A method for preparing alfaxalone using brexanolone

Abstract

The present invention discloses a method for preparing alfaxalone using brexanolone as a raw material. Alfaxalone is prepared through four steps: hydroxyl protection, fermentative hydroxylation, oxidation, and deprotection. In the preparation method of the present invention, using brexanolone as a raw material, which has the advantageous configuration of 5α-hydrogen and 3α-hydroxy group, hydroxyl group is introduced at the 11-position by using biological fermentative hydroxylation during the reaction process. In the preparation method, there is no need to use chemical reagents and catalysts with high costs, avoiding defects such as low yield and difficult purification of alfaxalone caused by the generation of 3β-hydroxy isomers in common preparation methods. The reaction conditions of the present invention are mild, the operation is simple and convenient, and the efficient and low-cost preparation of the drug alfaxalone can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of organic compounds and relates to a method for preparing alfaxalone from brexanolone. Background Art

[0002] Alfaxalone, chemically named 5α-pregnane-3α-hydroxy-11,20-dione, is a progesterone without endocrine hormone activity and is used as a neuroactive steroid anesthetic for intravenous general anesthetics and sedatives. Alfaxalone has no accumulation when administered repeatedly. Compared with propofol, its safety has been improved. Its potency in sedation and anesthesia is twice that of the currently leading propofol in the industry, causes less blood pressure drop than propofol, and has neuroprotective effects on the fetal and adult brains at normal anesthetic doses. Alfaxalone is widely used as an intravenous anesthetic all over the world.

[0003] Willam R. Nes et al. reported the synthesis of alfaxalone in 1951 (New W. R et al., J. Am. Chem. Soc. 1951. 73. 4765), but the synthesis of its substrate is relatively cumbersome and difficult, which is not conducive to industrialization, as shown in Scheme 1.

[0004]

[0005] Scheme 1

[0006] The method for preparing alfaxalone disclosed in 1973 (U.S. Patent US3714352), but the preparation method has low yield, lack of selectivity, requires large-scale purification, and is not easy to expand.

[0007] In 2010, Bandyopadhyaya A. K. et al. reported a method for constructing the structure of 3α-hydroxy by using the method of configuration inversion to prepare the main structure of alfaxalone (Bandyopadhyaya A. K. et al. Bioorg. Med. Chem. Lett. 2010. 20. 6680), but the yield is only 32%, as shown in Scheme 2.

[0008]

[0009] Scheme 2

[0010] In 2013, Barbora Slavíkova et al. (Barbora Slavíkova.et al., J.Med.Chem. 2013, 56, 2323) reported the construction of the 3α-hydroxy structure by reduction with sodium borohydride, but the formation of isomers could not be avoided, and the yield was 56%, as shown in Scheme 3.

[0011]

[0012] Scheme 3

[0013] Drawbridge pharmaceuticals disclosed its process patent WO2020006596 in 2020 and entered China as CN112384525A, as shown in Scheme 4.

[0014]

[0015] Scheme 4

[0016] This method uses asymmetric transfer hydrogenation to prepare alfaxalone, still producing the 3β-hydroxy isomer. By adding TBSCl and separating alfaxalone by column chromatography, the yield is 50 - 60%. This method has a low yield, uses heavy metals in the final step, and has low synthesis efficiency.

[0017] In view of this, there is an urgent market demand for new methods for the rapid preparation of neuroactive steroid anesthetics, hoping for relatively few chemical steps and high purity for industrial application.

[0018] The company disclosed a method for preparing the drug brexanolone using pregnenolone acetate in 2020 (CN112501235A). Using the raw material pregnenolone acetate from natural products with a single 5α-configuration and high stereoselectivity of enzyme-catalyzed reduction of the 3-keto group during the reaction, the efficient and low-cost preparation of the drug brexanolone can be achieved, as shown in Scheme 5.

[0019]

[0020] Scheme 5

[0021] Based on this, the present invention makes full use of the single stereoconfiguration of the 5α-hydrogen and 3α-hydroxy of brexanolone, and uses it as a raw material to develop a process route for preparing alfaxalone. Summary of the Invention

[0022] The technical problem to be solved by the present invention is to provide a method for preparing alfaxalone using brexanolone as a raw material. In the preparation method of the present invention, brexanolone is used as a raw material, and alfaxalone is prepared through four steps: 3-hydroxy protection, biocatalytic 11-hydroxylation, oxidation, and deprotection.

[0023] The reaction process of the above preparation method of the present invention is shown in Reaction Scheme (I):

[0024]

[0025] Reaction Scheme (I)

[0026] Among them, R is selected from an ester group or a silyl ether group.

[0027] The method of the present invention includes the following steps:

[0028] (1) Hydroxy protection: In a first solvent, brexanolone represented by the compound of formula (1) and a reagent for protecting the hydroxyl group react under alkaline conditions to obtain a compound of formula (2); the reaction process is shown in Reaction Scheme (A):

[0029]

[0030] (2) Hydroxylation by biocatalytic method: The compound of formula (2) obtained in the above step (1) is fermented using Rhizopus nigricans RN-M246 (Feng Kui et al., Chemistry & Bioengineering, 2012, 29, 6, 77-79) to prepare a compound of formula (3) in one step; the reaction process is shown in Reaction Scheme (B):

[0031]

[0032] (3) Oxidation reaction: The compound of formula (3) obtained in the above step (2) is dissolved in a second solvent, and the temperature is controlled during the reaction in the presence of an oxidizing agent and a catalyst to obtain a compound of formula (4); the reaction process is shown in Reaction Scheme (C):

[0033]

[0034] (4) Deprotection reaction: The compound of formula (4) obtained in the above step (3) is dissolved in a third solvent, and deprotection is carried out under the conditions of an alkali or a catalyst to obtain the target product alfaxalone; the reaction process is shown in Reaction Scheme (D):

[0035]

[0036] In step (1) of the present invention, when the hydroxy protecting group R is an ester group, the hydroxy protection reaction is specifically as follows: The compound of formula (1) reacts with the reagent for protecting the hydroxyl group in the first solvent under the action of a base to obtain a compound of formula (2).

[0037] Among them, the ester group is selected from one or more of C2-C10 straight-chain ester groups (such as ethyl ester, propyl ester, butyl ester, etc.), isobutyl ester group, isopentyl ester group, phenyl ester group, p-methoxyphenyl ester group, etc.; preferably, it is an ethyl ester group.

[0038] Among them, the molar ratio of the compound of formula (1), the reagent for protecting the hydroxyl group, and the base is 1:(1-4):(0.05-5); preferably, it is 1:3:0.1.

[0039] Among them, the first solvent is one or more of ethyl acetate, dichloromethane, chloroform, DMF, toluene, tetrahydrofuran, 2-methyltetrahydrofuran, etc.; preferably, it is ethyl acetate.

[0040] Among them, the base is selected from one or more of triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, etc.; preferably, it is DMAP.

[0041] Among them, the reagent for protecting the hydroxyl group is selected from one or more of C2-C10 straight-chain acyl chlorides or straight-chain acid anhydrides, isobutyryl chloride, isovaleryl chloride, benzoyl chloride, p-methoxybenzoyl chloride, etc.; preferably, it is acetyl chloride or acetic anhydride.

[0042] Among them, the temperature of the reaction is 0-50 °C; preferably, it is 45 °C.

[0043] Among them, the reaction time is 2-24 h; preferably, it is 4 h.

[0044] In step (1) of the present invention, when the hydroxyl protecting group R is a silyl ether group, the hydroxyl protection reaction is specifically as follows: The compound of formula (1) reacts with the reagent for protecting the hydroxyl group in the first solvent under the action of a base to obtain the compound of formula (2).

[0045] Among them, the silyl ether group is selected from one or more of trimethylsilyl ether group (TMS), tert-butyldimethylsilyl ether group (TBS), etc.; preferably, it is tert-butyldimethylsilyl ether group (TBS).

[0046] Among them, the molar ratio of the compound of formula (1), the reagent for protecting the hydroxyl group, and the base is 1:(2-4):(4-8); preferably, it is 1:2.5:4.

[0047] Among them, the first solvent is one or more of DMF, dichloromethane, chloroform, carbon tetrachloride, etc.; preferably, it is dichloromethane.

[0048] Among them, the base is selected from one or more of triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, etc.; preferably, it is imidazole.

[0049] Among them, the reagent for protecting the hydroxyl group is selected from one or more of trimethylchlorosilane (TMSCl), tert-butyldimethylchlorosilane (TBSCl), etc.; preferably, it is TBSCl.

[0050] Among them, the temperature of the reaction is 0 - 50 °C; preferably, it is 25 °C.

[0051] Among them, the reaction time is 2 - 24 h; preferably, it is 12 h.

[0052] In step (2) of the present invention, the fermentation strain is Rhizopus nigricans RN-M246, which catalyzes the 11α-hydroxylation reaction of the compound of formula (2). The fermentation medium is optimized to be conducive to the growth and transformation of the bacterial cells, and the compound of formula (3) is effectively prepared.

[0053] In step (2), in 1 L of water, the components and weights of the fermentation medium are 10 - 30 g of glucose, 20 - 30 g of peptone, 10 - 20 g of cold-pressed soybean powder, 1 - 5 g of ammonium citrate tribasic, and 2 - 5 g of dipotassium hydrogen phosphate; preferably, the components and weights of the fermentation medium are 30 g of glucose, 20 g of peptone, 10 g of cold-pressed soybean powder, 1 g of ammonium citrate tribasic, and 5 g of dipotassium hydrogen phosphate.

[0054] In step (2), the pH of the fermentation medium is 5.0 - 6.5; preferably, the pH of the fermentation medium is 6.5.

[0055] In step (2), the inoculum amount of Rhizopus nigricans is 25% - 30%; preferably, the inoculum amount of Rhizopus nigricans is 30%.

[0056] In step (2), Rhizopus nigricans is obtained through conventional slant culture and seed culture to obtain the bacteria for fermentation.

[0057] In step (3), the second solvent is selected from one or several of dichloromethane, chloroform, acetone, toluene, ethyl acetate, 2-methyltetrahydrofuran, etc.; preferably, it is dichloromethane.

[0058] In step (3), the oxidant is selected from one or more of sodium hypochlorite, chromium(III) oxide, PCC, PDC, sodium dichromate, potassium dichromate, etc.; preferably, it is sodium hypochlorite.

[0059] In step (3), the catalyst is selected from one or more of tetramethylpiperidine oxide (TEMPO), 4-hydroxy-tetramethylpiperidine oxide, 4-benzoyloxy-tetramethylpiperidine oxide, etc.; preferably, it is tetramethylpiperidine oxide (TEMPO).

[0060] In step (3), the mass ratio of the compound of formula (3), the oxidant, and the catalyst is 1:(3.5 - 4.5):(0.03 - 0.05); preferably, it is 1:3.75:0.03.

[0061] In step (3), the temperature of the oxidation reaction is 0°C - 25°C; preferably, it is 10°C.

[0062] In step (3), the time of the oxidation reaction is 2 - 5 h; preferably, it is 2.5 h.

[0063] In step (4), when the hydroxyl protecting group R is an ester group, the hydrolysis reaction is specifically as follows: the compound of formula (4) undergoes a hydrolysis reaction in the presence of a base in the third solvent to obtain alfaxalone.

[0064] Among them, the base is selected from one or more of LiOH, KOH, NaOH, t-BuOK, K2CO3, etc.; preferably, it is K2CO3.

[0065] Among them, the molar ratio of the compound of formula (4) to the base is 1:(0.5 - 2); preferably, it is 1:1.3.

[0066] Among them, the third solvent is selected from one or two of methanol, ethanol, etc.; preferably, it is methanol.

[0067] Among them, the temperature of the hydrolysis reaction is 10 - 75°C; preferably, it is 65°C.

[0068] Among them, the time of the hydrolysis reaction is 0.3 - 12 h; preferably, it is 2 h.

[0069] In step (4), when the hydroxyl protecting group R is a silyl ether group, the deprotection reaction is specifically as follows: the compound of formula (4) undergoes a deprotection reaction in the presence of a catalyst in the third solvent to obtain alfaxalone.

[0070] Among them, the catalyst is selected from one or more of tetrabutylammonium fluoride TBAF, tetrabutylammonium fluoride trihydrate TBAF . 3H2O, boron trifluoride diethyl etherate, acetic acid, ethyl acetate solution of hydrogen chloride, etc.; preferably, it is TBAF . 3H2O.

[0071] Among them, the mass ratio of the compound of formula (4) to the catalyst is 1:(1 - 6); preferably, it is 1:4.

[0072] Among them, the third solvent is selected from one or two of tetrahydrofuran, water, etc.; preferably, it is tetrahydrofuran.

[0073] Among them, the temperature of the deprotection reaction is 10-75 °C; preferably, it is 25 °C.

[0074] Among them, the time of the deprotection reaction is 2-48 h; preferably, it is 24 h.

[0075] In the present invention, by using the unique 3α-hydroxy and 5α-hydrogen stereoconfigurations in the structure of brexanolone, through the combination of chemical conversion and biological fermentation methods, alfaxalone was efficiently prepared. During the reaction process, whether it is 3α-hydroxy protection, obtaining 11-hydroxy substitution by biological fermentation, hydroxy oxidation, or deprotection reaction, the reaction conditions are safe and mild, without involving strong base or strong acid systems, and will not affect the configurations of 3α-hydroxy and 5α-hydrogen, and its unique stereoconfiguration is maintained, avoiding the problems of isomer generation and difficult separation caused by the existing chemical reduction of carbonyl or configuration inversion methods.

[0076] The beneficial effects of the present invention are as follows: Using brexanolone as a raw material and utilizing its single configuration of 3α-hydroxy and 5α-hydrogen, it overcomes the 3β-hydroxy isomers introduced by reducing 3-keto groups in the existing methods, avoiding the losses and cost increases caused by separation. Using biological fermentation methods to construct 11-hydroxy substituents, without the need to use costly chemical reagents and catalysts, avoiding the defects of generating a lot of waste and harsh reaction conditions in the existing preparation methods. The reaction conditions of the present invention are mild, the operation is simple and convenient, and it can realize the efficient, low-cost, and environmentally friendly preparation of the drug alfaxalone. Specific Embodiments

[0077] Combined with the following specific embodiments, the invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content. (The results shown in the examples are all obtained under optimized experimental conditions and have potential application values).

[0078] Fermentation Hydroxylation HPLC Detection

[0079] The fermentation broth was centrifuged at high speed, the supernatant was taken and diluted with methanol, and the diluted solution was filtered through a 0.45 μm organic filter membrane as a detection sample for HPLC analysis. Chromatographic conditions: C18 alkylsilyl-bonded reversed-phase column; the mobile phase was methanol-water (3:2, volume ratio); the flow rate was 0.8 mL·min -1 , the column temperature was 25 °C; the wavelength of the ultraviolet detector was 242 nm.

[0080] Preparation of Bacterial Suspension in Example 1

[0081] Add an appropriate amount of sterile water to the Rhizopus nigricans slant, wash down the spores, filter with gauze, and shake well to make 1×10 7 ~1×108 per mL -1 Rhizopus nigricans spore suspension.

[0082] Example 2 Shake flask transformation

[0083] Add 75 mL of fermentation medium and 10 mmol / L of the substrate compound of formula (2) to a 500 mL baffled shake flask. After sterilization, ultrasonicate, and inoculate with 4% of the Rhizopus nigricans spore suspension prepared in Example 1 of the present invention. Incubate at 28 °C and 160 r·min -1 for 24 h, then increase the rotation speed to 220 r·min -1 and continue the transformation for 48 h. -1

[0084] Example 3 Preparation of the compound of formula (2) (acetyl protection)

[0085] Add ethyl acetate (76 mL) and the compound of formula (1) (6.36 g, 20 mmol) to a flask, stir until dissolved and clear. Add DMAP (0.245 g, 2 mmol) and acetic anhydride (6.13 g, 60 mmol), and react at 45 °C for 4 h. After monitoring the reaction to completion by TLC, add water (20 mL) to quench the reaction, extract with ethyl acetate (100 mL), wash the organic phase with water and saturated brine, and concentrate under reduced pressure to obtain the crude product of the compound of formula (2) (6.9 g of white solid, molar yield 95.8%).

[0086] Example 4 Preparation of the compound of formula (2) (TBS protection)

[0087] Add DCM (100 mL) and the compound of formula (1) (6.36 g, 20 mmol) to a flask, stir until dissolved and clear. Add TBSCl (9.85 g, 65.35 mmol) and imidazole (7.12 g, 104.56 mmol), and react at 25 °C for 12 h. After monitoring the reaction to completion by TLC, add water (100 mL) and stir for 10 min, extract with DCM (80 mL), separate the layers, wash the organic phase with water and saturated NaCl, concentrate under reduced pressure, add methanol / water, and slurry at 25 °C for 3 h, then filter to obtain the crude product of the compound of formula (2) (8.30 g of white solid, molar yield 96.2%).

[0088] Example 5 Preparation of the compound of formula (3) (acetyl protection)

[0089] In 1 L of water, 10 g of the fermentation substrate, the acetyl-protected compound of formula (2), and the components of the fermentation medium are 30 g of glucose, 20 g of peptone, 10 g of cold-pressed soybean powder, 1 g of ammonium citrate, 5 g of dipotassium hydrogen phosphate, and the pH is 6.5. After the first-stage seed culture of Rhizopus nigricans for 24 hours, it is inoculated into the above fermentation medium at an inoculation amount of 30%. A 30 L fermenter is used for the transformation, the liquid filling volume is 80%, the tank pressure is 0.05 MPa, the air flow rate is 40 m 3 / hr, the temperature is 28 °C, the stirring speed is 180 rpm, the transformation is carried out for 72 hours, samples are taken for HPLC analysis, the substrate transformation is complete, the fermentation broth is inactivated at 85 °C, cooled to room temperature, and filtered by suction. The filter cake is extracted with ethyl acetate, concentrated to obtain white crystals, the crude product yield is 88.2%, and the content of the acetyl-protected compound of formula (3) is 80.2%.

[0090] Example 6 Preparation of the compound of formula (3) (TBS protection)

[0091] In 1 L of water, 10 g of the fermentation substrate, the TBS-protected compound of formula (2), and the components of the fermentation medium are 30 g of glucose, 20 g of peptone, 10 g of cold-pressed soybean powder, 1 g of ammonium citrate, 5 g of dipotassium hydrogen phosphate, and the pH is 6.5. After the first-stage seed culture of Rhizopus nigricans for 24 hours, it is inoculated into the above fermentation medium at an inoculation amount of 30%. A 30 L fermenter is used for the transformation, the liquid filling volume is 80%, the tank pressure is 0.05 MPa, the air flow rate is 40 m 3 / hr, the temperature is 28 °C, the stirring speed is 180 rpm, the transformation is carried out for 72 hours, samples are taken for HPLC analysis, the substrate transformation is complete, the fermentation broth is inactivated at 85 °C, cooled to room temperature, and filtered by suction. The filter cake is extracted with ethyl acetate, concentrated to obtain white crystals, the crude product yield is 90.5%, and the content of the TBS-protected compound of formula (3) is 81.5%.

[0092] Example 7 Preparation of the compound of formula (4) (acetyl protection)

[0093] Add 16 g of water into a 500 mL three-necked flask. While stirring, sequentially add 2 g of sodium bicarbonate, 200 g of dichloromethane, and 40 g of the compound of formula (3) protected by acetyl. After stirring until completely dissolved, lower the temperature. When the internal temperature of the reaction solution reaches 10 °C, add 1.2 g of tetramethylpiperidine oxide (TEMPO). Uniformly add dropwise 150 g of 10% sodium hypochlorite solution, control the dropping time within 1 - 2 h, and control the dropping temperature within 6 - 15 °C. After the dropping is completed, keep the temperature at 10 - 15 °C for heat preservation reaction for 2 - 5 h. Take a sample for TLC analysis until the raw materials are completely reacted. After the reaction is completed, add 30 g of 20% sodium thiosulfate solution to quench the reaction, stir for 10 minutes, let it stand for liquid separation (the emulsion layer is separated into the aqueous phase for extraction). The aqueous phase is extracted once with dichloromethane, stirred for 30 minutes, let it stand for liquid separation. The dichloromethane organic phases are combined and washed three times with 80 g * 3 of saturated sodium chloride solution, stirred for 10 minutes, and let it stand for liquid separation. The organic phase is concentrated under reduced pressure to a large amount of crystallization with dichloromethane, and then entrained twice with water until the dichloromethane is completely concentrated. Add 200 g of water, stir for 20 minutes, filter by suction, and the filter cake is dried at 75 °C with hot air circulation for 6 - 12 h to obtain 38.5 g of the compound of formula (4) protected by acetyl as a pale yellow to off-white powder, with a yield of 96.2%.

[0094] Example 8 Preparation of the compound of formula (4) (protected by TBS)

[0095] Add 16 g of water into a 500 mL three-necked flask. While stirring, sequentially add 2 g of sodium bicarbonate, 200 g of dichloromethane, and 40 g of the compound of formula (3) protected by TBS. After stirring until completely dissolved, lower the temperature. When the internal temperature of the reaction solution reaches 10 °C, add 1.2 g of tetramethylpiperidine oxide (TEMPO). Uniformly add dropwise 150 g of 10% sodium hypochlorite solution, control the dropping time within 1 - 2 h, and control the dropping temperature within 6 - 15 °C. After the dropping is completed, keep the temperature at 10 - 15 °C for heat preservation reaction for 2 - 5 h. Take a sample for TLC analysis until the raw materials are completely reacted. After the reaction is completed, add 30 g of 20% sodium thiosulfate solution to quench the reaction, stir for 10 minutes, let it stand for liquid separation (the emulsion layer is separated into the aqueous phase for extraction). The aqueous phase is extracted once with dichloromethane, stirred for 30 minutes, let it stand for liquid separation. The dichloromethane organic phases are combined and washed three times with 80 g * 3 of saturated sodium chloride solution, stirred for 10 minutes, and let it stand for liquid separation. The organic phase is concentrated under reduced pressure to a large amount of crystallization with dichloromethane, and then entrained twice with water until the dichloromethane is completely concentrated. Add 200 g of water, stir for 20 minutes, filter by suction, and the filter cake is dried at 75 °C with hot air circulation for 6 - 12 h to obtain 38.2 g of the compound of formula (4) protected by TBS as a pale yellow to off-white powder, with a yield of 95.5%.

[0096] Example 9 Hydrolysis of the compound of formula (4) to prepare alfaxalone (removal of acetyl protection)

[0097] Add methanol (43 mL) and K2CO3 (2.07 g, 15 mmol) into a flask. After dissolution and clarification, add the compound of formula (4) (3.74 g, 10 mmol) under N2 protection. Heat the temperature to 65 °C and react for 2 h. After monitoring the completion of the reaction by TLC, cool it to 25 °C, add 2 mol / L dilute hydrochloric acid to adjust the pH to 7 - 8, distill off methanol under reduced pressure, add water (20 mL), stir at 25 °C for 2 h, and filter by suction; add water (20 mL) to the filter cake again, stir at 25 °C for 2 h, filter by suction, and dry to obtain refined alfaxalone (3.2 g of white solid, molar yield 96.4%). 1 1H NMR (CDCl3, 400 MHz): δ 0.57 (s, 3H); 1.00 (s, 3H); 2.10 (s, 3H); 2.57 and 2.50 (m, 2H); 2.73 (t, 1H, J = 9.0); 4.05 (m, 1H).

[0098] Example 10 Preparation of alfaxalone by hydrolysis of the compound of formula (4) (TBS removal)

[0099] Add tetrahydrofuran (25 mL) and the compound of formula (4) (4.46 g, 10 mmol) into a flask. After dissolution and clarification, add TBAF . ·3H2O (15.7 g, 50 mmol) under N2 protection. React at 25 °C for 24 h. After monitoring the completion of the reaction by TLC, quench with saturated ammonium chloride, distill off tetrahydrofuran under reduced pressure, add dichloromethane (150 mL) and water (120 mL) for extraction, separate the layers, wash the organic phase successively with saturated aqueous NaHCO3 solution, 2N dilute hydrochloric acid, water, and saturated NaCl, and concentrate under reduced pressure to obtain the crude product of alfaxalone. Add 80% ethanol (10 mL) to the filter cake, slurry at 25 °C for 2 h, and filter by suction to obtain refined alfaxalone (3.18 g of white solid, molar yield 95.8%).

[0100] The method of the present invention efficiently prepares alfaxalone using chirally pure brexanolone as the raw material. During the preparation process, there is no need to use costly chemical reagents and catalysts, which provides a basis for further enhancing the role of the drug alfaxalone in protecting human health.

[0101] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, all changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope.

Claims

1. A method for preparing alfaxalone using brexanolone, characterized in that, The reaction process of the described method is shown in Reaction Scheme (I): Wherein, R is selected from acetyl, isobutyryl, isovaleryl, benzoyl, trimethylsilyl ether group TMS, tert-butyldimethylsilyl ether group TBS; The steps are as follows: (1) Hydroxy protection reaction: In a first solvent, brexanolone shown by the compound of formula (1) and a reagent for protecting the hydroxy group undergo a hydroxy protection reaction under alkaline conditions to obtain a compound of formula (2); the reaction process is shown in Reaction Scheme (A): (2) Fermentation hydroxylation reaction: The compound of formula (2) obtained in the step (1) is fermented by Rhizopus nigricans RN-M246 in one step to prepare a compound of formula (3); the components and weights of the fermentation medium are 10 - 30 g of glucose, 20 - 30 g of peptone, 10 - 20 g of cold-pressed soybean powder, 1 - 5 g of ammonium citrate tribasic, and 2 - 5 g of dipotassium hydrogen phosphate; the reaction process is shown in Reaction Scheme (B): (3) Oxidation reaction: The compound of formula (3) obtained in the step (2) is dissolved in a second solvent, and a temperature-controlled reaction is carried out in the presence of an oxidizing agent and a catalyst to obtain a compound of formula (4); the reaction process is shown in Reaction Scheme (C): (4) Deprotection reaction: The compound of formula (4) obtained in the step (3) is dissolved in a third solvent, and deprotection is carried out under the conditions of an alkali or a catalyst to obtain the target product alfaxalone; the reaction process is shown in Reaction Scheme (D):

2. The method according to claim 1, characterized in that, In step (1), the hydroxy protection reaction specifically is: The compound of formula (1) reacts with a reagent for protecting the hydroxy group in the solvent under the action of an alkali to obtain a compound of formula (2).

3. The method according to claim 2, characterized in that When R is acetyl, isobutyryl, isovaleryl, benzoyl, in step (1), the first solvent is selected from one or more of ethyl acetate, dichloromethane, chloroform, DMF, toluene, tetrahydrofuran, 2-methyltetrahydrofuran; and / or, the alkali is selected from one or more of triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP; and / or, the reagent for protecting the hydroxy group is selected from one or more of acetyl chloride, isobutyryl chloride, isovaleryl chloride, benzoyl chloride, acetic anhydride; and / or, the molar ratio of the compound of formula (1), the reagent for protecting the hydroxy group, and the alkali is 1:1 - 4:0.05 - 5; and / or, the reaction temperature is 0 - 50 °C; and / or, the reaction time is 2 - 24 h; and / or, when R is trimethylsilyl ether group TMS, tert-butyldimethylsilyl ether group TBS, in step (1), the first solvent is one or more of DMF, dichloromethane, chloroform, carbon tetrachloride; and / or, the alkali is selected from one or more of triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP; and / or, the reagent for protecting the hydroxy group is selected from one or two of trimethylchlorosilane TMSCl, tert-butyldimethylchlorosilane TBSCl; and / or, the molar ratio of the compound of formula (1), the reagent for protecting the hydroxy group, and the alkali is 1:2 - 4:4 - 8; and / or, the reaction temperature is 0 - 50 °C; and / or, the reaction time is 2 - 24 h.

4. The method according to claim 1, wherein In step (2), the pH of the fermentation medium is 5.0 - 6.5; and / or, the inoculum amount of the Rhizopus nigricans is 25% - 30%.

5. The method according to claim 1, characterized in that, In step (3), the second solvent is selected from one or more of dichloromethane, chloroform, acetone, toluene, ethyl acetate, 2-methyltetrahydrofuran; and / or, the oxidant is selected from one or more of sodium hypochlorite, chromium(III) oxide, PCC, PDC, sodium dichromate, potassium dichromate; and / or, the catalyst is selected from one or more of TEMPO (tetramethylpiperidine N-oxide), 4-hydroxy-TEMPO, 4-benzyloxy-TEMPO; and / or, the mass ratio of the compound of formula (3), the oxidant, and the catalyst is 1:3.5 - 4.5:0.03 - 0.05; and / or, the temperature of the oxidation reaction is 0°C - 25°C.

6. The method according to claim 1, characterized in that, When R is acetyl, isobutyryl, isovaleryl, benzoyl, in step (4), the deprotection reaction is specifically: the compound of formula (4) undergoes a deprotection reaction in the third solvent under the action of a base to obtain alphaxalone.

7. The method according to claim 6, wherein The base is selected from one or more of LiOH, KOH, NaOH, t-BuOK, K2CO3; and / or, the molar ratio of the compound of formula (4) to the base is 1:0.5 - 2; and / or, the third solvent is selected from one or two of methanol and ethanol; and / or, the temperature of the deprotection reaction is 10 - 75°C; and / or, the time of the deprotection reaction is 0.3 - 12 h.

8. The method according to claim 1, wherein When R is trimethylsilyl ether group TMS or tert-butyldimethylsilyl ether group TBS, in step (4), the deprotection reaction is specifically: the compound of formula (4) undergoes a deprotection reaction in the third solvent under the action of a catalyst to obtain alphaxalone.

9. The method according to claim 8, wherein, The catalyst is selected from one or more of tetrabutylammonium fluoride TBAF, tetrabutylammonium fluoride trihydrate TBAF·3H2O, boron trifluoride diethyl etherate, acetic acid, ethyl acetate solution of hydrogen chloride; and / or, the mass ratio of the compound of formula (4) to the catalyst is 1:1 - 6; and / or, the third solvent is selected from one or two of tetrahydrofuran and water; and / or, the temperature of the deprotection reaction is 10 - 75°C; and / or, the time of the deprotection reaction is 2 - 48 h.

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