Selective preparation process of B crystal form dexamethasone

Abstract

The invention discloses a directional preparation process of B crystal form dexamethasone. The process comprises the following steps: by taking 16-dehydropregnanone propionate as a starting material, sequentially performing catalytic dehydrogenation by using steroid 1, 4-dehydrogenase from bacillus subtilis, performing catalytic dehydrogenation by using potassium methyltrifluoroborate / copper acetate / 2, 4-dihydroxyprogesterone propionate as a starting material, and performing catalytic hydrogenation by using potassium methyltrifluoroborate / The preparation method comprises the following steps: preparing dexamethasone free alkali by key steps of stereoselective methylation of a 2, 2 '-bipyridine system, catalysis of 11 beta-hydroxylation by immobilized CYP11B1 monooxygenase, epoxidation of peroxybenzoic acid, ring opening of hydrogen fluoride-ethanol and the like, and finally directionally crystallizing in a system solution consisting of 0.5% malic acid and isopropanol-water in a volume ratio of 3: 1 to obtain the high-purity B crystal form dexamethasone.

Description

Technical Field

[0001] This invention relates to the field of dexamethasone preparation technology, specifically a selective preparation process for B-type dexamethasone. Background Technology

[0002] Dexamethasone, a highly effective glucocorticoid, plays an irreplaceable role in clinical anti-inflammatory, anti-allergic, and immunosuppressive therapy. It exists in several solid forms, with the B-form considered the most ideal in the pharmaceutical industry due to its superior chemical stability, solubility, and bioavailability. However, the traditional synthetic route for dexamethasone is lengthy, typically involving more than ten chemical reactions, and generally suffers from poor selectivity, low overall yield, use of highly toxic reagents, and the generation of large amounts of waste. Particularly in key steps, such as dehydrogenation at C-1,2, fluorination at C-9α, and hydroxylation at C-11β, traditional chemical methods often involve harsh conditions and numerous side reactions, resulting in low purity of the final product and difficulty in achieving directional control of the B-form. The introduction of the C-11β hydroxyl group is one of the core challenges in the synthetic process; chemical methods usually require multiple protection and deprotection steps, leading to complex routes. While early microbial fermentation methods offer stereoselectivity, they suffer from drawbacks such as low strain activity, long fermentation cycles, high cell membrane mass transfer resistance, and difficulties in product separation, hindering large-scale application. In the final crystallization and purification stage, traditional single-solvent recrystallization methods lack selectivity for crystal forms and easily yield A crystal form or other mixed crystal forms, which cannot meet the requirements of high-end formulations for uniformity of active pharmaceutical ingredient crystal form. Summary of the Invention

[0003] 1) Technical problems to be solved The purpose of this invention is to overcome the shortcomings of the prior art and provide a selective preparation process for B-type dexamethasone.

[0004] (ii) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a selective preparation process for B-type dexamethasone, comprising the following steps: Step 1: Using 16-dehydropregnane propionate as a starting material, steroid 1,4-dehydrogenase derived from Bacillus subtilis was used for catalytic dehydrogenation at 33-37°C and pH 7.1-7.3 for 10-14 hours to obtain Δ 1 , 4 , 16 -Pregnestrin-3β-ol-20-ketopropionate; Step 2: The Δ 1 , 4 , 16-Pregnatrin-3β-ol-20-ketopropionate is stereoselectively introduced with an α-methyl group via a system of potassium methyltrifluoroborate, copper acetate, and 2,2-bipyridine at 52–58 °C for 4–6 hours to yield 16α-methyl-Δ 1 , 4 -Pregnadien-3β-ol-20-ketopropionate; Step 3: The 16α-methyl-Δ 1 , 4 -Pregnadien-3β-ol-20-ketopropionate was catalyzed by immobilized CYP11B1 monooxygenase to introduce an 11β-hydroxy group, and the reaction was carried out for 22-26 hours to obtain 16α-methyl-11β-hydroxy-Δ 1 , 4 -Pregnadien-3β-ol-20-ketopropionate; Step 4: The 16α-methyl-11β-hydroxy-Δ 1 , 4 Pregnadien-3β-ol-20-ketopropionate is epoxidized with oxybenzoic acid at 0-5°C for 1.5-2.5 hours, and then ring-opened with hydrogen fluoride-ethanol at 23-27°C for 2-3 hours to obtain dexamethasone free base; Step 5: Mix isopropanol and water at a volume ratio of 2.5:1 to 3.5:1 to obtain mixture A. Then add malic acid at 0.3% to 0.7% of the mass of mixture A to form a system solution. Add the free base of dexamethasone to the system solution at a temperature of 4 to 6°C and crystallize for 5 to 7 hours to obtain dexamethasone in crystal form B.

[0005] Furthermore, the 16-dehydropregnane propionate is prepared by esterification of diosgenin with propionic anhydride and elimination reaction catalyzed by sodium hydroxide; specifically, the steps include: Pretreatment was performed using diosgenin with a purity of 97%~99%: the diosgenin was pulverized into 70~90 mesh particles, extracted with petroleum ether under reflux at 60~90℃ for 1.5~2.5 hours to remove fat-soluble impurities, filtered and dried to a moisture content of 0.3%~0.7%; then ultrasonically cleaned with 65%~75% ethanol aqueous solution for 20~50 minutes to remove water-soluble impurities, filtered and vacuum dried. Propionic anhydride esterification reaction: Pretreated diosgenin and propionic anhydride are mixed at a molar ratio of 1:2.5 to 1:3.5, and then pyridine catalyst of 4% to 6% by mass of diosgenin is added; the reaction is carried out under reflux in an oil bath at 105 to 115°C for 5 to 7 hours with a stirring speed of 180 to 220 rpm; then the reaction solution is cooled to room temperature, and 4 to 6 times the volume of ice water is slowly added to quench the reaction. After stirring for 25 to 35 minutes, the mixture is filtered, and the filter cake is washed with ice water 2 to 4 times until the pH reaches 6.5 to 7.5. The esterified product is then obtained by vacuum drying. Sodium hydroxide-catalyzed elimination reaction: The esterification product is dissolved in 8-12 times its mass of anhydrous ethanol, and 15%-25% sodium hydroxide aqueous solution is added, with a molar ratio of 1.8-2.2 times that of the esterification product; the reaction is refluxed in a water bath at 75-85℃ for 3-5 hours, with a stirring speed of 130-170 rpm. The reaction solution was cooled to room temperature, and the pH was adjusted to 4.5-6.5 with hydrochloric acid. The solution was concentrated under reduced pressure to 25%-35% of its original volume. It was extracted with 2.5-3.5 times its volume of ethyl acetate. The organic phase was washed 1-3 times with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain crude 16-dehydropregnenol propionate. The crude product was added to ethanol and water in a volume ratio of 0.8:1 to 1.2:1 for recrystallization, filtered, and then dried under vacuum to obtain the pure product.

[0006] Furthermore, the reaction conditions for steroid 1,4-dehydrogenase in step one are: temperature 33-37℃, pH 7.1-7.3, NAD+ ion concentration 0.4-0.6 mmol, and reaction time 10-14 hours; The method for preparing the steroid 1,4-dehydrogenase includes the following steps: Bacillus subtilis was fermented on a culture medium, and then the fermentation broth was obtained by filtration. The supernatant of the fermentation broth was collected by centrifugation, and purified by ammonium sulfate fractionation, dialysis, and ion exchange chromatography to obtain steroidal 1,4-dehydrogenase with a specific activity of 95-105 units per milligram. The preparation of the reaction system includes: A buffer solution was prepared using 45–55 mmol Tris-HCl, wherein the buffer solution had a pH of 7.1–7.3 and contained 8–12 mmol magnesium chloride. Prepare a dilution of 16-dehydropregnane propionate with a concentration of 8-12 g / L. Then add 4-6 units of enzyme per milligram of substrate to the dilution. At the same time, add glucose dehydrogenase at 1.5-2.5 units of substrate per milligram and glucose at 8-12 g / L as a coenzyme regeneration system. The reaction conditions include: maintaining the reaction temperature at 33~37℃ by constant temperature control in a water bath; adjusting the pH to 7.1~7.3 in real time using 0.05~0.15 molar sodium hydroxide or hydrochloric acid; and stirring speed of 110~130 rpm. After the reaction was completed, the enzyme protein was removed by ultrafiltration with a 0.20-0.25 micrometer membrane; the filtrate was extracted with ethyl acetate 2-4 times, the organic phases were combined and washed with saturated brine 1-3 times, and dried with anhydrous sodium sulfate. Concentrate to dryness under reduced pressure to obtain Δ 1 , 4 , 16 -Pregnestrin-3β-ol-20-ketopropionate crude product; The crude product was recrystallized from isopropanol to obtain the pure product.

[0007] Furthermore, in step two, the solvent for the methylation reaction is dimethyl sulfoxide, the reaction temperature is 52-58°C, and the reaction time is 4-6 hours. The conditions for the methylation reaction in step two include: Reaction system configuration: with Δ 1 , 4 , 16 Pregnattrien-3β-ol-20-ketopropionate was used as a substrate, and 1.1 to 1.3 times the molar amount of potassium methyltrifluoroborate, 0.08 to 0.12 times the molar amount of copper divalent acetate, and 0.08 to 0.12 times the molar amount of 2,2-bipyridine were added. Solvent preparation: Anhydrous dimethyl sulfoxide, used in an amount of 8 to 12 times the substrate mass; Reaction conditions: Under a nitrogen atmosphere, stir at 52~58℃ for 4~6 hours at a stirring speed of 160~200 rpm; Post-processing: After the reaction solution was cooled to room temperature, it was quenched with saturated ammonium chloride solution, extracted with ethyl acetate 2-4 times, and the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain the target product with a purity of 97%-99%. The mobile phase for silica gel column chromatography was petroleum ether-ethyl acetate in a volume ratio of 4:1-6:1 to 2:1-4:1, with gradient elution.

[0008] Furthermore, the amount of 2,2-bipyridine used in step two is Δ 1 , 4 , 16 The amount of 2,2-bipyridine is 8% to 12% of the molar amount of the substrate and is added to the reaction system after being premixed with copper divalent acetate for 4 to 6 minutes.

[0009] Furthermore, in step three, the carrier for immobilizing CYP11B1 monooxygenase is sodium alginate-chitosan composite gel beads, with an enzyme loading of 45-55 units per gram of carrier. The immobilization carrier is sodium alginate-chitosan composite gel beads, and the preparation steps include: A 1.5%~2.5% sodium alginate solution by weight and a 0.8%~1.2% chitosan acetate solution by weight and volume are mixed at a volume ratio of 2.5:1~3.5:1, and then dropped into a 1.5%~2.5% calcium chloride solution and cured for 25~35 minutes to obtain composite gel beads with a diameter of 0.8~2.2 mm. Dissolve CYP11B1 enzyme with a specific activity of 45-55 units per milligram in 45-55 mmol phosphate buffer at pH 7.2-7.6, and then adsorb it with composite gel beads at 23-27℃ for 10-14 hours by shaking. Crosslinking with 0.4%~0.6% glutaraldehyde solution for 0.8~1.2 hours, followed by washing to obtain immobilized enzyme with an enzyme loading of 45~55 units per gram of carrier, which can be reused 4~6 times.

[0010] Furthermore, in step three, the coenzyme regeneration system consists of glucose dehydrogenase and 8-12 grams of glucose per liter, with a reaction time of 22-26 hours.

[0011] Furthermore, in step four, the epoxidation reaction temperature is 0-5℃, and the amount of peroxybenzoic acid used is 1.1-1.3 times the molar amount of the intermediate; the epoxidation reaction conditions include: The substrate is 16α-methyl-Δ 1 , 4 -Pregnadien-3β-ol-20-ketopropionate, with 1.1 to 1.3 molar amounts of peroxybenzoic acid; The solvent is anhydrous dichloromethane, and the amount used is 7 to 9 times the mass of the substrate. Reaction conditions: Stirred reaction at 0~5℃ for 1.5~2.5 hours; Post-processing: Quench with 8%~12% sodium sulfite solution, wash with saturated sodium bicarbonate solution, dry and concentrate to obtain epoxide intermediate with a purity of 95%~97%.

[0012] Further, in step four, the fluorination ring-opening system is a hydrogen fluoride-ethanol volume ratio of 1:5 to 1:7, with a reaction time of 2 to 3 hours and a temperature of 23 to 27°C; the fluorination system is a hydrogen fluoride-ethanol solution, wherein the volume ratio of hydrogen fluoride to ethanol is 1:5 to 1:7, the reaction conditions are stirring at 23 to 27°C for 2 to 3 hours, and the post-treatment is neutralized to pH 6.5 to 8.5 with 15% to 25% sodium hydroxide solution, and the fluorinated intermediate is obtained by ethyl acetate extraction with a fluorine content of 3.6% to 4.0%.

[0013] Furthermore, in step five, the crystallization temperature is 4~6℃, the cooling rate is 0.6~1.0 degrees Celsius per hour, and the stirring speed is 130~170 revolutions per minute; The crystallization conditions include: Solvent system: isopropanol-water volume ratio 2.5:1~3.5:1, containing 0.3%~0.7% malic acid; Dissolution: Dexamethasone free base with a purity of 93%~97% is dissolved in the solvent system at a concentration of 13~17 g / L, and heated to 48~52℃ until clear; Cooling: Cool to 4-6°C at a rate of 0.6-1.0°C per hour, stirring at 130-170 rpm, and grow crystals for 5-7 hours; Product: Dexamethasone in B-type crystal form, with a purity of 98%~100%.

[0014] Furthermore, in step five, B-type dexamethasone seed crystals are added, with the amount being 1.5% to 2.5% of the mass of the free dexamethasone alkali.

[0015] Furthermore, the X-ray powder diffraction pattern of the B-type dexamethasone has characteristic peaks at 2θ of 8.0°~8.4°, 12.3°~12.7°, 16.1°~16.5°, 19.9°~20.3°, and 24.4°~24.8°.

[0016] Furthermore, the intermediate after step three is protected with trimethylchlorosilane at C-3 hydroxyl group, with pyridine as solvent and temperature of 23-27°C; Protective reaction conditions include: The substrate is 16α-methyl-11β-hydroxy-Δ 1 , 4 -Pregnadien-3β-ol-20-ketopropionate, with 1.4 to 1.6 molar amounts of trimethylchlorosilane; The solvent is anhydrous pyridine, and the amount used is 5 to 7 times the amount of the substrate. Reaction conditions: Stirring at 23~27℃ for 0.8~1.2 hours; Post-processing: Quench with methanol and extract with petroleum ether to obtain the protected product with a purity of 96%~98%.

[0017] Furthermore, the protecting group removal reagent was tetrabutylammonium fluoride, CAS number 429-41-4, the solvent was tetrahydrofuran, and the reaction time was 0.8~1.2 hours; The removal conditions include: Add 0.8 to 1.2 moles of tetrabutylammonium fluoride in tetrahydrofuran solution, in a volume of 1.1 to 1.3 molar amounts; Reaction conditions: Stirring at 23~27℃ for 0.8~1.2 hours; Post-treatment: Quench with water and extract with diethyl ether to obtain the deprotected product with a purity of 97%~99%.

[0018] Furthermore, the dexamethasone free alkali after step four is decolorized with activated carbon at a temperature of 38-42°C for 25-35 minutes, with the amount of activated carbon being 0.8%-1.2% of the intermediate mass. Decolorization conditions for activated carbon include: The concentration of dexamethasone free alkaline solution is 8-12 g / L, and the solvent is isopropanol-water volume ratio of 2.5:1-3.5:1; The amount of activated carbon used is 0.8%~1.2% of the substrate weight, and the mixture is stirred at 38~42℃ for 25~35 minutes. The transmittance of the filtrate after filtration is 98%~100%, and the detection wavelength is 440~460 nanometers.

[0019] Furthermore, the crystallized product in step five is vacuum dried under the following conditions: temperature 48~52℃, vacuum degree -0.090~-0.100 MPa, and time 8~12 hours, to obtain B-type dexamethasone with a purity of 99.3%~99.7%.

[0020] This invention innovatively employs an immobilized CYP11B1 monooxygenase biocatalyst specifically for catalyzing the introduction of 11β-hydroxyl groups. This enzyme exhibits absolute stereoselectivity, efficiently and specifically generating the desired configuration in a single reaction, avoiding complex chemical protection and deprotection steps and fundamentally simplifying the synthetic route. Furthermore, by immobilizing the enzyme on self-prepared sodium alginate-chitosan composite gel beads, the enzyme's stability and operability are significantly improved, allowing for reuse at least five times, greatly reducing the cost per use of the enzyme catalyst and laying the foundation for continuous industrial production. The accompanying glucose-glucose dehydrogenase coenzyme regeneration system achieves efficient NADPH coenzyme recycling without the need for additional expensive coenzymes, further controlling production costs.

[0021] A potassium methyltrifluoroborate / copper acetate / 2,2'-bipyridine catalytic system was employed. In this system, 2,2'-bipyridine, acting as the key ligand, coordinates with the copper center, precisely guiding the methyl group to attack from the less sterically hindered α-face, thereby selectively generating the 16α-methyl isomer. This effectively suppresses the formation of diastereomers, ensuring the product purity in this step remains consistently above 98%, providing a high-quality intermediate for subsequent reactions.

[0022] Isopropanol-water (3:1 volume ratio) was used as a mixed solvent, with 0.5% malic acid added as a crystal form regulator. This mixed solvent system, by precisely controlling the polarity and hydrogen bonding of the solvent, altered the packing pattern of dexamethasone molecules during crystallization. Malic acid, through a specific interaction with dexamethasone molecules, preferentially stabilized the nuclei of the B-form. Combining slow, programmed cooling (0.8℃ / h) with seed crystal addition technology, the crystallization process was successfully guided to directionally generate the B-form, effectively suppressing the precipitation of the thermodynamically more stable A-form or other impurities. The final B-form dexamethasone obtained achieved a purity of over 99.5%. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1 10.0 g of 16-dehydropregnane propionate was weighed as the starting material and dissolved in 1.0 L of 50 mmol Tris-HCl buffer (pH 7.2) containing 10 mmol magnesium chloride. 50 units of steroidal 1,4-dehydrogenase (100 IU / mg specific activity), 20 units of glucose dehydrogenase, and 10 g / L glucose were added to the system as a coenzyme regeneration system. The reaction was carried out at 35°C and 120 rpm for 12 hours, with the pH maintained at 7.2 by adding a small amount of 0.1 mol sodium hydroxide solution. After the reaction, the enzyme protein was removed using a 0.22 μm ultrafiltration membrane. The filtrate was extracted three times with ethyl acetate, and the combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain Δ 1 , 4 , 16 9.2 g of crude pregnathitrien-3β-ol-20-ketopropionate was obtained, with a yield of 92%. 9.0 g of the product obtained in the previous step was dissolved in 90 mL of anhydrous dimethyl sulfoxide, followed by the sequential addition of 1.2 mol equivalents of potassium methyltrifluoroborate, 0.1 mol equivalents of copper divalent acetate, and 0.1 mol equivalents of 2,2'-bipyridine, which had been premixed with copper divalent acetate for 5 minutes. The reaction mixture was stirred at 55°C and 180 rpm for 5 hours under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was quenched with saturated ammonium chloride solution, extracted three times with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with a petroleum ether-ethyl acetate gradient from 5:1 to 3:1 (v / v) to give 16α-methyl-Δ 1 , 4 7.6 g of pregnadien-3β-ol-20-ketopropionate was obtained, with a yield of 84% and a purity of 98.5% as determined by HPLC. 7.0 g of this product was added to a reaction system containing immobilized CYP11B1 monooxygenase, which was carried by sodium alginate-chitosan composite gel beads with an enzyme loading of 50 units / g carrier. The system also contained glucose dehydrogenase and 10 g / L glucose for coenzyme regeneration. The reaction was carried out at 25°C and 150 rpm for 24 hours with shaking. After the reaction, the immobilized enzyme was recovered by filtration. The filtrate was extracted with ethyl acetate, dried, and concentrated to obtain 16α-methyl-11β-hydroxy-Δ 1 , 46.3 g of pregnadien-3β-ol-20-ketopropionate, 90% yield. 6.0 g of this intermediate was dissolved in 48 mL of anhydrous dichloromethane. The system was cooled to an ice bath at 0–5 °C, and then 1.2 mol equivalents of peroxybenzoic acid were slowly added dropwise. The reaction was stirred at this temperature for 2 hours. After the reaction was complete, excess oxidant was quenched with 10% sodium sulfite solution, the organic phase was washed with saturated sodium bicarbonate solution, dried, and concentrated to obtain an epoxide intermediate. This epoxide intermediate was dissolved in a hydrogen fluoride-ethanol mixture (hydrogen fluoride to ethanol volume ratio 1:6) and stirred at 25 °C for 2.5 hours. After the reaction was complete, the solution was carefully neutralized to pH 7.5 with 20% sodium hydroxide solution, extracted three times with ethyl acetate, and the organic phases were combined, dried, and concentrated to obtain 4.8 g of free dexamethasone base, 80% yield. Finally, 4.5 g of the obtained free dexamethasone base was dissolved in 300 mL of a solvent consisting of isopropanol and water in a volume ratio of 3:1, containing 0.5% by mass of malic acid. The solution was heated to 50°C until completely clear. 2% by mass of B-type dexamethasone seed crystals were added to this hot solution, and the temperature was then gradually reduced to 5°C at a slow rate of 0.8°C per hour, while stirring at 150 rpm for 6 hours to cultivate crystals. The solution was then filtered. The crystals were collected, washed with a small amount of pre-cooled isopropanol-water mixture, and then vacuum dried at 50°C and -0.095 MPa for 10 hours to obtain 4.1 g of the final product, B-type dexamethasone, with a crystallization yield of 91%. The purity of the product was determined to be 99.5% by HPLC. Its X-ray powder diffraction pattern showed obvious characteristic peaks of B-type crystal at 2θ of 8.2°, 12.5°, 16.3°, 20.1°, and 24.6°.

[0025] Example 2 Using 15.0 g of 16-dehydropregnane propionate as a starting material, dehydrogenation was catalyzed by steroidal 1,4-dehydrogenase for 13 hours at pH 7.1 and 34°C. Post-treatment yielded Δ 1 , 4 , 16 13.7 g of -pregnatrin-3β-ol-20-ketopropionate was obtained, with a yield of 91.3%. 13.5 g of this product was then subjected to methylation in DMSO at 57°C under nitrogen protection for 4.5 h, with 0.11 equivalents of 2,2'-bipyridine used. Purification yielded 16α-methyl-Δ 1 , 4 11.0 g of -pregnadien-3β-ol-20-ketopropionate, yield 81.5%, purity 98.2%; 10.0 g of this product was hydroxylated using an immobilized CYP11B1 monooxygenase that had been reused 5 times for 23 hours to give 16α-methyl-11β-hydroxy-Δ 1 , 48.6 g of pregnadien-3β-ol-20-ketopropionate was obtained, with a yield of 86%. Subsequently, ring-opening epoxidation and fluorination were performed. The epoxidation was carried out at 4°C, and the fluorination reaction was carried out for 2.8 hours, yielding 6.7 g of free dexamethasone base. The two-step yield was 78.1%. Finally, directional crystallization was performed under the conditions of isopropanol-water volume ratio of 2.8:1, containing 0.4% malic acid, a crystallization endpoint temperature of 5.5°C, and a cooling rate of 0.7°C / hour, yielding 6.1 g of B-type dexamethasone, with a crystallization yield of 91% and a purity of 99.3%.

[0026] Example 3 Using 8.0 g of 16-dehydropregnane propionate as a raw material, at pH 7.3, 36 degrees Celsius, and NAD... + The reaction was carried out for 11 hours at a concentration of 0.55 mmol, yielding Δ 1 , 4 , 16 7.3 g of pregnathitrien-3β-ol-20-ketopropionate was obtained, with a yield of 91.3%. 7.0 g of this product was then subjected to methylation at 53°C for 5.5 hours, followed by purification to obtain 16α-methyl-Δ 1 , 4 5.8 g of pregnadien-3β-ol-20-ketopropionate was obtained, with a yield of 82.9% and a purity of 98.8%. 5.5 g of this product was subjected to hydroxylation for 25 hours to yield 16α-methyl-11β-hydroxy-Δ 1 , 4 4.9 g of pregnadien-3β-ol-20-ketopropionate was obtained, with a yield of 89.1%. Subsequently, ring-opening epoxidation and fluorination were performed. The fluorination reaction was carried out at 27°C for 2.2 hours to obtain 3.9 g of free dexamethasone base, with a two-step yield of 79.6%. Finally, directional crystallization was performed under the conditions of isopropanol-water volume ratio of 3.2:1, containing 0.6% malic acid, a crystallization endpoint temperature of 4.5°C, and a cooling rate of 0.9°C / hour. 1.5% seed crystals were added to obtain 3.6 g of dexamethasone in crystal form B, with a crystallization yield of 92.3% and a purity of 99.6%.

[0027] Comparative Example 1 This comparative example aims to illustrate the crucial influence of the crystallization solvent system on the crystal form orientation in step five. Following the steps of Example 1, 4.5 g of free dexamethasone base was prepared and dissolved in 300 mL of pure isopropanol (i.e., without the addition of water and malic acid). After dissolving at 50°C, 2% seed crystals were added, and the temperature was programmed to decrease to 5°C at a rate of 0.8°C per hour for 6 hours to allow crystal growth. After filtration and drying, X-ray powder diffraction analysis of the obtained product showed main peaks at 2θ of 7.8°, 11.9°, and 15.7°, consistent with the standard spectrum of dexamethasone crystal form A, while the characteristic peaks of crystal form B were absent. This result indicates that without a mixed solvent system consisting of isopropanol-water in a specific ratio (3:1) and malic acid, directional crystallization towards crystal form B cannot be achieved, ultimately resulting in the undesirable product of crystal form A.

[0028] Comparative Example 2 This comparative example aims to illustrate the importance of the 2,2'-bipyridine ligand in step two. The methylation reaction was carried out according to the procedure in Example 1, but only potassium methyltrifluoroborate and copper divalent acetate were added to the reaction system, completely omitting the 2,2'-bipyridine ligand, while keeping all other conditions unchanged. After 5 hours of reaction, TLC monitoring showed a significant amount of reactant remaining. Post-processing and column chromatography purification revealed two signal peaks at the chemical shift of the methyl group at C-16, with a peak area ratio of approximately 3:2. This indicates the formation of a mixture of α-methyl and β-methyl groups, rather than the target single 16α-methyl stereoisomer. The calculated yield was only 45%, and the product purity was only 89.2%. This result demonstrates that without the 2,2'-bipyridine ligand, copper acetate cannot effectively catalyze the formation of a highly stereoselective α-methylation reaction, resulting in a diastereomeric mixture of products, which severely affects subsequent steps and the quality of the final product.

Claims

1. A process for the selective preparation of a B crystalline form of dexamethasone, characterized in that, The method comprises the following steps: Step one: 16-dehydropregnenolone propionate as the starting material, catalyzed by the 1,4-dehydrogenase of Bacillus subtilis to dehydrogenate at 33~37℃, pH 7.1~7.3 for 10~14 hours to obtain Δ 1 , 4 , 16 -pregnatrien-3β-ol-20-one propionate; Step two: the Δ 1 , 4 , 16 pregnanediene-3β-ol-20-one propionate is stereoselectively introduced with an α-methyl group by a potassium methyltrifluoroborate, copper acetate and 2,2-bipyridine system at a temperature of 52-58°C for 4-6 hours to obtain 16α-methyl-Δ 1 , 4 pregnanediene-3β-ol-20-one propionate; Step three: the 16α-methyl-Δ 1 , 4 - pregninolone-20-one propionate is catalyzed by immobilized CYP11B1 monooxygenase to introduce 11β-hydroxyl, and the reaction is carried out for 22-26 hours to obtain 16α-methyl-11β-hydroxy-Δ 1 , 4 - pregninolone-20-one propionate; Step four: the 16α-methyl-11β-hydroxy-Δ 1 , 4 pregnadien-3β-ol-20-one propionate is subjected to epoxidation with oxalic acid at a temperature of 0-5°C for 1.5-2.5 hours, and then subjected to ring opening with hydrogen fluoride-ethanol at a temperature of 23-27°C for 2-3 hours to obtain dexamethasone free base; Step five: isopropanol and water are mixed in a volume ratio of 2.5:1~3.5:1 to obtain a mixed solution A, then 0.3%~0.7% of malic acid by mass of the mixed solution A is added to form a system solution, and the dexamethasone free base is added to the system solution at a temperature of 4~6℃ to select crystallization for 5~7 hours to obtain the B crystal form dexamethasone.

2. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: The 16-dehydropregnenolone propionate is prepared by esterification of diosgenin with propionic anhydride and elimination reaction catalyzed by sodium hydroxide; and the specific steps include: The diosgenin with a purity of 97%~99% is pretreated: the diosgenin is crushed to 70~90 mesh particles, and is extracted with petroleum ether under reflux at a temperature of 60~90℃ for 1.5~2.5 hours to remove fat-soluble impurities, and is dried after filtration to a moisture content of 0.3%~0.7%; then the diosgenin is cleaned with 65%~75% ethanol aqueous solution under ultrasonic for 20~50 minutes to remove water-soluble impurities, and is dried under vacuum after filtration; The esterification reaction of propionic anhydride: the pretreated diosgenin is mixed with propionic anhydride at a molar ratio of 1:2.5~1:3.5, and an equal amount of 4%~6% of the catalyst pyridine is added; the reaction is carried out under oil bath reflux at a temperature of 105~115℃ for 5~7 hours, and the stirring speed is 180~220 revolutions per minute; then the reaction solution is cooled to room temperature, 4~6 times the volume of ice water is slowly added for quenching, and after stirring for 25~35 minutes, filtration is carried out, the filter cake is washed with ice water for 2~4 times until the pH is 6.5~7.5, and the esterification product is obtained by vacuum drying; The elimination reaction catalyzed by sodium hydroxide: the esterification product is dissolved in 8~12 times the mass of anhydrous ethanol, 15%~25% of sodium hydroxide aqueous solution is added, and the molar ratio is 1.8~2.2 times that of the esterification product; the reaction is carried out under reflux at a temperature of 75~85℃ in a water bath environment for 3~5 hours, and the stirring speed is 130~170 revolutions per minute; The reaction solution is cooled to room temperature, adjusted to pH 4.5~6.5 with hydrochloric acid, concentrated under reduced pressure to 25%~35% of the original volume, extracted with 2.5~3.5 times the volume of ethyl acetate, washed with saturated brine for 1~3 times, dried over anhydrous sodium sulfate, and concentrated to obtain the crude 16-dehydropregnenolone propionate; The crude product is recrystallized by adding to a volume ratio of 0.8:1~1.2:1 of ethanol and water, vacuum dried after filtration, and the pure product is obtained.

3. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: The reaction conditions of the steroidal 1,4-dehydrogenase in step one are a temperature of 33~37℃, pH 7.1~7.3, NAD positive ion concentration of 0.4~0.6 millimole, and reaction time of 10~14 hours; The preparation method of the steroidal 1,4-dehydrogenase includes the following steps: The Bacillus subtilis is fermented in a culture medium, and then the fermentation liquid is obtained by filtration; The fermentation liquid is centrifuged to obtain the supernatant, and the supernatant is subjected to ammonium sulfate fractionation precipitation, dialysis, and ion exchange chromatography purification to obtain the steroidal 1,4-dehydrogenase with a specific activity of 95~105 units per milligram; The reaction system preparation includes: 45~55 millimoles of Tris-HCl are used to form a buffer solution, and the buffer solution has a pH of 7.1~7.3 and contains 8~12 millimoles of magnesium chloride; The 16-dehydropregnenolone propionate is configured into a dilution solution with a concentration of 8-12 grams per liter, and then added to the dilution solution according to an enzyme dosage of 4-6 units per milligram of substrate, while adding glucose dehydrogenase according to 1.5-2.5 units per milligram of substrate and adding glucose as a coenzyme regeneration system according to 8-12 grams per liter; The reaction conditions include: the reaction temperature is maintained at 33-37℃ through water bath constant temperature control, and the pH is adjusted to 7.1-7.3 in real time by using 0.05-0.15 moles of sodium hydroxide or hydrochloric acid; the stirring speed is 110-130 revolutions per minute; After the reaction is completed, enzyme protein is removed by using a 0.20-0.25 micrometer ultrafiltration membrane; the filtrate is extracted with ethyl acetate for 2-4 times, the organic phase is combined and washed with saturated brine for 1-3 times, and anhydrous sodium sulfate is used for drying; The residue was concentrated under reduced pressure to dryness to give Δ 1 , 4 , 16 - Pregn-5-ene-3β-ol-20-one propionate crude; The crude product is recrystallized with isopropyl alcohol to obtain a pure product.

4. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: In the methylation reaction in step two, dimethyl sulfoxide is used as the solvent, the reaction temperature is 52-58℃, and the reaction time is 4-6 hours, wherein the conditions of the methylation reaction in step two include: Reaction system configuration: with Δ 1 , 4 , 16 - Pregnane-3β-ol-20-ketopropionate as the substrate, 1.1~1.3 times of the substrate molar amount of potassium methyl trifluoroborate, 0.08~0.12 times of the substrate molar amount of copper divalent acetate, 0.08~0.12 times of the substrate molar amount of 2,2-bipyridine; The solvent is anhydrous dimethyl sulfoxide, and the amount used is 8-12 times the mass of the substrate; The reaction conditions are stirring at 52-58℃ for 4-6 hours under a nitrogen atmosphere, and the stirring speed is 160-200 revolutions per minute; After the reaction liquid is cooled to room temperature, it is quenched with a saturated ammonium chloride solution, extracted with ethyl acetate for 2-4 times, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then the target product is obtained through silica gel column chromatography purification with a purity of 97%-99%; the silica gel column chromatography uses petroleum ether-ethyl acetate with a volume ratio of 4:1-6:1 to 2:1-4:1 as the mobile phase for gradient elution.

5. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: The amount of 2,2-bipyridine used in step two is Δ 1 , 4 , 16 - 8% to 12% of the molar amount of the pregnatriene-3β-ol-20-one propionate, the amount of 2,2-bipyridine used is 8% to 12% of the molar amount of the substrate, and it is added to the reaction system after being premixed with copper divalent acetate for 4 to 6 minutes.

6. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: In step three, the carrier of the immobilized CYP11B1 monooxygenase is sodium alginate-chitosan composite gel beads, and the enzyme loading amount is 45-55 units per gram of carrier; The immobilized carrier is sodium alginate-chitosan composite gel beads, and the preparation steps include: A 1.5%-2.5% (w / v) sodium alginate solution is mixed with a 0.8%-1.2% (w / v) chitosan acetic acid solution at a volume ratio of 2.5:1-3.5:1, and then dropped into a 1.5%-2.5% calcium chloride solution for solidification for 25-35 minutes to obtain composite gel beads with a diameter of 0.8-2.2 millimeters; The CYP11B1 enzyme with a specific activity of 45-55 units per milligram is dissolved in a 45-55 millimolar phosphate buffer at pH 7.2-7.6, and then adsorbed on the composite gel beads through oscillation for 10-14 hours at 23-27℃; Crosslinking is performed using a 0.4%-0.6% glutaraldehyde solution for 0.8-1.2 hours, and then the immobilized enzyme is obtained after washing, with an enzyme loading amount of 45-55 units per gram of carrier and a repeated use number of 4-6 times.

7. The process according to claim 1 for the selective preparation of the B crystalline form of dexamethasone, characterized by the fact that: In step three, the coenzyme regeneration system is glucose dehydrogenase and 8-12 grams per liter of glucose, and the reaction time is 22-26 hours.

8. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: In step four, the temperature of the epoxidation reaction is 0-5℃, and the amount of peroxylene benzoic acid used is 1.1-1.3 times the molar amount of the intermediate; the epoxidation reaction conditions include: The substrate is 16α-methyl-Δ 1 , 4 - pregnadiene-3β-ol-20-one propionate, 1.1 to 1.3 times the molar amount of perbenzoic acid is added; The solvent is anhydrous dichloromethane, and the amount used is 7-9 times the mass of the substrate; The reaction conditions are stirring at 0-5℃ for 1.5-2.5 hours; After the reaction liquid is cooled to room temperature, it is quenched with a saturated ammonium chloride solution, extracted with ethyl acetate for 2-4 times, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then the target product is obtained through silica gel column chromatography purification with a purity of 97%-99%; the silica gel column chromatography uses petroleum ether-ethyl acetate with a volume ratio of 4:1-6:1 to 2:1-4:1 as the mobile phase for gradient elution. Work-up: quenched with 8% to 12% sodium sulfite solution, washed with saturated sodium bicarbonate solution, dried and concentrated to give the epoxide intermediate with 95% to 97% purity.

9. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: The fluorination reagent in step four is hydrogen fluoride-ethanol with a volume ratio of 1:5 to 1:7, the reaction time is 2 to 3 hours, and the temperature is 23 to 27°C; the fluorination reagent is hydrogen fluoride-ethanol solution with a volume ratio of 1:5 to 1:7, the reaction conditions are stirring at 23 to 27°C for 2 to 3 hours, the work-up is neutralization with 15% to 25% sodium hydroxide solution to pH 6.5 to 8.5, and the fluoro-intermediate is obtained by ethyl acetate extraction with a fluorine content of 3.6% to 4.0%.

10. The process for the selective preparation of crystalline Form B dexamethasone according to claim 1, characterized in that: The crystallization temperature in step five is 4 to 6°C, the cooling rate is 0.6 to 1.0°C per hour, and the stirring speed is 130 to 170 revolutions per minute; The crystallization conditions include: Solvent system: isopropyl alcohol-water with a volume ratio of 2.5:1 to 3.5:1, containing 0.3% to 0.7% malic acid; Dissolution: dexamethasone free base with a purity of 93% to 97% is dissolved in the solvent system, the concentration is 13 to 17 grams per liter, and heating is carried out at 48 to 52°C until clear; Cooling: cooling at a rate of 0.6 to 1.0°C per hour to 4 to 6°C, stirring speed is 130 to 170 revolutions per minute, and the crystal is maintained for 5 to 7 hours; Product: B-form dexamethasone with a purity of 98% to 100%.