Chemical synthesis method of 20 alpha-hydroxyprogesterone

Abstract

The invention provides a chemical synthesis method of 20 alpha-hydroxyprogesterone, and belongs to the technical field of organic synthesis. The invention provides a simple and efficient synthesis path, and high-purity 20 alpha-hydroxyprogesterone can be obtained with good yield and extremely high stereoselectivity and specificity. The method is easy and convenient to operate and good in reproducibility, and effectively overcomes the defects that a product is an isomer mixture, separation and purification are difficult, and the target product yield is low in a traditional method. The successful implementation of the invention provides a stable and reliable material source for obtaining sufficient high-purity 20 alpha-hydroxyprogesterone, powerfully supports the subsequent activity research, dosage form development and new drug creation of the compound, and has important significance in the field of health drugs for women, health protection and health protection. The compound shows important application value and potential in the field of research and development of novel progesterone drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis, and particularly relates to a chemical synthesis method of 20alpha-hydroxyprogesterone. BACKGROUND

[0002] Progesterone, as a key natural progestogen, plays an indispensable physiological role in maintaining female pregnancy, regulating menstrual cycle and protecting endometrium. It is widely used in the prevention and treatment of threatened abortion, abnormal uterine bleeding and related gynecological diseases in clinic. However, progesterone itself has limitations such as low oral bioavailability and rapid inactivation in liver, which to some extent restricts the maximization of its clinical application effect.

[0003] 20-hydroxyprogesterone is one of the main metabolites of progesterone in vivo, and the carbonyl group on the 20-position carbon of the progesterone molecule is reduced to a hydroxyl group, thereby forming a pair of diastereoisomers of 20alpha-hydroxyprogesterone and 20beta-hydroxyprogesterone. Preliminary studies have shown that such metabolites not only retain part of the progestogenic biological activity, but also introduce an easily further chemically modified hydroxyl functional group into the molecule, providing an important structural basis for the development of new progestin derivatives. In particular, the 20alpha-isomer, whose biological activity spectrum is closer to that of progesterone itself, is believed to have potential research and development value in neuroprotection, regulation of reproductive endocrine and the like.

[0004] Therefore, establishing a synthesis process capable of directing and efficiently preparing high-purity 20alpha-hydroxyprogesterone becomes a key prerequisite for promoting the compound from basic research to practical application. Existing chemical synthesis routes either rely on multi-step transformation from natural progesterone or use the strategy of reduction and selective oxidation, but these methods generally face problems such as complicated steps, unsatisfactory total yield, mixture of alpha and beta isomers in the final product with an unsatisfactory ratio, and high cost of isomer separation and purification. These technical bottlenecks make it difficult to obtain 20alpha-hydroxyprogesterone standard or raw material meeting the purity and quantity required for in-depth pharmacological and clinical research on a large scale, which seriously hinders the comprehensive exploration of its biological activity and the full exploitation of its potential medicinal value. SUMMARY

[0005] The present application aims to provide a chemical synthesis method of 20alpha-hydroxyprogesterone, so as to solve the technical problem that high-purity 20alpha-hydroxyprogesterone cannot be efficiently and selectively synthesized by the prior art.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a chemical synthesis method of 20alpha-hydroxyprogesterone, which comprises the following steps: In a reaction system consisting of an azo reagent, a phosphine reagent and an organic solvent, 20β-hydroxyprogesterone is subjected to an esterification reaction with a nucleophile to generate a 20α-hydroxyprogesterone-carboxylic acid ester intermediate; Subsequently, the 20α-hydroxyprogesterone-carboxylic acid ester intermediate is subjected to a hydrolysis reaction in an alkaline alcohol solution to obtain 20α-hydroxyprogesterone.

[0007] Preferably, the nucleophile is benzoic acid.

[0008] Preferably, the nucleophile is p-nitrobenzoic acid.

[0009] Preferably, the azo reagent is N,N,N',N'-tetramethylazodicarbonamide.

[0010] Preferably, the phosphine reagent is tributylphosphine.

[0011] Preferably, the organic solvent for the esterification reaction is benzene.

[0012] Preferably, the azo reagent is diethyl azodicarboxylate, the phosphine reagent is triphenylphosphine, and the organic solvent is tetrahydrofuran.

[0013] Preferably, the reaction temperature for the esterification reaction is 20-80℃. The reaction time for the esterification reaction is 20-30 hours. The base in the alkaline alcohol solution is potassium carbonate. The alcohol in the alkaline alcohol solution is methanol. The reaction temperature for the hydrolysis reaction is 40-80℃. The reaction time for the hydrolysis reaction is 1-3 hours. In the hydrolysis reaction, the reaction concentration of the 20α-hydroxyprogesterone-carboxylic acid ester intermediate is 0.01-1M.

[0014] Preferably, in the esterification reaction, the molar ratio of 20β-hydroxyprogesterone, azo reagent, phosphine reagent and nucleophile is 1:1.0-1.5:1.0-1.5:1.0-1.5.

[0015] Preferably, after the hydrolysis reaction, the reaction solution is extracted with ethyl acetate, dried with anhydrous sodium sulfate, and concentrated to obtain the final product 20α-hydroxyprogesterone.

[0016] The present application has the following advantages: The present application provides a simple and efficient synthesis path, which can obtain high-purity 20alpha-hydroxy progesterone with good yield and extremely high stereoselectivity. The method is simple to operate and has good reproducibility, and effectively overcomes the defects of traditional methods, such as product isomer mixture, difficult separation and purification, low yield of target product, etc. The successful implementation of the present application provides a stable and reliable material source for obtaining sufficient high-purity 20alpha-hydroxy progesterone, and strongly supports the subsequent activity research, dosage form development and new drug creation of the compound, and shows important application value and potential in the research and development field of female health drugs, especially new progestin drugs. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Synthesis route map of 20alpha-DHP-carboxylate; Figure 2 Synthesis route map of 20alpha-DHP; Figure 3 Nuclear magnetic resonance hydrogen spectrum comparison chart of 20-DHP-carboxylate key characteristic peaks, wherein: a: peak position of C-19 methyl proton in 20-DHP structure; b, c: peak position of C-20 hydroxyl proton in 20-DHP structure before and after reaction; Figure 4 Nuclear magnetic resonance hydrogen spectrum chart of Mitsunobu reaction system under different reaction temperature conditions; Figure 5 High performance liquid chromatogram of hydrolysis reaction system under different reaction temperatures, wherein: a: 20alpha-DHP standard; b: 20beta-DHP standard; c: 40 DEG C; d: 60 DEG C; e: 80 DEG C; f: 100 DEG C; g: 120 DEG C; h: 150 DEG C; Figure 6 Nuclear magnetic resonance hydrogen spectrum chart of 20beta-DHP; Figure 7 Nuclear magnetic resonance carbon spectrum chart of 20beta-DHP; Figure 8 Infrared spectrum chart of 20beta-DHP; Figure 9 Mass spectrum chart of 20beta-DHP; Figure 10 Nuclear magnetic resonance hydrogen spectrum chart of 20alpha-DHP-BA; Figure 11 Nuclear magnetic resonance carbon spectrum chart of 20alpha-DHP-BA; Figure 12 Infrared spectrum chart of 20alpha-DHP-BA; Figure 13 Mass spectrum chart of 20alpha-DHP-BA; Figure 14 The 1H NMR spectrum of 20α-DHP-PNB is shown. Figure 15 The image shows the carbon NMR spectrum of 20α-DHP-PNB. Figure 16 The infrared spectrum of 20α-DHP-PNB; Figure 17 The mass spectrum of 20α-DHP-PNB is shown. Figure 18 The image shows the 1H NMR spectrum of 20α-DHP. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Materials and methods used in the embodiments Instruments: SHBⅢ circulating water multi-purpose vacuum pump (Zhengzhou Changcheng Science & Industry Trade Co., Ltd.), 85-2 temperature-controlled magnetic stirrer (Jintan Medical Instrument Factory), UV-2550 ultraviolet spectrophotometer (Shimadzu Corporation, Japan), AB-135S electronic analytical balance (Mettler-Toledo GmbH, Germany), DF32 infrared drying oven (Beijing Xingzheng Instrument Equipment Factory), IRFressige 21 Fourier transform infrared spectrophotometer (Shimadzu Corporation, Japan), WATERS 2696-2487-2998 high-performance liquid chromatograph (equipped with diode array detector and fluorescence detector, Waters Corporation, USA), AVANCE NEO600 superconducting nuclear magnetic resonance spectrometer (Bruker AG, Switzerland), ThermoFisher Scientific Ultimate 3000 / Q-Exactive ultra-high performance liquid chromatography / high resolution mass spectrometry / mass spectrometry system (Thermo Fisher Scientific), laboratory ultrapure water system (Xiamen Shuhuoquan Artificial Intelligence Co., Ltd.)

[0020] Reagents: 20β-hydroxyprogesterone (20β-DHP, Toronto Research Chemicals, Canada, purity ≥ 98%), 20α-hydroxyprogesterone (20α-DHP, TLC Pharma Chem, USA, purity ≥ 99%), tributylphosphine (TBP, Shanghai Maclean's Co., Ltd.), benzoic acid (PhCOOH, BA, Shanghai Maclean's Co., Ltd.), N,N,N',N'-tetramethylazodicarbonamide (TMAD, Shanghai Shaoyuan Reagent Co., Ltd.), p-nitrobenzoic acid (4-Nitrobenzoic acid, p-O2N-PhCOOH, PNB, Shanghai Aladdin Co., Ltd.), triphenylphosphine (TPP, Shanghai Merrill Co., Ltd.), tetrahydrofuran (THF, Shanghai Maclean Co., Ltd.), benzene (Benzene, Shanghai Maclean Co., Ltd.), diethyl azodicarbonate (DEAD, Shanghai Aladdin Co., Ltd.), anhydrous potassium carbonate (K2CO3, Tianjin Beilian Fine Chemicals Development Co., Ltd.), methanol (MeOH, Shanghai Aladdin Co., Ltd.), petroleum ether (PE, Beijing Innocare Technology Co., Ltd.), ethyl acetate (EA, Beijing Innocare Technology Co., Ltd.). All reagents were of analytical grade; the chromatographic reagents were methanol and acetonitrile (Sigma-Aldrich (Shanghai) Trading Co., Ltd.). Example 1

[0021] 20α-hydroxyprogesterone was synthesized using TMAD, TBP, benzene, and benzoic acid as the reaction system. N,N,N',N'-Tetramethylazodicarbonamide (TMAD, 1.5 equivalents) and tributylphosphine (TBP, 1.5 equivalents) were dissolved in 2 mL of benzene, and the mixture was stirred in an ice bath for 5 minutes until the reaction system decolorized. Benzoic acid (BA, 1.5 equivalents) was then added, and the mixture was stirred at room temperature for 20 minutes. Subsequently, 20β-hydroxyprogesterone (1.0 equivalent, 1.6 mmol) was added, and the reaction was carried out at 60 °C for 24 hours. After the reaction was complete, the reaction system was concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography using a petroleum ether:ethyl acetate ratio of 3:1 to give the 20α-hydroxyprogesterone-benzoic acid ester intermediate.

[0022] Take 20 mg of the 20α-hydroxyprogesterone-benzoate intermediate obtained in the previous step and dissolve it in 2 mL of methanol. Add potassium carbonate (11 equivalents, 0.192 mmol) and react at 60 °C for 2 hours. After the reaction is complete, add 10 mL of ethyl acetate to the reaction system and wash successively with distilled water (10 mL × 2) and saturated sodium chloride solution (10 mL × 1). After drying the organic phase with anhydrous sodium sulfate, concentrate by rotary evaporation to obtain the final product 20α-hydroxyprogesterone. Example 2

[0023] 20α-hydroxyprogesterone was synthesized using TMAD, TBP, benzene, and p-nitrobenzoic acid as the reaction system. N,N,N',N'-Tetramethylazodicarbonamide (TMAD, 1.5 equivalents) and tributylphosphine (TBP, 1.5 equivalents) were dissolved in 2 mL of benzene, and the mixture was stirred in an ice bath for 5 minutes until the reaction system decolorized. Then, p-nitrobenzoic acid (PNB, 1.5 equivalents) was added, and the mixture was stirred at room temperature for 20 minutes. Subsequently, 20β-hydroxyprogesterone (1.0 equivalent, 1.6 mmol) was added, and the reaction was carried out at 60 °C for 24 hours. After the reaction was complete, the reaction system was concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain the 20α-hydroxyprogesterone-p-nitrobenzoate intermediate.

[0024] Take 20 mg of the 20α-hydroxyprogesterone-p-nitrobenzoate intermediate obtained in the previous step and dissolve it in 2 mL of methanol. Add potassium carbonate (11 equivalents, 0.192 mmol) and react at 60 °C for 2 hours. After the reaction is complete, add 10 mL of ethyl acetate to the reaction system and wash successively with distilled water (10 mL × 2) and saturated sodium chloride solution (10 mL × 1). After drying the organic phase with anhydrous sodium sulfate, concentrate by rotary evaporation to obtain the final product 20α-hydroxyprogesterone. Example 3

[0025] 20α-hydroxyprogesterone was synthesized using DEAD, TPP, THF, and benzoic acid as the reaction system. Diethyl azodicarbonate (DEAD, 1.0 equivalent) and triphenylphosphine (TPP, 1.0 equivalent) were dissolved in 2 mL of tetrahydrofuran (THF) and stirred in an ice bath for 5 minutes. Benzoic acid (BA, 1.0 equivalent) was then added, and the mixture was stirred at room temperature for 20 minutes. Subsequently, 20β-hydroxyprogesterone (1.0 equivalent, 1.6 mmol) was added, and the reaction was carried out at room temperature for 24 hours. After the reaction was complete, the reaction mixture was concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography using a petroleum ether:ethyl acetate ratio of 3:1 to give the 20α-hydroxyprogesterone-benzoate intermediate.

[0026] Take 20 mg of the 20α-hydroxyprogesterone-benzoate intermediate obtained in the previous step and dissolve it in 2 mL of methanol. Add potassium carbonate (11 equivalents, 0.192 mmol) and react at 60 °C for 2 hours. After the reaction is complete, add 10 mL of ethyl acetate to the reaction system and wash successively with distilled water (10 mL × 2) and saturated sodium chloride solution (10 mL × 1). After drying the organic phase with anhydrous sodium sulfate, concentrate by rotary evaporation to obtain the final product 20α-hydroxyprogesterone. Example 4

[0027] The synthetic method provided by this invention utilizes the Mitsunobu reaction, in the presence of an azo-phosphine reagent, to invert the configuration of 20β-DHP, which then undergoes esterification with a nucleophile to generate the intermediate 20α-DHP-carboxylic acid ester. Subsequently, under alkaline conditions, the 20α-DHP-carboxylic acid ester is hydrolyzed to yield the final product 20α-DHP.

[0028] The synthetic route for 20α-DHP-carboxylic acid esters is as follows: Figure 1 As shown. Azo and phosphine reagents were dissolved in 2 mL of solvent and stirred in an ice bath for 5 min until the reaction system decolorized. Then, a nucleophile (carboxylic acid) was added, and the mixture was stirred at room temperature for 20 min. Subsequently, 20β-DHP (1 eq, 1.6 mmol) was added, and the reaction was allowed to proceed for 24 h. After the reaction was complete, the reaction system was evaporated to dryness to obtain the crude product. 20α-DHP-carboxylic acid ester was obtained by silica gel column chromatography, with PE-EA as the elution system. Elution was monitored by TLC under the following conditions: PE : EA = 3 : 1 (v / v). Colorimetric conditions: UV 245 nm; colorimetric reagent: iodine (I₂).

[0029] To improve yield, the types of synthetic reagents were investigated in this invention. Nucleophiles included BA and PNB; azo-phosphine reagent combinations included DEAD-TPP and TMAD-TBP; the solvent used with DEAD-TPP was THF, while the solvent used with TMAD-TBP was Benzene. Specific combination schemes are shown in Table 1.

[0030] Table 1 Screening of reagents for the synthesis of 20α-DHP-carboxylic acid esters

[0031] a: TMAD (1.5 eq), TBP (1.5 eq), nucleophile (1.5 eq), 60℃, 24 h; b: DEAD (1 eq), TPP (1 eq), nucleophile (1 eq), rt, 24 h.

[0032] Subsequently, based on the determination of the types of nucleophiles and azo-phosphine reagents, the reaction temperatures (60 ℃, 80 ℃, 120 ℃) ​​were screened.

[0033] The product 20α-DHP-benzoate (20α-DHP-BA) was hydrolyzed using K2CO3 / MeOH to obtain the final product 20α-DHP. The synthetic route is as follows: Figure 2 As shown.

[0034] To improve the yield, this invention investigated the effects of reaction temperature, reaction time, and solvent addition on the yield of 20α-DHP. The specific operation was as follows: A certain volume (0.5 mL, 1 mL, 2 mL, 4 mL, 6 mL) of MeOH was added to 20 mg of 20α-DHP-carboxylic acid ester. After dissolution, K₂CO₃ (11 eq, 0.192 mmol) was added, and the reaction was carried out at certain temperatures (40 ℃, 60 ℃, 80 ℃, 100 ℃, 120 ℃, 150 ℃) for certain times (1 h, 2 h, 3 h). After the reaction was completed, 10 mL of EA was added to the reaction system, and the mixture was washed successively with distilled water (10 mL*2) and saturated NaCl solution (10 mL*1). The organic phase was dried over anhydrous Na₂SO₄, evaporated to dryness, and concentrated to obtain the final product. TLC conditions: PE : EA = 2 : l (v / v). Colorimetric conditions: UV 245 nm; colorimetric reagent: iodine (I₂).

[0035] Determination of product structure Infrared spectroscopy analysis Weigh an appropriate amount of sample and determine the infrared spectrum (IR) using the KBr pellet method.

[0036] High-resolution mass spectrometry analysis Weigh an appropriate amount of the sample and dissolve it in anhydrous ethanol. Measure the sample using high-resolution mass spectrometry (HRMS) in positive ion mode.

[0037] Nuclear magnetic resonance spectroscopy analysis and determination of the α / β configuration ratio dr(α / β) of the intermediate product Weigh 5 mg and 20 mg of the intermediate products obtained after the Mitsunobu reaction, dissolve them separately in 0.5 mL of deuterated chloroform, and determine the 1H NMR spectrum (1H NMR spectrum). 1 H-NMR and carbon spectroscopy (H-NMR) 13 C-NMR).

[0038] The protons at the same position in the chemical structures of the α-configuration intermediate and the β-configuration intermediate are... 1 The ratio of the integral area in H-NMR is the dr(α / β) value.

[0039] High-performance liquid chromatography (HPLC) analysis of 20α-DHP and 20β-DHP and determination of the final product dr(α / β) value The peak areas of 20α-DHP and 20β-DHP in the final product were determined by HPLC analysis, and the α / β peak area ratio was given as dr(α / β) value.

[0040] Chromatographic conditions: Inertsil ODS-3 column (250 mm × 4.6 mm, 5 μm), isocratic elution, mobile phase: methanol:acetonitrile:water = 35:35:30 (v / v / v), flow rate: 0.8 mL / min, detection wavelength: 241 nm, injection volume: 10 μL.

[0041] result Screening of synthetic conditions for 20α-DHP-carboxylic acid esters Determination of synthetic reagents Using yield as the evaluation index, the synthetic reagents in the Mitsunobu reaction were screened according to the four schemes in Table 1, and the resulting products... 1 H-NMR such as Figure 3 As shown in the figure. The results indicate that after the four reaction schemes, the hydrogen proton signal peak at the C-20 position (δ 3.73) in the 20β-DHP structure disappeared, while multiple signal peaks appeared at a low field of around δ 5.2. This indicates that 20β-DHP underwent an esterification reaction with the nucleophile BA or PNB, and the resulting product simultaneously possesses the steroidal core structure of 20-DHP and the benzene ring skeleton structure of BA or PNB. This causes the hydrogen proton signal peak at the C-20 position to shift to a lower field under the influence of the aromatic acid conjugated structure. Compared to the signal peak at δ 0.80 for the methyl proton at C-19 in 20β-DHP, this characteristic peak shifts to a lower field in the products of schemes A, C, and D, with chemical shifts of 0.76, 0.77, and 0.76, respectively, indicating a β-configuration inversion to the α-configuration. The corresponding products are 20α-DHP-benzoic acid ester (20α-DHP-BA), 20α-DHP-p-nitrobenzoic acid ester (20α-DHP-PNB), and 20α-DHP-BA, respectively. However, in scheme B, the product still retains the signal peak at δ 0.80, indicating that no configuration inversion occurred in scheme B, and the product is 20β-DHP-p-nitrobenzoic acid ester (20β-DHP-PNB).

[0042] 1 Preliminary H-NMR results demonstrate that schemes A, C, and D can achieve configuration inversion from β to α, while scheme B cannot. Calculations show that the yields for schemes A, C, and D are 40%, 17%, and 14%, respectively, indicating that compared to PNB, BA can achieve configuration inversion of 20β-DHP while also undergoing esterification to generate 20α-DHP-BA, resulting in a higher yield. Therefore, this invention determines scheme A as the optimal reagent combination, using Benzene as the solvent, and reacting 20β-DHP with the nucleophile BA in the presence of the azo-phosphine reagent TMAD-TBP to generate 20α-DHP-BA.

[0043] Determination of reaction temperature Based on the determined synthetic reagents, this invention investigated the reaction temperature in Scheme A. The products obtained after reacting the system at 60℃, 80℃, and 120℃ for 24 h were... 1 H-NMR such as Figure 4 As shown, the results indicate that when the reaction temperature is 60℃, only the proton signal peak at δ 5.20 appears in the spectrum, suggesting that only the product 20α-DHP-BA is formed at this temperature. Above this temperature, a peak at δ 5.14 appears next to the δ 5.20 peak, indicating that at temperatures above 60℃, 20β-DHP-benzoate (20β-DHP-BA) is present in the reaction system while 20α-DHP-BA is being formed. The dr(α / β) value of the product was calculated based on the peak area ratio of δ 5.20 and δ 5.14, and the results are shown in Table 2. This shows that at a reaction temperature of 60℃, the β configuration in the reaction system is completely inverted to the α configuration, and dr(α / β) = 1. Above 60℃, the byproduct 20β-DHP-BA appears, and the proportion of the byproduct increases with higher temperatures. It can be seen that the diastereoselectivity of this reaction is greatly affected by temperature. Under high temperature conditions, the racemization trend of the α configuration of the product is more obvious. Therefore, this invention selects 60 °C as the optimal reaction temperature for this step.

[0044] Table 2. Effect of reaction temperature on the dr(α / β) value of the products in the Mitsunobu reaction system.

[0045] Screening of 20α-DHP synthesis conditions Determination of reaction temperature 20α-DHP-BA undergoes hydrolysis under alkaline conditions. HPLC chromatograms of the products at different reaction temperatures are shown below. Figure 5 The dr(α / β) ratios of the products, calculated from the HPLC peak areas, are shown in Table 3. Within the temperature range of 40-150℃, dr(α / β) initially increased and then decreased with increasing reaction temperature. Especially after reaching 100℃, the proportion of 20α-DHP in the product decreased significantly, while the highest dr(α / β) ratio of 9:1 was observed at 60℃. This indicates that excessively high or low reaction temperatures will promote racemization of the product to varying degrees, thus hindering the formation of 20α-DHP. Therefore, the optimal reaction temperature for this step was determined to be 60℃.

[0046] Table 3. Effect of reaction temperature on the dr(α / β) value of the products in the hydrolysis reaction system.

[0047] Determination of reaction time Based on a reaction temperature of 60 °C, the effect of reaction time on the diastereoselectivity of this step was investigated. HPLC results (Table 4) showed that dr(α / β) first increased and then decreased with increasing reaction time. At 2 h of reaction, the proportion of 20α-DHP in the product was the highest, reaching 95%. Therefore, the reaction time for this step was determined to be 2 h.

[0048] Table 4. Effect of reaction time on the dr(α / β) value of the products in the hydrolysis reaction system.

[0049] Screening of reaction concentration Based on a reaction temperature of 60 °C and a reaction time of 2 h, this invention investigated the effect of reaction concentration on the hydrolysis reaction dr(α / β), and the results are shown in Table 5. The data show that as the solvent volume of the reaction system increases (0.5 mL-6 mL), the reaction concentration decreases (0.388 M→0.032 M), and the dr(α / β) value first increases and then decreases, rising from 74 / 26 to 95 / 5, and then decreasing to 93 / 7. The highest dr(α / β) corresponds to a reaction concentration of 0.097 M, indicating that the reaction concentration has a significant impact on the dr(α / β) value of this hydrolysis step; concentrations above or below 0.097 M both increase the proportion of 20β-DHP in the product.

[0050] Table 5. Effect of reaction concentration on the dr(α / β) value in the hydrolysis reaction.

[0051] Verification Experiment The optimal reaction conditions were selected according to the above scheme, and 20α-DHP was synthesized to verify the reproducibility of the synthesis method. n =3). HPLC results showed that the dr(α / β) values ​​of the products obtained in the three groups of experiments were 94 / 6, 95 / 5, and 95 / 5, respectively, indicating that the purity of the obtained 20α-DHP was 94.67 ± 0.58%, the hydrolysis yield was 16.7 ± 0.3%, and the RSD was less than 2%, indicating that the method had good reproducibility. The results are as follows: Table 6 Validation Experiment Results

[0052] Structural characterization 20β-DHP IR ν(KBr): 3526, 2947, 2870, 1674, 1612 cm -1 . 1H-NMR (600 MHz, CDCl3) δ 5.73 (s, 1H), 3.73 (dq, J = 9.7, 6.2 Hz, 1H), 2.44−2.31 (m, 4H), 2.27 (ddd, J = 14.6, 4.3, 2.4 Hz, 1H), 2.12 (dt, J = 12.8, 3.4 Hz, 1H), 2.03(ddd, J = 13.4, 5.1, 3.1 Hz, 1H), 1.84 (ddt, J = 12.7, 5.6, 2.6 Hz, 1H), 1.70(d, J = 4.8 Hz, 1H), 1.69−1.62 (m, 2H), 1.55−1.50 (m, 2H), 1.45 (qd, J =13.1, 4.0 Hz, 1H), 1.34 (q, J = 9.3 Hz, 1H), 1.27−1.21 (m, 1H), 1.19 (s, 4H), 1.17 (d, J = 4.8 Hz, 1H), 1.16−1.13 (m, 6H), 1.09−1.02 (m, 2H), 0.99−0.92 (m,1H), 0.80 (s, 3H) . 13 C-NMR(150 MHz, CDCl3) δ 199.57, 171.36, 123.80, 70.14,58.26, 55.64, 53.70, 41.62, 38.59, 38.56, 35.66, 35.21, 33.94, 32.86, 31.94,25.55, 24.05, 23.50, 20.70, 17.35, 12.53. ESI-MS m / z: 317.24 ([M+H] + C 21 H 32 O2 theoretical value 316.24). 20β-DHP 1 H-NMR ( Figure 6 ), 13 C-NMR ( Figure 7 ), IR ( Figure 8 MS ( Figure 9 (See attached image for details.)

[0053] 20α-DHP-BA White powder, yield 40%, IR ν(KBr): 2947, 1706, 1672, 1271, 717 cm⁻¹ -1 . 1 H-NMR(600 MHz, CDCl3) δ 7.64 – 7.51 (m, 2H), 7.46 (dt, J = 28.1, 7.7 Hz, 3H), 5.74(s, 1H), 5.20 (dt, J = 12.5, 6.2 Hz, 1H), 2.41 (ddt, J = 29.5, 17.9, 4.3 Hz,3H), 2.28 (dt, J = 14.2, 3.6 Hz, 1H), 2.07 – 2.00 (m, 1H), 1.95 (ddt, J =23.1, 15.8, 5.1 Hz, 2H), 1.85 (ddd, J = 12.3, 5.6, 2.8 Hz, 1H), 1.70 (qd, J =17.7, 15.3, 8.9 Hz, 4H), 1.60 – 1.54 (m, 2H), 1.49 – 1.40 (m, 2H), 1.39 –1.31 (m, 6H), 1.31 – 1.16 (m, 12H), 1.00 – 0.92 (m, 1H), 0.91 – 0.83 (m, 1H), 0.77 (s, 3H). 13 C-NMR (151 MHz, CDCl3) δ 199.52, 171.22, 165.97, 132.71,129.49, 128.30, 123.88, 77.24, 77.02, 76.81, 73.40, 73.22, 55.79, 55.71,53.80, 41.79, 38.61, 35.29, 33.97, 31.97, 25.40, 24.01, 20.73, 20.70, 19.99,17.37.. ESI-MS m / z: 421.27 ([M+H] + C 28 H 36 O3 (theoretical value: 420.27).

[0054] 1The basic core structures of 20α-DHP and benzoic acid are preserved in the ¹H NMR. The hydrogen at position C-20 of the steroidal ring of 20α-DHP, influenced by the ester bond formed by the condensation of the -COOH group on the benzoic acid structure and the -OH group on the 20β-DHP structure, shifts from δ 3.73 to a lower field, resulting in a single multiplet at δ 5.2. 13 In C-NMR, a characteristic peak of -OCO- appears at δ 171.12, and the peaks at δ 132.72, 130.85, 129.58, 128.35, and 123.85 are signal peaks generated by the five C atoms in the benzene ring structure; in the IR spectrum, at 1706 cm⁻¹... -1 The peak caused by the stretching vibration of -OCO- indicates that 20-DHP is successfully linked to benzoic acid via an ester bond. 20α-DHP-BA 1 H-NMR ( Figure 10 ), 13 C-NMR ( Figure 11 ), IR ( Figure 12 MS ( Figure 13 (See attached image for details.)

[0055] Intermediate product 20α-DHP-PNB Yellow powdery solid, yield 17%. IR ν(KBr): 2958, 2355, 1716, 1525, 1263 cm⁻¹ -1 . 1 H-NMR (600 MHz, CDCl3) δ 8.36 – 8.32 (m, 2H), 8.30 – 8.26 (m, 2H), 8.20 –8.17 (m, 2H), 7.90 – 7.85 (m, 1H), 5.75 – 5.70 (m, 1H), 5.23 (dq, J = 8.6, 6.2 Hz, 1H), 4.32 (q, J = 7.1 Hz, 1H), 4.23 (dh, J= 14.1, 7.2 Hz, 2H), 2.47– 2.33 (m, 4H), 2.33 – 2.25 (m, 1H), 2.06 – 2.00 (m, 1H), 1.86 (ddt, J =15.7, 5.5, 3.3 Hz, 1H), 1.76 – 1.64 (m, 3H), 1.64 – 1.54 (m, 3H), 1.35 – 1.13 (m, 17H), 1.09 – 1.01 (m, 1H), 1.00 – 0.93 (m, 1H), 0.78 (s, 3H). 13 C-NMR (151MHz, CDCl3) δ 171.12, 163.84, 150.49, 136.05, 130.73, 123.58, 74.53, 55.09,53.79, 42.32, 38.57, 33.96, 31.99, 25.41, 24.18, 20.94, 19.95, 17.36, 12.62.ESI-MS m / z: 466.2588 ([M+H] + C 28 H 35 Theoretical value of NO5: 465.25.

[0056] 1 The multiplet at the C20 hydrogen position on the 20-DHP steroid ring in H-NMR shifts to a lower field and appears at δ 5.23; 13 In C-NMR, a characteristic peak of -OCO- appears at δ 171.12; in the IR spectrum, at 1716 cm⁻¹... -1 The presence of a peak caused by the -OCO- stretching vibration confirms the successful ester bond connection between 20-DHP and PNB. 20α-DHP-PNB 1 H-NMR ( Figure 14 ), 13 C-NMR ( Figure 15 ), IR ( Figure 16 MS ( Figure 17 (See attached image for details.)

[0057] 20α-DHP IR ν(KBr): 3526, 2947, 2870, 1674, 1612 cm -1 . 1 H-NMR (600 MHz, CDCl3) δ 5.73 (s, 1H), 3.72 (dq, J= 8.1, 6.2 Hz, 1H), 2.44−2.31 (m, 4H), 2.28 (ddd, J = 14.7, 4.3, 2.4 Hz, 1H), 2.02 (ddd, J = 13.4, 5.0, 3.1 Hz, 1H),1.93 (ddq, J = 12.2, 9.9, 3.2 Hz, 2H), 1.88−1.82 (m, 1H), 1.70 (tdd, J =13.1, 6.5, 3.3 Hz, 2H), 1.63−1.59 (m, 1H), 1.59−1.51 (m, 3H), 1.42 (qd, J =13.2, 4.1 Hz, 1H), 1.34 (q, J = 9.0 Hz, 1H), 1.23 (d, J = 6.2 Hz, 4H), 1.19(s, 4H), 1.15 (dd, J = 13.1, 4.0 Hz, 1H), 1.05 (ddd, J = 10.6, 5.5, 2.8 Hz,2H), 0.94 (ddd, J = 12.3, 10.7, 4.2 Hz, 1H), 0.72 (s, 3H)..20α-DHP 1 See H-NMR spectrum Figure 18 20α-DHP 13 The C-NMR (150 MHz, CDCl3), IR, and ESI-MS m / z data are almost identical to those of 20β-DHP and will not be shown here.

[0058] In summary, this invention provides a novel synthetic method for preparing high-purity 20α-DHP from 20β-DHP as the starting material, via configuration inversion and esterification through the Mitsunobu reaction, followed by mild alkaline hydrolysis. In the first step of the Mitsunobu reaction, 20β-DHP reacts with the nucleophile BA under the action of the azo-phosphine reagent TMAD-TBP at 60°C for 24 h, achieving configuration inversion of the C-20 hydroxyl group and efficiently synthesizing the 20α-DHP-BA intermediate with a yield of 40% and dr(α / β) = 1. In the second step of the hydrolysis reaction, 20α-DHP-BA is hydrolyzed in K2CO3 / MeOH solution. When the reaction concentration is 0.097 M, the reaction is carried out at 60°C for 2 h, yielding a purity of 94.67 ± 0.58% and a yield of 16.7 ± 0.3%, with RSDs all less than 1%, demonstrating good reproducibility.

[0059] As demonstrated by the above embodiments, this invention provides a novel method for the directional synthesis of high-purity 20α-hydroxyprogesterone based on the Mitsunobu-hydrolysis strategy. Through ingenious reaction design, this method successfully achieves efficient and highly selective configurational inversion from 20β-hydroxyprogesterone to 20α-hydroxyprogesterone. Stable and reproducible process conditions were established through optimization of key parameters such as reaction reagents, temperature, time, and concentration. Ultimately, a 20α-hydroxyprogesterone product with a high diastereomeric ratio and excellent chemical purity was successfully obtained. This method has a clear route and is feasible, laying a solid technological foundation for the large-scale preparation of high-purity 20α-hydroxyprogesterone and significantly promoting the subsequent development and application research of this compound.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chemical synthesis method for 20α-hydroxyprogesterone, characterized in that, The method includes the following steps: In a reaction system consisting of an azo reagent, a phosphine reagent, and an organic solvent, 20β-hydroxyprogesterone is esterified with a nucleophilic reagent to generate a 20α-hydroxyprogesterone-carboxylic acid ester intermediate. The 20α-hydroxyprogesterone-carboxylic acid ester intermediate was then hydrolyzed in an alkaline alcohol solution to obtain 20α-hydroxyprogesterone.

2. The method according to claim 1, characterized in that, The nucleophile is benzoic acid.

3. The method according to claim 1, characterized in that, The nucleophile is p-nitrobenzoic acid.

4. The method according to claim 1, characterized in that, The azo reagent is N,N,N',N'-tetramethylazodicarbonamide.

5. The method according to claim 1, characterized in that, The phosphine reagent is tributylphosphine.

6. The method according to claim 1, characterized in that, The organic solvent used in the esterification reaction is benzene.

7. The method according to claim 1, characterized in that, The azo reagent is diethyl azodicarbonate, the phosphine reagent is triphenylphosphine, and the organic solvent is tetrahydrofuran.

8. The method according to claim 1, characterized in that, The reaction temperature for the esterification reaction is 20℃~80℃; The esterification reaction takes 20-30 hours. The alkali in the alkaline alcohol solution is potassium carbonate; The alcohol in the alkaline alcohol solution is methanol; The reaction temperature for the hydrolysis reaction is 40℃~80℃; The hydrolysis reaction takes 1 to 3 hours. In the hydrolysis reaction, the reaction concentration of the 20α-hydroxyprogesterone-carboxylic acid ester intermediate is 0.01 M to 1 M.

9. The method according to claim 1, characterized in that, In the esterification reaction, the molar ratio of 20β-hydroxyprogesterone, azo reagent, phosphine reagent and nucleophilic reagent is 1:1.0-1.5:1.0-1.5:1.0-1.

5.

10. The method according to any one of claims 1 to 9, characterized in that, After the hydrolysis reaction was completed, the reaction solution was extracted with ethyl acetate, dried with anhydrous sodium sulfate, and concentrated to obtain the final product 20α-hydroxyprogesterone.

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