Synthesis method of stable isotope labeled 17alpha-hydroxypregnenolone-2, 3, 4-13C3

Abstract

The invention relates to a synthesis method of stable isotope labeled 17alpha-hydroxypregnenolone 2, 3, 4-13C3, which comprises the following steps: S1, dissolving sodium cyanide in a solvent system, adding a phase transfer catalyst and dehydroepiandrosterone 2, 3, 4-13C3, stirring for catalytic reaction, and filtering to obtain a cyanohydrin intermediate I; s2, dissolving the obtained cyanohydrin intermediate I in another solvent system, adding methyl magnesium bromide under the protection of inert atmosphere, and stirring to carry out Grignard reaction; and S3, after the Grignard reaction in the S2 is finished, adding excessive sulfuric acid aqueous solution, stirring, carrying out hydrolysis reaction, and extracting, washing, drying, carrying out rotary evaporation and purifying the obtained product to obtain the 17alpha-hydroxypregnenolone 2, 3, 4-13C3 product. Compared with the prior art, the purity of the prepared 17alpha-hydroxypregnenolone-2, 3, 4-13C3 product is greater than or equal to 98%, the abundance is greater than or equal to 99atom% 13C, the abundance is not obviously diluted, and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of the synthesis of isotope-labeled products, and relates to a method for synthesizing stable isotope-labeled 17α-hydroxypregnenolone-2,3,4- 13 C3. Background Art

[0002] Steroid hormones are a series of hormones formed from cholesterol in the human body through different enzymes, including neurosteroid hormones, progestogens, androgens, estrogens, corticosteroids, etc., which play an important regulatory role in human growth, development, reproduction, metabolism, etc. Pregnenolone is one of the most abundant neurosteroids and is the precursor of many neurosteroids found in the brain, including dehydroepiandrosterone (DHEA), progesterone, testosterone, estrogen, cortisol, etc. Changes in its content in the human body will directly affect the levels of downstream hormones; in addition, pregnenolone can regulate brain myelination, neuroinflammation, neurotransmission, and neuroplasticity, etc., and its content is closely related to human cognition, aging, addiction, immunity, etc. Therefore, accurately detecting its content has important reference value in the diagnosis of diseases, anti-aging research, immune support research, etc.

[0003] The most commonly used methods for detecting pregnenolone such as 17α-hydroxypregnenolone in traditional clinical medicine are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Due to the large differences in content in the human body (ng / mL - pg / mL), the traditional ELISA and RIA detection methods require a large amount of samples, take a long time, and are limited by the cross-reaction of antibodies, often resulting in inaccurate quantification and false positive results, thus affecting the diagnosis and treatment of related diseases; in addition, the detection of pregnenolone such as 17α-hydroxypregnenolone is prone to confusion with other substances with similar structures in the human body. Therefore, the traditional clinical medicine detection methods cannot meet the detection requirements of high sensitivity and high recognition. In contrast, the clinical stable isotope dilution mass spectrometry (clinical mass spectrometry) is more discriminatory and accurate, and the concentration accuracy can reach pg / mL, which is a sensitivity that cannot be achieved by traditional clinical medicine detection methods. Internationally, clinical mass spectrometry medicine has been vigorously developed as early as 10 years ago and has replaced or partially replaced traditional biochemical diagnostic analysis methods in many clinical detection fields, showing broad development space. 17α-hydroxypregnenolone-2,3,4- 13 C3, this stable isotope-labeled pregnenolone, is the core of the detection of this type of hormone by clinical stable isotope dilution mass spectrometry.

[0004] Currently, detection reagents for pregnenolone represented by 17α-hydroxypregnenolone-2,3,4- 13 C3 cannot be independently synthesized in China, and the related reagents are expensive and in short supply. For example, 17α-hydroxypregnenolone-20,21-13 The price of C2,16,16-d2 is as high as 6,600 yuan / mg. This has severely restricted the scientific research and detection progress of clinical mass spectrometry of steroid hormones such as pregnenolone in China, the exploration of new detection methods, the production of test kits, the expansion of actual clinical detection use, as well as research on anti-aging and immune support, etc.

[0005] There is no literature report on the synthesis process of 17α-hydroxy pregnenolone-2,3,4- 13 C3 at home and abroad. Therefore, only the synthesis reports of natural abundance 17α-hydroxy pregnenolone can be referred to. Two synthesis methods have been reported in the literature on the synthesis of natural abundance 17α-hydroxy pregnenolone using dehydroepiandrosterone as the raw material.

[0006] One is the research by Thiele, M et al. in 2015. Using n-butyllithium as the catalyst, trimethylsilylacetylene reacts with dehydroepiandrosterone to obtain the intermediate (3β,17α)-21-(trimethylsilyl)pregna-5-ene-20-yne-3,17-diol. Then, this intermediate undergoes alkynylhydroxymercuration reaction in a newly prepared mercury sulfate (prepared from mercuric oxide) / sulfuric acid aqueous solution system to form methyl ketone (acetyl group), that is, the Kuchelov Reaction. Then, the TMS group is removed to obtain a hydroxyl group, and finally 17α-hydroxy pregnenolone is obtained. Although the reported yield of this method is relatively high, it uses n-butyllithium as the catalyst with poor reaction stability and requires harsh reaction conditions such as -40°C. The overall reaction operation is complex. Using toxic heavy metal ions to catalyze the reaction is not conducive to environmentally friendly chemistry and also increases the post-treatment cost.

[0007] The other is the research by Schor, Laura et al. in 1990. Using dehydroepiandrosterone as the raw material, it reacts with lithium acetylide ethylenediamine complex in an acetylene gas atmosphere to obtain an alkynediol intermediate. Then, the hydroxyl group is protected with formic acid to obtain a double methyl ester-protected alkynyl intermediate. Then, the double-protected alkynyl intermediate reacts with N-bromoacetamide in a sodium acetate, acetic acid, and water system to obtain a 17α-hydroxy pregnenolone double-protected intermediate. Finally, hydrolysis and deprotection are carried out to obtain 17α-hydroxy pregnenolone. This reaction uses a group protection strategy, involving the protection and deprotection processes of two groups, resulting in a relatively long overall synthesis step, a complex process, and a low yield. The need to use an acetylene atmosphere during the reaction increases the instability of the reaction and the lack of safety. In addition, the lithium acetylide ethylenediamine complex used is expensive, and the raw material cost is relatively high. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3. As the first labeled synthesis method for this kind of pregnenolone hormone, the obtained 17α-hydroxy pregnenolone-2,3,4-13 The purity of the C3 product is ≥98%, and the abundance is ≥99 atom%. 13 For C, no obvious dilution of the abundance occurs, etc.

[0009] The object of the present invention can be achieved by the following technical solutions:

[0010] A method for synthesizing stable isotope-labeled 17α-hydroxypregnenolone-2,3,4- 13 C3, which uses 13 C3 triple-labeled dehydroepiandrosterone as a raw material, and through specific phase transfer catalysis technology, Grignard reaction and cyanoacetylation technology, 17α-hydroxypregnenolone-2,3,4- 13 C3 is innovatively synthesized. Specifically, the synthesis method of the present invention includes the following steps:

[0011] S1. Dissolve sodium cyanide in a solvent system, add a phase transfer catalyst and dehydroepiandrosterone-2,3,4- 13 C3, stir for catalytic reaction, and filter to obtain cyanohydrin intermediate Ⅰ;

[0012] S2. Dissolve the obtained cyanohydrin intermediate Ⅰ in another solvent system, and add methylmagnesium bromide under the protection of an inert atmosphere (such as an inert atmosphere of argon, nitrogen, etc.), and stir for Grignard reaction;

[0013] S3. After the Grignard reaction in S2 is completed, add an excessive amount of sulfuric acid aqueous solution and stir for hydrolysis reaction. The obtained product is extracted, washed, dried, rotary evaporated and purified to obtain 17α-hydroxypregnenolone-2,3,4- 13 C3 product.

[0014] The 17α-hydroxypregnenolone-2,3,4- 13 C3 prepared by the present invention has the following structure:

[0015]

[0016] Furthermore, the solvent system in S1 is a mixture of solvent A and water in a volume ratio of 1:1 to 10. Preferably, the solvent A is one or a combination of several of dichloromethane, chloroform, dichloroethane, n-hexane, cyclohexane, petroleum ether, ethyl acetate, ether, benzene, toluene, and xylene. Specifically, the volume ratio of solvent A to water can be values such as 1:1, 1:10, 1:5, etc.

[0017] Furthermore, in S1, the molar ratio of dehydroepiandrosterone-2,3,4- 13 C3 to sodium cyanide is 1:1 to 20.

[0018] Further, in S1, the phase transfer catalyst is one or a combination of several of cyclodextrin, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogensulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride, benzyltriethylammonium chloride, tetradecyltrimethylammonium chloride, pyridine, and tributylamine.

[0019] Further, in S1, the addition amount of the phase transfer catalyst is 0.5% - 5% of the mass of dehydroepiandrosterone-2,3,4- 13 C3;

[0020] The temperature of the catalytic reaction is 0 - 90 °C, which can be 0 °C, room temperature, or other temperature values that require additional heating such as 60 °C, 90 °C, etc.

[0021] Further, in S2, the solvent system uses solvent B, which is selected from one or a combination of several of anhydrous acetonitrile, anhydrous 1,3-dimethyl-2-imidazolidinone, anhydrous dimethylformamide, anhydrous dimethyl sulfoxide, anhydrous hexamethylphosphoric triamide, anhydrous diethyl ether, and anhydrous tetrahydrofuran.

[0022] Further, in S2, the methylmagnesium bromide is 1 - 10 times the molar amount of the cyanohydrin intermediate I.

[0023] Further, in S2, the temperature of the Grignard reaction is 0 - 90 °C, which can be 0 °C, room temperature, or other temperature values that require additional heating such as 60 °C, 90 °C, etc.

[0024] Further, in S3, the mass concentration of the sulfuric acid aqueous solution is 1% - 20%, specifically it can be 1%, 5%, 10%, 20%, etc.

[0025] The temperature of the hydrolysis reaction is 0 - 90 °C, which can be 0 °C, room temperature, or other temperature values that require additional heating such as 60 °C, 90 °C, etc.

[0026] Further, in S3, the extractant used in the extraction process is solvent C, which is one or a combination of several of dichloromethane, chloroform, dichloroethane, n-hexane, cyclohexane, petroleum ether, ethyl acetate, diethyl ether, benzene, toluene, and xylene.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The obtained 17α-hydroxypregnenolone-2,3,4- 13 C3 product has a purity ≥ 98% and an abundance ≥ 99 atom% 13 C, and the abundance is not significantly diluted. The product can be used as an internal standard reagent for stable isotope dilution mass spectrometry detection in related hormone scientific research, clinical, and biomedical fields, as well as raw materials for disease screening kits for clinical mass spectrometry, etc.

[0029] (2) Compared with the existing methods for synthesizing products with natural abundance, it also avoids the cumbersome and complex protection steps, has mild reaction conditions, a simple synthesis route, greatly improves the yield and efficiency, and increases the added value of related labeled products;

[0030] (3) Link the dehydroepiandrosterone-2,3,4- 13 C3 product with the 17α-hydroxypregnenolone-2,3,4- 13 C3 product to realize the upstream and downstream relationship of the products, which is more conducive to the serial development of labeled steroid hormone products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the overall synthesis route diagram of 17α-hydroxypregnenolone-2,3,4- 13 C3 labeling of the present invention;

[0032] Figure 2 1H NMR spectrum of the cyanohydrin intermediate prepared in Example 1;

[0033] Figure 3 13C NMR spectrum of the cyanohydrin intermediate prepared in Example 1;

[0034] Figure 4 1H NMR of 17α-hydroxypregnenolone-2,3,4- 13 C3 prepared in Example 1;

[0035] Figure 5 13C NMR of 17α-hydroxypregnenolone-2,3,4- 13 C3 prepared in Example 1;

[0036] Figure 6 Liquid mass spectrometry of 17α-hydroxypregnenolone-2,3,4- 13 C3 prepared in Example 1;

[0037] Figure 7 Synthesis process route diagram of Comparative Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0040] As used herein, the selection scope of the terms "and / or", "or / and", and "and / or" includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items. The said any and all combinations include combinations of any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR".

[0041] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0042] In this application, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum value and the maximum value of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum value and the maximum value of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0043] In this article, only some numerical ranges are specifically disclosed. However, any lower limit can be combined with any upper limit to form a range not explicitly recorded; and any lower limit can be combined with other lower limits to form a range not explicitly recorded. Similarly, any upper limit can be combined with any other upper limit to form a range not explicitly recorded. In addition, each separately disclosed point or single numerical value itself can be used as a lower limit or an upper limit and combined with any other point or single numerical value or combined with other lower limits or upper limits to form a range not explicitly recorded.

[0044] For the temperature parameter in this application, unless otherwise specified, it allows both constant temperature treatment and treatment within a certain temperature range. The said constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0045] In this article, the "suitable" in "suitable combination method", "suitable method", "any suitable method", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0046] In this application, terms such as "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as limiting the scope of protection of this application.

[0047] In this application, "optionally", "optional", "option" mean that it is optional, that is, it refers to any one of the two parallel options of "having" or "not having". If "optional" appears in multiple places in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0048] In the description of the application, the meaning of "a variety of" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0049] Unless otherwise specified, all formulations and tests in this article occur in an environment of 25 °C.

[0050] In this article, "comprising", "including", "containing", "having" or other variants are intended to cover non-closed inclusion, and no distinction is made between these terms. The term "including" means that other steps and components can be added without affecting the final result. The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional components, ingredients, steps, or limitations described herein. In this article, no distinction is made between the terms "efficacy", "performance", "effect", and "function".

[0051] If there is no special instruction, all embodiments and optional embodiments of this application can be combined with each other to form a new technical solution. If there is no special instruction, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0052] If there is no special instruction, all steps of this application can be carried out sequentially or randomly, and preferably sequentially.

[0053] In the following examples, dehydroepiandrosterone-2,3,4- 13 C3 was purchased from Sigma-Aldrich, with an abundance ≥ 99 atom% 13 C, product number: 929980.

[0054] If there are no specially described raw materials or processing techniques, it means that they are all conventional commercially available raw materials or conventional processing techniques in the art.

[0055] Example 1:

[0056] This example provides a method for synthesizing stable isotope-labeled 17α-hydroxypregnenolone-2,3,4- 13 C3, with reference to Figure 1As shown in the figure, it includes the following steps:

[0057] In a 50 mL single-necked flask, add 3.4 mmol of sodium cyanide dissolved in a mixed solvent composed of 20 mL of dichloromethane and 20 mL of water, stir evenly, add 5 mg of cyclodextrin (β-CD) and 3.4 mmol of dehydroepiandrosterone-2,3,4- 13 C3, that is, the molar ratio of the two raw materials is 1:1, stir and react at 25 °C for 12 h; after the reaction is completed, filter with a suction funnel to obtain 0.65 g of solid product, which is the cyanohydrin intermediate Ⅰ.

[0058] The structure of the cyanohydrin intermediate Ⅰ was characterized by 1H NMR, 13C NMR and mass spectrometry: 1 1H NMR (500 MHz, CDCl3), δ: 0.715~0.785 (s, 3H, —C H 3), 0.844~0.895 [m, 1H, —C H (C)—], 0.912~0.925 (s, 3H, —C H 3), 0.936~1.192 [m, 2H, —C H (C)—], 1.201~1.354 [m, 2H, —CH2— 13 C H 2— 13 CH(OH)— 13 CH2—], 1.357~2.025 (m, 12H, —C H 2—), 2.175~2.385 [m, 2H, — 13 CH2— 13 CH(OH)— 13 C H 2—], 3.043~3.406 [m, 1H, — 13 CH2— 13 C H (OH)— 13 CH2—], 2.597~4.007 [m, 1H, CN—C(C)2—O H , 5.209~5.259 [m, 1H, — 13 CH2— 13 CH(OH)— 13 CH2—C(C)=C H —]; 13CNMR (120 MHz, CDCl3), δ: 141.772, 123.515, 120.531, 80.275, 70.162 - 70.738*, 51.423, 49.858, 46.791, 42.548 - 42.860*, 37.541, 37.138, 36.754, 35.825, 33.547, 31.774 - 32.067*, 29.945, 21.973, 20.491, 19.685, 13.704.

[0059] Another 100 mL two - necked flask was taken, 2.0 mmol of cyanohydrin intermediate Ⅰ and 30 mL of anhydrous tetrahydrofuran were added. Under nitrogen protection, 1.33 mL of methylmagnesium bromide diethyl ether solution (concentration 3 mol / L, effective content 4 mmol, the amount of substance is 2 times that of cyanohydrin intermediate Ⅰ) was added, and the reaction was stirred at 0 °C for 12 h; after the reaction, 40 mL of 1% sulfuric acid aqueous solution was added, and the reaction was stirred at 80 °C for 1 d; after the reaction, it was extracted with 100 mL of n - hexane, the organic phase was washed with 100 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, rotary evaporated and mixed with silica gel, and the product was passed through a silica gel column, and the mobile phase was ethyl acetate:n - hexane = 1:1, to obtain 17α - hydroxypregnenolone - 2,3,4 - 13 0.60 g of white solid of C3, purity ≥ 98%, abundance ≥ 99 atom% 13 C, yield 90.0%.

[0060] 17α - hydroxypregnenolone - 2,3,4 - 13 The structure of 17α - hydroxypregnenolone - 2,3,4 - C3 was characterized by 1H - NMR, 13C - NMR and mass spectrometry, and its abundance was characterized by liquid chromatography - mass spectrometry: 1 1H - NMR (500 MHz, CDCl3), δ: 0.708 - 0.785 (s, 3H, — C H 3), 0.815 - 0.884 [m, 1H, — C H (C)—], 0.908 - 0.926 (s, 3H, — C H 3), 0.963 - 1.135 [m, 2H, — C H (C)—], 1.147 - 1.213 (s, 3H, — CO — C H 3), 1.253 - 1.352 [m, 2H, — CH2 — 13 C H 2 — 13 CH(OH) — 13 CH2 — ], 1.378 - 2.019 (m, 12H, — C H 2 — ), 2.121 - 2.386 [m, 2H, — 13 CH2 — 13CH(OH)— 13 C H 2—], 3.053 to 3.376 [m, 1H, — 13 CH2— 13 C H (OH)— 13 CH2—], 4.495 to 4.577 [m, 1H, CH3CO—C(R)2—O H , 5.210 to 5.272 [m, 1H, — 13 CH2— 13 CH(OH)— 13 CH2—C(C)=C H —]; 13 13C NMR (120 MHz, CDCl3), δ: 220.206, 142.097, 120.541, 70.609, 70.169 to 70.810*, 51.417, 50.299, 47.348, 42.579 to 42.865*, 37.527, 37.272, 36.754, 35.829, 31.716 to 32.074*, 31.516, 30.771, 30.738, 21.974, 20.491, 19.684, 13.706. LC-MS, m / z: [M+H] + , [M+Na] + Found 336, 358; calculated 335.

[0061] Example 2

[0062] This example provides a method for synthesizing stable isotope-labeled 17α-hydroxypregnenolone-2,3,4- 13 13C3, comprising the following steps:

[0063] In a 50 mL single-necked flask, 6.8 mmol of sodium cyanide was dissolved in a mixed solvent composed of 20 mL of dichloromethane and 20 mL of water and stirred evenly. 5 mg of cyclodextrin and 3.4 mmol of dehydroepiandrosterone-2,3,4- 13 13C3 were added, and the molar ratio of the raw materials was 2:1. The reaction was stirred at 25 °C for 12 h; after the reaction was completed, it was filtered through a suction funnel to obtain 0.60 g of cyanohydrin intermediate I.

[0064] Take another 100 mL two-necked flask, add 1.9 mmol of cyanohydrin intermediate Ⅰ and 30 mL of anhydrous tetrahydrofuran, add 1.27 mL of methylmagnesium bromide diethyl ether solution (concentration 3 mol / L, effective content 3.8 mmol, the amount of substance is 2 times that of cyanohydrin intermediate Ⅰ) under nitrogen protection, and stir and react at 0 °C for 12 h; after the reaction, add 40 mL of 1% sulfuric acid aqueous solution, and stir and react at 80 °C for 1 d; after the reaction, extract with 100 mL of n-hexane, wash the organic phase with 100 mL of saturated aqueous sodium chloride solution, dry with anhydrous sodium sulfate, filter, rotary evaporate and mix with silica gel, and the product is passed through a silica gel column, and the mobile phase is ethyl acetate:n-hexane = 1:1, to obtain 17α-hydroxy pregnenolone-2,3,4- 13 0.56 g of C3 white solid, purity ≥ 98%, abundance ≥ 99 atom% 13 C, yield 87.9%.

[0065] Example 3:

[0066] This example provides a method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, including the following steps:

[0067] In a 50 mL single-necked flask, add 3.4 mmol of sodium cyanide dissolved in a mixed solvent composed of 20 mL of dichloromethane and 20 mL of water, stir evenly, add 5 mg of cyclodextrin (i.e., β-CD) and 3.4 mmol of dehydroepiandrosterone-2,3,4- 13 C3, the molar ratio of raw material feeding is 1:1, stir and react at 25 °C for 12 h; after the reaction, filter with a suction funnel to obtain 0.65 g of cyanohydrin intermediate Ⅰ.

[0068] Take another 100 mL two-necked flask, add 2.0 mmol of cyanohydrin intermediate Ⅰ and 30 mL of anhydrous ether, add 1.33 mL of methylmagnesium bromide diethyl ether solution (concentration 3 mol / L, effective content 4 mmol, the amount of substance is 2 times that of cyanohydrin intermediate Ⅰ) under nitrogen protection, and stir and react at 0 °C for 12 h; after the reaction, add 40 mL of 1% sulfuric acid aqueous solution, and stir and react at 80 °C for 1 d; after the reaction, extract with 100 mL of n-hexane, wash the organic phase with 100 mL of saturated aqueous sodium chloride solution, dry with anhydrous sodium sulfate, filter, rotary evaporate and mix with silica gel, and the product is passed through a silica gel column, and the mobile phase is ethyl acetate:n-hexane = 1:1, to obtain 17α-hydroxy pregnenolone-2,3,4- 13 0.62 g of C3 white solid, purity ≥ 98%, abundance ≥ 99 atom% 13 C, yield 92.5%.

[0069] Example 4:

[0070] This example provides a method for synthesizing stable isotope-labeled 17α-hydroxypregnenolone-2,3,4- 13 C3, comprising the following steps:

[0071] In a 50 mL single-necked flask, 3.4 mmol of sodium cyanide was dissolved in a mixed solvent composed of 20 mL of dichloromethane and 20 mL of water and stirred evenly. 5 mg of tetrabutylammonium bromide and 3.4 mmol of dehydroepiandrosterone-2,3,4- 13 C3 were added. The molar ratio of the raw materials was 1:1, and the reaction was stirred at 25 °C for 12 h. After the reaction was completed, filtration was carried out using a suction funnel to obtain 0.67 g of cyanohydrin intermediate I.

[0072] Another 100 mL two-necked flask was taken, 2.0 mmol of cyanohydrin intermediate I and 30 mL of anhydrous tetrahydrofuran were added. Under nitrogen protection, 1.33 mL of methylmagnesium bromide diethyl ether solution (concentration 3 mol / L, effective content 4 mmol, the amount of substance was 2 times that of cyanohydrin intermediate I) was added, and the reaction was stirred at 0 °C for 12 h. After the reaction was completed, 40 mL of 1% sulfuric acid aqueous solution was added, and the reaction was stirred at 80 °C for 1 d. After the reaction was completed, extraction was carried out with 100 mL of n-hexane. The organic phase was washed with 100 mL of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, rotary evaporated and silica gel was added for mixing. The product was passed through a silica gel column, and the mobile phase was ethyl acetate:n-hexane = 1:1, to obtain 0.61 g of 17α-hydroxypregnenolone-2,3,4- 13 C3 white solid, with a purity ≥ 98% and an abundance ≥ 99 atom% 13 C, and the yield was 91.0%.

[0073] Comparative Example 1:

[0074] Since the synthesis of 17α-hydroxypregnenolone-2,3,4- 13 C3 disclosed in the present invention was reported for the first time, the comparative example took the synthesis of natural abundance 17α-hydroxypregnenolone by Schor, Laura et al. in 1990 as an example. The synthesis route is as Figure 7 .

[0075] Dissolve 3β-hydroxyandrosta-5-en-17-one (8) (1.09 g) in tetrahydrofuran (10 mL). Under an acetylene atmosphere, quickly add lithium acetylide-ethylenediamine (3.4 g) to the reaction system, and react at room temperature for 24 h. After the reaction, slowly add water until no more solid precipitates, and purify by column chromatography to obtain an orange solid. Then neutralize with dilute hydrochloric acid and extract with dichloromethane to obtain 1.05 g of crude product. Purify by column chromatography again, and recrystallize the purified product in a methanol-water system to obtain the ethynyl derivative (9) (730 mg). Stir (9) (370 mg) and p-toluenesulfonic acid (20 mg) in formic acid (50 mL) at room temperature for 18 h, and then pour the mixture into water. After extraction with dichloromethane, obtain the crude product, and separate and purify the crude product by column chromatography to obtain compound (10a). Stir (10a) (58 mg), sodium acetate (140 mg), and N-bromoacetamide (160 mg) in glacial acetic acid (12 mL) and water (1.2 mL) at room temperature for 40 min. Add zinc powder (170 mg), and reflux the mixture for 40 min until the solution becomes colorless. Dilute with water (15 mL) and extract with dichloromethane to obtain a yellow oil (49 mg), and recrystallize in n-hexane to obtain (11a). Add (11a) (50 mg) and sodium bicarbonate (50 mg) to methanol (10 mL), stir at room temperature for 30 min. After the reaction, neutralize the reaction system with dilute hydrochloric acid and extract to obtain 17α-hydroxypregnenolone (42 mg).

[0076] It can be seen that this method adopts a group protection strategy, involving the protection and deprotection processes of two groups, resulting in a relatively long overall synthesis step. Multiple intermediates need to be purified by column chromatography, the process is complex, and the yield is low. An acetylene atmosphere needs to be used during the reaction, which increases the instability of the reaction and the safety is insufficient. In addition, the lithium acetylide-ethylenediamine complex used is expensive, and the raw material cost is high.

[0077] On the basis of the above Examples 1 to 4, the present invention can also adjust the types of raw materials such as solvent A and phase transfer catalyst X, the addition amounts of the corresponding raw materials, and process conditions such as the reaction temperature and time, and it is speculated that stable isotope-labeled 17α-hydroxypregnenolone-2,3,4- 13 C3 can also be obtained.

[0078] Example 5

[0079] Compared with Example 1, most of them are the same, except that solvent A is adjusted to toluene.

[0080] Example 6

[0081] Compared with Example 1, most of them are the same, except that the ratio of solvent A to water is adjusted to volume ratio solvent A: water = 1:10.

[0082] Example 7

[0083] Compared with Example 1, most of them are the same, except that the dosage of phase transfer catalyst X is adjusted to 5% of the mass of dehydroepiandrosterone-2,3,4- 13 C3.

[0084] Example 8

[0085] Compared with Example 1, most of them are the same, except that the temperature of the phase transfer catalytic reaction is adjusted to 0 °C.

[0086] Example 9

[0087] Compared with Example 1, most of them are the same, except that the time of the phase transfer catalytic reaction is adjusted to 5 days.

[0088] Example 10

[0089] Compared with Example 1, most of them are the same, except that methylmagnesium bromide is adjusted to 10 times the molar amount of the cyanohydrin intermediate.

[0090] Example 11

[0091] Compared with Example 1, most of them are the same, except that the temperature of the Grignard reaction is adjusted to 60 °C.

[0092] Example 12

[0093] Compared with Example 1, most of them are the same, except that the time of the Grignard reaction is adjusted to 2 days.

[0094] Example 13

[0095] Compared with Example 1, most of them are the same, except that the concentration of the sulfuric acid aqueous solution is adjusted to 20% by mass of sulfuric acid.

[0096] Example 14

[0097] Compared with Example 1, most of them are the same, except that the time of the hydrolysis reaction is adjusted to 10 days.

[0098] Example 15

[0099] Compared with Example 1, most of them are the same, except that the temperature of the hydrolysis reaction is adjusted to 10 °C.

[0100] Example 16

[0101] Compared with Example 1, most of them are the same, except that solvent C is adjusted to toluene.

[0102] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, It includes the following steps: S1. Dissolve sodium cyanide in a solvent system, add a phase transfer catalyst and dehydroepiandrosterone-2,3,4- 13 C3, stir for catalytic reaction, and filter to obtain cyanohydrin intermediate Ⅰ; S2. Dissolve the obtained cyanohydrin intermediate I in another solvent system, add methylmagnesium bromide under the protection of an inert atmosphere, and stir for a Grignard reaction; After the Grignard reaction in S3 and S2 is completed, an excessive amount of sulfuric acid aqueous solution is added and stirred for hydrolysis reaction. The obtained product is extracted, washed, dried, rotary evaporated, and purified to obtain 17α-hydroxy pregnenolone-2,3,4- 13 C3 product.

2. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, The solvent system in S1 is a mixture of solvent A and water in a volume ratio of 1:1 to 10, and the solvent A is one or a combination of several of dichloromethane, chloroform, dichloroethane, n-hexane, cyclohexane, petroleum ether, ethyl acetate, diethyl ether, benzene, toluene, and xylene.

3. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S1, dehydroepiandrosterone-2,3,4- 13 The molar ratio of C3 to sodium cyanide is 1:1 to 20.

4. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S1, the phase transfer catalyst is one or a combination of several of cyclodextrin, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogensulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride, benzyltriethylammonium chloride, tetradecyltrimethylammonium chloride, pyridine, and tributylamine.

5. A method for synthesizing a stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S1, the addition amount of the phase transfer catalyst is 0.5% to 5% of the mass of dehydroepiandrosterone-2,3,4- 13 C3; The temperature of the catalytic reaction is 0 to 90 °C.

6. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, The solvent system in S2 uses solvent B, which is selected from one or a combination of several of anhydrous acetonitrile, anhydrous 1,3-dimethyl-2-imidazolidinone, anhydrous dimethylformamide, anhydrous dimethyl sulfoxide, anhydrous hexamethylphosphoric triamide, anhydrous diethyl ether, and anhydrous tetrahydrofuran.

7. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S2, the methylmagnesium bromide is 1 to 10 times the molar amount of the cyanohydrin intermediate I.

8. A method for synthesizing a stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S2, the temperature of the Grignard reaction is 0 to 90 °C.

9. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S3, the mass concentration of the sulfuric acid aqueous solution is 1% to 20%; The temperature of the hydrolysis reaction is 0 to 90 °C.

10. A method for synthesizing stable isotope-labeled 17α-hydroxy pregnenolone-2,3,4- 13 C3, characterized in that, In S3, the extractant used in the extraction process is solvent C, which is one or a combination of several of dichloromethane, chloroform, dichloroethane, n-hexane, cyclohexane, petroleum ether, ethyl acetate, diethyl ether, benzene, toluene, and xylene.

Images