CN115724899A - Preparation method of high-purity cholesterol - Google Patents

Preparation method of high-purity cholesterol Download PDF

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CN115724899A
CN115724899A CN202211454023.0A CN202211454023A CN115724899A CN 115724899 A CN115724899 A CN 115724899A CN 202211454023 A CN202211454023 A CN 202211454023A CN 115724899 A CN115724899 A CN 115724899A
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cholesterol
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肖剑波
王涛
万定建
孟宪志
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Shanghai Qizheng Pharmaceutical Technology Co ltd
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Abstract

The invention belongs to the technical field of synthesizing cholesterol, and provides a preparation method of high-purity cholesterol, which comprises the steps of carrying out sulfonation reaction on 21-hydroxy-20-methyl pregn-4-ene-3-ketone, a sulfonyl compound and alkali in a solvent; performing 3-carbonyl protection reaction on the sulfonation reaction product, triethyl orthoformate and p-toluenesulfonic acid; performing addition reaction on the 3-carbonyl protection reaction product, the Grignard reagent and cuprous salt; the 3-bit protecting group of the addition reaction product is subjected to acetylation reaction under the conditions of acetic acid and acetic anhydride; and performing reduction reaction on the acetylation reaction product to obtain cholesterol. The invention has simple process, high synthesis yield and low cost; reduces impurities in the product, improves the purity of cholesterol, and has high safety of the synthesis method. Meanwhile, because the reaction steps are optimized, the post-reaction treatment is very simple, complicated purification steps such as column chromatography and the like are avoided, a high-purity product can be obtained only by pulping and crystallization, and the method is suitable for large-scale production.

Description

Preparation method of high-purity cholesterol
Technical Field
The invention relates to the technical field of synthetic cholesterol, in particular to a preparation method of high-purity cholesterol.
Background
Cholesterol is a derivative of cyclopentane multi-hydrogen phenanthrene, widely exists in animal bodies, and is an essential substance indispensable to animal tissue cells. It not only participates in the formation of cell membrane, but also is a raw material for synthesizing bile acid, vitamin D and steroid hormone. At present, commercial cholesterol is mainly used as a pharmaceutical adjuvant, a cosmetic additive, a raw material for producing liquid crystal, a raw material for producing vitamin D3 and the like.
At present, the source of cholesterol is animals, and the cholesterol is extracted by organic solvent after the brain and spinal tendon of pig, cattle and sheep are saponified. Since many diseases now found are transmitted from animals to humans, there is a need to develop a safer method of cholesterol synthesis for human health and safety.
The chemical synthesis of cholesterol has been reported mainly as follows:
the patent No. CN1772760A adopts a method for synthesizing cholesterol by using dioscin as a raw material, and a synthetic route is as follows, and the synthetic route carries out 6 steps of reactions to synthesize the cholesterol with the total molar yield of 61 percent. However, the route has the disadvantages of high raw material price, complex steps, high reagent toxicity, high pollution, large consumption of concentrated hydrochloric acid and zinc powder for ring-opening reaction, high consumption and poor economical efficiency.
Figure BDA0003952712660000021
Patent No. CN105218610A adopts a method for synthesizing cholesterol by using stigmasterol degradation products as raw materials, and the synthetic route is as follows:
Figure BDA0003952712660000022
the synthesis technology takes 21-hydroxy-20-methyl pregn-4-ene-3-ketone as a raw material to carry out 5 steps of reaction, the total molar yield is 67 percent, in the hydrogenation reaction, palladium-carbon catalyst is adopted for selective hydrogenation, the raw material cost is high, the selective reducibility is poor, the product is not easy to purify, and the yield is low.
Patent CN104961788A discloses synthesis of cholesterol with total molar yield of 80% by 2 steps of reaction with pregnenolone as raw material. The synthesis route is as follows, and the route adopts a noble metal rhodium catalyst and a chiral phosphine ligand, is expensive and is not suitable for industrial production.
Figure BDA0003952712660000031
Patent CN106632565 discloses the synthesis of cholesterol with total molar yield of 70% by 4-step reaction using stigmasterol as raw material. The synthetic route is as follows, which uses O 3 The process difficulty is increased, higher requirements are put on monitoring reaction and equipment, and the economy and the safety are poor.
Figure BDA0003952712660000032
Patent 114524856A discloses a method for synthesizing cholesterol by using BA as raw material, and the synthetic route is as follows. The Witting reaction of the synthesis technology adopts the triphenylphosphine reaction, and the reaction product is not easy to purify.
In the hydrogenation reaction, raneyNi is used as a catalyst, the selective reducibility is poor, and the yield is not high.
Figure BDA0003952712660000041
Patent WO2021120127 discloses a process for the synthesis of cholesterol using BA as starting material, the synthetic route of which is as follows. In the synthetic route, the Grignard reagent coupling reaction needs 10 times of Grignard reagent to react due to the influence of 3-position hydroxyl, so that the reaction has high danger and does not meet the atom economy requirement.
Figure BDA0003952712660000042
Therefore, the research on the preparation method of the high-purity cholesterol, which has the advantages of low cost, environmental protection, good safety, easy purification of products, yield improvement and reduction selection, has important significance.
Disclosure of Invention
The invention aims to provide a preparation method of high-purity cholesterol in order to overcome the defects of the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of high-purity cholesterol, which comprises the following steps:
1) Carrying out sulfonation reaction on 21-hydroxy-20-methylpregna-4-en-3-one, a sulfonyl compound and alkali in a solvent to obtain a 21-sulfonyl compound;
2) Carrying out 3-carbonyl protection reaction on a 21-sulfonyl compound, triethyl orthoformate and p-toluenesulfonic acid in a solvent to obtain 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene;
3) Performing addition reaction on 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene, a Grignard reagent and a cuprous salt in a solvent to obtain a cholesterol crude product;
4) The 3-protecting group of the cholesterol crude product is subjected to acetylation reaction under the conditions of acetic acid and acetic anhydride to obtain 3-acetyl-3, 5-diencholestane;
5) 3-acetyl-3, 5-diene cholestane and a reducing agent are subjected to reduction reaction or 3-acetyl-3, 5-diene cholestane and ketoreductase are subjected to reduction reaction to obtain cholesterol.
Preferably, the sulfonyl compound in step 1) is p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride or fluorosulfonyl fluoride, the base is triethylamine or diisopropylethylamine, and the solvent is dichloromethane or dichloroethane.
Preferably, the mass ratio of the 21-hydroxy-20-methylpregna-4-en-3-one, the sulfonyl compound and the base in the step 1) is 0.8-1.2: 0.9 to 1.2:0.6 to 0.9, the temperature of the sulfonation reaction is between 5 ℃ below zero and 5 ℃, and the time of the sulfonation reaction is between 8 and 16 hours.
Preferably, the solvent in step 2) is absolute ethyl alcohol or methanol; the mass ratio of the 21-sulfonyl compound to the triethyl orthoformate to the p-toluenesulfonic acid is 100:100 to 500:0.3 to 0.8; the temperature of the 3-bit carbonyl protection reaction is-5 to 5 ℃, and the reaction time is 8 to 16 hours.
Preferably, the Grignard reagent in step 3) is isoamyl magnesium bromide or isoamyl lithium bromide; the cuprous salt is cuprous bromide, cuprous bromide dimethyl sulfide, cuprous chloride, cuprous iodide or dilithium tetrachlorocuprate.
Preferably, the mass molar ratio of the 21-sulfonyl-20-methyl pregna-3-ethoxy-3, 5-diene to the Grignard reagent is 200-350 g:1mol of the active component; the mole ratio of the grignard reagent to the cuprous salt is 3-10: 0.1 to 1; the temperature of the addition reaction is-5 ℃.
Preferably, in the step 4), the mass-to-volume ratio of the cholesterol crude product to the acetic acid to the acetic anhydride is 150 to 250g: 60-140 mL: 60-140 mL, the temperature of acetylation reaction is 60-140 ℃, and the time of acetylation reaction is 2-4 h.
Preferably, the 3-acetyl-3, 5-diene cholestane in the step 5) and a reducing agent are subjected to reduction reaction in an organic solvent to obtain cholesterol; the reducing agent is sodium borohydride, potassium borohydride, zinc borohydride or calcium borohydride, and the organic solvent is ethylene glycol dimethyl ether or tetrahydrofuran; the molar ratio of the 3-acetyl-3, 5-diene cholestane to the reducing agent is 1:0.25 to 1.
Preferably, the 3-acetyl-3, 5-diencholestane and the ketoreductase in the step 5) are subjected to reduction reaction in a phosphate buffer solution of methyl tert-butyl ether, and the pH value of a reaction solution is 7 to 7.5 in the reduction reaction process; the temperature of the reduction reaction is 40-50 ℃, and the time of the reduction reaction is 42-55 h.
Preferably, the volume ratio of the methyl tert-butyl ether to the phosphate buffer solution in the phosphate buffer solution of the methyl tert-butyl ether is 1-2: 1 to 2, and the pH value of the phosphate buffer solution is 7 to 7.5.
The beneficial effects of the invention include:
the method takes 21-hydroxy-20-methylpregna-4-en-3-one (4-BA) as an initial raw material, and obtains the cholesterol through 21-position hydroxyl sulfonylation, triethyl orthoformate reaction to protect 3-position carbonyl, cuprous salt catalyzed Grignard reagent substitution reaction, de-ethoxylation protection, acetylation and sodium borohydride reduction reaction. The method has the advantages of simple process, high synthesis yield, low cost, environment-friendly process and suitability for industrial production, and the reaction can be completed only by 1 equivalent of Grignard reagent. The method of the invention reduces the impurities in the product, improves the purity of cholesterol, has high safety of the synthetic method, avoids the risk of virus infection and improves the safety of clinical use. Meanwhile, because the reaction steps are optimized, the post-reaction treatment is very simple, complicated purification steps such as column chromatography and the like are avoided, a high-purity product can be obtained only by pulping and crystallization, and the method is suitable for large-scale production.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of a 21-sulfonyl compound in example 1;
FIG. 2 is a nuclear magnetic hydrogen spectrum of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene from example 1.
Detailed Description
The invention provides a preparation method of high-purity cholesterol, which comprises the following steps:
1) Carrying out sulfonation reaction on 21-hydroxy-20-methylpregna-4-en-3-one, a sulfonyl compound and alkali in a solvent to obtain a 21-sulfonyl compound;
2) Performing 3-carbonyl protection reaction on a 21-sulfonyl compound, triethyl orthoformate and p-toluenesulfonic acid in a solvent to obtain 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene;
3) Performing addition reaction on 21-sulfonyl-20-methyl pregna-3-ethoxy-3, 5-diene, a Grignard reagent and a cuprous salt in a solvent to obtain a cholesterol crude product;
4) Acetylation of 3-protecting group of the cholesterol crude product in acetic acid and acetic anhydride condition to obtain 3-acetyl-3, 5-diencholestane;
5) The 3-acetyl-3, 5-diencholestane and a reducing agent are subjected to reduction reaction or the 3-acetyl-3, 5-diencholestane and ketoreductase are subjected to reduction reaction to obtain the cholesterol.
In the present invention, the sulfonyl compound of step 1) is preferably p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride or fluorosulfonyl fluoride, the base is preferably triethylamine or diisopropylethylamine, and the solvent is preferably dichloromethane or dichloroethane.
In the present invention, the mass ratio of the 21-hydroxy-20-methylpregna-4-en-3-one, the sulfonyl compound and the base in step 1) is preferably 0.8 to 1.2:0.9 to 1.2:0.6 to 0.9, more preferably 0.9 to 1.1:0.95 to 1.1:0.65 to 0.85, more preferably 1:1 to 1.05:0.7 to 0.8.
The mass-volume ratio of the hydroxy-20-methylpregna-4-en-3-one and the solvent in step 1) of the invention is preferably 0.8-1.2 g:3 to 4mL, more preferably 0.9 to 1.1g:3.2 to 3.8mL, more preferably 0.95 to 1.05g: 3.4-3.6 mL.
In the invention, the temperature of the sulfonation reaction in the step 1) is preferably-5 ℃, more preferably-3 ℃, and even more preferably-1 ℃; the sulfonation reaction time is preferably 8 to 16 hours, more preferably 10 to 14 hours, and still more preferably 11 to 13 hours.
In the invention, the solvent in the step 2) is preferably absolute ethyl alcohol or methanol; the mass ratio of the 21-sulfonyl compound, triethyl orthoformate and p-toluenesulfonic acid is preferably 100:100 to 500:0.3 to 0.8, more preferably 100: 200-400: 0.4 to 0.7, more preferably 100:260 to 350 parts of: 0.5 to 0.6; the mass-to-volume ratio of the 21-sulfonyl compound to the solvent is preferably 100g:300 to 700mL, more preferably 100g:400 to 600mL, more preferably 100g: 450-550 mL.
In the invention, the temperature of the 3-carbonyl protection reaction in the step 2) is preferably-5 ℃, more preferably-3 ℃, and more preferably-1 ℃; the time for the protection reaction of the carbonyl group at the 3-position is preferably 8 to 16 hours, more preferably 10 to 14 hours, and still more preferably 11 to 13 hours.
The Grignard reagent in the step 3) of the invention is preferably isoamyl magnesium bromide or isoamyl lithium bromide; the cuprous salt is preferably cuprous bromide, cuprous dimethyl sulfide bromide, cuprous chloride, cuprous iodide or dilithium tetrachlorocuprate.
In the invention, the mass molar ratio of the 21-sulfonyl-20-methyl pregna-3-ethoxy-3, 5-diene to the Grignard reagent is preferably 200 to 350g:1mol, more preferably 220 to 320g:1mol, more preferably 250 to 280g:1mol of the active component; the mole ratio of the grignard reagent to the cuprous salt is preferably 3 to 10:0.1 to 1, more preferably 4 to 9:0.3 to 0.8, more preferably 5 to 7:0.4 to 0.6; the temperature of the addition reaction is preferably-5 to 5 ℃, more preferably-3 to 3 ℃, and still more preferably-1 to 1 ℃.
In step 4) of the present invention, the mass-to-volume ratio of the cholesterol crude product, acetic acid and acetic anhydride is preferably 150 to 250g: 60-140 mL:60 to 140mL, more preferably 170 to 230g: 80-120 mL:80 to 120mL, more preferably 180 to 220g: 90-110 mL: 90-110 mL; the temperature of the acetylation reaction is preferably 60 to 140 ℃, more preferably 80 to 120 ℃, and even more preferably 90 to 110 ℃; the time for the acetylation reaction is preferably 2 to 4 hours, more preferably 2.5 to 3.5 hours, and still more preferably 3 hours.
In the invention, the 3-acetyl-3, 5-diencholestane in the step 5) and a reducing agent are subjected to reduction reaction in an organic solvent to obtain cholesterol; the reducing agent is preferably sodium borohydride, potassium borohydride, zinc borohydride or calcium borohydride, and the organic solvent is preferably ethylene glycol dimethyl ether or tetrahydrofuran; the molar ratio of the 3-acetyl-3, 5-diencholestane to the reducing agent is preferably 1:0.25 to 1, more preferably 1:0.4 to 0.8, more preferably 1:0.5 to 0.7.
The 3-acetyl-3, 5-diene cholestane and the ketoreductase are subjected to reduction reaction in a phosphate buffer solution of methyl tert-butyl ether in the step 5), and the mass ratio of the 3-acetyl-3, 5-diene cholestane to the ketoreductase is preferably 45-55: 0.2 to 0.4, more preferably 47 to 52:0.25 to 0.35, more preferably 49 to 50:0.3.
in the reduction reaction process of the invention, the pH value of the reaction solution is preferably 7 to 7.5, more preferably 7.1 to 7.4, and even more preferably 7.2 to 7.3; the temperature of the reduction reaction is preferably 40 to 50 ℃, more preferably 42 to 48 ℃, and even more preferably 44 to 46 ℃; the time for the reduction reaction is preferably 42 to 55 hours, more preferably 45 to 52 hours, and still more preferably 48 to 50 hours.
In the phosphate buffer of methyl tert-butyl ether, the volume ratio of methyl tert-butyl ether to phosphate buffer is preferably 1-2: 1 to 2, more preferably 1:1; the pH of the phosphate buffer is preferably 7 to 7.5, more preferably 7.1 to 7.4, and still more preferably 7.2 to 7.3.
The process route for synthesizing the cholesterol by taking 21-hydroxy-20-methyl pregn-4-ene-3-one (4-BA) as a raw material is as follows:
Figure BDA0003952712660000091
the technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
100g of 21-hydroxy-20-methylpregn-4-en-3-one, 350mL of dichloromethane and 72g of triethylamine were added to a 1L three-necked flask, mixed with stirring, 106g of p-toluenesulfonyl chloride (same mass per addition) was added in three portions in an ice water bath (0 ℃) and the reaction was stirred at 0 ℃ for 12 hours. After the reaction is completed, 300mL of water is added, stirred for 2min, and liquid is separated. The organic layer was washed with 10% by mass brine, dried, suction filtered, concentrated to dryness, slurried with 200mL of methanol, dried and air dried to obtain 136.7g of 21-sulfonyl compound.
106.68g of 21-sulfonyl compound and 500mL of absolute ethanol were put into a 1L one-neck flask, mixed with stirring, and then 128g of triethyl orthoformate was added, and 0.5g of p-toluenesulfonic acid monohydrate was added under ice water bath (0 ℃ C.) and stirred for 12 hours. After completion of the reaction by TLC detection, the reaction solution was poured into 1.5L of ice NaHCO 3 Aqueous solution (NaHCO) 3 5%) of the crude product, filtering, rinsing the filter cake twice with deionized water, and air-drying to obtain 110.4g of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene.
1L of 1mol/L isoamyl magnesium bromide is added into a 3L three-neck flask, 200mL of 0.5mol/L dilithium tetrachlorocuprate and 280g of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene solution in 500mL of tetrahydrofuran are added dropwise in an ice water bath (0 ℃), TLC detection reaction is finished, 1mol/L hydrochloric acid is added for quenching reaction, ethyl acetate is added for extraction, and liquid separation is carried out. And drying and evaporating an organic layer to obtain a crude product, adding 560mL of methanol, 10g of p-toluenesulfonic acid and 50mL of water into the crude product, stirring, separating out a solid, performing suction filtration, washing with water, washing with methanol, and drying to obtain 200g of a crude cholesterol product.
200g of the cholesterol crude product was dissolved in 100mL of acetic acid and 100mL of acetic anhydride, heated to 110 ℃ and refluxed for 3 hours, and the reaction was detected by TLC to be complete. Evaporating to dryness to recover acetic anhydride, and crystallizing the crude product with methanol to obtain 177.8g of 3-acetyl-3, 5-diencholestane pure product.
1mol of 3-acetyl-3, 5-diencholestane is dissolved in 800mL of tetrahydrofuran, a newly prepared calcium borohydride solution (the concentration of calcium borohydride is 1 mol/L) with the total amount of 0.5mol is added in three times (the mass of each addition is the same) at 25 ℃, and the reaction is stopped. Adding 10mL of methanol to decompose the residual calcium borohydride, adding 1mol/L hydrochloric acid to adjust the pH value to 5, adding ethyl acetate to separate liquid, drying and evaporating an organic layer to obtain a white solid, and crystallizing ethanol to obtain 367g of a pure cholesterol product.
In this example, the molar yield of the 21-sulfonyl compound was 93%. The nuclear magnetic hydrogen spectrum of the 21-sulfonyl compound is shown in FIG. 1, and HNMR (400MHz, CDCl3) delta: 7.78 (m, 2H), 7.34 (m, 2H), 5.72 (s, 1H), 3.96 (m, 1H), 3.79 (m, 1H), 2.45 (s, 3H), 0.91 (d, 3H), 0.88 (d, 3H), 0.68 (s, 3H).
In this example, the molar yield of 21-sulfonyl-20-methylpregna-3-ethoxy-3,5-diene was 97.8%. The nuclear magnetic hydrogen spectrum of 21-sulfonyl-20-methyl pregna-3-ethoxy-3, 5-diene is shown in FIG. 2, HNMR (400MHz, CDCl3) delta: 7.78 (m, 2H), 7.35 (m, 2H), 5.20 (s, 1H), 5.11 (s, 1H), 4.00 (m, 1H), 3.77 (m, 1H), 3.76 (m, 2H), 2.45 (s, 3H), 1.17 (d, 3H), 0.91 (d, 3H), 0.88 (d, 3H), 0.68 (s, 3H).
In this example, the molar yield of 3-acetyl-3, 5-diencholestane was 84.6%; the purity of the cholesterol was 99.5% by HPLC and the molar yield of the cholesterol was 95%.
Example 2
The procedure for the preparation of 3-acetyl-3, 5-diencholestane from 21-hydroxy-20-methylpregn-4-en-3-one was as described in example 1.
5.04g of 3-acetyl-3, 5-diencholestane was dissolved in 40mL of methyl tert-butyl ether in phosphate buffer, methyl tert-butyl ether at a volume ratio of 1 to phosphate buffer solution, and the pH of the phosphate buffer solution (from Shanghaitan chemical Co., ltd.) was 7.0. Adding 30mg of ketoreductase, 2g of glucose, 70mg of NAD and 70mg of NAD (P) +, glucose dehydrogenase (CDX-901, 30mg, 66U/mg), reacting for 48h at 45 ℃, adding calcium hydroxide suspension in batches in the reaction to keep the pH value of the reaction system at 7.0, separating liquid after the reaction is finished, adding methyl tert-butyl ether into a water layer for extraction, combining organic layers, drying and evaporating to obtain a white solid, and crystallizing by ethanol to obtain 3.98g of pure cholesterol.
In this example, the purity of cholesterol was 99.3% by HPLC, and the molar yield of cholesterol was 87%.
Example 3
90g of 21-hydroxy-20-methylpregn-4-en-3-one, 320mL of dichloroethane and 65g of diisopropylethylamine were added to a 1L three-necked flask, mixed with stirring, 95g of fluorosulfonyl fluoride (same mass per addition) was added in two portions in an ice water bath (0 ℃) and reacted with stirring at 0 ℃ for 10 hours. After the reaction is completed, 260mL of water is added, stirred for 2min, and liquid is separated. And washing the organic layer with 10% by mass of saline, drying, filtering, concentrating, pulping with 180mL of methanol, drying, and airing to obtain the 21-sulfonyl compound.
A1L single-neck flask was charged with 98g of 21-sulfonyl compound and 400mL of methanol, mixed with stirring, and then added with 115g of triethyl orthoformate, and 0.42g of p-toluenesulfonic acid monohydrate was added under ice water bath (0 ℃ C.) and stirred for 10 hours. After the reaction was complete by TLC, the reaction mixture was poured into 1.4L of ice NaHCO 3 Aqueous solution (NaHCO) 3 5%) of the crude product, filtering, rinsing the filter cake twice with deionized water, and air drying to obtain 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene.
1L of 0.9mol/L isoamyl lithium bromide is added into a 3L three-neck flask, 200mL of 0.48mol/L cuprous bromide and 260g of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene solution in 480mL tetrahydrofuran are added dropwise in an ice water bath (0 ℃), after TLC detection reaction is finished, 1mol/L hydrochloric acid is added for quenching reaction, ethyl acetate is added for extraction, and liquid separation is carried out. And drying and evaporating the organic layer to obtain a crude product, adding 520mL of methanol, 9g of p-toluenesulfonic acid and 45mL of water into the crude product, stirring, separating out a solid, performing suction filtration, washing with water, washing with methanol, and drying to obtain a cholesterol crude product.
180g of cholesterol crude product is dissolved by 85mL of acetic acid and 85mL of acetic anhydride, heated to 107 ℃ and refluxed for 2.5h, and the reaction is detected by TLC to be finished. Evaporating to dryness to recover acetic anhydride, and crystallizing the crude product with methanol to obtain pure 3-acetyl-3, 5-diencholestane.
1mol of 3-acetyl-3, 5-diencholestane is dissolved in 750mL of ethylene glycol dimethyl ether, sodium borohydride solution (the concentration of sodium borohydride is 1 mol/L) with the total amount of 0.45mol is added in two times (the mass of each addition is the same) at 25 ℃, and the reaction is finished by spotting. Adding 10mL of methanol to decompose the residual sodium borohydride, adding 1mol/L hydrochloric acid to adjust the pH value to 5, adding ethyl acetate to separate liquid, drying and evaporating an organic layer to obtain a white solid, and crystallizing ethanol to obtain a pure cholesterol product.
In this example, the molar yield of the 21-sulfonyl compound was 92.3%; the molar yield of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene was 97.5%; the molar yield of 3-acetyl-3, 5-diencholestane is 84.2%; the purity of the cholesterol was 99.3% by HPLC and the molar yield of the cholesterol was 94.7%.
Example 4
110g of 21-hydroxy-20-methyl pregn-4-ene-3-one, 370mL of dichloromethane and 80g of diisopropyl ethylamine were added to a 1L three-necked flask, mixed with stirring, 105g of trifluoromethanesulfonic anhydride (same mass per addition) was added in two portions in an ice water bath (0 ℃), and the reaction was stirred at 0 ℃ for 13 hours. After the reaction is completed, 320mL of water is added, stirred for 3min, and liquid is separated. And washing the organic layer by using saline with the mass concentration of 12%, drying, filtering, concentrating, pulping by using 210mL of methanol, drying, and airing to obtain the 21-sulfonyl compound.
105g of 21-sulfonyl compound and 550mL of anhydrous ethanol were put into a 1L one-neck flask, mixed with stirring, and then 135g of triethyl orthoformate was added, and 0.58g of p-toluenesulfonic acid monohydrate was added under an ice-water bath (0 ℃ C.) and stirred for 13 hours. After the reaction was complete by TLC, the reaction solution was poured into 1.6L of ice NaHCO 3 Aqueous solution (NaHCO) 3 5.5%) and filtering, and filtering cakeRinsing twice with deionized water, and then drying to obtain 21-sulfonyl-20-methyl pregna-3-ethoxy-3, 5-diene.
1.1L of 1mol/L isoamyl magnesium bromide is added into a 3L three-neck flask, 200mL of 0.54mol/L cuprous bromide dimethyl sulfide and a solution of 300g of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene dissolved in 520mL of tetrahydrofuran are added dropwise in an ice water bath (0 ℃), TLC detection reaction is finished, 1mol/L hydrochloric acid is added for quenching reaction, ethyl acetate is added for extraction, and liquid separation is carried out. And drying and evaporating an organic layer to obtain a crude product, adding 580mL of methanol, 12g of p-toluenesulfonic acid and 55mL of water into the crude product, stirring, separating out a solid, performing suction filtration, washing with water, washing with methanol, and drying to obtain a cholesterol crude product.
200g of the cholesterol crude product was dissolved in 110mL of acetic acid and 110mL of acetic anhydride, heated to 113 ℃ and refluxed for 3.5h, and the reaction was detected by TLC to be complete. Evaporating to dryness to recover acetic anhydride, and crystallizing the crude product with methanol to obtain pure 3-acetyl-3, 5-diencholestane.
4.9g 3-acetyl-3, 5-diencholestane was dissolved in 40mL methyl tert-butyl ether in phosphate buffer, the volume ratio of methyl tert-butyl ether to phosphate buffer solution was 1, and the pH of the phosphate buffer solution (from Shanitan chemical Co., ltd.) was 7.2. Adding 28mg ketoreductase, 1.9g glucose, 70mg NAD and 70mg NAD (P) +, glucose dehydrogenase (CDX-901, 30mg, 66U/mg), reacting at 47 ℃ for 45h, adding calcium hydroxide suspension in batches to keep the pH value of the reaction system at 7.2, separating after the reaction is finished, adding methyl tert-butyl ether into a water layer for extraction, combining organic layers, drying and evaporating to obtain a white solid, and crystallizing by ethanol to obtain a pure cholesterol product.
In this example, the molar yield of the 21-sulfonyl compound was 92.7%; the molar yield of 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene was 97.3%; the molar yield of 3-acetyl-3, 5-diencholestane is 84.5%; the purity of the cholesterol was 99.4% by HPLC and the molar yield of the cholesterol was 89.2%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for preparing high-purity cholesterol is characterized by comprising the following steps:
1) Carrying out sulfonation reaction on 21-hydroxy-20-methylpregna-4-en-3-one, a sulfonyl compound and alkali in a solvent to obtain a 21-sulfonyl compound;
2) Carrying out 3-carbonyl protection reaction on a 21-sulfonyl compound, triethyl orthoformate and p-toluenesulfonic acid in a solvent to obtain 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene;
3) Performing addition reaction on 21-sulfonyl-20-methyl pregna-3-ethoxy-3, 5-diene, a Grignard reagent and a cuprous salt in a solvent to obtain a cholesterol crude product;
4) Acetylation of 3-protecting group of the cholesterol crude product in acetic acid and acetic anhydride condition to obtain 3-acetyl-3, 5-diencholestane;
5) The 3-acetyl-3, 5-diencholestane and a reducing agent are subjected to reduction reaction or the 3-acetyl-3, 5-diencholestane and ketoreductase are subjected to reduction reaction to obtain the cholesterol.
2. The method according to claim 1, wherein the sulfonyl compound in step 1) is p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride or fluorosulfonyl fluoride, the base is triethylamine or diisopropylethylamine, and the solvent is dichloromethane or dichloroethane.
3. The method according to claim 1 or 2, wherein the mass ratio of the 21-hydroxy-20-methylpregna-4-en-3-one, the sulfonyl compound and the base in step 1) is 0.8 to 1.2:0.9 to 1.2:0.6 to 0.9, the temperature of the sulfonation reaction is between 5 ℃ below zero and 5 ℃, and the time of the sulfonation reaction is between 8 and 16 hours.
4. The method according to claim 3, wherein the solvent of step 2) is absolute ethanol or methanol; the mass ratio of the 21-sulfonyl compound to the triethyl orthoformate to the p-toluenesulfonic acid is 100:100 to 500:0.3 to 0.8; the temperature of the 3-bit carbonyl protection reaction is-5 to 5 ℃, and the reaction time is 8 to 16 hours.
5. The method according to claim 4, wherein the Grignard reagent of step 3) is isopentyl magnesium bromide or isopentyl lithium bromide; the cuprous salt is cuprous bromide, cuprous bromide dimethyl sulfide, cuprous chloride, cuprous iodide or dilithium tetrachlorocuprate.
6. The preparation method according to claim 5, wherein the mass molar ratio of the 21-sulfonyl-20-methylpregna-3-ethoxy-3, 5-diene to Grignard reagent is 200-350 g:1mol; the mole ratio of the grignard reagent to the cuprous salt is 3-10: 0.1 to 1; the temperature of the addition reaction is-5 ℃.
7. The preparation method according to any one of claims 4 to 6, wherein in step 4), the mass-to-volume ratio of the cholesterol crude product, acetic acid and acetic anhydride is 150 to 250g: 60-140 mL: 60-140 mL, the temperature of acetylation reaction is 60-140 ℃, and the time of acetylation reaction is 2-4 h.
8. The process according to claim 7, wherein the 3-acetyl-3, 5-diencholestane of step 5) is subjected to a reduction reaction with a reducing agent in an organic solvent to obtain cholesterol; the reducing agent is sodium borohydride, potassium borohydride, zinc borohydride or calcium borohydride, and the organic solvent is ethylene glycol dimethyl ether or tetrahydrofuran; the molar ratio of the 3-acetyl-3, 5-diene cholestane to the reducing agent is 1:0.25 to 1.
9. The process according to claim 7, wherein the 3-acetyl-3, 5-cholestane and ketoreductase in step 5) are subjected to a reduction reaction in a phosphate buffer solution of methyl t-butyl ether, wherein the pH of the reaction solution is 7 to 7.5 during the reduction reaction; the temperature of the reduction reaction is 40-50 ℃, and the time of the reduction reaction is 42-55 h.
10. The method according to claim 9, wherein the volume ratio of the methyl tert-butyl ether to the phosphate buffer in the phosphate buffer of the methyl tert-butyl ether is 1 to 2:1 to 2, and the pH value of the phosphate buffer solution is 7 to 7.5.
CN202211454023.0A 2022-11-21 2022-11-21 Preparation method of high-purity cholesterol Pending CN115724899A (en)

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CN114315947A (en) * 2021-11-17 2022-04-12 上海敏韬医药科技有限公司 Novel method for synthesizing cholesterol and 25-hydroxycholesterol by using 22-sterol as raw material
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CN113651866A (en) * 2021-08-02 2021-11-16 上海敏韬医药科技有限公司 Novel method for synthesizing cholesterol by taking 21-hydroxy-20-methyl pregn-4-ene-3-one as raw material
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