stirring. During the acetylation reaction, the 3-keto group undergoes enolization under acidic conditions followed by esterification to form a 3-acetoxy group, simultaneously forming a 3,5-diene. The reaction formula is as follows: .

[0029] In this invention, the acetylation reaction preferably further includes: subjecting the obtained reaction solution to water precipitation, solid-liquid separation, and drying the obtained solid. The volume ratio of water used for water precipitation to acetic anhydride is preferably 4-6:1, specifically 5:1; the solid-liquid separation is preferably filtration.

[0030] After obtaining 3-acetoxypregn-3,5-diene-11,20-dione, the present invention mixes the 3-acetoxypregn-3,5-diene-11,20-dione, a solubilizer, a buffer solution, and a biological enzyme system to carry out an enzymatic reaction to obtain 3β-hydroxypregn-5-diene-11,20-dione; the biological enzyme system includes biological enzymes, coenzymes, and coenzyme regeneration, and the biological enzymes include ketone reductase and hydrolase.

[0031] In this invention, the co-solvent is preferably tert-butanol. The mass ratio of the 3-acetoxypregn-3,5-diene-11,20-dione to the volume ratio of the co-solvent is preferably 1 g: 3~3.5 mL, specifically 1 g: 3 mL.

[0032] In this invention, the buffer solution is preferably a phosphate buffer solution, the pH of which is preferably 6.5-7.5, and the concentration is preferably 100 mmol / L. The mass ratio of the 3-acetoxypregn-3,5-diene-11,20-dione to the volume of the buffer solution is preferably 1 g:7 mL.

[0033] In this invention, the ketone reductase is preferably a steroid 3β-ketone reductase, which can reduce the ketone group at the C3 position to a β-hydroxyl group; the hydrolase is preferably a steroid side chain hydrolase, which can hydrolyze the steroid side chain.

[0034] In this invention, the preferred mass ratio of the 3-acetoxypregn-3,5-diene-11,20-dione, steroid 3β-keto reductase, and steroid side-chain hydrolase is 1:0.2~0.3:0.1~0.2, specifically 1:0.2:0.1. The enzyme dosage described in this invention results in a high raw material conversion rate.

[0035] In this invention, the coenzyme is preferably nicotinamide adenine dinucleotide (coenzyme I); the mass ratio of 3-acetoxypregn-3,5-diene-11,20-dione to the coenzyme is preferably 1:0.002~0.005, specifically 1:0.002.

[0036] In this invention, the coenzyme regeneration preferably includes glucose and glucose dehydrogenase; the mass ratio of 3-acetoxypregn-3,5-diene-11,20-dione, glucose and glucose dehydrogenase is preferably 1:0.4~0.8:0.02~0.04, specifically 1:0.4:0.02.

[0037] In this invention, the preferred temperature for the enzymatic reaction is 18-25°C, specifically 18-20°C, and the preferred time is until the raw material conversion rate is ≥98% as determined by HPLC, specifically 13 hours, with a raw material conversion rate of 99.6%. The enzymatic reaction temperature described in this invention results in a high raw material conversion rate. During the enzymatic reaction, the pH of the solution is preferably controlled at 6.5-7.5, specifically 6.8-7.0; the reaction formula is as follows: .

[0038] In this invention, the enzymatic reaction preferably further includes: cooling the obtained reaction solution, collecting the precipitated solid, dissolving it in methanol, and performing water precipitation.

[0039] After obtaining 3β-hydroxypregn-5-ene-11,20-dione, the present invention mixes the 3β-hydroxypregn-5-ene-11,20-dione, a hydrogenation catalyst, and an organic solvent, and introduces hydrogen gas into the resulting liquid to carry out a hydrogenation reaction to obtain 3β-hydroxy-5α-pregnane-11,20-dione.

[0040] In this invention, the hydrogenation catalyst is preferably one of palladium / calcium carbonate and palladium / carbon, and the palladium content in palladium / calcium carbonate and palladium / carbon is preferably 1~5wt%, specifically 2%; the mass ratio of 3β-hydroxypregn-5-ene-11,20-dione to the hydrogenation catalyst is preferably 1:0.03~0.5, specifically 1:0.05 or 1:0.08.

[0041] In this invention, the organic solvent is preferably ethanol; the mass ratio of the 3β-hydroxypregn-5-ene-11,20-dione to the volume ratio of the organic solvent is preferably 1g:1~20mL, specifically 1g:10mL.

[0042] In this invention, the flow rate of hydrogen gas introduced is preferably 8-12 mL / min, specifically 10 mL / min. The temperature of the hydrogenation reaction is preferably 20-50°C, specifically 40°C, and the reaction time is preferably until the reaction of the raw materials is complete as detected by thin-layer chromatography; the reaction formula is as follows: .

[0043] In this invention, the hydrogenation reaction preferably further includes: solid-liquid separation of the obtained reaction liquid, concentration and crystallization of the obtained liquid, and drying of the obtained crystal.

[0044] After obtaining 3β-hydroxy-5α-pregnane-11,20-dione, the present invention mixes the 3β-hydroxy-5α-pregnane-11,20-dione, a nucleophilic precursor, a proton donor, a phosphorus reagent, an azocarboxylic acid ester compound, and an organic solvent to carry out a transposition reaction to obtain alfasal.

[0045] In this invention, the preferred method for mixing the 3β-hydroxy-5α-pregnane-11,20-dione, nucleophilic reagent precursor, proton donor, phosphorus reagent, azocarboxylic acid ester compound, and organic solvent is to first mix the organic solvent, phosphorus reagent, and azocarboxylic acid ester compound, and then add the 3β-hydroxy-5α-pregnane-11,20-dione, nucleophilic reagent precursor, and proton donor.

[0046] In this invention, the organic solvent preferably includes one or more of tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane and diethyl ether, and the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the volume ratio of the organic solvent is preferably 1g:1~20mL, specifically 1g:5mL.

[0047] In this invention, the phosphorus reagent preferably includes one or more of triphenylphosphine, tributylphosphine, trimethoxyphosphine, and triethyl phosphite; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the phosphorus reagent is preferably 1:1 to 2, specifically 1:1. The azocarboxylic acid ester compound preferably includes one or more of diethyl azodicarboxylate, di-tert-butyl azodicarboxylate, and dibenzyl azodicarboxylate; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the volume ratio of the azocarboxylic acid ester compound is preferably 1 g:0.5 to 2 mL, specifically 1 g:1 mL. The phosphorus reagent reacts with the azocarboxylic acid ester compound to form a quaternary phosphonium salt-hydrazine intermediate, the formation of which is a prerequisite for the transposition reaction.

[0048] In this invention, the nucleophilic reagent precursor preferably includes one or more of benzoic acid, sodium benzoate, p-nitrobenzoic acid, sodium p-nitrobenzoate, p-fluorobenzoic acid, sodium p-fluorobenzoate, o-fluorobenzoic acid, and sodium o-fluorobenzoate; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the nucleophilic reagent precursor is preferably 1:0.5~2, specifically 1:0.5.

[0049] In this invention, the proton donor preferably includes one or more of trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid and p-toluenesulfonic acid; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the volume of the proton donor is preferably 1g:0.2~1.5mL, specifically 1g:0.2mL.

[0050] In this invention, the temperature of the transposition reaction is preferably 20~50℃, specifically 40℃, and the time is preferably until the reaction of the raw materials is complete as detected by thin-layer chromatography; the reaction formula is as follows: .

[0051] In this invention, the reaction preferably further includes: concentrating the obtained reaction solution, mixing and stirring the obtained concentrate, ethanol, and sodium hydroxide, performing water precipitation, and refining the obtained solid with methanol. The stirring time is preferably 2-5 hours, specifically 3 hours; ethanol is the solvent; the mass ratio of 3β-hydroxy-5α-pregnane-11,20-dione to sodium hydroxide is preferably 1:0.1-1, and sodium hydroxide is an alkaline hydrolysis reagent. The mass ratio of 3β-hydroxy-5α-pregnane-11,20-dione to methanol is preferably 1 g:1-15 mL; the methanol refining specifically involves: first dissolving the solid in a large amount of methanol by heating, then concentrating and distilling off a portion of the methanol, then cooling and crystallizing, and finally filtering.

[0052] The method for preparing alfasalol provided by this invention has low environmental pollution, high reaction selectivity, few byproducts, and high yield, making it suitable for large-scale industrial production.

[0053] To further illustrate the present invention, the preparation method of alfasalol provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0054] In the embodiments of the present invention, the biological enzymes, coenzymes and other raw and auxiliary materials used were all obtained commercially from Shangke Biopharmaceutical (Shanghai) Co., Ltd., including steroid 3β-keto reductase: ES-KRED-121, steroid side chain hydrolase: ES-PLE-107, coenzyme I: NAD, and glucose dehydrogenase: ES-GDH-110.

[0055] Example 1 Alfasalone was prepared according to the following synthetic reaction: .

[0056] 1. Preparation of 3-acetoxypregn-3,5-diene-11,20-dione 20 g of 11-ketoprogesterone, 200 mL of acetic anhydride, and 2 g of p-toluenesulfonic acid were added to a reaction flask. The mixture was stirred and the temperature was controlled at 30 °C until the reaction was complete and the reaction was detected by thin-layer chromatography. 1000 mL of water was added for water precipitation, and the mixture was filtered and dried to obtain 22 g of 3-acetoxypregn-3,5-diene-11,20-dione (compound 2), with a yield of 110%.

[0057] 2. Preparation of 3β-hydroxypregn-5-ene-11,20-dione Compound 2 (22g) was added to 66mL of tert-butanol and 154mL of 100mM phosphatase buffer (pH 6.5). The mixture was stirred thoroughly for 25-30 minutes. Then, 4.4g of steroid 3β-keto reductase, 2.2g of steroid side-chain hydrolase, 44mg of coenzyme I (nicotinamide adenine dinucleotide), 8.8g of glucose, and 440mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 18-20℃, with the pH controlled at 6.8-7.0 using 10wt% sodium carbonate aqueous solution. Samples were taken after 13 hours, and the conversion rate was 99.6%. The temperature was then lowered to 8℃. The solid was collected by filtration at 10℃. After drying, 60 mL of methanol was added to dissolve the solid. This process was repeated three times. The solid was then concentrated under reduced pressure until crystals precipitated. Water precipitation was performed (8-10℃, 2 h) to precipitate more material. The solid was filtered and dried to obtain 19.2 g of 3β-hydroxypregn-5-ene-11,20-dione (compound 3) (yield 87.3%), with a purity of 99.8% (the molar ratio of 3β-hydroxyl to 3α-hydroxyl in the product was 99.8:0.2, and the selectivity of 3β-hydroxyl was >99%).

[0058] 3. Preparation of 3β-hydroxy-5α-pregnane-11,20-dione 19.2 g of 3β-hydroxypregn-5-en-11,20-dione (compound 3) was added to 200 mL of ethanol, along with 1.5 g of palladium / calcium carbonate (2 wt% palladium). The reaction was carried out at 40 °C under hydrogen gas until the reaction was complete as confirmed by thin-layer chromatography. The mixture was filtered to obtain a filtrate, which was then concentrated, crystallized, filtered again, and dried to yield 18.2 g of 3β-hydroxy-5α-pregnane-11,20-dione (compound 4), with a yield of 94.8%. The molar ratio of 5α-H to 5β-H in the product was 99.5:0.5, and the selectivity for 5α-H was >99%.

[0059] 4. Preparation of Alpha Salmon Add 19 g of triphenylphosphine and 19 mL of diethyl azodicarbonate to 100 mL of tetrahydrofuran, then add 18.2 g of 3β-hydroxy-5α-pregnane-11,20-dione, 9.5 g of sodium benzoate, and 4 mL of trifluoroacetic acid. Maintain the temperature at 40 °C and stir until the reaction is complete as detected by thin-layer chromatography. Concentrate the tetrahydrofuran, add 150 mL of methanol and 5 g of sodium hydroxide, stir for 3 hours, add water for precipitation, filter, and purify with methanol to obtain 17.2 g of alfasalol with an HPLC purity of 99.9% (e.g., [missing information]). Figure 1 (As shown); based on 20g of 11-ketoprogesterone, the total yield was 86%.

[0060] Example 2 The only difference from Example 1 is that the amount of steroidal side-chain hydrolase used in step 2 is reduced. The operation steps are as follows: Compound 2 (20g) was added to 60mL of tert-butanol and 140mL of 100mM phosphatase buffer solution at pH 6.5. The mixture was stirred thoroughly for 25-30min. Then, 4.0g of steroid 3β-keto reductase, 1.8g of steroid side-chain hydrolase, 40mg of coenzyme I (nicotinamide adenine dinucleotide), 8.0g of glucose, and 400mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 18-20℃, and the pH was controlled at 6.8-7.0 with 10wt% sodium carbonate aqueous solution. After 13h, the conversion rate was measured to be 92.2%, and the product conversion rate decreased significantly.

[0061] Example 3 The only difference from Example 1 is that the amount of steroid 3β-keto reductase used in step 2 is reduced. The operation steps are as follows: Compound 2 (20g) was added to 60mL of tert-butanol and 140mL of 100mM phosphatase buffer solution at pH 6.5. The mixture was stirred thoroughly for 25-30min. Then, 3.8g of steroid 3β-keto reductase, 2.0g of steroid side-chain hydrolase, 40mg of coenzyme I (nicotinamide adenine dinucleotide), 8.0g of glucose, and 400mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 18-20℃, and the pH was controlled at 6.8-7.0 with 10wt% sodium carbonate aqueous solution. After 13h, the conversion rate was measured to be 85.4%, indicating a significant decrease in product conversion rate.

[0062] Example 4 The only difference from Example 1 is that the amount of steroidal side-chain hydrolase used in step 2 is increased. The operation steps are as follows: Compound 2 (20g) was added to 60mL of tert-butanol and 140mL of 100mM phosphatase buffer solution at pH 6.5. The mixture was stirred thoroughly for 25-30min. Then, 4.0g of steroid 3β-keto reductase, 4.2g of steroid side-chain hydrolase, 40mg of coenzyme I (nicotinamide adenine dinucleotide), 8.0g of glucose, and 400mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 18-20℃, and the pH was controlled at 6.8-7.0 with 10wt% sodium carbonate aqueous solution. After 13h, the conversion rate was measured to be 89.3%, indicating a significant decrease in product conversion rate.

[0063] Example 5 The only difference from Example 1 is that the amount of steroid 3β-keto reductase used in step 2 is increased. The operation steps are as follows: Compound 2 (20g) was added to 60mL of tert-butanol and 140mL of 100mM phosphatase buffer solution at pH 6.5. The mixture was stirred thoroughly for 25-30min. Then, 6.2g of steroid 3β-keto reductase, 2.0g of steroid side-chain hydrolase, 40mg of coenzyme I (nicotinamide adenine dinucleotide), 8.0g of glucose, and 400mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 18-20℃, and the pH was controlled at 6.8-7.0 with 10wt% sodium carbonate aqueous solution. After 13h, the conversion rate was measured to be 93.4%, indicating a significant decrease in product conversion rate.

[0064] Example 6 The only difference from Example 1 is that the temperature of the enzymatic conversion is lowered in step 2. The operation steps are as follows: Compound 2 (20g) was added to 60mL of tert-butanol and 140mL of 100mM phosphatase buffer solution at pH 6.5. The mixture was stirred thoroughly for 25-30min. Then, 4.0g of steroid 3β-keto reductase, 2.0g of steroid side-chain hydrolase, 40mg of coenzyme I (nicotinamide adenine dinucleotide), 8.0g of glucose, and 400mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 15-17℃, and the pH was controlled at 6.8-7.0 with 10wt% sodium carbonate aqueous solution. After 13h, the conversion rate was measured to be 83.6%, indicating a significant decrease in product conversion rate.

[0065] Example 7 The only difference from Example 1 is that the temperature of the enzymatic conversion is increased in step 2. The operation steps are as follows: Compound 2 (20g) was added to 60mL of tert-butanol and 140mL of 100mM phosphatase buffer solution at pH 6.5. The mixture was stirred thoroughly for 25-30min. Then, 4.0g of steroid 3β-keto reductase, 2.0g of steroid side-chain hydrolase, 40mg of coenzyme I (nicotinamide adenine dinucleotide), 8.0g of glucose, and 400mg of glucose dehydrogenase were added sequentially. The reaction was carried out using a biological enzymatic method at 26-27℃, and the pH was controlled at 6.8-7.0 with 10wt% sodium carbonate aqueous solution. After 13h, the conversion rate was measured to be 88.7%, indicating a significant decrease in product conversion rate.

[0066] Comparative Example 1 The alfasalon intermediate was prepared according to the following synthetic reaction: .

[0067] Add 20g of 11-ketoprogesterone to 200mL of ethanol, add 1.5g of palladium / calcium carbonate (palladium content 2wt%), control the temperature at 30℃ and pass hydrogen gas through the mixture until the reaction of the raw materials is complete as detected by thin-layer chromatography, filter, and perform detection.

[0068] Comparative Example 1 used 11-ketoprogesterone as a raw material and hydrogenated it with a Pd / calcium carbonate catalyst to obtain an intermediate with a molar ratio of 5α-H to 5β-H of 1:1.

[0069] Comparative Example 2 According to WO2020 / 006596, alfasalone was prepared according to the following synthetic reaction: .

[0070] 3,11,20-trione-5α-H-pregnane and potassium bicarbonate were dissolved in ethanol, deoxygenated, and then RuCl[(S,S)-Tsdpen] (p-isopropyltoluene) was added. The mixture was heated to 56°C for 20 hours. The mixture was filtered and evaporated to give crude 3α-hydroxy-5α-pregnane-11,20-dione.

[0071] Comparative Example 2 used compound a as a raw material, and reduced the 3-keto group with ruthenium chloride to obtain alfasalone and the isomer 3β-hydroxyalfasalone in a molar ratio of 80:20, with a stereoselectivity of only 80% for 3α-OH.

[0072] Directly hydrogenating 11-ketoprogesterone at the C5 position results in only about 50% of the 5α-H atoms being 5β-H atoms. However, the C3 and C4 double bonds in 3-acetoxypregn-3,5-diene-11,20-dione are difficult to hydrogenate directly. This invention first constructs a C3β-hydroxyl group, then prepares an α-hydrogen at the C5 position, and finally transposes the C3β-hydroxyl group to an α-hydroxyl group. This method is highly specific and yields a high alpha-hydroxyl yield.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing alfasal, characterized in that, Includes the following steps: An acetylation reaction was carried out by mixing 11-ketoprogesterone, acetic anhydride and an acidic catalyst to obtain 3-acetoxypregn-3,5-diene-11,20-dione. The 3-acetoxypregn-3,5-diene-11,20-dione, a solubilizer, a buffer solution, and a biological enzyme system are mixed and subjected to an enzymatic reaction to obtain 3β-hydroxypregn-5-diene-11,20-dione; the biological enzyme system includes a biological enzyme, a coenzyme, and a coenzyme regenerator, and the biological enzyme includes a ketone reductase and a hydrolase. The 3β-hydroxypregn-5-ene-11,20-dione, a hydrogenation catalyst, and an organic solvent were mixed, and hydrogen gas was introduced into the resulting liquid to carry out a hydrogenation reaction to obtain 3β-hydroxy-5α-pregnane-11,20-dione. The 3β-hydroxy-5α-pregnane-11,20-dione, nucleophilic precursor, proton donor, phosphorus reagent, azocarboxylic acid ester compound, and organic solvent were mixed and subjected to a transposition reaction to obtain alfasal.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the 3-acetoxypregn-3,5-diene-11,20-dione, ketone reductase, and hydrolase is 1:0.2~0.3:0.1~0.

2.

3. The preparation method according to claim 1 or 2, characterized in that, The temperature for the enzymatic reaction is 18~25℃.

4. The preparation method according to claim 1, characterized in that, The coenzyme is nicotinamide adenine dinucleotide; the mass ratio of 3-acetoxypregn-3,5-diene-11,20-dione to the coenzyme is 1:0.002~0.

005.

5. The preparation method according to claim 1, characterized in that, The coenzyme regeneration includes glucose and glucose dehydrogenase; the mass ratio of 3-acetoxypregn-3,5-diene-11,20-dione, glucose and glucose dehydrogenase is 1:0.4~0.8:0.02~0.

04.

6. The preparation method according to claim 1, characterized in that, The mass ratio of the 11-ketoprogesterone to the volume ratio of the acetic anhydride is 1 g: 2~10 mL.

7. The preparation method according to claim 1 or 6, characterized in that, The acidic catalyst includes one or more of p-toluenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid; the mass ratio of 11-ketoprogesterone to the acidic catalyst is 1:0.1~0.5; and the temperature of the acetylation reaction is 20~50℃.

8. The preparation method according to claim 1, characterized in that, The hydrogenation catalyst is one of palladium / calcium carbonate and palladium / carbon; the mass ratio of the 3β-hydroxypregn-5-ene-11,20-dione to the hydrogenation catalyst is 1:0.03~0.5; the temperature of the hydrogenation reaction is 20~50℃.

9. The preparation method according to claim 1, characterized in that, The phosphorus reagent includes one or more of triphenylphosphine, tributylphosphine, trimethoxyphosphine, and triethyl phosphite; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the phosphorus reagent is 1:1~2; the azocarboxylic acid ester compound includes one or more of diethyl azodicarboxylate, di-tert-butyl azodicarboxylate, and dibenzyl azodicarboxylate; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the volume ratio of the azocarboxylic acid ester compound is 1g:0.5~2mL.

10. The preparation method according to claim 1, characterized in that, The nucleophilic precursor includes one or more of benzoic acid, sodium benzoate, p-nitrobenzoic acid, sodium p-nitrobenzoate, p-fluorobenzoic acid, sodium p-fluorobenzoate, o-fluorobenzoic acid, and sodium o-fluorobenzoate; the mass ratio of the 3β-hydroxy-5α-pregnane-11,20-dione to the nucleophilic precursor is 1:0.5~2; the temperature of the transposition reaction is 20~50℃.

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