A stable isotope-labeled cholester-3,4- 13 Synthesis of C2

Abstract

The present application relates to a kind of stable isotope labeled cholesterol-3,4- 13 The synthesis method of C2, comprising the following steps: (1) take cyclohexanone intermediate II and 13 C double labeled ester compound in alkali and solvent, stirring reaction, obtain acetylation labeled intermediate III;(2) acetylation labeled intermediate III and mixed acid reaction, obtain carbon ring reconfiguration reaction intermediate IV;(3) carbon ring reconfiguration reaction intermediate IV in acetic anhydride, stirring reaction, obtain esterification reaction intermediate V;(4) esterification reaction intermediate V and sodium borohydride in solvent system reaction, after reaction, adjust system pH to be acidic, extraction, wash organic phase, dry, spin evaporation and remove solution, purification, obtain cholesterol-3,4- 13 C2 product, i.e. target product. The present application breaks the monopoly of cholesterol-3,4- 13 C2 which is one of the core labeled reagents for steroid detection necessary for vitamin detection, tumor screening, etc., fills the blank in the domestic related field, etc.

Description

Technical Field

[0001] This invention belongs to the field of isotope synthesis technology, and relates to a stable isotope labeling method for cholesterol-3,4- 13 Methods for synthesizing C2. Background Technology

[0002] Steroid hormones are a series of hormones formed from cholesterol in the human body through various enzymes. These include progesterone, androgens, estrogens, and corticosteroids, and play important regulatory roles in human growth, development, reproduction, and metabolism. Disorders of these hormone metabolisms are the cause of many diseases, and accurate detection of their changes is of significant diagnostic value.

[0003] Cholesterol is a type of steroid hormone and a precursor to many steroids. It possesses the steroid structure of cholesterol-5-enol and is widely distributed in animal bodies. Closely related to cell membrane formation, it is one of the bioactive substances required for the synthesis of bile acids, steroid hormones, and vitamin D, and is an essential substance for human and animal tissue cells. Numerous epidemiological studies have confirmed a close relationship between cholesterol levels and the incidence of coronary heart disease; elevated cholesterol levels in the human body are also a contributing factor to atherosclerotic cardiovascular disease. Recent studies have found that cholesterol levels in the human body are associated with various cancers, such as breast cancer, stomach cancer, colorectal cancer, prostate cancer, and kidney cancer; in addition, its levels are also related to gallstones, fatty liver, and diabetes, among other diseases.

[0004] In traditional clinical medicine, the most commonly used methods for detecting cholesterol and other steroid hormones are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). However, due to the significant variation in cholesterol levels within the human body (from ng / mL to pg / mL), traditional ELISA and RIA methods require large sample volumes, are time-consuming, and are often limited by antibody cross-reactivity, frequently resulting in inaccurate quantification and false positives, thus affecting the diagnosis and treatment of related diseases. Furthermore, the detection of cholesterol and other steroid hormones is easily confused with other substances in the body with similar structures. Therefore, traditional clinical medical methods cannot meet the requirements for high sensitivity and high relevance. In contrast, clinical stable isotope dilution mass spectrometry (clinical mass spectrometry) offers greater relevance and accuracy, with concentration precision reaching pg / mL, a sensitivity unattainable by traditional clinical medical methods. Internationally, the development of clinical mass spectrometry medicine began as early as 10 years ago. In 2014, the Journal of Clinical Endocrinology and Metabolism stated that the detection of steroid hormones such as cholesterol should be replaced by clinical mass spectrometry instead of traditional methods. Furthermore, in 2015, it was stated that only data from clinical mass spectrometry should be recognized. Since 2018, clinical mass spectrometry in China has also entered a period of rapid development, replacing or partially replacing traditional biochemical diagnostic analysis methods in several clinical testing areas, demonstrating vast potential for growth.

[0005] Stable isotope-labeled steroid hormone reagents are the core of stable isotope dilution mass spectrometry detection of these hormones in clinical applications. Therefore, cholesterol-3,4- 13 C2 is the core component of clinical mass spectrometry detection for cholesterol in the diagnosis and treatment of various diseases, including coronary heart disease, atherosclerosis, tumors, fatty liver, gallstones, and diabetes.

[0006] Currently, cholesterol-3,4- 13 C2-based steroid diagnostic reagents, essential for vitamin testing and tumor screening, cannot be synthesized domestically. Their production and supply are monopolized by developed countries, such as CIL (Cambridge Isotope) and Sigma-Aldrich in the United States. These reagents are expensive and in limited supply. (Cholesterol-3,4-) 13 The price of C2 is as high as 70,000 yuan / g. This has severely restricted the progress of clinical mass spectrometry research on cholesterol and other steroid hormones in my country, the exploration of new detection methods, the production of reagent kits, and the expansion of its practical clinical use.

[0007] There is currently no cholesterol-3,4- in China. 13 Regarding the synthesis of C2, the only reported case from abroad was the one published in 1982 by the Sun-Shine and Yuan teams.13 C3,4- cholesterol was synthesized using C-dip acyl chloride as a building block and employing a group protection strategy. 13 The C2 method. The acyl chloride-labeled building blocks used in this method have poor stability and low yield, making it expensive. 13 The utilization rate of raw material C is reduced, resulting in higher costs. In addition, the synthesis method adopts a group protection strategy, which involves the protection and deprotection processes of two groups, leading to a longer overall synthesis process and greater complexity.

[0008] Only some foreign countries have cholesterol-3,4- 13 C2 has similar structural compounds 13 Reports on C-labeling introduction strategies: ISA GJ et al. reported an ozone oxidation degradation strategy in 2000, using... 13 C-Yelde is used as an introducing reagent to react with the oxidative cleavage intermediate of α,β-unsaturated carbonyl double bonds to prepare progesterone. However, this oxidation process involves numerous side reactions, is lengthy, and uses precious metal catalysis, leading to a sharp increase in cost. In 2018, BERTHONNEAU C. et al. reported a strategy of introducing benzenethiophenol into the A ring for activation and re-oxidation in J. Org. Chem., employing... 13 C-Wittig reagent is used as a carbon source to introduce activating groups into the steroid skeleton; however, this method involves long experimental steps, complex oxidation operations, and the non-corresponding selective reduction of carbonyl groups produces complex mixtures, making purification difficult and resulting in low preparation efficiency. Summary of the Invention

[0009] The purpose of this invention is to provide a stable isotope-labeled cholesterol-3,4- 13 The synthesis method of C2 aims to break the foreign monopoly on cholesterol-3,4-, one of the core marker reagents for steroid detection, which is essential for vitamin testing and tumor screening. 13 C2's monopoly fills a gap in related domestic fields.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] A stable isotope labeling cholesterol-3,4- 13 The method for synthesizing C2 includes the following steps:

[0012] (1) The cyclic carbonyl intermediate II and 13 C-labeled ester compounds were reacted with stirring in a base and solvent to give acetylated labeled intermediate III;

[0013] (2) The acetylated labeled intermediate III reacts with a mixed acid to obtain the carbocyclic reconstruction reaction intermediate IV;

[0014] (3) The carbocyclic reconstruction reaction intermediate IV was reacted in acetic anhydride by stirring to obtain the esterification reaction intermediate V;

[0015] (4) Esterification intermediate V reacts with sodium borohydride in a solvent system. After the reaction is complete, the pH of the system is adjusted to acidic, the organic phase is extracted, washed, dried, and dissolved by rotary evaporation to obtain cholesterol-3,4- 13 Product C2 is the target product;

[0016] The structural formula of the cyclic carbonyl intermediate II is:

[0017] Wherein, R is -CH(CH3)CH2CH2CH2CH(CH3)2.

[0018] Furthermore, the cyclic carbonyl intermediate II is prepared by the following method:

[0019] (A) 4-Cholesterol-3-one was subjected to ring-opening oxidation in an organic solvent system under the action of an oxidant to obtain ring-opening oxidized carboxylic acid intermediate I;

[0020] (B) The cyclization intermediate I of the carboxylic acid was subjected to a cyclization reaction in acetic acid solvent in the presence of sodium acetate and under inert gas protection to obtain the cyclized carbonyl intermediate II.

[0021] Furthermore, in step (A), the organic solvent system is a mixture of isopropanol and water, the oxidant is a mixture of potassium permanganate and sodium periodate; the molar ratio of 4-cholesten-3-one to the oxidant is 1:2 to 10; the oxidation reaction temperature is 25 to 100°C; and the oxidation reaction time is 2 to 8 hours.

[0022] Furthermore, in step (2), the molar ratio of the ring-opening oxidative carboxylic acid intermediate I to sodium acetate is 1:1 to 5, specifically 1:1, 1:5, or 1:2, etc.; the ring-closure reaction temperature is 100 to 150°C; and the ring-closure reaction time is 1 to 2 days.

[0023] Furthermore, in step (1), the cyclic carbonyl intermediate II, 13 The molar ratio of the C-labeled ester compound is 1:1 to 10, and the amount added can be any intermediate value such as 1:1, 1:10, or 1:5; the molar ratio of the cyclohexyl carbonyl intermediate II to the base is 1:1 to 10, and the amount added can be any intermediate value such as 1:1, 1:10, or 1:5; the mass-volume ratio of the cyclohexyl carbonyl intermediate II to the solvent is 1:10 to 20, and can be any intermediate value such as 1:10, 1:20, or 1:15.

[0024] Furthermore, in step (1), the alkali used is a mixture of one or more of the following: ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, sodium hydride, and potassium hydride.

[0025] Furthermore, in step (1), the solvent used is one or a mixture of several of the following: tetrahydrofuran, methanol, ethanol, acetone, diethyl ether, dichloromethane, chloroform, n-hexane, petroleum ether, tert-butanol, cyclohexane, ethyl acetate, and dimethyl sulfoxide.

[0026] Furthermore, in step (1), the temperature of the stirring reaction is -20 to 200°C, and the time is 0.5 to 10.5 days.

[0027] Furthermore, in step (1), the 13 The structure of C-labeled ester compounds is as follows:

[0028]

[0029] Wherein, X is an alkyl, cycloalkyl, or aryl group.

[0030] Furthermore, in step (2), the mixed acid is a mixture of acetic acid and hydrochloric acid.

[0031] Furthermore, in step (2), the ratio of the amount of acetylated labeled intermediate III to acetic acid and hydrochloric acid added is 7g:(120-160)mL:(25-35)mL, wherein the mass fraction of hydrochloric acid is 36.5%.

[0032] Furthermore, in step (2), the reaction temperature is 40-60℃ and the time is 1-3 days.

[0033] Furthermore, in step (3), the ratio of the amount of carbocyclic reconstruction reaction intermediate IV to acetic anhydride added is (4.0-4.2) g: 70 mL.

[0034] Furthermore, in step (3), the temperature of the stirring reaction is 15-25°C and the time is 18-30h.

[0035] Furthermore, in step (4), the solvent system used is a mixed solvent system of methanol and tetrahydrofuran.

[0036] Furthermore, in step (4), the ratio of the amount of esterification reaction intermediate V, methanol, tetrahydrofuran and sodium borohydride added is (2.0~2.5)g: (25~35)mL: (25~35)mL: 0.4g.

[0037] Furthermore, in step (4), the pH is adjusted to 3.0 using hydrochloric acid.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) This invention adopts 13 C-labeled ester compounds serve as labeling building blocks, which are chemically stable and offer high yields in labeled synthesis. Introducing these building blocks in a later step minimizes the consumption of expensive raw materials, making it more economical and better mitigating the risk of product dilution.

[0040] (2) A semi-synthetic strategy is adopted, with abundant and readily available raw materials at low cost, reducing synthetic steps and improving synthetic efficiency; the reaction route is scalable, allowing for subsequent development of three-carbon labeled cholesterol or "transplantation" into the development of other labeled steroid clinical diagnostic reagents with similar structures, such as... 13 C-labeled progesterone, pregnenolone, testosterone, 17-hydroxyprogesterone, and androstenedione, etc., form a series of compounds.

[0041] (3) The obtained cholesterol-3,4- 13 C2 product purity ≥98%, abundance ≥98% 13 C. No significant dilution was observed. The product can be used as an internal standard reagent for stable isotope dilution mass spectrometry in the clinical and biopharmaceutical fields, as well as a raw material for disease screening kits for clinical mass spectrometry. Attached Figure Description

[0042] Figure 1 This is a flowchart of the synthesis process of the present invention. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0044] In this invention, ethyl acetate-1,2- 13 C2 was purchased from Sigma-Aldrich and has an abundance of ≥99 atom%. 13 C, Product No. 283819; Acetyl chloride - 13 C2 was purchased from Sigma-Aldrich and has an abundance of ≥99 atom%. 13 C, Product No. 293164; Phenyl acetate-1,2- 13 C2, Cyclohexyl acetate-1,2- 13 C2 is composed of acetyl chloride- 13 It is prepared from C2, and the preparation method is as follows:

[0045]

[0046] In a 500 mL single-necked flask, 86 mmol of phenol or cyclohexanol, 79 mmol of acetyl chloride, and 150 mL of cyclohexane were added, and the mixture was stirred and refluxed at 25 °C for 6 h. After the reaction was complete, phenyl acetate-1,2- was obtained by rotary evaporation. 13 C2 or cyclohexyl acetate-1,2- 13 C2.

[0047] Unless otherwise specified, all other raw materials or processing techniques are commercially available materials or conventional processing techniques in the field.

[0048] Example 1:

[0049] This embodiment provides a stable isotope-labeled cholesterol-3,4- 13 The method for synthesizing C2 includes the following steps:

[0050] 1. Preparation of cyclic carbonyl intermediate II

[0051] In a 2L single-necked flask, add (52mmol) 4-cholesten-3-one, (62mmol) sodium carbonate, and 800mL isopropanol. After stirring and dissolving at 70℃, add dropwise a solution prepared from (7.5mmol) potassium permanganate, (292mmol) sodium periodate, and 400mL water. Control the dropping temperature at 70℃±5℃ and the dropping rate at 20mL / min. After the dropping is completed, continue to keep the reaction at the temperature for 5h. After the reaction was complete, the mixture was cooled, filtered, and the filter cake was washed with 100 mL of 5% sodium hydroxide solution, followed by 200 mL of dichloromethane. The organic phase was separated, and the aqueous phase was retained. Most of the isopropanol was removed by rotary evaporation. The pH of the aqueous phase was adjusted to 3.0, and then extracted with 250 mL of dichloromethane. The organic phase was washed with 300 mL of water, dried over anhydrous Na₂SO₄, filtered, and dissolved by rotary evaporation to obtain ring-opening oxidized carboxylic acid intermediate I. In a 500 mL two-necked flask, (37 mmol) of ring-opening oxidized carboxylic acid intermediate I, (91 mmol) of sodium acetate, and 150 mL of acetic acid were added. Under nitrogen protection, the mixture was heated to 135 °C and refluxed with stirring for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain cyclic carbonyl intermediate II.

[0052] 2. Cholesterol-3,4- 13 Preparation of C2

[0053] In a 250 mL single-necked flask, add 10.5 g (27 mmol) of intermediate II and 3.7 g (27 mmol) of phenyl acetate-1,2- in a molar ratio of 1:1. 13C2, then add 4.3g (108mmol) sodium hydride (60%) and 100mL anhydrous tetrahydrofuran, and stir at 25℃ for 4d; after the reaction is completed, rotary evaporate silica gel to mix the sample, and pass the product through silica gel column with ethyl acetate: n-hexane (5%-15% gradient, time 40min) as the mobile phase to obtain intermediate III 7.0g.

[0054] In a separate 250 mL single-necked flask, add 7.0 g (16 mmol) of intermediate III, 140 mL of acetic acid, and 28 mL of hydrochloric acid (36.5%). Stir the mixture at 50 °C for 2 days. After the reaction is complete, add 200 mL of water and extract the organic phase with 200 mL of ethyl acetate. Dry the sample with anhydrous sodium sulfate, and stir with silica gel by rotary evaporation. Pass the product through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 4.1 g of intermediate IV, with a yield of 66.3%.

[0055] In a separate 250 mL single-necked flask, 4.1 g (11 mmol) of intermediate IV and 70 mL of acetic anhydride were added, and the mixture was stirred at 20 °C for 24 h. After the reaction was complete, the product was stirred with silica gel by rotary evaporation. The product was then passed through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 2.3 g of intermediate V, with a yield of 49.3%.

[0056] In a separate 250 mL single-necked flask, add 2.3 g (5 mmol) of intermediate V, 30 mL of methanol, and 30 mL of tetrahydrofuran. Stir the reaction mixture at 60 °C. Add 0.4 g (10 mmol) of sodium borohydride and continue the reaction at 60 °C for 24 h. After the reaction is complete, add 20 mL of water and dilute hydrochloric acid (5%) to a pH of 3.0. Extract with 100 mL of dichloromethane, wash the organic phase with 100 mL of saturated brine, dry with anhydrous sodium sulfate, and stir with rotary evaporation onto silica gel. Filter the product through a silica gel column with ethyl acetate:n-hexane in a 1:6 ratio (with 2% isopropanol) as the mobile phase to obtain cholesterol-3,4- 13 C2 white solid, 1.8 g, purity ≥ 98%, abundance ≥ 98 atom%. 13 C, yield 80.3%.

[0057] Example 2

[0058] This embodiment provides a stable isotope-labeled cholesterol-3,4- 13 The method for synthesizing C2 includes the following steps:

[0059] 1. Preparation of cyclic carbonyl intermediate II

[0060] In a 2L single-necked flask, add (52mmol) 4-cholesten-3-one, (62mmol) sodium carbonate, and 800mL isopropanol. After stirring and dissolving at 70℃, add dropwise a solution prepared from (7.5mmol) potassium permanganate, (292mmol) sodium periodate, and 400mL water. Control the dropping temperature at 70℃±5℃ and the dropping rate at 20mL / min. After the dropping is completed, continue to keep the reaction at the temperature for 5h. After the reaction was complete, the mixture was cooled, filtered, and the filter cake was washed with 100 mL of 5% sodium hydroxide solution, followed by 200 mL of dichloromethane. The organic phase was separated, and the aqueous phase was retained. Most of the isopropanol was removed by rotary evaporation. The pH of the aqueous phase was adjusted to 3.0, and then extracted with 250 mL of dichloromethane. The organic phase was washed with 300 mL of water, dried over anhydrous NaSO4, filtered, and dissolved by rotary evaporation to obtain ring-opening oxidized carboxylic acid intermediate I. In a 500 mL two-necked flask, (37 mmol) of ring-opening oxidized carboxylic acid intermediate I, (91 mmol) of sodium acetate, and 150 mL of acetic acid were added. Under nitrogen protection, the mixture was heated to 135 °C and refluxed with stirring for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain cyclic carbonyl intermediate II.

[0061] 2. Cholesterol-3,4- 13 Preparation of C2

[0062] In a 250 mL single-necked flask, add 10.5 g (27 mmol) of intermediate II and 2.4 g (27 mmol) of ethyl acetate-1,2- in a molar ratio of 1:1. 13 C2 was then added along with 4.3 g (108 mmol) of sodium hydroxide (60%) and 100 mL of anhydrous tetrahydrofuran. The mixture was stirred at 25 °C for 4 days. After the reaction was complete, the product was mixed with silica gel by rotary evaporation and passed through a silica gel column with ethyl acetate: n-hexane (5%-15% gradient, time 40 min) as the mobile phase to obtain 7.2 g of intermediate III.

[0063] In a separate 250 mL single-necked flask, add 7.0 g (16 mmol) of intermediate III, 140 mL of acetic acid, and 28 mL of hydrochloric acid (36.5%). Stir the mixture at 50 °C for 2 days. After the reaction is complete, add 200 mL of water and extract the organic phase with 200 mL of ethyl acetate. Dry the sample with anhydrous sodium sulfate, and stir with silica gel by rotary evaporation. Pass the product through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 4.1 g of intermediate IV, with a yield of 66.3%.

[0064] In a separate 250 mL single-necked flask, 4.1 g (11 mmol) of intermediate IV and 70 mL of acetic anhydride were added, and the mixture was stirred at 20 °C for 24 h. After the reaction was complete, the product was stirred with silica gel by rotary evaporation. The product was then passed through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 2.3 g of intermediate V, with a yield of 49.3%.

[0065] In a separate 250 mL single-necked flask, add 2.3 g (5 mmol) of intermediate V, 30 mL of methanol, and 30 mL of tetrahydrofuran. Stir the reaction mixture at 60 °C. Add 0.4 g (10 mmol) of sodium borohydride and continue the reaction at 60 °C for 24 h. After the reaction is complete, add 20 mL of water and dilute hydrochloric acid (5%) to a pH of 3.0. Extract with 100 mL of dichloromethane, wash the organic phase with 100 mL of saturated brine, dry with anhydrous sodium sulfate, and stir with rotary evaporation onto silica gel. Filter the product through a silica gel column with ethyl acetate:n-hexane in a 1:6 ratio (with 2% isopropanol) as the mobile phase to obtain cholesterol-3,4- 13 C2 white solid, 1.8 g, purity ≥ 98%, abundance ≥ 98 atom%. 13 C, yield 80.3%.

[0066] Example 3

[0067] This embodiment provides a stable isotope-labeled cholesterol-3,4- 13 The method for synthesizing C2 includes the following steps:

[0068] 1. Preparation of cyclic carbonyl intermediate II

[0069] In a 2L single-necked flask, add (52mmol) 4-cholesten-3-one, (62mmol) sodium carbonate, and 800mL isopropanol. After stirring and dissolving at 70℃, add dropwise a solution prepared from (7.5mmol) potassium permanganate, (292mmol) sodium periodate, and 400mL water. Control the dropping temperature at 70℃±5℃ and the dropping rate at 20mL / min. After the dropping is completed, continue to keep the reaction at the temperature for 5h. After the reaction was complete, the mixture was cooled, filtered, and the filter cake was washed with 100 mL of 5% sodium hydroxide solution, followed by 200 mL of dichloromethane. The organic phase was separated, and the aqueous phase was retained. Most of the isopropanol was removed by rotary evaporation. The pH of the aqueous phase was adjusted to 3.0, and then extracted with 250 mL of dichloromethane. The organic phase was washed with 300 mL of water, dried over anhydrous NaSO4, filtered, and dissolved by rotary evaporation to obtain ring-opening oxidized carboxylic acid intermediate I. In a 500 mL two-necked flask, (37 mmol) of ring-opening oxidized carboxylic acid intermediate I, (91 mmol) of sodium acetate, and 150 mL of acetic acid were added. Under nitrogen protection, the mixture was heated to 135 °C and refluxed with stirring for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain cyclic carbonyl intermediate II.

[0070] 2. Cholesterol-3,4- 13 Preparation of C2

[0071] In a 250 mL single-necked flask, add 10.5 g (27 mmol) of intermediate II and 3.9 g (27 mmol) of cyclohexyl acetate-1,2- in a molar ratio of 1:1. 13C2 was then added along with 4.3 g (108 mmol) of sodium hydroxide (60%) and 100 mL of anhydrous tetrahydrofuran. The mixture was stirred at 25 °C for 4 days. After the reaction was complete, the product was mixed with silica gel by rotary evaporation and passed through a silica gel column with ethyl acetate: n-hexane (5%-15% gradient, time 40 min) as the mobile phase to obtain 7.4 g of intermediate III.

[0072] In a separate 250 mL single-necked flask, add 7.0 g (16 mmol) of intermediate III, 140 mL of acetic acid, and 28 mL of hydrochloric acid (36.5%). Stir the mixture at 50 °C for 2 days. After the reaction is complete, add 200 mL of water and extract the organic phase with 200 mL of ethyl acetate. Dry the sample with anhydrous sodium sulfate, and stir with silica gel by rotary evaporation. Pass the product through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 4.1 g of intermediate IV, with a yield of 66.3%.

[0073] In a separate 250 mL single-necked flask, 4.1 g (11 mmol) of intermediate IV and 70 mL of acetic anhydride were added, and the mixture was stirred at 20 °C for 24 h. After the reaction was complete, the product was stirred with silica gel by rotary evaporation. The product was then passed through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 2.3 g of intermediate V, with a yield of 49.3%.

[0074] In a separate 250 mL single-necked flask, add 2.3 g (5 mmol) of intermediate V, 30 mL of methanol, and 30 mL of tetrahydrofuran. Stir the reaction mixture at 60 °C. Add 0.4 g (10 mmol) of sodium borohydride and continue the reaction at 60 °C for 24 h. After the reaction is complete, add 20 mL of water and dilute hydrochloric acid (5%) to a pH of 3.0. Extract with 100 mL of dichloromethane, wash the organic phase with 100 mL of saturated brine, dry with anhydrous sodium sulfate, and stir with rotary evaporation onto silica gel. Filter the product through a silica gel column with ethyl acetate:n-hexane in a 1:6 ratio (with 2% isopropanol) as the mobile phase to obtain cholesterol-3,4- 13 C2 white solid, 1.8 g, purity ≥ 98%, abundance ≥ 98 atom%. 13 C, yield 80.3%.

[0075] Example 4

[0076] This embodiment provides a stable isotope-labeled cholesterol-3,4- 13 The method for synthesizing C2 includes the following steps:

[0077] 1. Preparation of cyclic carbonyl intermediate II

[0078] In a 2L single-necked flask, add (52mmol) 4-cholesten-3-one, (62mmol) sodium carbonate, and 800mL isopropanol. After stirring and dissolving at 70℃, add dropwise a solution prepared from (7.5mmol) potassium permanganate, (292mmol) sodium periodate, and 400mL water. Control the dropping temperature at 70℃±5℃ and the dropping rate at 20mL / min. After the dropping is completed, continue to keep the reaction at the temperature for 5h. After the reaction was complete, the mixture was cooled, filtered, and the filter cake was washed with 100 mL of 5% sodium hydroxide solution, followed by 200 mL of dichloromethane. The organic phase was separated, and the aqueous phase was retained. Most of the isopropanol was removed by rotary evaporation. The pH of the aqueous phase was adjusted to 3.0, and then extracted with 250 mL of dichloromethane. The organic phase was washed with 300 mL of water, dried over anhydrous NaSO4, filtered, and dissolved by rotary evaporation to obtain ring-opening oxidized carboxylic acid intermediate I. In a 500 mL two-necked flask, (37 mmol) of ring-opening oxidized carboxylic acid intermediate I, (91 mmol) of sodium acetate, and 150 mL of acetic acid were added. Under nitrogen protection, the mixture was heated to 135 °C and refluxed with stirring for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain cyclic carbonyl intermediate II.

[0079] 2. Cholesterol-3,4- 13 Preparation of C2

[0080] In a 250 mL single-necked flask, add 10.5 g (27 mmol) of intermediate II and 3.9 g (27 mmol) of cyclohexyl acetate-1,2- in a molar ratio of 1:1. 13 C2, then add 4.3 g (108 mmol) sodium hydroxide and 100 mL anhydrous tetrahydrofuran, and stir at 25 °C for 4 days. After the reaction is complete, the sample is mixed with silica gel by rotary evaporation, and the product is passed through a silica gel column with ethyl acetate: n-hexane (5%-15% gradient, time 40 min) as the mobile phase to obtain 7.5 g of intermediate III.

[0081] In a separate 250 mL single-necked flask, add 7.0 g (16 mmol) of intermediate III, 140 mL of acetic acid, and 28 mL of hydrochloric acid (36.5%). Stir the mixture at 50 °C for 2 days. After the reaction is complete, add 200 mL of water and extract the organic phase with 200 mL of ethyl acetate. Dry the sample with anhydrous sodium sulfate, and stir with silica gel by rotary evaporation. Pass the product through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 4.1 g of intermediate IV, with a yield of 66.3%.

[0082] In a separate 250 mL single-necked flask, 4.1 g (11 mmol) of intermediate IV and 70 mL of acetic anhydride were added, and the mixture was stirred at 20 °C for 24 h. After the reaction was complete, the product was stirred with silica gel by rotary evaporation. The product was then passed through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 2.3 g of intermediate V, with a yield of 49.3%.

[0083] In a separate 250 mL single-necked flask, add 2.3 g (5 mmol) of intermediate V, 30 mL of methanol, and 30 mL of tetrahydrofuran. Stir the reaction mixture at 60 °C. Add 0.4 g (10 mmol) of sodium borohydride and continue the reaction at 60 °C for 24 h. After the reaction is complete, add 20 mL of water and dilute hydrochloric acid (5%) to a pH of 3.0. Extract with 100 mL of dichloromethane, wash the organic phase with 100 mL of saturated brine, dry with anhydrous sodium sulfate, and stir with rotary evaporation onto silica gel. Filter the product through a silica gel column with ethyl acetate:n-hexane in a 1:6 ratio (with 2% isopropanol) as the mobile phase to obtain cholesterol-3,4- 13 C2 white solid, 1.8 g, purity ≥ 98%, abundance ≥ 98 atom%. 13 C, yield 80.3%.

[0084] Example 5

[0085] Compared to Example 1, most aspects are the same, except for adjusting intermediate II in step 2 and 13 The molar ratio of C-labeled ester compounds is 1:10.

[0086] Example 6

[0087] Compared with Example 1, most of the contents are the same, except that the type of solvent used in step 2 is changed from anhydrous tetrahydrofuran to anhydrous methanol.

[0088] Example 7

[0089] Compared with Example 1, most of the process is the same, except that the type of solvent and reaction temperature in step 2 are adjusted. The solvent is changed from anhydrous tetrahydrofuran to DMSO, and the reaction temperature is adjusted from 25°C to 200°C.

[0090] Example 8

[0091] Compared with Example 1, most of them are the same, except that the reaction time in step 2 is adjusted from 4 days to 8 days.

[0092] Example 9

[0093] It is almost identical to Example 1, except that the molar ratio of intermediate II and base in step 2 is adjusted to 1:10.

[0094] Example 10

[0095] The process is largely the same as in Example 1, except that the mass-to-volume ratio of intermediate II and solvent in step 2 is adjusted to 1:20.

[0096] Comparative Example 1:

[0097] Referring to the adoption published by Sun-Shine and Yuan's team in 1982 13C3,4- cholesterol was synthesized using C-dip acyl chloride as a building block and employing a group protection strategy. 13 The C2 method (SUN SHINE, YUAN. Synthesis of 3,4-13C2 steroids[J]. Steroids, 1982, 39(3):279-289.) uses existing processes to prepare cholesterol-3,4- 13 C2:

[0098] 1. Preparation of cyclic carbonyl intermediate II

[0099] In a 3L single-necked flask, add (86mmol) 4-cholesten-3-one, (102mmol) sodium carbonate, and 1300mL isopropanol. After stirring and dissolving at 70℃, add dropwise a solution prepared from (12mmol) potassium permanganate, (483mmol) sodium periodate, and 660mL water. Control the dropping temperature at 70℃±5℃ and the dropping rate at 20mL / min. After the dropping is completed, continue to keep the reaction at the temperature for 5h. After the reaction was complete, the mixture was cooled, filtered, and the filter cake was washed with 165 mL of 5% sodium hydroxide solution, followed by 330 mL of dichloromethane. The organic phase was separated, and the aqueous phase was retained. Most of the isopropanol was removed by rotary evaporation. The pH of the aqueous phase was adjusted to 3.0, and then extracted with 420 mL of dichloromethane. The organic phase was washed with 500 mL of water, dried over anhydrous Na₂SO₄, filtered, and dissolved by rotary evaporation to obtain 24.9 g of ring-opening oxidized carboxylic acid intermediate I. In a 1 L two-necked flask, (61 mmol) of ring-opening oxidized carboxylic acid intermediate I, (150 mmol) of sodium acetate, and 247 mL of acetic acid were added. Under nitrogen protection, the mixture was heated to 135 °C and refluxed with stirring for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain 17.3 g of cyclic carbonyl intermediate II, with a yield of 74.2%.

[0100] 2. Preparation of acetylated labeling intermediate III

[0101] In a 500 mL single-necked flask, under nitrogen protection, intermediate II (45 mmol) and N-isopropylcyclohexylamine were added in a 1:1 molar ratio. The mixture was cooled to -20 °C and stirred for 1 h. Subsequently, the reaction system was cooled to -70 °C and 3.6 g (45 mmol) of acetyl chloride solution was slowly added dropwise. 13 The reaction mixture was stirred in 100 mL of anhydrous tetrahydrofuran solution of C2 at -70 °C for 1 h. After the reaction was complete, 100 mL of hydrochloric acid containing 45 mmol HCl was added, and the mixture was slowly brought back to room temperature. The mixture was then extracted with an organic solvent, washed, dried, and the solvent was evaporated to obtain 7.1 g of intermediate III, with a yield of 37%.

[0102] 3. Cholesterol-3,4- 13 Preparation of C2

[0103] A 250 mL single-necked flask was filled with 7.0 g (16 mmol) of acetylated labeled intermediate III, 140 mL of acetic acid, and 28 mL of hydrochloric acid (36.5%). The mixture was stirred at 50 °C for 2 days. After the reaction was complete, 200 mL of water and 200 mL of ethyl acetate were added to extract the organic phase. The extract was dried over anhydrous sodium sulfate, and the sample was stirred with rotary evaporation onto silica gel. The product was then passed through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to give 4.1 g of intermediate IV, with a yield of 66.3%.

[0104] In a separate 250 mL single-necked flask, 4.1 g (11 mmol) of intermediate IV and 70 mL of acetic anhydride were added, and the mixture was stirred at 20 °C for 24 h. After the reaction was complete, the product was stirred with silica gel by rotary evaporation. The product was then passed through a silica gel column with ethyl acetate:n-hexane in a mobile phase of 1:10 to obtain 2.3 g of intermediate V, with a yield of 49.3%.

[0105] In a separate 250 mL single-necked flask, add 2.3 g (5 mmol) of intermediate V, 30 mL of methanol, and 30 mL of tetrahydrofuran. Stir the reaction mixture at 60 °C. Add 0.4 g (10 mmol) of sodium borohydride and continue the reaction at 60 °C for 24 h. After the reaction is complete, add 20 mL of water and dilute hydrochloric acid (5%) to a pH of 3.0. Extract with 100 mL of dichloromethane, wash the organic phase with 100 mL of saturated brine, dry with anhydrous sodium sulfate, and stir with rotary evaporation onto silica gel. Filter the product through a silica gel column with ethyl acetate:n-hexane in a 1:6 ratio (with 2% isopropanol) as the mobile phase to obtain cholesterol-3,4- 13 C2 white solid, 1.8 g, purity ≥ 98%, abundance ≥ 98 atom%. 13 C, yield 80.3%.

[0106] As can be seen from step 2 of Comparative Example 1, the Sun-Shine and Yuan team published their findings in 1982 using... 13 C3,4- cholesterol was synthesized using C-dip acyl chloride as a building block and employing a group protection strategy. 13 The C2 method has many shortcomings in the introduction of marked blocks: (1) Firstly, it adopts 13 (1) C-labeled acyl chloride is used as the labeling reagent, which is chemically very unstable and has poor process stability; (2) Due to the high chemical reactivity of acyl chloride, the entire labeling reaction is carried out under very harsh conditions, such as -70℃; (3) The yield of the labeling reaction is only 37%, and the atom utilization rate is poor. In contrast, the labeling of the building blocks in Example 1 using phenyl acetate-1,2- 13 C2 is chemically stable, and the labeling introduction reaction conditions are mild, resulting in good process stability. The labeling introduction reaction yield is as high as 66.3%, with high atom utilization.

[0107] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A stable isotope-labeled cholesterol-3,4- 13 The method for synthesizing C2 is characterized by, The process route of the synthesis method is as follows: ， The synthesis method specifically includes the following steps: (1) Take the cyclic carbonyl intermediate II and 13 C-labeled ester compounds were reacted with stirring in a base and solvent to give acetylated labeled intermediate III; (2) The acetylated labeled intermediate III reacts with a mixed acid to obtain the carbocyclic reconstruction reaction intermediate IV; (3) The carbocyclic reconstruction reaction intermediate IV was reacted in acetic anhydride by stirring to obtain the esterification reaction intermediate V; (4) Esterification intermediate V reacts with sodium borohydride in a solvent system. After the reaction is complete, the pH of the system is adjusted to acidic, extracted, the organic phase is washed, dried, dissolved by rotary evaporation, and purified to obtain cholesterol-3,4- 13 Product C2 is the target product; The structural formula of the cyclic carbonyl intermediate II is: Where R is -CH(CH3)CH2CH2CH2CH(CH3)2; In step (1), the cyclic carbonyl intermediate II, 13 The molar ratio of C-labeled ester compounds is 1:1~10; The molar ratio of the cyclic carbonyl intermediate II to the base is 1:1~10; The mass-to-volume ratio of the cyclic carbonyl intermediate II to the solvent is 1 g: 10~20 mL; In step (1), the 13 The structure of C-labeled ester compounds is as follows: ， Wherein, X is phenyl, ethyl, or cyclohexyl.

2. A stable isotope-labeled cholesterol-3,4- according to claim 1 13 The method for synthesizing C2 is characterized by, The cyclic carbonyl intermediate II was prepared by the following method: (A) 4-Cholesterol-3-one was subjected to ring-opening oxidation in an organic solvent system under the action of an oxidant to obtain ring-opening oxidized carboxylic acid intermediate I; (B) The cyclic oxidation intermediate I of carboxylic acid was subjected to a cyclization reaction in acetic acid solvent in the presence of sodium acetate and under inert gas protection to obtain the cyclic carbonyl intermediate II.

3. A stable isotope-labeled cholesterol-3,4- according to claim 2 13 The method for synthesizing C2 is characterized by, In step (A), the organic solvent system is a mixture of isopropanol and water, and the oxidant is a mixture of potassium permanganate and sodium periodate. The molar ratio of 4-cholesten-3-one to the oxidant used is 1:2~10; The oxidation reaction temperature is 25~100℃; The oxidation reaction time is 2-8 hours; In step (B), the molar ratio of ring-opening oxidized carboxylic acid intermediate I to sodium acetate is 1:1~5; The cyclization reaction takes place at a temperature of 100~150 ℃; The ring-closing reaction takes 1 to 2 days.

4. A stable isotope-labeled cholesterol-3,4- according to claim 1 13 The method for synthesizing C2 is characterized by, In step (1), the alkali used is ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, or a mixture of one or more of these. The solvent used is one or a mixture of several of the following: tetrahydrofuran, methanol, ethanol, acetone, diethyl ether, dichloromethane, chloroform, n-hexane, petroleum ether, tert-butanol, cyclohexane, ethyl acetate, and dimethyl sulfoxide.

5. A stable isotope-labeled cholesterol-3,4- according to claim 1 13 The method for synthesizing C2 is characterized by, In step (1), the temperature of the stirring reaction is -20 ~ 200 ℃ and the time is 0.5 ~ 10.5 days.

6. A stable isotope-labeled cholesterol-3,4- according to claim 1 13 The method for synthesizing C2 is characterized by, In step (2), the mixed acid is a mixture of acetic acid and hydrochloric acid; The ratio of acetylated labeled intermediate III to acetic acid and hydrochloric acid was 7 g : (120~160) mL : (25-35) mL, wherein the mass fraction of hydrochloric acid was 36.5%. The reaction temperature is 40-60℃, and the reaction time is 1-3 days.

7. A stable isotope-labeled cholesterol-3,4- according to claim 1 13 The method for synthesizing C2 is characterized by, In step (3), the ratio of the amount of carbocyclic reconstruction reaction intermediate IV to acetic anhydride added is (4.0~4.2) g: 70 mL; The temperature for the stirring reaction is 15~25℃, and the time is 18~30h.

8. A stable isotope-labeled cholesterol-3,4- according to claim 1 13 The method for synthesizing C2 is characterized by, In step (4), the solvent system used is a mixed solvent system of methanol and tetrahydrofuran; The ratio of the amount of intermediate V in the esterification reaction, methanol, tetrahydrofuran and sodium borohydride added is (2.0~2.5) g : (25~35) mL : (25~35) mL : 0.4 g; The pH was adjusted to 3.0 using hydrochloric acid.

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