CN117946196A - Stable isotope labeled cholesterol-3, 4-13C2Is synthesized by the method of (2) - Google Patents

Stable isotope labeled cholesterol-3, 4-13C2Is synthesized by the method of (2) Download PDF

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CN117946196A
CN117946196A CN202410105583.8A CN202410105583A CN117946196A CN 117946196 A CN117946196 A CN 117946196A CN 202410105583 A CN202410105583 A CN 202410105583A CN 117946196 A CN117946196 A CN 117946196A
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reaction
cholesterol
ring
stable isotope
solvent
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刘前
白少飞
雷雯
孙雯
范若宁
赵诚
喻静兰
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Shanghai Research Institute of Chemical Industry SRICI
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Shanghai Research Institute of Chemical Industry SRICI
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Abstract

The invention relates to a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2, which comprises the following steps: (1) Taking and reacting a cyclic carbonyl intermediate II and 13 C double-labeled ester compound in alkali and a solvent under stirring to obtain an acetylation-labeled intermediate III; (2) Reacting the acetylation marked intermediate III with mixed acid to obtain a carbocycle reconstruction reaction intermediate IV; (3) The carbocycle reconstruction reaction intermediate IV is in acetic anhydride and stirred for reaction to obtain an esterification reaction intermediate V; (4) And (3) reacting the esterification reaction intermediate V with sodium borohydride in a solvent system, adjusting the pH of the system to be acidic after the reaction is finished, extracting, washing an organic phase, drying, rotary steaming to remove the solvent, and purifying to obtain a cholesterol-3, 4- 13C2 product, namely the target product. The invention breaks monopoly of cholesterol-3, 4- 13C2 which is one of the necessary steroid detection core marking reagents for vitamin detection, tumor screening and the like abroad, fills up the blank of the related fields in China and the like.

Description

Synthesis method of stable isotope labeled cholesterol-3, 4- 13C2
Technical Field
The invention belongs to the technical field of isotope synthesis, and relates to a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2.
Background
Steroid hormones are a series of hormones formed by cholesterol through different enzymes in the human body, including progestogens, androgens, estrogens, corticosteroids, etc., which play an important role in regulating the growth, development, reproduction, metabolism, etc. of the human body. Such hormonal metabolic disorders are the cause of many diseases, and accurate detection of their changes is of great reference value in the diagnosis of diseases.
Cholesterol (Cholesterol), which is one of steroid hormones, is a "female parent" of a series of steroids, has a steroid structure of cholest-5-en-ol, is widely present in animals, is closely related to the formation of cell membranes, is one of bioactive substances required for synthesis of bile acids, sterols, vitamin D, and the like, and is an essential substance for human animal tissue cells. Numerous epidemiological studies have demonstrated that cholesterol levels are closely related to the onset of coronary heart disease and that elevated levels in humans are also one of the contributors to atherosclerotic cardiovascular disease. In recent years, it has been found that the cholesterol content in humans is associated with various tumors, such as breast cancer, stomach cancer, colorectal cancer, prostate cancer and kidney cancer; in addition, the content of the extract is also related to various diseases such as gall-stone, fatty liver, diabetes and the like.
The most common methods for the detection of steroid hormones such as cholesterol in traditional clinical medicine are enzyme-linked immunosorbent assay (ELISA) and Radioimmunoassay (RIA). Because the content in human bodies is very different (ng/mL-pg/mL), the traditional ELISA and RIA detection methods have large sample size and long time consumption, are limited by the cross reaction of antibodies, often have inaccurate quantification and false positive results, and further influence the diagnosis and treatment of related diseases; in addition, the detection of steroid hormones such as cholesterol is easily confused with other substances having similar structures in the human body. Therefore, the conventional clinical medicine detection method cannot meet the detection requirements of high sensitivity and high recognition. In contrast, the stable isotope dilution mass spectrometry (clinical mass spectrometry) for clinical use has more recognition and accuracy, the concentration accuracy can reach pg/mL, and the sensitivity cannot be achieved by the traditional clinical medical detection method. The international development of clinical mass spectrometry has been advanced as early as 10 years ago, and the journal Journal of Clinical endocrinology and Metabolism of 2014 has indicated that the detection of steroid hormones such as cholesterol should be replaced by clinical mass spectrometry instead of the conventional detection method, and later in 2015, only the relevant data of clinical mass spectrometry should be approved. The Chinese clinical mass spectrum also enters the rapid development period after 2018, and the traditional biochemical diagnosis analysis method is replaced or partially replaced in a plurality of clinical detection fields, so that a wide development space is displayed.
The stable isotope labeled steroid hormone reagent is the core of the clinical detection of the hormone by using a stable isotope dilution mass spectrometry, so that cholesterol-3, 4- 13C2 is the core of the clinical mass spectrometry detection of cholesterol applied to diagnosis and treatment of various diseases such as coronary heart disease, atherosclerosis, tumor, fatty liver, gall-stone and diabetes.
At present, cholesterol-3, 4- 13C2, which is representative, is a detection reagent of steroid necessary for vitamin detection and tumor screening, cannot be autonomously synthesized in China, the production and supply of the detection reagent are monopolized in developed countries, such as CIL (sisal bridge isotope) company, sigma-Aldrich company and the like in the United states, the related reagent is expensive and limited in supply, and the cholesterol-3, 4- 13C2 is as high as 70000 yuan/g. The method forms serious restrictions on the development of clinical mass spectrometry detection of cholesterol and other steroid hormones in China, the exploration of novel detection methods, the production of kits, the expansion of practical use of clinical detection and the like.
There is no synthesis report of cholesterol-3, 4- 13C2 in China, and only a method for synthesizing cholesterol-3, 4- 13C2 by using 13 C double-labeled acyl chloride as a labeled building block and adopting a group protection strategy published by Sun-Shine and Yuan team in 1982 is reported abroad. The acyl chloride marked building blocks adopted by the method have poor stability and lower yield, so that the utilization rate of expensive 13 C raw materials is reduced, and the cost is higher; in addition, the synthesis method adopts a group protection strategy, and relates to the protection and deprotection processes of two groups, so that the overall synthesis steps are longer, and the process is complex.
There are only a few reports of 13 C marker introduction strategies for compounds with similar structures to cholesterol-3, 4- 13C2 abroad: ISA g.j. equal to Tetrahedron reported an ozonation degradation strategy in 2000, and 13 C-ylide was used as an introduction reagent to react with an alpha, beta-unsaturated carbonyl double bond oxidative cleavage intermediate to prepare progesterone. However, the oxidation process has a plurality of side reactions and long process, and the noble metal is used for catalysis, so that the cost is increased sharply; BERTHONNEAU C et al, J.org.chem.2018, reported a strategy for activation and reoxidation of thiophenol introduced into the A ring, using 13 C-Wittig reagent as carbon source, introducing an activating group into the steroid skeleton; however, the method has long experimental steps, complex oxidation operation, difficult purification caused by complex mixture generated after non-corresponding selective reduction of carbonyl, and low preparation efficiency.
Disclosure of Invention
The invention aims to provide a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2, so as to break monopoly of cholesterol-3, 4- 13C2 which is one of steroid detection core labeling reagents necessary for vitamin detection, tumor screening and the like in foreign countries, and fill the blank of the related fields in China.
The aim of the invention can be achieved by the following technical scheme:
A synthesis method of stable isotope labeled cholesterol-3, 4- 13C2 comprises the following steps:
(1) Stirring and reacting the cyclic carbonyl intermediate II and 13 C double-labeled ester compound in alkali and solvent to obtain an acetylation-labeled intermediate III;
(2) Reacting the acetylation marked intermediate III with mixed acid to obtain a carbocycle reconstruction reaction intermediate IV;
(3) The carbocycle reconstruction reaction intermediate IV is in acetic anhydride and stirred for reaction to obtain an esterification reaction intermediate V;
(4) The esterification reaction intermediate V reacts with sodium borohydride in a solvent system, the pH of the system is regulated to be acidic after the reaction is finished, the organic phase is extracted, washed, dried, distilled to remove solvent in a rotary way, and purified to obtain cholesterol-3, 4- 13C2, namely a target product;
The structural formula of the ring-closing carbonyl intermediate II is as follows:
Wherein R is-CH (CH 3)CH2CH2CH2CH(CH3)2).
Further, the cyclic carbonyl intermediate II is prepared by the following method:
(A) 4-cholesten-3-one is subjected to ring opening oxidation reaction in an organic solvent system under the action of an oxidant to obtain a ring opening oxidation carboxylic acid intermediate I;
(B) And (3) ring-opening oxidation carboxylic acid intermediate I is subjected to ring-closing reaction in acetic acid solvent under the protection of inert gas in the presence of sodium acetate to obtain a ring-closing carbonyl intermediate II.
Further, in the step (A), the organic solvent system is a mixed solvent of isopropanol and water, and the oxidant is a compound of potassium permanganate and sodium periodate; the molar ratio of the 4-cholesten-3-one and the oxidant is 1:2-10; the oxidation reaction temperature is 25-100 ℃; the oxidation reaction time is 2-8 hours.
Further, in the step (2), the molar ratio of the ring-opened oxidized carboxylic acid intermediate I to the sodium acetate is 1:1-5, specifically may be 1:1,1:5, or 1:2, etc.; the temperature of the ring closing reaction is 100-150 ℃; the ring closing reaction time is 1-2 days.
Further, in the step (1), the molar ratio of the cyclic carbonyl intermediate II to the 13 C double-labeled ester compound is 1:1-10, and the addition amount can be any intermediate point value such as 1:1,1:10, or 1:5; the molar ratio of the ring-closing carbonyl intermediate II to the alkali is 1:1-10, and the addition amount can be any intermediate point value such as 1:1,1:10, or 1:5; the mass volume ratio of the ring-closing carbonyl intermediate II to the solvent is 1:10-20, and can be intermediate point values of 1:10,1:20, 1:15 and the like.
Further, in the step (1), the alkali is one or a mixture of a plurality of ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, sodium hydride and potassium hydride.
Further, in the step (1), the solvent is one or a mixture of a plurality of tetrahydrofuran, methanol, ethanol, acetone, diethyl ether, methylene dichloride, chloroform, normal hexane, petroleum ether, tertiary butanol, cyclohexane, ethyl acetate and dimethyl sulfoxide.
Further, in the step (1), the temperature of the stirring reaction is between-20 and 200 ℃ and the time is between 0.5 and 10.5 days.
Further, in the step (1), the 13 C double-labeled ester compound has a structure as follows:
wherein X is alkyl, cycloalkyl or aryl.
Further, in the step (2), the mixed acid is a mixture of acetic acid and hydrochloric acid.
Further, in the step (2), the ratio of the added amount of the acetylated labeled intermediate III to acetic acid and hydrochloric acid was 7g: (120-160) mL: (25-35) mL, wherein the mass fraction of the hydrochloric acid is 36.5%.
Further, in the step (2), the reaction temperature is 40-60 ℃ and the reaction time is 1-3 days.
Further, in the step (3), the ratio of the addition amount of the carbocycle reconstitution reaction intermediate IV to acetic anhydride is (4.0 to 4.2) g:70mL.
In the step (3), the temperature of the stirring reaction is 15-25 ℃ and the time is 18-30 h.
Further, in the step (4), the solvent system is a mixed solvent system of methanol and tetrahydrofuran.
Further, in the step (4), the ratio of the addition amounts of the esterification intermediate V, methanol, tetrahydrofuran and sodium borohydride is (2.0 to 2.5) g: (25-35) mL: (25-35) mL:0.4g.
Further, in the step (4), the pH is adjusted to 3.0 with hydrochloric acid.
Compared with the prior art, the invention has the following advantages:
(1) The invention adopts 13 C double-labeled ester compounds as the labeled introduced building blocks, the chemical property of the labeled building blocks is stable, and the labeling synthesis yield is higher; the marked building blocks are introduced in the later steps, so that the consumption of valuable raw materials is reduced as much as possible, the economic applicability is stronger, and the risk of dilution of the abundance of the product can be better avoided.
(2) The semi-synthesis strategy is adopted, so that the raw materials are rich and easy to obtain, the price is low, the synthesis steps are reduced, and the synthesis efficiency is improved; the reaction route is expandable, and can be used for developing three-carbon marked cholesterol or 'transplanted' in other marked steroid clinical diagnosis reagents with similar structures, such as 13 C marked progesterone, pregnenolone, testosterone, 17-hydroxy progesterone, androstenedione and the like, so as to form a series of compounds.
(3) The purity of the obtained cholesterol-3, 4- 13C2 product is more than or equal to 98%, the abundance is more than or equal to 98atom percent 13 C, obvious dilution does not occur, and the product can be used as an internal standard reagent for detecting stable isotope dilution mass spectrometry in the clinical and biomedical fields, a raw material of a disease screening kit for clinical mass spectrometry, and the like.
Drawings
FIG. 1 is a synthetic process scheme of the present invention.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
In the invention, ethyl acetate-1, 2- 13C2 is purchased from Sigma-Aldrich company, the abundance is more than or equal to 99atom percent 13 C, and the product number is 283819; acetyl chloride- 13C2 is purchased from Sigma-Aldrich company, and the abundance is greater than or equal to 99atom percent 13 C, and the product number is 293164; phenyl acetate-1, 2- 13C2 and cyclohexyl acetate-1, 2- 13C2 were prepared from acetyl chloride- 13C2 by the following method:
Into a 500mL one-necked flask, phenol (86 mmol) or cyclohexanol, (79 mmol) acetyl chloride and 150mL cyclohexane were added, and the mixture was refluxed at 25℃for 6 hours. After the reaction is finished, phenyl acetate-1, 2- 13C2 or cyclohexyl acetate-1, 2- 13C2 is obtained after rotary evaporation.
The remainder, unless specifically stated, is indicative of a conventional commercially available feedstock or conventional processing technique in the art.
Example 1:
the embodiment provides a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2, which comprises the following steps:
1. preparation of cyclic carbonyl intermediate II
Into a 2L single-neck flask, 4-cholesten-3-one (52 mmol), sodium carbonate (62 mmol) and isopropyl alcohol (800 mL) are added, and after stirring and dissolving at 70 ℃, a solution prepared from potassium permanganate (7.5 mmol), sodium periodate (292 mmol) and water (400 mL) is dripped, the dripping temperature is controlled to be 70+/-5 ℃, the dripping speed is controlled to be 20mL/min, and the heat preservation reaction is continued for 5h after the dripping is finished. After the reaction is finished, cooling, suction filtering, washing a filter cake with 100mL of 5% sodium hydroxide solution, washing with 200mL of dichloromethane, separating to remove an organic phase, reserving a water phase, removing most of isopropanol by rotary evaporation, adjusting the pH of the water phase to 3.0, extracting with 250mL of dichloromethane, washing the organic phase with 300mL of water, drying with anhydrous Na 2SO4, filtering, rotary evaporation to remove the solution, and obtaining a ring-opening oxidized carboxylic acid intermediate I; in a 500mL two-necked flask, the ring-opened carboxylic acid intermediate I (37 mmol), sodium acetate (91 mmol) and acetic acid (150 mL) were added, and the mixture was heated to 135℃under nitrogen protection, followed by stirring and refluxing for 24 hours. And after the reaction is finished, removing the solvent by rotary evaporation to obtain a cyclic carbonyl intermediate II.
2. Preparation of cholesterol-3, 4- 13C2
Into a 250mL single-neck flask, 10.5g (27 mmol) of intermediate II and 3.7g (27 mmol) of phenyl acetate-1, 2- 13C2 in a molar ratio of 1:1 were added, and 4.3g (108 mmol) of sodium hydrogen (sodium hydride) (60%) and 100mL of anhydrous tetrahydrofuran were further added, and the mixture was stirred at 25℃for 4d; after the reaction is finished, the sample is mixed by steaming silica gel, the product passes through a column by silica gel, and the mobile phase is ethyl acetate: n-hexane (5% -15% gradient, time 40 min) 7.0g of intermediate III.
A250 mL single-necked flask was additionally charged with 7.0g (16 mmol) of intermediate III, 140mL of acetic acid, 28mL of hydrochloric acid (36.5%) and reacted at 50℃with stirring for 2d. After the reaction is finished, 200mL of water and 200mL of ethyl acetate are added to extract an organic phase, anhydrous sodium sulfate is dried, silica gel is steamed in a rotating way to mix a sample, the product passes through a column through silica gel, the mobile phase is ethyl acetate and n-hexane are 1:10, 4.1g of intermediate IV is obtained, and the yield is 66.3%.
Another 250mL single-neck flask was charged with 4.1g (11 mmol) of intermediate IV and 70mL of acetic anhydride, and the reaction was stirred at 20℃for 24 hours. After the reaction is finished, the silica gel is distilled, the product is subjected to column chromatography through silica gel, the mobile phase is ethyl acetate and n-hexane is 1:10, and 2.3g of intermediate V is obtained, and the yield is 49.3%.
A250 mL single-necked flask was further prepared, 2.3g (5 mmol) of intermediate V, 30mL of methanol and 30mL of tetrahydrofuran were added, the mixture was stirred at 60℃to react, 0.4g (10 mmol) of sodium borohydride was added, and the reaction was continued at 60℃for 24 hours. After the reaction, 20mL of water, diluted hydrochloric acid (5%) until the pH is 3.0, 100mL of dichloromethane are added for extraction, 100mL of saturated saline water is used for washing the organic phase, anhydrous sodium sulfate is used for drying, the rotary evaporation silica gel is used for sample mixing, the product passes through a silica gel column, and the mobile phase is ethyl acetate: n-hexane is 1:6 (2% isopropyl alcohol is added) to obtain 1.8g of cholesterol-3, 4- 13C2 white solid, the purity is more than or equal to 98%, the abundance is more than or equal to 98atom% 13 C, and the yield is 80.3%.
Example 2
The embodiment provides a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2, which comprises the following steps:
1. preparation of cyclic carbonyl intermediate II
Into a 2L single-neck flask, 4-cholesten-3-one (52 mmol), sodium carbonate (62 mmol) and isopropyl alcohol (800 mL) are added, and after stirring and dissolving at 70 ℃, a solution prepared from potassium permanganate (7.5 mmol), sodium periodate (292 mmol) and water (400 mL) is dripped, the dripping temperature is controlled to be 70+/-5 ℃, the dripping speed is controlled to be 20mL/min, and the heat preservation reaction is continued for 5h after the dripping is finished. After the reaction is finished, cooling, suction filtering, washing a filter cake by 100mL of 5% sodium hydroxide solution, washing by 200mL of dichloromethane, separating to remove an organic phase, reserving a water phase, removing most of isopropanol by rotary evaporation, adjusting the pH of the water phase to 3.0, extracting by 250mL of dichloromethane, washing the organic phase by 300mL of water, drying by anhydrous NaSO 4, filtering, rotary evaporation, and removing the solution to obtain a ring-opening oxidized carboxylic acid intermediate I; in a 500mL two-necked flask, the ring-opened carboxylic acid intermediate I (37 mmol), sodium acetate (91 mmol) and acetic acid (150 mL) were added, and the mixture was heated to 135℃under nitrogen protection, followed by stirring and refluxing for 24 hours. And after the reaction is finished, removing the solvent by rotary evaporation to obtain a cyclic carbonyl intermediate II.
2. Preparation of cholesterol-3, 4- 13C2
Into a 250mL single-necked flask, 10.5g (27 mmol) of intermediate II and 2.4g (27 mmol) of ethyl acetate-1, 2- 13C2 in a molar ratio of 1:1 were charged, and then 4.3g (108 mmol) of sodium hydrogen (60%) and 100mL of anhydrous tetrahydrofuran were added thereto, followed by stirring at 25℃for 4 days. After the reaction is finished, the sample is mixed by steaming silica gel, the product passes through a column by silica gel, and the mobile phase is ethyl acetate: n-hexane (5% -15% gradient, 40 min) to give intermediate III 7.2g.
A250 mL single-necked flask was additionally charged with 7.0g (16 mmol) of intermediate III, 140mL of acetic acid, 28mL of hydrochloric acid (36.5%) and reacted at 50℃with stirring for 2d. After the reaction is finished, 200mL of water and 200mL of ethyl acetate are added to extract an organic phase, anhydrous sodium sulfate is dried, silica gel is steamed in a rotating way to mix a sample, the product passes through a column through silica gel, the mobile phase is ethyl acetate and n-hexane are 1:10, 4.1g of intermediate IV is obtained, and the yield is 66.3%.
Another 250mL single-neck flask was charged with 4.1g (11 mmol) of intermediate IV and 70mL of acetic anhydride, and the reaction was stirred at 20℃for 24 hours. After the reaction is finished, the silica gel is distilled, the product is subjected to column chromatography through silica gel, the mobile phase is ethyl acetate and n-hexane is 1:10, and 2.3g of intermediate V is obtained, and the yield is 49.3%.
A250 mL single-necked flask was further prepared, 2.3g (5 mmol) of intermediate V, 30mL of methanol and 30mL of tetrahydrofuran were added, the mixture was stirred at 60℃to react, 0.4g (10 mmol) of sodium borohydride was added, and the reaction was continued at 60℃for 24 hours. After the reaction, 20mL of water, diluted hydrochloric acid (5%) until the pH is 3.0, 100mL of dichloromethane are added for extraction, 100mL of saturated saline water is used for washing the organic phase, anhydrous sodium sulfate is used for drying, the rotary evaporation silica gel is used for sample mixing, the product passes through a silica gel column, and the mobile phase is ethyl acetate: n-hexane is 1:6 (2% isopropyl alcohol is added) to obtain 1.8g of cholesterol-3, 4- 13C2 white solid, the purity is more than or equal to 98%, the abundance is more than or equal to 98atom% 13 C, and the yield is 80.3%.
Example 3
The embodiment provides a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2, which comprises the following steps:
1. preparation of cyclic carbonyl intermediate II
Into a 2L single-neck flask, 4-cholesten-3-one (52 mmol), sodium carbonate (62 mmol) and isopropyl alcohol (800 mL) are added, and after stirring and dissolving at 70 ℃, a solution prepared from potassium permanganate (7.5 mmol), sodium periodate (292 mmol) and water (400 mL) is dripped, the dripping temperature is controlled to be 70+/-5 ℃, the dripping speed is controlled to be 20mL/min, and the heat preservation reaction is continued for 5h after the dripping is finished. After the reaction is finished, cooling, suction filtering, washing a filter cake by 100mL of 5% sodium hydroxide solution, washing by 200mL of dichloromethane, separating to remove an organic phase, reserving a water phase, removing most of isopropanol by rotary evaporation, adjusting the pH of the water phase to 3.0, extracting by 250mL of dichloromethane, washing the organic phase by 300mL of water, drying by anhydrous NaSO 4, filtering, rotary evaporation, and removing the solution to obtain a ring-opening oxidized carboxylic acid intermediate I; in a 500mL two-necked flask, the ring-opened carboxylic acid intermediate I (37 mmol), sodium acetate (91 mmol) and acetic acid (150 mL) were added, and the mixture was heated to 135℃under nitrogen protection, followed by stirring and refluxing for 24 hours. And after the reaction is finished, removing the solvent by rotary evaporation to obtain a cyclic carbonyl intermediate II.
2. Preparation of cholesterol-3, 4- 13C2
Into a 250mL single-necked flask, 10.5g (27 mmol) of intermediate II and 3.9g (27 mmol) of cyclohexyl acetate-1, 2- 13C2 in a molar ratio of 1:1 were charged, and then 4.3g (108 mmol) of sodium hydrogen (60%) and 100mL of anhydrous tetrahydrofuran were added thereto, and the mixture was stirred at 25℃for 4 days. After the reaction is finished, the sample is mixed by steaming silica gel, the product passes through a column by silica gel, and the mobile phase is ethyl acetate: n-hexane (5% -15% gradient, 40 min) 7.4g of intermediate III.
A250 mL single-necked flask was additionally charged with 7.0g (16 mmol) of intermediate III, 140mL of acetic acid, 28mL of hydrochloric acid (36.5%) and reacted at 50℃with stirring for 2d. After the reaction is finished, 200mL of water and 200mL of ethyl acetate are added to extract an organic phase, anhydrous sodium sulfate is dried, silica gel is steamed in a rotating way to mix a sample, the product passes through a column through silica gel, the mobile phase is ethyl acetate and n-hexane are 1:10, 4.1g of intermediate IV is obtained, and the yield is 66.3%.
Another 250mL single-neck flask was charged with 4.1g (11 mmol) of intermediate IV and 70mL of acetic anhydride, and the reaction was stirred at 20℃for 24 hours. After the reaction is finished, the silica gel is distilled, the product is subjected to column chromatography through silica gel, the mobile phase is ethyl acetate and n-hexane is 1:10, and 2.3g of intermediate V is obtained, and the yield is 49.3%.
A250 mL single-necked flask was further prepared, 2.3g (5 mmol) of intermediate V, 30mL of methanol and 30mL of tetrahydrofuran were added, the mixture was stirred at 60℃to react, 0.4g (10 mmol) of sodium borohydride was added, and the reaction was continued at 60℃for 24 hours. After the reaction, 20mL of water, diluted hydrochloric acid (5%) until the pH is 3.0, 100mL of dichloromethane are added for extraction, 100mL of saturated saline water is used for washing the organic phase, anhydrous sodium sulfate is used for drying, the rotary evaporation silica gel is used for sample mixing, the product passes through a silica gel column, and the mobile phase is ethyl acetate: n-hexane is 1:6 (2% isopropyl alcohol is added) to obtain 1.8g of cholesterol-3, 4- 13C2 white solid, the purity is more than or equal to 98%, the abundance is more than or equal to 98atom% 13 C, and the yield is 80.3%.
Example 4
The embodiment provides a synthesis method of stable isotope labeled cholesterol-3, 4- 13C2, which comprises the following steps:
1. preparation of cyclic carbonyl intermediate II
Into a 2L single-neck flask, 4-cholesten-3-one (52 mmol), sodium carbonate (62 mmol) and isopropyl alcohol (800 mL) are added, and after stirring and dissolving at 70 ℃, a solution prepared from potassium permanganate (7.5 mmol), sodium periodate (292 mmol) and water (400 mL) is dripped, the dripping temperature is controlled to be 70+/-5 ℃, the dripping speed is controlled to be 20mL/min, and the heat preservation reaction is continued for 5h after the dripping is finished. After the reaction is finished, cooling, suction filtering, washing a filter cake by 100mL of 5% sodium hydroxide solution, washing by 200mL of dichloromethane, separating to remove an organic phase, reserving a water phase, removing most of isopropanol by rotary evaporation, adjusting the pH of the water phase to 3.0, extracting by 250mL of dichloromethane, washing the organic phase by 300mL of water, drying by anhydrous NaSO 4, filtering, rotary evaporation, and removing the solution to obtain a ring-opening oxidized carboxylic acid intermediate I; in a 500mL two-necked flask, the ring-opened carboxylic acid intermediate I (37 mmol), sodium acetate (91 mmol) and acetic acid (150 mL) were added, and the mixture was heated to 135℃under nitrogen protection, followed by stirring and refluxing for 24 hours. And after the reaction is finished, removing the solvent by rotary evaporation to obtain a cyclic carbonyl intermediate II.
2. Preparation of cholesterol-3, 4- 13C2
Into a 250mL single-necked flask, 10.5g (27 mmol) of intermediate II and 3.9g (27 mmol) of cyclohexyl acetate-1, 2- 13C2 in a molar ratio of 1:1 were charged, and then 4.3g (108 mmol) of sodium hydroxide and 100mL of anhydrous tetrahydrofuran were added thereto to stir the mixture at 25℃for 4d. After the reaction is finished, the sample is mixed by steaming silica gel, the product passes through a column by silica gel, and the mobile phase is ethyl acetate: n-hexane (5% -15% gradient, 40 min) 7.5g of intermediate III.
A250 mL single-necked flask was additionally charged with 7.0g (16 mmol) of intermediate III, 140mL of acetic acid, 28mL of hydrochloric acid (36.5%) and reacted at 50℃with stirring for 2d. After the reaction is finished, 200mL of water and 200mL of ethyl acetate are added to extract an organic phase, anhydrous sodium sulfate is dried, silica gel is steamed in a rotating way to mix a sample, the product passes through a column through silica gel, the mobile phase is ethyl acetate and n-hexane are 1:10, 4.1g of intermediate IV is obtained, and the yield is 66.3%.
Another 250mL single-neck flask was charged with 4.1g (11 mmol) of intermediate IV and 70mL of acetic anhydride, and the reaction was stirred at 20℃for 24 hours. After the reaction is finished, the silica gel is distilled, the product is subjected to column chromatography through silica gel, the mobile phase is ethyl acetate and n-hexane is 1:10, and 2.3g of intermediate V is obtained, and the yield is 49.3%.
A250 mL single-necked flask was further prepared, 2.3g (5 mmol) of intermediate V, 30mL of methanol and 30mL of tetrahydrofuran were added, the mixture was stirred at 60℃to react, 0.4g (10 mmol) of sodium borohydride was added, and the reaction was continued at 60℃for 24 hours. After the reaction, 20mL of water, diluted hydrochloric acid (5%) until the pH is 3.0, 100mL of dichloromethane are added for extraction, 100mL of saturated saline water is used for washing the organic phase, anhydrous sodium sulfate is used for drying, the rotary evaporation silica gel is used for sample mixing, the product passes through a silica gel column, and the mobile phase is ethyl acetate: n-hexane is 1:6 (2% isopropyl alcohol is added) to obtain 1.8g of cholesterol-3, 4- 13C2 white solid, the purity is more than or equal to 98%, the abundance is more than or equal to 98atom% 13 C, and the yield is 80.3%.
Example 5
Most of the same as in example 1, except that the molar ratio of intermediate II and 13 C of step 2 to the di-labeled ester compound was adjusted to 1:10.
Example 6
In comparison with example 1, the same procedure was followed except that the solvent type used in step 2 was changed from anhydrous tetrahydrofuran to anhydrous methanol.
Example 7
In comparison with example 1, the same procedure was repeated except that the type of solvent used in step 2 and the reaction temperature were adjusted, the solvent was changed from anhydrous tetrahydrofuran to DMSO, and the reaction temperature was adjusted from 25℃to 200 ℃.
Example 8
In comparison with example 1, the reaction time was adjusted from 4d to 8d, except that the reaction time in step 2 was adjusted.
Example 9
Most of the same as in example 1, except that the molar ratio of intermediate II to base in step 2 was adjusted to 1:10.
Example 10
The process is largely identical to example 1, except that the mass to volume ratio of intermediate II to solvent in step 2 is adjusted to 1:20.
Comparative example 1:
Referring to the method of synthesizing cholesterol-3, 4- 13C2 using 13 C double labeled acyl chloride as labeled block and using group protection strategy published by Sun-Shine and Yuan team in 1982 (SUN SHINE, YUAN.Synthesis of 3,4-13C2 sterois [ J ]. Steroids,1982,39 (3): 279-289 ]) cholesterol-3, 4- 13C2 was prepared using prior art techniques:
1. preparation of cyclic carbonyl intermediate II
Into a 3L single-neck flask, (86 mmol) 4-cholesten-3-one, (102 mmol) sodium carbonate and 1300mL isopropanol are added, after stirring and dissolving at 70 ℃, a solution prepared from (12 mmol) potassium permanganate, (483 mmol) sodium periodate and 660mL water is dripped, the dripping temperature is controlled to be 70+/-5 ℃, the dripping speed is controlled to be 20mL/min, and the heat preservation reaction is continued for 5h after the dripping is finished. After the reaction is finished, cooling, suction filtering, washing a filter cake with 165mL of 5% sodium hydroxide solution, washing with 330mL of dichloromethane, separating to remove an organic phase, reserving a water phase, removing most of isopropanol by rotary evaporation, adjusting the pH of the water phase to 3.0, extracting with 420mL of dichloromethane, washing the organic phase with 500mL of water, drying with anhydrous Na 2SO4, filtering, rotary evaporation to remove the solution, and obtaining 24.9g of ring-opening oxidized carboxylic acid intermediate I; in a 1L two-necked flask, 61mmol of the ring-opening oxidized carboxylic acid intermediate I, (150 mmol) of sodium acetate and 247mL of acetic acid were added, and the mixture was heated to 135℃under nitrogen protection, stirred and refluxed for 24 hours. After the reaction, the solvent was distilled off by rotary evaporation to obtain 17.3g of a cyclic carbonyl intermediate II, with a yield of 74.2%.
2. Preparation of acetylated marked intermediate III
In a 500mL single-neck flask, adding (45 mmol) of intermediate II and N-isopropyl cyclohexylamine in a molar ratio of 1:1 under the protection of nitrogen, cooling to-20 ℃ and stirring for 1h, then cooling the reaction system to-70 ℃ and slowly dripping 100mL anhydrous tetrahydrofuran solution dissolved with 3.6g (45 mmol) of acetyl chloride- 13C2, and keeping stirring at-70 ℃ for reacting for 1h. After the reaction, 100mL of hydrochloric acid in which 45mmol of HCl was dissolved was added, and the reaction mixture was slowly returned to room temperature, followed by extraction with an organic solvent, washing, drying, and spin-drying of the solvent to obtain 7.1g of intermediate III in 37% yield.
3. Preparation of cholesterol-3, 4- 13C2
A250 mL single-necked flask was charged with 7.0g (16 mmol) of the acetylated intermediate III, 140mL of acetic acid, 28mL of hydrochloric acid (36.5%) and reacted at 50℃with stirring for 2d. After the reaction is finished, 200mL of water and 200mL of ethyl acetate are added to extract an organic phase, anhydrous sodium sulfate is dried, silica gel is steamed in a rotating way to mix a sample, the product passes through a column through silica gel, the mobile phase is ethyl acetate and n-hexane are 1:10, 4.1g of intermediate IV is obtained, and the yield is 66.3%.
Another 250mL single-neck flask was charged with 4.1g (11 mmol) of intermediate IV and 70mL of acetic anhydride, and the reaction was stirred at 20℃for 24 hours. After the reaction is finished, the silica gel is distilled, the product is subjected to column chromatography through silica gel, the mobile phase is ethyl acetate and n-hexane is 1:10, and 2.3g of intermediate V is obtained, and the yield is 49.3%.
A250 mL single-necked flask was further prepared, 2.3g (5 mmol) of intermediate V, 30mL of methanol and 30mL of tetrahydrofuran were added, the mixture was stirred at 60℃to react, 0.4g (10 mmol) of sodium borohydride was added, and the reaction was continued at 60℃for 24 hours. After the reaction, 20mL of water, diluted hydrochloric acid (5%) until the pH is 3.0, 100mL of dichloromethane are added for extraction, 100mL of saturated saline water is used for washing the organic phase, anhydrous sodium sulfate is used for drying, the rotary evaporation silica gel is used for sample mixing, the product passes through a silica gel column, and the mobile phase is ethyl acetate: n-hexane is 1:6 (2% isopropyl alcohol is added) to obtain 1.8g of cholesterol-3, 4- 13C2 white solid, the purity is more than or equal to 98%, the abundance is more than or equal to 98atom% 13 C, and the yield is 80.3%.
As can be seen from step 2 of comparative example 1, the method of using 13 C double labeled acyl chloride as labeled block and using radical protection strategy for synthesizing cholesterol-3, 4- 13C2 published by Sun-Shine and Yuan team in 1982 has many disadvantages in the introduction of labeled block: (1) Firstly, 13 C marked acyl chloride is adopted as a marked introduction reagent, the chemical property is very unstable, and the process stability is poor; (2) Since acyl chlorides are too reactive in chemistry, the entire label introduction reaction is caused to proceed under very severe conditions, such as-70 ℃ and the like; (3) The yield of the label introduction reaction is only 37%, and the atom utilization rate is poor. In contrast, compared with the embodiment 1, the marked building block is introduced by phenyl acetate-1, 2- 13C2, the chemical property is stable, the marked introduction reaction condition is mild, and the process stability is better; the yield of the label introduction reaction is up to 66.3%, and the atom utilization rate is high.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims (10)

1. A method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2, which is characterized by comprising the following steps:
(1) Taking and reacting a cyclic carbonyl intermediate II and 13 C double-labeled ester compound in alkali and a solvent under stirring to obtain an acetylation-labeled intermediate III;
(2) Reacting the acetylation marked intermediate III with mixed acid to obtain a carbocycle reconstruction reaction intermediate IV;
(3) The carbocycle reconstruction reaction intermediate IV is in acetic anhydride and stirred for reaction to obtain an esterification reaction intermediate V;
(4) The esterification reaction intermediate V reacts with sodium borohydride in a solvent system, the pH of the system is regulated to be acidic after the reaction is finished, the organic phase is extracted, washed, dried, distilled to remove solvent in a rotary way, and purified to obtain cholesterol-3, 4- 13C2, namely a target product;
The structural formula of the ring-closing carbonyl intermediate II is as follows:
Wherein R is-CH (CH 3)CH2CH2CH2CH(CH3)2).
2. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein the cyclic carbonyl intermediate ii is prepared by the following method:
(A) 4-cholesten-3-one is subjected to ring opening oxidation reaction in an organic solvent system under the action of an oxidant to obtain a ring opening oxidation carboxylic acid intermediate I;
(B) And (3) ring-opening oxidation carboxylic acid intermediate I is subjected to ring-closing reaction in acetic acid solvent under the protection of inert gas in the presence of sodium acetate to obtain a ring-closing carbonyl intermediate II.
3. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 2, wherein in step (a), the organic solvent system is a mixed solvent of isopropanol and water, and the oxidant is a compound of potassium permanganate and sodium periodate;
The molar ratio of the 4-cholesten-3-one to the oxidant is 1:2-10;
The oxidation reaction temperature is 25-100 ℃;
The oxidation reaction time is 2-8 hours;
In the step (B), the molar ratio of the ring-opening oxidized carboxylic acid intermediate I to the sodium acetate is 1:1-5;
The temperature of the ring closing reaction is 100-150 ℃;
the ring closing reaction time is 1-2 days.
4. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in the step (1), the molar ratio of the cyclic carbonyl intermediate II to the 13 C double-labeled ester compound is 1:1-10;
the molar ratio of the ring-closing carbonyl intermediate II to the alkali is 1:1-10;
the mass volume ratio of the ring-closing carbonyl intermediate II to the solvent is 1 g:10-20 mL.
5. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in step (1), the base is one or a mixture of several of ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, sodium hydride, and potassium hydride;
the solvent is one or more of tetrahydrofuran, methanol, ethanol, acetone, diethyl ether, dichloromethane, chloroform, n-hexane, petroleum ether, tert-butanol, cyclohexane, ethyl acetate, and dimethyl sulfoxide.
6. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in the step (1), the temperature of stirring reaction is-20-200 ℃ and the time is 0.5-10.5 days.
7. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in step (1), the 13 C double-labeled ester compound has the structure:
wherein X is alkyl, cycloalkyl or aryl.
8. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in step (2), the mixed acid is a mixture of acetic acid and hydrochloric acid;
the ratio of the added amount of the acetylation marked intermediate III to acetic acid and hydrochloric acid was 7g: (120-160) mL: (25-35) mL, wherein the mass fraction of the hydrochloric acid is 36.5%;
The reaction temperature is 40-60 ℃ and the reaction time is 1-3 days.
9. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in the step (3), the ratio of the addition amount of carbocycle reconstitution reaction intermediate iv to acetic anhydride is (4.0-4.2) g:70mL;
the temperature of the stirring reaction is 15-25 ℃ and the time is 18-30 h.
10. The method for synthesizing stable isotope labeled cholesterol-3, 4- 13C2 according to claim 1, wherein in step (4), the solvent system used is a mixed solvent system of methanol and tetrahydrofuran;
the ratio of the addition amount of the esterification reaction intermediate V, methanol, tetrahydrofuran and sodium borohydride is (2.0-2.5) g: (25-35) mL: (25-35) mL:0.4g;
The pH was adjusted to 3.0 using hydrochloric acid.
CN202410105583.8A 2024-01-25 2024-01-25 Stable isotope labeled cholesterol-3, 4-13C2Is synthesized by the method of (2) Pending CN117946196A (en)

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