CN114315947A

CN114315947A - Novel method for synthesizing cholesterol and 25-hydroxycholesterol by using 22-sterol as raw material

Info

Publication number: CN114315947A
Application number: CN202111369991.7A
Authority: CN
Inventors: 王涛; 李民强; 孟宪志; 万定建; 李佳霖; 罗觅纯
Original assignee: Shanghai Mintao Pharmaceutical Technology Co ltd
Current assignee: Wang Tao
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2022-04-12
Anticipated expiration: 2041-11-17
Also published as: CN114315947B

Abstract

The invention provides a method for synthesizing cholesterol and 25-hydroxycholesterol, which comprises the steps of 1) reacting 22-hydroxy-20-methylpregn-4-en-3-one with pyrrolidine or ethylene glycol to protect a 3-carbonyl group, then sulfonylating the 22-hydroxyl group, carrying out copper-catalyzed Grignard reagent substitution reaction, and carrying out sodium borohydride reduction reaction to obtain the cholesterol or 25-hydroxycholesterol. The synthesis method has the advantages of simple process, high yield, low cost and environment-friendly process, and is suitable for industrial production.

Description

Novel method for synthesizing cholesterol and 25-hydroxycholesterol by using 22-sterol as raw material

Technical Field

The invention relates to a synthesis method of a steroid compound, in particular to a synthesis method of cholesterol and 25-hydroxycholesterol.

Background

Cholesterol, also known as cholesterol, is a derivative of cyclopentapyrophenanthrene, and is widely found in animals. It 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, 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 discovered now are transmitted to human beings by animals, especially the occurrence of European mad cow disease at the end of last reagent and Streptococcus suis infection at the beginning of this century, people are in doubt of the cholesterol safety of the traditional preparation method, and a safer cholesterol synthesis method is needed.

Patent No. CN1772760A adopts a method for synthesizing cholesterol from diosgenin as raw material, and the synthetic route is as follows:

the synthesis route has low yield, large consumption of raw and auxiliary materials, large pollution and low economy.

Patent No. CN105218610A adopts a method for synthesizing cholesterol by taking a stigmasterol degradation product as a raw material, and the synthetic route is as follows:

in the synthesis technology, 3-carbonyl-4-ene pregna-22-aldehyde is used as a raw material to synthesize the cholesterol, and a palladium-carbon catalyst is adopted in a hydrogenation reaction, so that the cost is high, the selective reducibility is poor, the product is not easy to purify, and the yield is low.

Patent WO2021120127 discloses a method for synthesizing cholesterol using BA as raw material, the synthetic route is as follows:

in the synthetic route, the Grignard reagent Oldham reaction needs 10 times of Grignard reagent to react due to the influence of 3-position hydroxyl, so that the reaction is very dangerous and does not accord with atom economy.

Disclosure of Invention

The invention aims to provide a method for synthesizing cholesterol and 25-hydroxycholesterol, which has simple process and high yield, takes 22-hydroxy-20-methylpregn-4-en-3-one (4-BA) as a raw material to synthesize the cholesterol and 25-hydroxycholesterol, and the synthetic route is as follows:

a method for synthesizing cholesterol and 25-hydroxycholesterol by using 22-hydroxy-20-methyl pregn-4-en-3-one (4-BA) as a raw material comprises the following steps:

1) protecting the 3-position carbonyl group: the 22-hydroxy-20-methyl pregna-4-ene-3-ketone is catalyzed by p-toluenesulfonic acid and reacts with pyrrolidine or ethylene glycol at the reaction temperature of 10-120 ℃ for 3-5 hours, then the reaction is stopped by sodium bicarbonate, and 22-hydroxy-20-methyl pregna-3-pyrrolidine-3, 5-diene or 22-hydroxy-20-methyl pregna-3-ethylene glycol-5-ene is obtained by elutriation and filtration.

2) And (3) sulfonation reaction: reacting 22-hydroxy-20-methyl pregna-3-tetrahydropyrrole-3, 5-diene or 21-hydroxy-20-methyl pregna-3-ethylene glycol-5-ene with p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride and fluorosulfonyl fluoride in dichloromethane or dichloroethane under the condition of triethylamine or diisopropylethylamine, washing with water, extracting, and concentrating to obtain 22-sulfonyl compound.

3) And (3) substitution reaction: adding the 22-sulfonyl compound and a Grignard reagent isoamyl magnesium bromide in a tetrahydrofuran solvent for reaction, quenching with acid, separating liquid, and concentrating to obtain the cholestane with 3 protected positions or the cholestane with 3 protected positions and 25-hydroxyl.

4) Acetylation reaction: cholestane protected at position 3 or cholestane protected at position 3 with 25-hydroxy group. Performing exchange reaction on protecting groups at 3-position under the conditions of acetic acid and acetic anhydride, evaporating ethanol to dryness and crystallizing to obtain 3-acetyl-3, 5-diencholestane and 3-acetyl-25 hydroxyl-3, 5-diencholestane.

5) Reduction reaction: dissolving 3-acetyl-3, 5-diene cholestane or 3-acetyl-25-hydroxy-3, 5-diene cholestane in glycol dimethyl ether and tetrahydrofuran, and reacting with sodium borohydride, potassium borohydride, zinc borohydride, calcium borohydride or reductase to obtain cholesterol or 25-hydroxy cholesterol.

Further, in the above method, the weight ratio of each reactant in the 3 carbonyl protection reaction in the step 1) is: 21-hydroxy-20-methylpregn-4-en-3-one: tetrahydropyrrole or ethylene glycol 1.0: 1.0-5.0: 1.0 to 5.0.

Further, in the above method, the 3-carbonyl protection reaction in step 1) is performed in an organic solvent, and the solvent is one of ethanol, methanol, and toluene.

Further, in the above method, in the sulfonation reaction in the step 2), triethylamine, diisopropylethylamine, etc. are used as a base to react with p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, and fluorosulfonyl fluoride in a dichloromethane solvent to obtain 22-sulfonate.

Further, in the above method, the substitution reaction in step 3) is carried out using reagents such as isopentyl magnesium bromide, isopentyl lithium bromide, 3-trimethylsiloxy-3-methylbutyl magnesium bromide, and the like. The molar ratio of reactants is as follows: 22-sulfonic acid ester: isoamyl magnesium bromide ═ 1: 3 to 10.

Further, in the above method, the grignard reagent substitution reaction in step 3) requires a cuprous salt as a catalyst, the cuprous salt is cuprous bromide, cuprous bromide dimethyl sulfide, cuprous chloride, cuprous iodide, and lithium tetrachlorocupric chloride, and the reaction molar ratio is 1: 0.1 to 1.

Further, in the above method, in the step 5), the 3-acetyl-3, 5-diencholestane or 3-acetyl-25-hydroxy-3, 5-diencholestane is dissolved in ethylene glycol dimethyl ether and tetrahydrofuran, sodium borohydride is added, and the reaction product is extracted and crystallized with ethanol to obtain cholesterol or 25-hydroxycholesterol. 3-acetyl-3, 5-diencholestane: sodium borohydride molar ratio 1.0: 0.25 to 1.

Further, in the above method, the reduction reaction of 3-acetyl-25-hydroxy-3, 5-diencholestane or 3-acetyl-3, 5-diencholestane in step 5) is carried out in methyl tert-butyl ether phosphate buffer solution by carbonyl reductase to obtain cholesterol or 25-hydroxycholesterol, and calcium hydroxide suspension is added dropwise during the reduction reaction to maintain the pH of the reaction solution at about 7-7.5.

Further, cholesterol or 25-hydroxycholesterol obtained by the above method.

The invention has the following beneficial effects:

compared with the method that the dioscorea sapogenin is used as the starting raw material, the method has the advantages that a large amount of concentrated hydrochloric acid and zinc powder are consumed to carry out ring-opening reaction, so that the defects of large consumption, large pollution and low economy are caused; and the defects of high raw material cost, poor selective reducibility effect, difficult product purification and low yield in the reaction taking the stigmasterol as the raw material. The invention takes 21-hydroxy-20-methyl pregn-4-alkene-3-ketone (4-BA) as a starting material to synthesize cholesterol or 25-hydroxycholesterol, and the cholesterol or 25-hydroxycholesterol is obtained through 3 carbonyl protection, sulfonylation reaction, isopentyl magnesium bromide format reagent substitution reaction, acetylation reaction and reduction reaction. The synthesis method has simple reaction process and high yield. Low production cost, environment-friendly process and more suitability for industrial implementation.

Drawings

FIG. 1 is a reaction scheme of a method for synthesizing cholesterol and 25-hydroxycholesterol by using 21-hydroxy-20-methyl pregn-4-en-3-one (4-BA) as a raw material, which is disclosed by the invention;

FIG. 2 is a nuclear magnetic hydrogen spectrum of a carbonyl pyrrolidine protecting compound at position 3 in the example of the present invention;

FIG. 3 is a nuclear magnetic hydrogen spectrum of a carbonyl ethylene glycol protective compound at position 3 in an example of the present invention;

FIG. 4 shows nuclear magnetic hydrogen spectra of 22-p-toluenesulfonate-20-methylpregna-3-en-5-ol in example of the present invention;

FIG. 5 shows nuclear magnetic hydrogen spectra of 3-acetyl 3, 5-diencholestane in examples of the present invention;

FIG. 6 is a nuclear magnetic hydrogen spectrum of cholesterol in an example of the present invention;

FIG. 7 shows nuclear magnetic hydrogen spectrum of 25-hydroxycholesterol in example of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The synthesis method of cholesterol and 25-hydroxycholesterol is shown in figure 1, and comprises the following steps:

1. protection of 3-carbonyl tetrahydropyrrole

1000g of 22-hydroxy-20-methylpregna-4-en-3-one and 5000ml of anhydrous methanol are put into a 10L three-neck flask, 200g of pyrrolidine is dripped under the condition of heating reflux, the reaction liquid turns turbid again, the reaction is cooled to room temperature after the dripping is finished, and 1050g of 22-hydroxy-20-methylpregna-3-pyrrolidine-3, 5-diene is obtained after filtration and drying. The molar yield was 90.4%.

HNMR (500MHz, CDCl3) delta: 5.09(m,1H),4.78(m,1H),3.13(m,4H),1.87(m,4H),1.04 (d,3H),1.00(d,3H),0.72(s, 3H). The spectrum is shown in figure 2.

2. Carbonyl glycol protection at the 3 position

1000g of 22-hydroxy-20-methyl pregn-4-ene-3-one and 2000ml of toluene are put into a 5000ml three-neck flask, 300ml of ethylene glycol and 200g of anhydrous copper sulfate are added, heating reflux water separation is carried out, TLC detection reaction is finished, copper sulfate is filtered, toluene is evaporated, and 980g of 22-hydroxy-20-methyl pregn-3-ethylene glycol-5-ene is obtained after methanol crystallization of a concentrate. The molar yield was 86.5%.

HNMR (400MHz, CDCl3) delta 5.34(m,1H),4.0(m,2H),3.95(m,2H),1.04(d,3H), 0.97(d,3H), 0.65(s, 3H). The spectrum is shown in figure 3.

3. 1000g of 22-hydroxy-20-methyl pregna-3-tetrahydropyrrole-3, 5-diene and 2000ml of pyridine are put into a three-neck flask for synthesizing 22-p-toluenesulfonate-20-methyl pregna-3-tetrahydropyrrole-3, 5-diene, the temperature is reduced to 0 ℃ in an ice water bath, 900g of p-toluenesulfonyl chloride is added in batches, stirring is continued for 30min after the addition is finished, and the TLC detection reaction is finished. Pouring the reaction solution into 4000ml of ice water to separate out a large amount of white solid, performing suction filtration, pulping the solid methanol and drying to obtain 1100g of product. The molar yield was 78.4%.

4. 1000g of 22-hydroxy-20-methyl pregna-3-ethylene glycol-5-ene and 2000ml of pyridine are put into a three-neck flask for synthesizing 22-p-toluenesulfonate-20-methyl pregna-3-ethylene glycol-5-ene, the temperature is reduced to 0 ℃ in an ice water bath, 900g of p-toluenesulfonyl chloride is added in batches, stirring is continued for 30min after the addition is finished, and the TLC detection reaction is finished. Pouring the reaction solution into 4000ml of ice water to separate out a large amount of white solid, performing suction filtration, pulping the solid methanol and drying to obtain 1000g of product. The molar yield was 70.8%.

HNMR (500MHz, CDCl3) delta 7.80(m,2H),7.33(m,2H),5.34(m,1H),4.0(m,2H),3.95 (m,2H),1.04(d,3H), 0.97(d,3H), 0.65(s, 3H). The spectrum is shown in figure 4.

5. Synthesis of 3-acetyl 3, 5-diencholestane

Adding 1000ml of 1mol/L isoamyl magnesium bromide into a 3000ml three-neck flask, dropwise adding 200ml of 0.5mol/L copper lithium tetrachloride under an ice-water bath, dropwise adding 270g of 22-p-toluenesulfonate-20-methylpregna-3-tetrahydropyrrole-3, 5-diene solution in tetrahydrofuran under the ice-water bath, after TLC detection reaction is finished, adding 1mol/L hydrochloric acid for quenching reaction, adding ethyl acetate for extraction, and separating liquid. The organic layer was dried and evaporated to dryness to obtain 180g of crude product which was directly used in the next reaction.

180g of the crude product are dissolved in 100ml of acetic acid and 100ml of acetic anhydride and heated to reflux for reaction for 3 hours. The reaction was completed by TLC detection. Evaporating to dryness to recover acetic anhydride, and crystallizing crude methanol to obtain 160g of 3-acetyl 3, 5-diencholestane pure product. The molar yield was 74.6%.

HNMR(500MHz,CDCl3)δ:5.69(m,1H),5.40(m,1H),2.13(s,3H),1.02(d,3H),0.92 (d,3H),0.87(d,3H),0.86(d,3H)，0.70(s,3H)。

6. Synthesis of 3-acetyl 3, 5-diencholestane

Adding 1000ml of 1mol/L isoamyl magnesium bromide into a 3000ml three-neck flask, dropwise adding 200ml of 0.5mol/L copper lithium tetrachloride under an ice-water bath, dropwise adding 270g of 22-p-toluenesulfonate-20-methylpregna-3-glycol-3, 5-diene solution in tetrahydrofuran under the ice-water bath, after TLC detection reaction is finished, adding 1mol/L hydrochloric acid for quenching reaction, adding ethyl acetate for extraction, and separating liquid. The organic layer was dried and evaporated to dryness to give 173g of crude product which was directly charged to the next reaction.

173g of crude product is dissolved in 100ml of acetic acid and 100ml of acetic anhydride and reacted for 3 hours under reflux. The reaction was completed by TLC detection. Evaporating to dryness to recover acetic anhydride, and crystallizing crude methanol to obtain 146g of pure 3-acetyl 3, 5-diencholestane. The molar yield was 67%.

HNMR (500MHz, CDCl3) delta: 5.69(m,1H),5.40(m,1H),2.13(s,3H),1.02(d,3H),0.92 (d,3H),0.87(d,3H),0.86(d,3H), 0.70(s, 3H). The spectrum is shown in figure 5.

7. Synthesis of 3-acetyl 3, 5-diene-25-hydroxycholestane

3000ml of three-neck flask is added with 1000ml of 1 mol/L3-trisilicon hydroxyl-3-methyl butyl magnesium bromide, 200ml of 0.5mol/L copper lithium tetrachloride is dripped under an ice water bath, 270g of 22-p-toluenesulfonate-20-methyl pregna-3-tetrahydropyrrole-3, 5-diene solution in tetrahydrofuran is dripped under the ice water bath, 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. The organic layer was dried to dryness to give 164g of crude product which was directly charged to the next reaction.

164g of the crude product was dissolved in 100ml of acetic acid and 100ml of acetic anhydride and reacted under reflux for 3 hours. The reaction was completed by TLC detection. Evaporating to dryness to recover acetic anhydride, and crystallizing crude methanol to obtain 138g of pure 3-acetyl 3, 5-diene-25 hydroxycholestanol product. The molar yield was 62%.

Synthesis of 8, 3-acetyl 3, 5-diene-25-hydroxycholestane

3000ml of three-neck flask is added with 1000ml of 1 mol/L3-trimethylsilyl hydroxy-3-methylbutyl magnesium bromide, 200ml of 0.5mol/L copper lithium tetrachloride is dripped under an ice-water bath, 270g of 22-p-toluenesulfonate-20-methylpregna-3-ethanediol-3, 5-diene solution in tetrahydrofuran is dripped under the ice-water bath, and the TLC detection reaction is finished. Adding 1mol/L hydrochloric acid to quench the reaction, adding ethyl acetate to extract, and separating liquid. The organic layer was dried and evaporated to dryness to obtain 151g of crude product which was directly used in the next reaction.

151g of the crude product are dissolved in 100ml of acetic acid and 100ml of acetic anhydride and reacted for 3 hours under reflux. The reaction was completed by TLC detection. Evaporating to dryness to recover acetic anhydride, and crystallizing crude methanol to obtain 119g of pure 3-acetyl 3, 5-diene-25 hydroxycholestanol product. The molar yield was 52.7%.

9. Synthesis of cholesterol

4260g of 3-acetyl 3, 5-diencholestane is dissolved in 8000ml of tetrahydrofuran, 340g of newly prepared calcium borohydride solution is added in portions at room temperature, and the reaction is stopped. Adding 100ml 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 3670g of a pure product. Purity > 99% by HPLC. The molar yield was 95%. HNMR (500MHz, CDCl3) delta: 5.35(m,1H),3.51(m,1H),3.35(m,1H),1.01(d,3H),0.92(d,3H),0.87(d,3H), 0.86(d,3H), 0.68(s, 3H). The spectrum is shown in figure 6.

10. Synthesis of 25-hydroxycholesterol

5140g of 3-acetyl 3, 5-diene 25-hydroxycholestane is dissolved in 8000ml of tetrahydrofuran, 340g of freshly prepared calcium borohydride solution are added in portions at room temperature, and the reaction is stopped. Adding 100ml 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, drying and evaporating an organic layer to obtain a white solid, and crystallizing ethanol to obtain 4017g of a pure product. Purity > 99% by HPLC. The molar yield was 85.82%. HNMR (500MHz, CDCl3) delta: 5.35(m,1H),3.51(m,1H),1.21(d,6H),1.01(d,3H), 0.93(d,3H), 0.68(s, 3H).

11. 25-hydroxycholesterol (enzyme reduction)

5.14g of 3-acetyl 3, 5-diene 25-hydroxycholestane were dissolved in 20ml of methyl tert-butyl ether and 20ml of a phosphate buffer solution at pH 7.0. 30mg of ketoreductase, 2g of glucose, NAD and NAD (P)⁺70mg of each, glucose dehydrogenase (CDX-901,30mg, 66U/mg), preserving heat at 45 ℃ for reaction for 48h, adding calcium hydroxide suspension in batches in the reaction to keep the pH value at about 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 dryness to obtain a white solid, and crystallizing with ethanol to obtain a pure product of 3.98 g. Purity by HPLC>99 percent. The molar yield was 85%.

HNMR (500MHz, CDCl3) delta: 5.35(m,1H),3.51(m,1H),1.21(d,6H),1.01(d,3H), 0.93(d,3H), 0.68(s, 3H). The spectrum is shown in figure 7.

The comprehensive experiment result shows that the method for synthesizing the cholesterol and the 25-hydroxyl cholesterol by taking the 22-sterol as the raw material has the advantages of simple reaction process, high yield, low production cost, environment-friendly process and more suitability for industrial implementation.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

Claims

1. A method for synthesizing cholesterol and 25-hydroxycholesterol by taking 22-hydroxy-20-methyl pregn-4-en-3-one (4-BA) as a raw material is characterized in that a phytosterol fermentation product 22-hydroxy-20-methyl pregn-4-en-3-one (4-BA) is taken as a raw material, and the method comprises the following steps:

1) protecting the 3-position carbonyl group: catalyzing 22-hydroxy-20-methylpregna-4-en-3-one (4-BA) by using p-toluenesulfonic acid, reacting with pyrrolidine or ethylene glycol at the reaction temperature of 10-120 ℃ for 3-5 hours, stopping the reaction by using sodium bicarbonate, performing elutriation, and filtering to obtain 21-hydroxy-20-methylpregna-3-pyrrolidine-3, 5-diene or 21-hydroxy-20-methylpregna-3-ethylene glycol-5-ene;

2) and (3) sulfonation reaction: reacting 21-hydroxy-20-methyl pregna-3-tetrahydropyrrole-3, 5-diene or 21-hydroxy-20-methyl pregna-3-ethylene glycol-5-ene with p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride and fluorosulfonyl fluoride in dichloromethane or dichloroethane under the condition of triethylamine or diisopropylethylamine, washing with water, extracting, and concentrating to obtain 22-sulfonyl compound;

3) and (3) substitution reaction: adding a 22-sulfonyl compound and a Grignard reagent isoamyl magnesium bromide in a tetrahydrofuran solvent for reaction, quenching with acid, separating liquid, and concentrating to obtain 3-position protected cholestane or 3-position protected 25-hydroxy cholestane;

4) acetylation reaction: 3-position protected cholestane or 3-position protected 25-hydroxy cholestane, performing 3-position protecting group exchange reaction under the conditions of acetic acid and acetic anhydride, evaporating ethanol, and crystallizing to obtain 3-acetyl-3, 5-diencholestane or 3-acetyl-25-hydroxy-3, 5-diencholestane;

2. The method according to claim 1, wherein the weight ratio of each reactant in the 3-carbonyl protection reaction in the step 1) is as follows: 21-hydroxy-20-methylpregn-4-en-3-one: pyrrolidine or ethylene glycol = 1.0: 1.0-5.0: 1.0 to 5.0.

3. The method of claim 1, wherein the 3-carbonyl protection reaction of step 1) is performed in an organic solvent, and the solvent is one of ethanol, methanol and toluene.

4. The method of claim 1, wherein the sulfonation reaction in step 2) is performed by reacting triethylamine, diisopropylethylamine, etc. as a base with p-toluenesulfonyl chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, and fluorosulfonyl fluoride in a dichloromethane solvent to obtain 22-sulfonate.

5. The method as claimed in claim 1, wherein the substitution reaction of step 3) is carried out by using reagents such as a Grignard reagent of isoamyl magnesium bromide, isoamyl lithium bromide, 3-trimethylsiloxy-3-methylbutyl magnesium bromide and the like, and the molar ratio of the reactants is as follows: 22-sulfonic acid ester: isopentyl magnesium bromide = 1: 3 to 10.

6. The method of claim 1, wherein the grignard reagent substitution reaction in step 3) requires cuprous salt as a catalyst, the cuprous salt is cuprous bromide, cuprous dimethyl sulfide bromide, cuprous chloride, cuprous iodide, and lithium tetrachlorocupric divalent salt, and the reaction molar ratio is 1: 0.1 to 1.

7. The process of claim 1, wherein step 5) the 3-acetyl-3, 5-diencholestane or 3-acetyl-25-hydroxy-3, 5-diencholestane is dissolved in ethylene glycol dimethyl ether, tetrahydrofuran, sodium borohydride is added, the reaction product is crystallized by extraction with ethanol to yield cholesterol or 25-hydroxycholesterol, 3-acetyl-3, 5-diencholestane: sodium borohydride mole ratio = 1.0: 0.25 to 1.

8. The method of claim 1, wherein the step 5) of reducing 3-acetyl-25 hydroxy-3, 5-diencholestane or 3-acetyl-3, 5-diencholestane is performed by reducing with carbonyl reductase in methyl tert-butyl ether phosphate buffer solution to obtain cholesterol or 25-hydroxycholesterol, and calcium hydroxide suspension is added dropwise during the reduction reaction to maintain pH of the reaction solution at about 7-7.5.

9. Cholesterol or 25-hydroxycholesterol prepared by the process of any one of claims 1 to 8.