Method for synthesizing high-purity cholesterol

Abstract

The present invention discloses a method for synthesizing high-purity cholesterol, which uses plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnoral alcohol or BA, as raw material, and synthesizes the above cholesterol through steps such as oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation reduction, deprotection or hydrolysis, and the purity can reach 99% or more. Considering the shortcomings of traditional animal-derived cholesterol, the present invention synthesizes cholesterol from plant-derived raw material BA, which is not only safe and avoids the risk of pathogenic bacteria and virus infection, but also has a high synthesis yield, good product purity, environmentally friendly and convenient for industrialized production, and the present invention greatly reduces the level of impurities in the product, making it easy to obtain high-purity cholesterol and improving the safety of clinical use.

Description

[Technical field]

[0001] The present invention belongs to the technical field of organic chemical synthesis, and relates to a method for synthesizing high-purity cholesterol using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnoral alcohol or BA (Bisnoralcohol), as a raw material. [Background technology]

[0002] Cholesterol is an essential and important substance for animal tissue cells. It is not only involved in the formation of cell membranes, but also a raw material for the synthesis of bile acids and steroid hormones in the body. Currently, commercialized cholesterol is mainly used for the following purposes: 1. Pharmaceutical excipients-liposome additives, such as excipients for novel coronavirus mRNA vaccines (adding 30-50% cholesterol when producing liposomes can significantly increase the drug-carrying capacity and stability of liposomes); 2. Cosmetic additives; 3. Raw materials for producing liquid crystals; 4. Starting materials for producing vitamin D3. Currently, most of the commercialized cholesterol is derived from animal brain stems (brain stem cholesterol) or lanolin (lanolin cholesterol), both of which are animal-derived cholesterol. Research has found that animal-derived products are likely to contain animal pathogens and other harmful factors, and the safety of cholesterol has come to the forefront, especially with the outbreak of infectious diseases such as mad cow disease, swine Streptococcus, and avian influenza. For example, the upstream raw material for vitamin D3 production is cholesterol, and European and American countries have long banned the use of brain stem cholesterol as a raw material due to the risk of epidemics. China has also restricted the use of brainstem cholesterol as a raw material since July 1, 2020. Therefore, in order to ensure people's health and safety, it is urgent to develop a method for synthesizing high-purity cholesterol from plants.

[0003] The main methods reported for chemically synthesizing cholesterol are as follows: (1) Using diosgenin as the raw material, cholesterol was synthesized through six-step reactions with a total molar yield of 61% (CN1772760A, shown in Scheme 1). This scheme was not suitable for industrial production due to the relatively high price of raw materials, the complicated procedures, and the high toxicity and pollution of the reagents used. [ka]

[0004] (2) Using the stigmasterol decomposition product as the raw material, cholesterol was synthesized through a 5-step reaction with a total molar yield of 67% (as shown in CN105218610A, scheme 2). However, the applicant of the present invention found through experiments that this scheme had the following problems. When triethyl orthoformate was used to etherify and protect the carbonyl group at the C-3 position in the first step reaction of the patent, the selectivity was low, and it was easy to cause the reaction of the aldehyde in the side chain, resulting in the production of acetal (see "Comparative Example 1" in the contents of the invention for details). Therefore, there were serious doubts about the feasibility of this scheme. [ka]

[0005] (3) Using pregnenolone as a raw material, cholesterol was synthesized through a four-step reaction with a total molar yield of 72% (CN105218609A, as shown in Scheme 3). This scheme is not suitable for large-scale industrial production because the precious metal rhodium catalyst and chiral phosphine ligand used are expensive. [ka]

[0006] (4) Using pregnenolone as the raw material, cholesterol was synthesized through a two-step reaction with a total molar yield of 80% (CN104961788A, as shown in Scheme 4). This scheme also requires expensive noble metal rhodium catalyst and chiral phosphine ligand, and is not suitable for large-scale industrial production. [ka]

[0007] (5) Using stigmasterol as a raw material, cholesterol was synthesized through a five-step reaction with a total molar yield of 68% (CN105237603A, as shown in Scheme 5). In this scheme, O 3 However, the requirements for reaction monitoring and equipment are high, and the economical and safe aspects are not sufficient. [ka]

[0008] (6) Using stigmasterol as a raw material, cholesterol was synthesized through a four-step reaction with a total molar yield of 70% (CN106632565A, as shown in Scheme 6). 3 However, the use of these compounds makes the process more difficult, increases the requirements for reaction monitoring and equipment, and is therefore insufficient in terms of economy and safety. [ka]

[0009] (7) Using BA as the raw material, cholesterol was synthesized through a five-step reaction with a total molar yield of 78.5% (CN113248557A, as shown in Scheme 7). The purification in this scheme uses column chromatography purification, which is not suitable for industrial production, and the purity of cholesterol obtained in this scheme is not high (95%-96%), and it is difficult to meet the standards for pharmaceutical excipients without further purification. [ka]

[0010] Cholesterol derived from animals carries the risk of infection with diseases such as mad cow disease, swine Streptococcus pyogenes, and avian influenza. The chemical synthesis schemes of cholesterol reported so far have drawbacks such as complicated operations, high pollution, expensive catalysts, and low product purity, so the development of a safer and more efficient synthesis method for high-purity cholesterol using raw materials derived from plants is of great value. Summary of the Invention

[0011] In order to solve the shortcomings of the existing technology, the object of the present invention is to provide a method for synthesizing high-purity cholesterol from plants. The present invention uses 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnoral alcohol or BA, derived from plants, as raw material, and synthesizes said cholesterol through steps such as oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation reduction, deprotection or hydrolysis, and the purity can reach 99% or more. In the present invention, the starting material for synthesizing cholesterol, BA, is safe and economical, and the method for synthesizing cholesterol is simple to operate, has high yield, high purity, is environmentally friendly, and is convenient for industrial production.

[0012] The raw material BA (bisnorcohol) used in the present invention is a plant-derived green raw material obtained by fermenting plant sterols remaining in the oil refining process. The current annual production volume has reached 1,000 tons, and the price is low, which can fully avoid the risk of pathogenic bacteria and virus infection that can be present in the animal-derived cholesterol of the prior art.

[0013] In the synthesis method of the present invention, the raw material BA includes, but is not limited to, those obtained by biological fermentation of plant sterols or those obtained by chemical synthesis methods.

[0014] The method for synthesizing cholesterol using a BA raw material provided by the present invention comprises the steps of: (a) oxidizing BA represented by formula (1) in a first solvent to obtain a compound of formula (2); (b) subjecting the compound of formula (2) to a Wittig reaction in a second solvent to obtain a compound of formula (3); (c) acetylating the compound of formula (3) in a third solvent to obtain a compound of formula (4); (d) reducing the compound of formula (4) in a fourth solvent to obtain a compound of formula (5); (e) reacting the compound of formula (5) with a hydroxyl protection reaction in a fifth solvent to obtain a compound of formula (6); (f) selectively reducing the compound of formula (6) by hydrogenolysis in a sixth solvent to obtain a compound of formula (7); (g) deprotecting or hydrolyzing the compound of formula (7) in a seventh solvent to obtain cholesterol; Including, Here, the reaction process of the above method is as shown in Scheme (A). [ka]

[0015] where R is selected from an ester group, a silyl ether group, Preferably, R is a C2-C10 straight chain ester group, an isobutyl ester [ka] Isopentyl ester group [ka] Phenyl ester group [ka] p-Methoxyphenyl ester group [ka] Trimethylsilyl Ether Group [ka] and tert-butyldimethylsilyl ether group [ka] and More preferably, R is an ethyl ester. [ka] Propyl ester [ka] Butyl ester [ka] Isobutyl Ester [ka] Isopentyl ester [ka] Phenyl esters [ka] p-Methoxyphenyl ester [ka] Trimethylsilyl Ether Group [ka] and tert-butyldimethylsilyl ether group [ka] One or more selected from:

[0016] Note: In the compounds (3) to (6), the double bonds between the C-22 and C-23 positions are predominantly E-configured and predominantly Z-configured. The ratio of these two configurations is E / Z ≒ 87 / 13 ( 1 1 H NMR), and this ratio does not change without subsequent purification steps. In step (a) of the present invention, the oxidation reaction is specifically a reaction in which BA represented by formula (1) is oxidized with TEMPO, sodium hydrogen carbonate, tetrabutylammonium bromide and an oxidizing agent in a first solvent to obtain a compound of formula (2).

[0017] Here, the molar ratio of BA represented by formula (1), TEMPO, sodium hydrogen carbonate, tetrabutylammonium bromide, and the oxidizing agent is 1:(0-1):(0-20):(0-1):(1-5), and preferably 1:0.01:1.35:0.1:1.15.

[0018] Here, the oxidation reaction is carried out in the presence of an oxidizing agent, and the oxidizing agent is one or more selected from N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), 2-iodoxybenzoic acid (IBX), etc., and is preferably N-chlorosuccinimide (NCS).

[0019] Here, the first solvent is one or more selected from dichloromethane, tetrahydrofuran, toluene, dimethylsulfoxide, water, etc., and is preferably a mixed solvent of dichloromethane and water (volume ratio V / V=5 / 2). Here, the temperature of the oxidation reaction is 0 to 30°C, and preferably 0°C. Here, the time for the oxidation reaction is 3 to 8 hours, and preferably 6 hours.

[0020] In a particular embodiment, the synthesis step of the compound of formula (2) includes dissolving BA represented by formula (1) in a first solvent, and then adding TEMPO, sodium bicarbonate, tetrabutylammonium bromide and NCS to carry out an oxidation reaction to obtain the compound of formula (2).

[0021] In step (b) of the present invention, the Wittig reaction is specifically a reaction in which the compound of formula (2), 3,3-dimethylallyl halide, triphenylphosphine, and potassium tert-butoxide are subjected to a Wittig reaction in a second solvent to obtain the compound of formula (3).

[0022] Here, the molar ratio of the compound of formula (2), 3,3-dimethylallyl halide, triphenylphosphine, and potassium tert-butoxide is 1:(1-4):(1-4):(1-4), and preferably 1:1.3:1.3:1.3. Wherein, the second solvent is one or more of toluene, benzene, tetrahydrofuran, heptane, etc., preferably toluene.

[0023] Here, the 3,3-dimethylallyl halide is one or more selected from 3,3-dimethylallyl chloride, 3,3-dimethylallyl bromide, etc., and is preferably 3,3-dimethylallyl bromide. Here, the temperature of the Wittig reaction is -10 to 112°C, and preferably 10°C. Here, the time for the Wittig reaction is 0.5 to 9 hours, and preferably 0.5 hours.

[0024] In step (c) of the present invention, the acetylation reaction is specifically a reaction in which the compound of formula (3), acetyl chloride, acetic anhydride, and a base are acetylated in a third solvent to obtain the compound of formula (4).

[0025] Here, the molar ratio of the compound of formula (3), acetyl chloride, acetic anhydride, and base is 1:(0.5-62.5):(1-62.5):(0-6), and preferably 1:25:24:4. Here, the base is one or more selected from pyridine, triethylamine, DIPEA, DMAP, diisopropylamine, etc., and is preferably diisopropylamine. Here, the third solvent is one or more of acetic anhydride, acetyl chloride, ethyl acetate, dichloromethane, etc., and is preferably a mixed solvent of acetyl chloride and acetic anhydride. The temperature of the acetylation reaction is 40 to 110°C, and preferably 70°C. Here, the acetylation reaction time is 1 to 10 hours, preferably 2 to 10 hours, and more preferably 6 hours. Here, the acetylation reaction uses acetyl chloride and acetic anhydride as both reactants and solvents.

[0026] In a particular embodiment, the step of synthesizing the compound of formula (4) includes the step of adding acetyl chloride, acetic anhydride, and a base to the compound of formula (3) to perform an acetylation reaction to obtain the compound of formula (4).

[0027] In step (d) of the present invention, the reduction reaction is specifically a reaction in which the compound of formula (4) is reduced with a reducing agent in a fourth solvent to obtain a compound of formula (5).

[0028] Here, the molar ratio of the compound of formula (4) to the reducing agent is 1:(1 to 25), and preferably 1:4. Here, the fourth solvent is one or more of tetrahydrofuran, ethanol, water, dichloromethane, 2-methyltetrahydrofuran, isopropyl alcohol, acetic acid, methyl tert-butyl ether, etc., and is preferably a mixed solvent of tetrahydrofuran, ethanol, and water (volume ratio V / V / V=10 / 5 / 3). Here, the reducing agent is NaBH 4 and KBH 4 and the like, preferably NaBH 4 It is. Here, the temperature of the reduction reaction is 0 to 50°C, and preferably 25°C. Here, the reduction reaction time is 6 to 12 hours, and preferably 8 hours.

[0029] In one particular embodiment, the step of synthesizing the compound of formula (5) includes dissolving the compound of formula (4) in a fourth solvent and reducing it with a reducing agent to obtain the compound of formula (5).

[0030] In step (e) of the present invention, when the hydroxy-protecting group R is an ester group, the hydroxyl protection reaction is specifically a reaction in which the compound of formula (5) is reacted with a hydroxy-protecting reagent in a fifth solvent under the action of a base to obtain a compound of formula (6).

[0031] The ester group is a C2-C10 linear ester group (ethyl ester [ka] Propyl ester [ka] Butyl ester [ka] etc.), isobutyl ester [ka] Isopentyl ester [ka] Phenyl esters [ka] and p-methoxyphenyl ester [ka] and the like, preferably an ethyl ester.

[0032] Here, the molar ratio of the compound of the formula (5), the protecting reagent for the ester group, and the base is 1:(1-4):(0.05-5), and preferably 1:3:0.1. Wherein, the fifth solvent is one or more of ethyl acetate, dichloromethane, chloroform, DMF, toluene, tetrahydrofuran, 2-methyltetrahydrofuran, etc., and is preferably ethyl acetate. Here, the base is one or more selected from triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, etc., and is preferably DMAP. Here, the reaction temperature is 0 to 50°C, and preferably 45°C. Here, the reaction time is 2 to 24 hours, and preferably 4 hours.

[0033] In step (e) of the present invention, when the hydroxy-protecting group is a silyl ether group, the hydroxyl protection reaction is specifically a reaction in which a compound of formula (5) is reacted with a protecting reagent for a silyl ether group in a fifth solvent under the action of a base to obtain a compound of formula (6).

[0034] Here, the silyl ether group is a trimethylsilyl ether group. [ka] and tert-butyldimethylsilyl ether group [ka] and the like, and preferably a tert-butyldimethylsilyl ether group. [ka] It is.

[0035] Here, the molar ratio of the compound of the formula (5), the protecting reagent for the silyl ether group, and the base is 1:(2-4):(4-8), and preferably 1:2.5:4. Wherein, the fifth solvent is one or more of DMF, dichloromethane, chloroform, carbon tetrachloride, etc., and is preferably dichloromethane. Here, the base is one or more selected from triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, etc., and is preferably imidazole. Here, the reaction temperature is 0 to 50°C, and preferably 25°C. Here, the reaction time is 2 to 24 hours, and preferably 12 hours.

[0036] In one particular embodiment, the step of synthesizing the compound of formula (6) comprises dissolving the compound of formula (5) in a fifth solvent and reacting with a hydroxy protecting reagent under the action of a base to obtain the compound of formula (6).

[0037] In step (f) of the present invention, the selective hydrogenation reduction reaction is specifically a reaction in which the compound of formula (6) is subjected to a selective hydrogenation reduction reaction with a reducing agent in a sixth solvent under the action of a catalyst to obtain a compound of formula (7). Here, the catalyst is Raney Ni. Here, the reducing agent is H 2 It is. Here, the mass ratio of the compound of formula (6) to the catalyst Raney Ni is 1:(0.05 to 5), and preferably 1:1. Here, the sixth solvent is one or more selected from 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, isopropyl alcohol, etc., and is preferably ethyl acetate. Here, the temperature of the hydrogenation reduction reaction is 0 to 60°C, and preferably 30°C. Here, the reducing agent H in the hydrogenation reduction reaction 2 The pressure is 1 to 20 atm, and preferably 1 atm. Here, the time for the hydrogenation reduction reaction is 4 to 48 hours, preferably 4 to 30 hours, and more preferably 7 hours.

[0038] In one particular embodiment, the cholesterol synthesis step comprises dissolving the compound of formula (6) in a sixth solvent, adding Raney Ni, and 2 followed by selective hydrogenation to obtain a compound of formula (7).

[0039] In step (g) of the present invention, when the protecting group R is an ester group, the hydrolysis reaction is specifically a reaction in which the compound of formula (7) is hydrolyzed in a seventh solvent under the action of a base to obtain cholesterol.

[0040] Here, the base is LiOH, KOH, NaOH, t-BuOK, K 2 CO 3 and the like, preferably K 2 CO 3 It is. Here, the molar ratio of the compound of the formula (7) to the base is 1:(0.5 to 2), and preferably 1:1.3. Here, the seventh solvent is one or more selected from methanol, ethanol, etc., and is preferably methanol. The temperature of the hydrolysis reaction is 10 to 75°C, and preferably 65°C. Here, the hydrolysis reaction time is 0.3 to 12 hours, and preferably 2 hours. In step (g) of the present invention, when the protecting group R is a silyl ether group, the deprotection reaction is specifically a reaction in which the compound of formula (7) is deprotected in the seventh solvent under the action of a catalyst to obtain cholesterol. Here, the catalyst is tetrabutylammonium fluoride (TBAF), tetrabutylammonium fluoride trihydrate (TBAF·3H 2 The solvent is one or more selected from the group consisting of ethyl acetate solution of boron trifluoride etherate, acetic acid, and hydrogen chloride, preferably TBAF·3H 2 It is O. Here, the molar ratio of the compound of the formula (7) to the catalyst is 1:(1 to 6), and preferably 1:4. Here, the seventh solvent is one or more selected from tetrahydrofuran, water, etc., and is preferably tetrahydrofuran. The temperature for the deprotection reaction is 10 to 75°C, and preferably 25°C. Here, the time for the deprotection reaction is 2 to 48 hours, and preferably 24 hours.

[0041] In a particular embodiment, the step of synthesizing cholesterol includes dissolving the compound of formula (7) in a seventh solvent, adding a base or a catalyst, and carrying out a deprotection or hydrolysis reaction to obtain cholesterol.

[0042] The present invention also provides 20 compounds, the structures of which are as shown in the following formulas (6-2E), (6-3E), (6-4E), (6-5E), (6-6E), (6-2Z), (6-3Z), (6-4Z), (6-5Z), (6-6Z), (6'-2E), (6'-3E), (6'-4E), (6'-5E), (6'-6E), (6'-2Z), (6'-3Z), (6'-4Z), (6'-5Z), and (6'-6Z). [ka]

[0043] The beneficial effects of the present invention include the following: In the production method of high-purity cholesterol of the present invention, the commercialized raw material BA used is a raw material derived from plants, which avoids the risk of infection by pathogenic bacteria and viruses that may exist in raw materials derived from animals, is cheap and easy to obtain, and the obtained cholesterol is of high purity (99% or more), the synthesis process is simple, the yield is high, there are few side reactions, it is environmentally friendly, and it is convenient to achieve industrial production of high-purity cholesterol, which can solve the problems of existing cholesterol products such as low safety, low purity, high synthesis cost, and environmental friendliness. [Brief description of the drawings]

[0044] [Figure 1]FIG. 1 shows a gas chromatogram of the crude product of the compound of formula (7-1) obtained in Example 6 of the present invention. [Diagram 2] FIG. 2 shows a gas chromatogram of the purified product of the compound of formula (7-1) obtained in Example 6 of the present invention. [Diagram 3] FIG. 3 shows a gas chromatogram of the purified product of the compound of formula (7-2) obtained in Example 6 of the present invention. [Figure 4] FIG. 4 shows a gas chromatogram of the purified compound of formula (7-3) obtained in Example 6 of the present invention. [Diagram 5] FIG. 5 shows a gas chromatogram of the purified product of the compound of formula (7-4) obtained in Example 6 of the present invention. [Figure 6] FIG. 6 shows a gas chromatogram of the purified product of the compound of formula (7-5) obtained in Example 6 of the present invention. [Figure 7] FIG. 7 shows a gas chromatogram of the purified product of the compound of formula (7-6) obtained in Example 6 of the present invention. [Figure 8] FIG. 8 shows the single crystal structure of cholesterol. [Figure 9] FIG. 9 shows a gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-1). [Figure 10] FIG. 10 shows a gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-2). [Figure 11] FIG. 11 shows a gas chromatogram of cholesterol obtained by hydrolysis of the compound of formula (7-3). [Figure 12] FIG. 12 shows a gas chromatogram of cholesterol obtained by hydrolysis of the compound of formula (7-4). [Figure 13] FIG. 13 shows a gas chromatogram of cholesterol obtained by hydrolysis of the compound of formula (7-5). [Figure 14] FIG. 14 shows a gas chromatogram of cholesterol obtained by the deprotection reaction of the compound of formula (7-6). [Figure 15]FIG. 15 shows a gas chromatogram of the crude product of the compound of formula (7-1) obtained by hydrogenating the compound of formula (6-1) with a 10% Pd / C catalyst. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention will be described in more detail with reference to the following specific examples and drawings. The processes, conditions, experimental methods, etc. for carrying out the present invention are common knowledge or common general knowledge in the relevant field, except for the contents specifically described below, and the contents are not particularly limited to the present invention.

[0046] In the following examples, the structures of the compounds were determined by nuclear magnetic resonance and high-resolution mass spectrometry, the reagents were mainly provided by Shanghai Guoyue Chemical Reagent Company, the purification of the products was mainly carried out by slurrying and column chromatography, and the silica gel (200-300) was obtained from Qingdao Ocean Chemical Plant.

[0047] The present invention provides a method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one (BA) as a raw material, and the reaction process is as shown in the above scheme (A). Note that the present invention further includes the production of other by-products in the method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one (BA) as a raw material, and the specific reaction scheme is as shown in (A'). [ka]

[0048] where R is selected from an ester group, a silyl ether group, Note: In the compounds (3) to (6), the double bonds between the C-22 and C-23 positions are predominantly E-configured and predominantly Z-configured. The ratio of these two configurations is E / Z ≒ 87 / 13 ( 11 H NMR), and this ratio does not change unless a subsequent purification procedure is performed. The ratio of compound (5) (3β-OH) / (5') (3α-OH) is about 92 / 8 ( 1 1 H NMR), and the ratios of compounds (6) / (6') and (7) / (7') do not change unless a purification procedure is performed.

[0049] In the present invention, the compounds of the above formulae (3), (4), (5), (5'), (6) and (6') have a D-ring side chain double bond (double bond at C-22 and C-23 positions) introduced by the Wittig reaction that has a cis-trans configuration (E and Z configuration), and the ratio of the two configurations is E / Z ≈ 87 / 13, so this ratio will not change unless a subsequent purification procedure is performed. Compounds of formulae (5) (3β-OH) and (5') (3α-OH) can be obtained by treating compound (4) with NaBH 4 or KBH 4 The ratio of 3β-OH / 3α-OH in the resulting compounds (5) (3β-OH) and (5') (3α-OH) is about 92 / 8, and this ratio does not change unless a purification procedure is performed. Therefore, the compounds of formula (3), formula (4), formula (5), formula (5'), formula (6), formula (6'), formula (7) and formula (7') are not single substances but mixtures.

[0050] Example 1 Preparation of the compound of formula (2) [ka] In this example, the preparation of compound of formula (2) under different experimental conditions is shown.

[0051] (1) BA (24.79 g, 0.075 mol), TEMPO (118 mg, 0.750 mmol), dichloromethane (200 mL), sodium hydrogen carbonate (8.8 g, 0.105 mol), NCS (11.55 g, 86.5 mmol), tetrabutylammonium bromide (2.42 g, 7.5 mmol), and water (160 mL) were sequentially added to a flask, and the mixture was allowed to react at 0° C. for 6 hours. After detecting the completion of the reaction by TLC, sodium thiosulfate pentahydrate solution (5.6 g sodium thiosulfate pentahydrate / 110 mL water) was added, stirred at 5-10°C for 20 minutes, separated, the aqueous phase was extracted with dichloromethane (150 mL x 2), the organic layers were combined, washed with 1% sodium hydroxide solution (100 mL), separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound of formula (2) (24.0 g white solid, molar yield: 94.9%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.55 (s, 1H), 5.71 (s, 1H), 2.45 - 2.23 (m, 5H), 1.99 (t, J = 13.7 Hz, 2H), 1.91 - 1.78 (m, 2H), 1.68 (t, J = 10.2 Hz, 2H), 1.43 (m, 5H), 1.30 - 1.19 (m, 2H), 1.17 (s, 3H), 1.11 (d, J = 5.5 Hz, 3H), 1.06 - 0.89 (m, 3H), 0.75 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ 205.00, 199.65, 171.31, 123.99, 55.25, 53.84, 51.04, 49.54, 43.10, 39.39, 38.68, 35.80, 35.68, 34.06, 32.93, 32.05, 27.11, 24.64, 21.06, 17.48, 13.53, 12.44. HRMS(ESI): calcd for C 22 H 32 NaO 2 [M+Na] + , 351.2295, found 351.2292.

[0052] (2) BA (24.79 g, 0.075 mol), TEMPO (118 mg, 0.750 mmol), dichloromethane (200 mL), sodium hydrogen carbonate (8.8 g, 0.105 mol), NBS (17.8 g, 100 mmol), tetrabutylammonium bromide (2.42 g, 7.5 mmol), and water (160 mL) were sequentially added to a flask, and the mixture was allowed to react at 0° C. for 6 hours. After detecting the completion of the reaction by TLC, sodium thiosulfate pentahydrate solution (8 g sodium thiosulfate pentahydrate / 160 mL water) was added, stirred at 5-10°C for 20 minutes, separated, the aqueous phase was extracted with dichloromethane (150 mL x 2), the organic layers were combined, washed with 1% sodium hydroxide solution (100 mL), separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound of formula (2) (23.6 g white solid, molar yield: 95.2%).

[0053] (3) The compound BA (24.79 g, 75 mmol) of formula (1), IBX (42 g, 150 mmol), THF (250 mL) and DMSO (200 mL) were added to a flask in this order, and reacted at 25° C. for 5 hours. After detecting the completion of the reaction by TLC, water was added, suction filtered, the filtrate was concentrated under reduced pressure, and extracted by adding dichloromethane (400 mL) and water (300 mL). The organic phase was washed with 1% sodium hydroxide solution (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound of formula (2) (23.2 g of white solid, molar yield: 93.5%).

[0054] Example 2 Preparation of the compound of formula (3) [ka] In this example, the preparation of compound of formula (3) under different experimental conditions is shown.

[0055] (1) Triphenylphosphine (23.97g, 91.40mmol), 3,3-dimethylallyl bromide (13.62g, 91.40mmol), and 230mL of toluene were added to a flask, and the mixture was stirred at 80°C for 30 minutes to react, heated under reflux for 2 hours, and then cooled to 25°C. In an ice bath, potassium tert-butoxide (10.26g, 91.40mmol) was added, and the mixture was stirred for 0.5 hours. Then, the compound of formula (2) (23.09g, 70.30mmol) was added in batches, and the mixture was reacted at 10°C for 0.5 hours. After detecting the completion of the reaction by TLC, water (23mL) was added to quench the reaction, and 2M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (120mL x 2) and concentrated under reduced pressure to obtain a milky white solid. The solid was added to 50% ethanol (80 mL), slurried at 25° C. for 1 hour, filtered, and the cake was rinsed with 50% ethanol (13 mL) to give the compound of formula (3) (3E and 3Z, 3E / 3Z≈87 / 13, 24.9 g of white solid, molar yield: 93.14%). HRMS (ESI): calcd for C 27 H 40 NaO [M+Na] + ,403.2971, found 403.2967.

[0056] Note: In the present invention, the E / Z configuration ratio of the intermediate compounds of formula (4), formula (5) and formula (6) obtained by acetylating and reducing the compound of formula (3) is basically unchanged, and the double bond between C-22 and C-23 has E configuration as the main configuration and Z configuration as the minor configuration (3E / 3Z ≒ 87 / 13). The cis-trans isomer of the double bond between C-22 and C-23 in the compound of formula (6) is reduced by Raney nickel, deprotected or hydrolyzed to obtain the product cholesterol. Therefore, the E / Z configuration ratio of the corresponding compounds is not shown in the following examples.

[0057] (2) Triphenylphosphine (23.97g, 91.40mmol), 3,3-dimethylallyl bromide (13.62g, 91.40mmol), and 230mL of n-heptane were added to a flask, and the mixture was heated to reflux for 3 hours, then cooled to 25°C, potassium tert-butoxide (10.26g, 91.40mmol) was added in an ice bath, and the mixture was stirred for 0.5 hours, after which the compound of formula (2) (23.00g, 70.07mmol) was added in batches and reacted at 15°C for 1 hour. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, water (23mL) was added to quench the reaction, 2M HCl was added to adjust the pH to 6-7, and the organic phase was washed with water (120mL x 2) and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to 50% ethanol (70 mL), slurried at 25° C. for 1 hour, filtered, and the cake was rinsed with 50% ethanol (13 mL) to give compounds of formula (3) (3E and 3Z, 24.5 g of white solid, molar yield: 91.94%).

[0058] (3) Triphenylphosphine (23.97g, 91.40mmol), 3,3-dimethylallyl bromide (13.62g, 91.40mmol), and 230mL of toluene were added to a flask, and the mixture was stirred at 80°C for 30 minutes to react, heated under reflux for 2 hours, and then cooled to 25°C. In an ice bath, potassium tert-butoxide (11.27g, 100.5mmol) was added, and the mixture was stirred for 40 minutes. Then, the compound of formula (2) (23.09g, 70.30mmol) was added in batches and reacted at 10°C for 2 hours. After detecting the completion of the reaction by TLC, water (23mL) was added to quench the reaction, and 2M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (120mL x 2) and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to 50% ethanol (70 mL), slurried at 25° C. for 1 hour, filtered, and the cake was rinsed with 50% ethanol (13 mL) to give compounds of formula (3) (3E and 3Z, 24.73 g of white solid, molar yield: 92.5%).

[0059] (4) Triphenylphosphine (23.97g, 91.40mmol), 3,3-dimethylallyl bromide (13.62g, 91.40mmol), and 230mL of toluene were added to a flask, and the mixture was refluxed for 2 hours. The mixture was then cooled to 25°C, and potassium tert-butoxide (9.23g, 82.26mmol) was added in an ice bath, and the mixture was stirred for 0.5 hours. Then, the compound of formula (2) (15.00g, 45.70mmol) was added, and the mixture was refluxed for 2.5 hours. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, and water (23mL) was added to quench the reaction. 2M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (100mL x 2), and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to 50% ethanol (45 mL) and slurried at 25° C. for 1 hour to give compounds of formula (3) (3E and 3Z, 16.72 g of white solid, molar yield: 96.20%).

[0060] (5) Triphenylphosphine (23.97g, 91.40mmol), 3,3-dimethylallyl chloride (9.56g, 91.40mmol), and 230mL of toluene were added to a flask, and the mixture was refluxed for 10 hours. The mixture was then cooled to 25°C, and potassium tert-butoxide (9.23g, 82.26mmol) was added in an ice bath, and the mixture was stirred for 0.5 hours. Then, the compound of formula (2) (7.5g, 22.85mmol) was added, and the mixture was refluxed for 4.5 hours. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, and water (23mL) was added to quench the reaction. 2M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (100mL x 2), and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to 50% ethanol (45 mL) and slurried at 25° C. for 1 hour to give compounds of formula (3) (3E and 3Z, 6.26 g of white solid, molar yield: 72.04%).

[0061] Example 3 Preparation of compound of formula (4) [ka] In this example, the preparation of compound of formula (4) under different experimental conditions is shown.

[0062] (1) The compound of formula (3) (15g, 39.44mmol), acetic anhydride (16.09g, 157.76mmol) and acetyl chloride (3.08g, 39.44mmol) were added to a flask, and reacted at 70°C for 6 hours. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, concentrated under reduced pressure, cooled slightly, pyridine (2g) and methanol (75mL) were added, stirred to disperse, concentrated under reduced pressure, cooled slightly, methanol (75mL) was added, stirred for 10 minutes and refluxed, the reaction solution was cooled to 25°C, stirred for 30 minutes, filtered, the cake was rinsed with methanol (24mL), suction filtered and dried to obtain the compound of formula (4) (4E and 4Z, 15.50g of white solid, molar yield: 93.03%). HRMS (ESI): calcd for C 29 H 42 NaO 2 [M+Na] + ,445.3077, found 445.3081.

[0063] (2) The compound of formula (3) (15 g, 39.44 mmol), acetic anhydride (96.6 g, 946.2 mmol), acetyl chloride (77.4 g, 986 mmol) and DIPEA (30.58 g, 236.6 mmol) were added to a flask, and the mixture was reacted at 70 ° C for 3 hours. After the completion of the reaction was detected by TLC, the mixture was cooled to 25 ° C, concentrated under reduced pressure, cooled slightly, and the reaction solution was added to ice water (200 mL), stirred for 15 minutes, filtered, and the water was removed. The resulting solid was added to methanol (60 mL) and pyridine (1.5 mL), slurried at 25 ° C for 30 minutes, filtered, and the cake was rinsed with methanol (10 mL), suction filtered, and dried to obtain the compound of formula (4) (4E and 4Z, 16 g of white solid, molar yield: 96.04%).

[0064] (3) The compound of formula (3) (15 g, 39.44 mmol), acetic anhydride (96.3 g, 943.2 mmol), and acetyl chloride (74.1 g, 943.2 mmol) were added to a flask, and the mixture was refluxed at 65 ° C for 4 hours to react. After the completion of the reaction was detected by TLC, the mixture was cooled to 25 ° C, concentrated under reduced pressure, and cooled slightly. The reaction solution was added to ice water (300 mL), stirred for 15 minutes, filtered, and the water was removed. The resulting solid was added to methanol (60 mL) and pyridine (3 mL), slurried at 25 ° C for 30 minutes, filtered, and the cake was rinsed with methanol (7.5 mL), suction filtered, and dried to obtain the compound of formula (4) (4E and 4Z, 14.99 g of white solid, molar yield: 89.97%).

[0065] (4) The compound of formula (3) (15 g, 39.44 mmol), acetic anhydride (95.51 g, 936.23 mmol), acetyl chloride (73.49 g, 936.23 mmol) and diisopropylamine (11.98 g, 118.35 mmol) were added to a flask, and the mixture was refluxed at 60 ° C for 4 hours to react. After the completion of the reaction was detected by TLC, the mixture was cooled to 25 ° C, concentrated under reduced pressure, cooled slightly, and the reaction solution was added to ice water (300 mL), stirred for 15 minutes, filtered, and the water was removed. The resulting solid was added to methanol (60 mL) and pyridine (3 mL), slurried at 25 ° C for 30 minutes, filtered, and the cake was rinsed with methanol (7.5 mL), suction filtered, and dried to obtain the compound of formula (4) (4E and 4Z, 16.32 g of a white solid, molar yield: 97.96%).

[0066] Example 4 Preparation of crude product of compound of formula (5) [ka] This example shows the preparation of crude compound of formula (5) under different experimental conditions.

[0067] (1) The compound of formula (4) (9.80 g, 23.21 mmol) and a mixed solvent of tetrahydrofuran, ethanol and water (196 mL, V / V / V=10 / 5 / 3) were added to a flask, and sodium borohydride (3.51 g, 92.84 mmol) was added in batches, and the mixture was reacted at 30°C for 8 hours. After detecting the completion of the reaction by TLC, 6M hydrochloric acid was added to adjust the pH of the reaction solution to 7-8, and a large amount of solid was precipitated in the reaction solution, which was filtered. The filtrate was taken and the solvent was evaporated under reduced pressure, and a large amount of solid was precipitated. Dichloromethane (100 mL) and water (50 mL) were added for extraction, and the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain the crude product of the compound of formula (5) (8.71 g of pale yellow solid, molar yield: 98.09%), which was directly used in the next step reaction.

[0068] Note: The main components of the crude product of the compound of formula (5) obtained using sodium borohydride as a reducing agent are compounds (5) (5E and 5Z, 3β-OH) and (5') (5'E and 5'Z, 3α-OH). 1 According to H NMR, the ratio of compound (5) / (5') was 92 / 8. The ratio of 5(3β-OH) / 5'(3α-OH) in the crude product of compound (5) obtained in the following examples is basically the same as that in this example, so it is not shown hereafter.

[0069] (2) The compound of formula (4) (9.80 g, 23.21 mmol), tetrahydrofuran, ethanol and water mixed solvent (147 mL, V / V / V=10 / 5 / 2) were added to a flask, sodium borohydride (2.64 g, 69.63 mmol) was added in batches, and the mixture was reacted at 25 ° C for 10 hours. After detecting the completion of the reaction by TLC, 6 M hydrochloric acid was added to adjust the pH of the reaction solution to 7-8, a large amount of solid precipitated in the reaction solution, filtered, the filtrate was taken and the solvent was evaporated under reduced pressure, a large amount of solid precipitated, dichloromethane (100 mL) and water (50 mL) were added for extraction, separation was performed, the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain the crude product of the compound of formula (5) (8.79 g of pale yellow solid, molar yield: 98.9%), which was directly used in the next step reaction.

[0070] (3) The compound of formula (4) (9.80 g, 23.21 mmol), dichloromethane, a mixed solvent of methanol and water (95 mL, V / V / V=18 / 9 / 1) were added to a flask, sodium borohydride (1.76 g, 46.44 mmol) was added in batches, and the mixture was reacted at 25 ° C for 6 hours. After detecting the completion of the reaction by TLC, 3M hydrochloric acid was added to adjust the pH of the reaction solution to 7-8, a large amount of solids were precipitated in the reaction solution, filtered, the filtrate was taken and the solvent was evaporated under reduced pressure, a large amount of solids were precipitated, dichloromethane (100 mL) and water (50 mL) were added for extraction, separation was performed, the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain the crude product of the compound of formula (5) (8.77 g of pale yellow solid, molar yield: 98.76%), which was directly used in the next step reaction.

[0071] (4) The compound of formula (4) (9.80 g, 23.21 mmol), tetrahydrofuran, ethanol and water mixed solvent (200 mL, V / V / V=10 / 5 / 3) were added to a flask, and potassium borohydride (5.01 g, 92.84 mmol) was added in batches, and the mixture was reacted at 30°C for 8 hours. After detecting the completion of the reaction by TLC, 6M hydrochloric acid was added to adjust the pH of the reaction solution to 7-8, and a large amount of solid was precipitated in the reaction solution, which was filtered. The filtrate was taken and the solvent was evaporated under reduced pressure, and a large amount of solid was precipitated. Dichloromethane (100 mL) and water (50 mL) were added for extraction, and the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain the crude product of the compound of formula (5) (8.80 g of pale yellow solid, molar yield: 99.1%), which was directly used in the next step reaction. Note: The main components of the crude product of the compound of formula (5) obtained by reduction with potassium borohydride are compounds (5) (5E and 5Z, 3β-OH) and (5') (5'E and 5'Z, 3α-OH), among which the content of compound 5' (3α-OH) is higher. 1 According to H NMR, compound 5(3β-OH) / 5′(3α-OH)≒85 / 15.

[0072] Example 5 Preparation of crude product of compound of formula (6) 1. Preparation of crude product of compound of formula (6-1) [ka] Ethyl acetate (76 mL) and the crude product of the compound of formula (5) (7.6 g, 20 mmol) were added to a flask, and the mixture was stirred to dissolve transparently. DMAP (0.245 g, 2 mmol) and acetic anhydride (6.13 g, 60 mmol) were added, and the mixture was reacted at 45°C for 4 hours. After the completion of the reaction was detected by TLC, water (20 mL) was added to quench the reaction, and ethyl acetate (76 mL) was added for extraction. The organic phase was washed with water and saturated saline, and then with anhydrous Na 2 SO 4 and concentrated under reduced pressure to give the crude product of compound of formula (6-1) (8.3 g of white solid, molar yield: 97.8%).

[0073] Note: The main components of the crude product of the compound of formula (6-1) were compounds (6-1) (6-1E and 6-1Z) and (6'-1) (6'-1E and 6'-1Z), and the ratio of compounds (6-1) / (6'-1) was ≒ 92 / 8.

[0074] 2. Preparation of crude product of compound of formula (6-2) [ka] Ethyl acetate (50 mL) and the crude product of the compound of formula (5) (5 g, 13 mmol) were added to a flask, and the mixture was stirred to dissolve transparently. DMAP (0.159 g, 1.3 mmol) and propionic anhydride (5.07 g, 39 mmol) were added, and the mixture was reacted at 50° C. for 5 hours. After the completion of the reaction was detected by TLC, water (20 mL) was added to quench the reaction, and ethyl acetate (76 mL) was added for extraction. The organic phase was washed with water and saturated saline, and anhydrous Na 2 SO 4 The mixture was dried at 40° C., concentrated under reduced pressure, added with methanol / water, slurried at 25° C. for 5 hours, and filtered with suction to obtain the crude product of the compound of formula (6-2) (5.6 g of a white solid, molar yield: 98.2%). Note: The main components of the crude product of the compound of formula (6-2) were compounds (6-2) (6-2E and 6-2Z) and (6'-2) (6'-2E and 6'-2Z), and the ratio of compounds (6-2) / (6'-2) was ≒ 92 / 8.

[0075] 3. Preparation of crude product of compound of formula (6-3) [ka] Ethyl acetate (50 mL) and the crude product of the compound of formula (5) (5 g, 13 mmol) were added to a flask, and the mixture was stirred to dissolve transparently. DMAP (0.159 g, 1.3 mmol) and butyric anhydride (6.2 g, 39 mmol) were added, and the mixture was reacted at 55°C for 4 hours. After the completion of the reaction was detected by TLC, water (20 mL) was added to quench the reaction, and ethyl acetate (76 mL) was added for extraction. The organic phase was washed with water and saturated saline, and then with anhydrous Na 2 SO 4 The mixture was dried at 40° C., concentrated under reduced pressure, added with methanol / water, slurried at 25° C. for 5 hours, and filtered with suction to obtain the crude product of the compound of formula (6-3) (5.71 g of a white solid, molar yield: 97%). Note: The main components of the crude product of the compound of formula (6-3) were compounds (6-3) (6-3E and 6-3Z) and (6'-3) (6'-3E and 6'-3Z), and the ratio of compounds (6-3) / (6'-3) was ≒ 92 / 8.

[0076] 4. Preparation of crude product of compound of formula (6-4) [ka] Ethyl acetate (115 mL) and the crude product of the compound of formula (5) (7.65 g, 20 mmol) were added to a flask, and the mixture was stirred to dissolve transparently. DMAP (0.488 g, 4 mmol) and benzoic anhydride (13.5 g, 60 mmol) were added, and the mixture was reacted at 45°C for 4 hours. After the completion of the reaction was detected by TLC, the mixture was concentrated under reduced pressure, methanol / water was added, and the mixture was slurried at 25°C for 5 hours. The mixture was then suction filtered to obtain the crude product of the compound of formula (6-4) (9.34 g of a white solid, molar yield: 96%). Note: The main components of the crude product of the compound of formula (6-4) were compounds (6-4) (6-4E and 6-4Z) and (6'-4) (6'-4E and 6'-4Z), and the ratio of compounds (6-4) / (6'-4) was ≒ 92 / 8.

[0077] 5. Preparation of crude product of compound of formula (6-5) [ka] Add DCM (50 mL) and the crude product of the compound of formula (5) (5 g, 13.07 mmol) to a flask, stir to dissolve transparently, add triethylamine (2.64 g, 26.14 mmol) and DMAP (0.318 g, 2.61 mmol), add p-chlorobenzoyl chloride (3.4 g, 19.6 mmol) dropwise at 0°C, and dissolve in N 2 The reaction was allowed to proceed at 35°C for 12 hours while protecting the reaction solution with 100% NaHCO. After the completion of the reaction was detected by TLC, water (30 mL) was added to quench the reaction, the liquids were separated, and the organic phase was washed with saturated NaHCO. 3 The mixture was washed successively with aqueous solution, 2N dilute hydrochloric acid, water, and saturated NaCl, concentrated under reduced pressure, added with methanol / water, slurried at 25°C for 5 hours, and filtered with suction to obtain the crude product of the compound of formula (6-5) (6.4 g of a white solid, molar yield: 94.8%). Note: The main components of the crude product of the compound of formula (6-5) were compounds (6-5) (6-5E and 6-5Z) and (6'-5) (6'-5E and 6'-5Z), and the ratio of compounds (6-5) / (6'-5) was ≒ 92 / 8.

[0078] 6. Preparation of crude product of compound of formula (6-6) [ka] A flask was charged with DCM (100 mL) and the crude product of the compound of formula (5) (10 g, 26.14 mmol), which was dissolved by stirring until clear. TBSCl (9.85 g, 65.35 mmol) and imidazole (7.12 g, 104.56 mmol) were added, and the mixture was reacted at 25 ° C for 12 hours. After the completion of the reaction was detected by TLC, water (100 mL) was added, the mixture was stirred for 10 minutes, and the mixture was separated. The organic phase was washed with water and saturated NaCl, concentrated under reduced pressure, methanol / water was added, the mixture was slurried at 25 ° C for 3 hours, and the mixture was suction filtered to obtain the crude product of the compound of formula (6-6) (12.43 g of white solid, molar yield: 95%). Note: The main components of the crude product of the compound of formula (6-6) were compounds (6-6) (6-6E and 6-6Z) and (6'-6) (6'-6E and 6'-6Z), and the ratio of compounds (6-6) / (6'-6) was ≒ 92 / 8.

[0079] Example 6 Preparation of compound of formula (7) 1. Preparation of Compound of Formula (7-1) [ka] A flask was charged with ethyl acetate (75 mL), the crude product of the compound of formula (6-1) (5 g, 11.77 mmol), and stirred to dissolve transparently. Then, Raney Ni (5 g, wet weight, activated according to the standard procedure) was added and the mixture was cooled to 50° C. 2 (1 atm) at 35 ° C for 7 hours, and the end of the reaction was detected by gas chromatography. After removing Raney Ni by diatomaceous earth filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-1) (white solid, gas chromatography purity: 91.25%, see Figure 1), which was directly used in the purification step. The white solid was added to a mixed solution of methanol and ethyl acetate, heated to reflux until the solution became transparent, naturally cooled to 15 ° C, stirred for 5 hours, and suction filtered to obtain the compound of formula (7-1) (4.2 g of white solid, molar yield: 84%, gas chromatography purity: 99.40%, see Figure 2). 1 H NMR (600 MHz, CDCl 3) δ 5.40 (d, J = 4.9 Hz, 1H), 4.68 - 4.57 (m, 1H), 2.40 - 2.31 (m, 2H), 2.05 (s, 3H), 2.05 - 1.95 (m, 2H), 1.91 - 1.81 (m, 3H), 1.66 - 1.43 (m, 8H), 1.41 - 1.33 (m, 3H), 1.31 - 1.24 (m, 1H), 1.22 - 1.07 (m, 7H), 1.04 (s, 3H), 1.03 - 0.96 (m, 2H), 0.94 (d, J = 6.5 Hz, 3H), 0.89 (dd, J = 6.6, 2.8 Hz, 6H), 0.70 (s, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 170.55, 139.67, 122.66, 73.99, 56.69, 56.14, 50.04, 42.32, 39.74, 39.53, 38.13, 37.00, 36.60, 36.19, 35.80, 31.91, 31.87, 28.24, 28.02, 27.78, 24.29, 23.83, 22.83, 22.57, 21.45, 21.03, 19.32, 18.72, 11.86. Note: The main components of the crude product of the compound of formula (7-1) were compounds (7-1) and (7'-1), and the ratio thereof was compound (7-1) / (7'-1) ≈ 92 / 8.

[0080] 2. Preparation of Compound of Formula (7-2) [ka] A flask was charged with ethyl acetate (75 mL), the crude product of the compound of formula (6-2) (5 g, 11.4 mmol), and stirred to dissolve transparently. Then, Raney Ni (7.5 g, wet weight, activated according to the standard procedure) was added and H 2(1 atm) at 55 ° C for 7 hours, and the end of the reaction was detected by gas chromatography. After removing Raney Ni by diatomaceous earth filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-2) as a white solid, which was directly used in the purification step. The white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became transparent, naturally cooled to 15 ° C, stirred for 4 hours, and filtered by suction to obtain the compound of formula (7-2) (4.0 g of white solid, molar yield: 80%, gas chromatography purity: 98.35%, see Figure 3). 1 H NMR (600 MHz, CDCl 3 ) δ 5.39 (d, J = 4.9 Hz, 1H), 4.67 - 4.62 (m, 1H), 2.44 - 2.24 (m, 4H), 2.10 - 1.95 (m, 2H), 1.93 - 1.81 (m, 3H), 1.68 - 1.43 (m, 8H), 1.40 - 1.34 (m, 3H), 1.29-1.25 (m, 1H), 1.20-1.10 (m, 9H), 1.04 (s, 3H), 1.03 - 0.96 (m, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.89 (dd, J = 6.6, 2.8 Hz, 6H), 0.70 (s, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 173.96, 139.75, 122.59, 56.70, 56.14, 50.03, 42.32, 39.74, 39.53, 38.16, 37.01, 36.61, 36.19, 35.80, 31.91, 31.87, 28.24, 28.02, 27.94, 27.81, 24.29, 23.83, 22.83, 22.57, 21.04, 19.33, 18.72, 11.86, 9.19. Note: The main components of the crude product of the compound of formula (7-2) were compounds (7-2) and (7'-2), and the ratio thereof was compounds (7-2) / (7'-2) ≈ 92 / 8.

[0081] 3. Preparation of Compound of Formula (7-3) [ka] A flask was charged with ethyl acetate (75 mL), the crude product of the compound of formula (6-3) (5 g, 11 mmol), and stirred to dissolve transparently. Then, Raney Ni (5 g, wet weight, activated according to the standard procedure) was added and the mixture was cooled to 50° C. 2 (1 atm) at 35 ° C for 10 hours, and the end of the reaction was detected by gas chromatography. After removing Raney Ni by diatomaceous earth filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-3) as a white solid, which was directly used in the purification step. The white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became transparent, naturally cooled to 15 ° C, stirred for 5 hours, and filtered by suction to obtain the compound of formula (7-3) (4.1 g of white solid, molar yield: 82%, gas chromatography purity: 99.5%, see Figure 4). 1 H NMR (600 MHz, CDCl 3 ) δ 5.39 (d, J = 4.9 Hz, 1H), 4.74 - 4.58 (m, 1H), 2.33 (d, J = 7.1 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.09 - 1.95 (m, 2H), 1.93 - 1.80 (m, 3H), 1.68 - 1.65 (m, 2H), 1.65 - 1.41 (m, 7H), 1.41 - 1.31 (m, 3H), 1.31 - 1.24 (m, 1H), 1.23 - 1.06 (m, 7H), 1.04 (s, 3H), 1.03-0.99 (m, 2H), 0.99 - 0.92 (m, 7H), 0.89 (dd, J = 6.7, 2.7 Hz, 6H), 0.70 (s, 3H). 13 C NMR (151 MHz, CDCl 3) δ 173.14, 139.74, 122.59, 73.68, 56.70, 56.14, 50.03, 42.32, 39.74, 39.53, 38.18, 37.01, 36.60, 36.19, 35.80, 31.91, 31.87, 28.24, 28.02, 27.83, 24.29, 23.84, 22.83, 22.57, 21.04, 19.33, 18.72, 18.56, 13.65, 11.86. Note: The main components of the crude product of the compound of formula (7-3) were compounds (7-3) and (7'-3), and the ratio thereof was compounds (7-3) / (7'-3) ≒ 92 / 8.

[0082] 4. Preparation of Compound of Formula (7-4) [ka] In a flask, tetrahydrofuran (35 mL), ethyl acetate (35 mL), and the crude product of the compound of formula (6-4) (4.87 g, 10 mmol) were added and stirred to dissolve transparently. Then, Raney Ni (4.87 g, wet weight, activated according to the standard procedure) was added and the mixture was heated with H 2 (1 atm) at 35 ° C for 24 hours, and the end of the reaction was detected by gas chromatography. After removing Raney Ni by diatomaceous earth filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-4) as a white solid, which was directly used in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became transparent, naturally cooled to 15 ° C, stirred for 4 to 5 hours, and filtered by suction to obtain the compound of formula (7-4) (4.1 g of white solid, molar yield: 84.2%, gas chromatography purity: 98.08%, see Figure 5). 1 H NMR (600 MHz, CDCl 3) δ 8.11 - 7.99 (m, 2H), 7.59 - 7.51 (m, 1H), 7.43 (t, J = 7.8 Hz, 2H), 5.42 (dd, J = 5.1, 2.1 Hz, 1H), 4.89 - 4.84 (m, 1H), 2.47 (d, J = 8.0 Hz, 2H), 2.05-1.97 (m, 3H), 1.94 - 1.90 (m, 1H), 1.86-1.80 (m, 1H), 1.77 - 1.73 (m, 1H), 1.63 - 1.45 (m, 6H), 1.41 - 1.30 (m, 3H), 1.29 - 1.05 (m, 11H), 1.04 - 0.99 (m, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.87 (dd, J = 6.6, 2.8 Hz, 6H), 0.69 (s, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 166.01, 139.69, 132.72, 130.87, 129.55, 128.27, 122.80, 56.71, 56.15, 50.06, 42.34, 39.76, 39.54, 38.23, 37.05, 36.68, 36.20, 35.82, 31.95, 31.90, 28.25, 28.03, 27.90, 24.31, 23.85, 22.84, 22.58, 21.07, 19.40, 18.74, 11.88. Note: The main components of the crude product of the compound of formula (7-4) were compounds (7-4) and (7'-4), and the ratio of compounds (7-4) / (7'-4) was ≈92 / 8.

[0083] 5. Preparation of Compound of Formula (7-5) [ka] A flask was charged with tetrahydrofuran (39 mL), ethyl acetate (39 mL), and the crude product of the compound of formula (6-5) (5.16 g, 10 mmol). The mixture was stirred until it was dissolved and clear. Then, Raney Ni (10 g, wet weight, activated according to the standard procedure) was added and the mixture was heated with H 2(1 atm) at 30 ° C for 24 hours, and the end of the reaction was detected by gas chromatography. After removing Raney Ni by diatomaceous earth filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-5) as a white solid, which was directly used in the purification step. The white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became transparent, naturally cooled to 15 ° C, stirred for 4 hours, and filtered by suction to obtain the compound of formula (7-5) (4.23 g of white solid, molar yield: 82%, gas chromatography purity: 97.96%, see Figure 6). 1 H NMR (500 MHz, CDCl 3 ) δ 8.02 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 5.44 (d, J = 5.0 Hz, 1H), 4.88 - 4.82 (m, 1H), 3.88 (s, 3H), 2.47 (d, J = 7.9 Hz, 2H), 2.06 - 1.98 (m, 3H), 1.95 - 1.91 (m, 1H), 1.90 - 1.81 (m, 1H), 1.79 - 1.69 (m, 1H), 1.67 - 1.44 (m, 7H), 1.36 (d, J = 8.9 Hz, 3H), 1.30 - 1.10 (m, 7H), 1.09 (s, 3H), 1.07 - 0.99 (m, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.89 (dd, J = 6.6, 2.4 Hz, 6H), 0.71 (s, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 165.78, 163.20, 139.79, 131.55, 123.31, 122.69, 113.50, 60.41, 58.50, 56.71, 56.15, 55.42, 50.06, 42.33, 39.76, 39.53, 38.30, 37.07, 36.67, 36.20, 35.81, 31.95, 31.90, 28.25, 28.03, 27.95, 24.31, 23.84, 22.83, 22.58, 21.06, 19.40, 18.73, 18.46, 14.21, 11.87. Note: The main components of the crude product of the compound of formula (7-5) were compounds (7-5) and (7'-5), and the ratio thereof was compounds (7-5) / (7'-5) ≒ 92 / 8.

[0084] 6. Preparation of Compound of Formula (7-6) [ka] A flask was charged with ethyl acetate (75 mL), the crude product of the compound of formula (6-6) (5 g, 10 mmol), and stirred to dissolve transparently. Then, Raney Ni (5 g, wet weight, activated according to the standard procedure) was added and the mixture was cooled to 50° C. 2 (1 atm) at 35 ° C for 12 hours, and the end of the reaction was detected by gas chromatography. After removing Raney Ni by diatomaceous earth filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-6) as a white solid, which was directly used in the purification step. The white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became transparent, naturally cooled to 15 ° C, stirred for 4 hours, and filtered by suction to obtain the compound of formula (7-6) (4.05 g of white solid, molar yield: 81%, gas chromatography purity: 98.57%, see Figure 7). 1 H NMR (500 MHz, CDCl 3 ) δ 5.43 - 5.30 (m, 1H), 3.53 - 3.47 (m, 1H), 2.32 - 2.26 (m, 1H), 2.21 - 2.17 (m, 1H), 2.07 - 1.96 (m, 2H), 1.89 - 1.80 (m, 2H), 1.77 - 1.70 (m, 1H), 1.62 - 1.45 (m, 8H), 1.41 - 1.31 (m, 3H), 1.31 - 1.24 (m, 1H), 1.18 - 1.06 (m, 7H), 1.02 (s, 4H), 1.01 - 0.98 (m, 1H), 0.93 (d, J = 6.4 Hz, 3H), 0.91 (s, 9H), 0.89 (dd, J = 6.6, 2.4 Hz, 6H), 0.69 (s, 3H), 0.08 (s, 6H). 13C NMR (151 MHz, CDCl 3 ) δ 141.60, 121.19, 72.67, 56.82, 56.16, 50.23, 42.84, 42.34, 39.82, 39.53, 37.40, 36.61, 36.20, 35.79, 32.10, 31.96, 31.92, 28.25, 28.03, 25.96, 24.31, 23.82, 22.83, 22.57, 21.08, 19.44, 18.73, 18.28, 11.86. Note: The main components of the crude product of the compound of formula (7-6) were compounds (7-6) and (7'-6), and the ratio thereof was compounds (7-6) / (7'-6) ≒ 92 / 8.

[0085] Example 7 Production of cholesterol 1. Preparation of cholesterol by hydrolysis of the compound of formula (7-1) [ka] Add methanol (40 mL) and K 2 CO 3 (1.28 g, 12.1 mmol) was added and dissolved transparently, then N 2 Under protection, the compound of formula (7-1) (4g, 9.33mmol) was added, heated to 65℃ and reacted for 2 hours, and the completion of the reaction was detected by TLC, followed by cooling to 25℃, adding 2mol / L dilute hydrochloric acid to adjust the pH to 7-8, evaporating methanol under reduced pressure, adding water (20mL), stirring at 25℃ for 2 hours, suction filtering, adding water (20mL) to the cake, stirring at 25℃ for 2 hours, suction filtering, and drying to obtain purified cholesterol (CCDC 2099442, single crystal structure see Figure 8, 3.57g white solid, molar yield: 99.1%, gas chromatography purity: 99.10%, see Figure 9). mp: 147-149℃. 1 H NMR (600 MHz, CDCl 3) δ 5.36 - 5.34 (m, 1H), 3.55 - 3.49 (m, 1H), 2.35 - 2.19 (m, 2H), 2.06 - 1.92 (m, 2H), 1.86 - 1.79 (m, 3H), 1.68 - 1.19 (m, 14H), 1.19 - 1.03 (m, 7H), 1.01 (s, 3H), 0.98 - 0.93 (m, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.86 (dd, J = 6.6, 2.8 Hz, 6H), 0.68 (s, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 140.77, 121.73, 71.82, 56.78, 56.17, 50.14, 42.33, 42.32, 39.80, 39.53, 37.27, 36.52, 36.20, 35.80, 31.93, 31.92, 31.68, 28.25, 28.03, 24.31, 23.84, 22.84, 22.58, 21.10, 19.41, 18.73, 11.87.HRMS(ESI): calcd for C 27 H 46 NaO [M+Na] + ,409.3441, found 409.3121.

[0086] 2. Preparation of cholesterol by hydrolysis of the compound of formula (7-2) [ka] Add methanol (40 mL) and K 2 CO 3 (1.57 g, 11.4 mmol) was added and dissolved transparently, and then N 2Under protection, the compound of formula (7-2) (4g, 9mmol) was added, heated to 65°C and reacted for 2 hours, and the completion of the reaction was detected by TLC. After that, it was cooled to 25°C, and 2mol / L dilute hydrochloric acid was added to adjust the pH to 7-8, and methanol was evaporated under reduced pressure, water (20mL) was added, and the mixture was stirred at 25°C for 2 hours, suction filtered, and water (20mL) was added to the cake, and the mixture was stirred at 25°C for 2 hours, suction filtered, and dried to obtain purified cholesterol (3.46g white solid, molar yield: 98.8%, gas chromatography purity: 98.96%, see Figure 10).

[0087] 3. Preparation of cholesterol by hydrolysis of the compound of formula (7-3) [ka] Add methanol (40 mL) and K 2 CO 3 (1.57 g, 11.4 mmol) was added and dissolved transparently, and then N 2 Under protection, the compound of formula (7-3) (4g, 8.76mmol) was added, heated to 65°C and reacted for 2 hours, and the completion of the reaction was detected by TLC. After that, it was cooled to 25°C, and 2mol / L dilute hydrochloric acid was added to adjust the pH to 7-8, and methanol was evaporated under reduced pressure, water (20mL) was added, and the mixture was stirred at 25°C for 2 hours, suction filtered, and water (20mL) was added to the cake, and the mixture was stirred at 25°C for 2 hours, suction filtered, and dried to obtain purified cholesterol (3.33g white solid, molar yield: 98.5%, gas chromatography purity: 99.41%, see FIG. 11).

[0088] 4. Preparation of cholesterol by hydrolysis of the compound of formula (7-4) [ka] Methanol (25 mL) and KOH (0.31 g, 5.5 mmol) were added to the flask, and after dissolving, the mixture was diluted with N 2Under protection, the compound of formula (7-4) (2.45g, 5mmol) was added, and the mixture was reacted at 45°C for 6 hours. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, and 2mol / L dilute hydrochloric acid was added to adjust the pH to 5-6. Methanol was evaporated under reduced pressure, water (20mL) was added, and the mixture was stirred at 25°C for 2 hours, and then filtered by suction. 80% ethanol (10ml) was further added to the cake, and the mixture was stirred at 25°C for 2 hours, and then filtered by suction. The mixture was dried to obtain purified cholesterol (1.83g of white solid, molar yield: 94.8%, gas chromatography purity: 99.44%, see FIG. 12).

[0089] 5. Preparation of cholesterol by hydrolysis of the compound of formula (7-5) [ka] Methanol (25 mL) and KOH (0.31 g, 5.5 mmol) were added to the flask, and after dissolving, the mixture was diluted with N 2 Under protection, the compound of formula (7-5) (2.6g, 5mmol) was added, and the mixture was reacted at 45°C for 8 hours. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, and 2mol / L dilute hydrochloric acid was added to adjust the pH to 5-6. Methanol was evaporated under reduced pressure, water (20mL) was added, and the mixture was stirred at 25°C for 2 hours, and then filtered by suction. 80% ethanol (10ml) was further added to the cake, and the mixture was stirred at 25°C for 2 hours, and then filtered by suction and dried to obtain purified cholesterol (1.8g of white solid, molar yield: 93.3%, gas chromatography purity: 98.69%, see FIG. 13).

[0090] 6. Preparation of cholesterol by deprotection of the compound of formula (7-6) [ka] In a flask, tetrahydrofuran (25 mL) and the compound of formula (7-6) (2.5 g, 5 mmol) were added and dissolved until clear. 2 Under protection, TBAF・3H 2O (7.88 g, 25 mmol) was added and reacted at 25° C. for 24 hours. After the completion of the reaction was detected by TLC, the reaction was quenched by adding saturated ammonium chloride, tetrahydrofuran was evaporated under reduced pressure, dichloromethane (70 mL) and water (50 mL) were added for extraction, and the organic phase was separated and washed with saturated NaHCO 3 The mixture was washed successively with an aqueous solution, 2N dilute hydrochloric acid, water, and saturated NaCl, and concentrated under reduced pressure to obtain crude cholesterol. The cake was added with 80% ethanol (10 ml), slurried at 25° C. for 3 hours, and filtered with suction to obtain purified cholesterol (1.84 g of white solid, molar yield: 95.3%, gas chromatography purity: 98.6%, see FIG. 14 ).

[0091] The impurity structures that may be introduced during the reaction process of obtaining the compound of formula (7-1) by Raney Ni hydrogenation reduction of the compound of formula (6-1) are the compounds of formulae (8-1), (8-2), (8-3) and (8-4). [ka]

[0092] In the crude gas chromatogram of the compound of formula (7-1) obtained in Example 6 of the present invention (see FIG. 1), the retention times of the compounds of formulae (8-1), (8-2), (8-3) and (8-4) correspond to any one of 9.123 minutes (0.11%), 9.318 minutes (0.42%), 9.538 minutes (0.16%) and 10.324 minutes (0.39%).

[0093] The crude product of the compound of formula (7-1) of the present invention was purified by one recrystallization, and the purity increased from 91.25% to 99.40% (see FIG. 2 for gas chromatogram), during which the 3α-OR by-product was basically removed, and the content of impurities corresponding to the corresponding retention times was also reduced, respectively 9.168 min (0.19%), 9.382 min (0.12%), 9.576 min (0.05%), and 10.366 min (0.17%), and the molar yield of this recrystallization operation method was 84%. The gas chromatographic purity of cholesterol obtained by alkaline hydrolysis was 99.10% (see FIG. 9), which was basically consistent with the purity of the compound of formula (7-1).

[0094] Comparative Example 1 As reported in a patent document (Background Art Scheme 2, CN105218610A), stigmasterol decomposition products were used as raw materials to synthesize cholesterol through a five-step reaction with a total molar yield of 67%. The reaction of the first step in the technical scheme of this patent document is as shown in the following reaction scheme 1. [ka]

[0095] In reaction scheme 1 described in patent document CN105218610A, the compound of formula (02) after BA oxidation was used as the raw material, and ethanol was used as the solvent. Under the action of p-toluenesulfonic acid and triethyl orthoformate, the compound of formula (03) was obtained (molar yield: 97.50%) by heating to 40°C and maintaining for 4 hours.

[0096] According to the experimental method provided by the above patent document, the present invention uses ethanol as a solvent and the compound of formula (02) as a substrate, and reacts under the catalysis of p-toluenesulfonic acid and triethyl orthoformate at 40°C for 4 hours. After the raw materials are completely reacted as detected by TLC, the post-treatment is carried out according to the method of patent document (CN105218610A), and the compound of formula (03') (as shown in reaction formula 2) is obtained, but it does not match the compound described in the patent document. In the present invention, the amount of triethyl orthoformate is also reduced, but after the raw materials are completely reacted as detected by TLC, the result of reaction formula 1 described in patent document (CN105218610A) is not obtained, and the result of reaction formula 3 below is obtained. This indicates that when the carbonyl group at position 3 of the compound of formula (02) is protected according to the method reported in patent document CN105218610A, the aldehyde group at position C-22 is preferentially protected to form an acetal, thereby obtaining the compounds of formulae (03') and (03'') shown in reaction scheme 2 or reaction scheme 3, but not the compound of formula (03) described in patent document (CN105218610A). Apparently, the compounds of formulae (03') and (03'') shown in reaction scheme 2 or formula 3 cannot undergo the subsequent Wittig reaction. [ka]

[0097] Experimental method: Ethanol (4ml), triethyl orthoformate (2ml), compound of formula (02) (2.00g, 70.20mmol) and p-toluenesulfonic acid (20mg, 0.12mmol) were added to a flask, and the flask was kept at 40℃ for 4 hours to react. The raw materials were completely reacted as detected by TLC. Sodium acetate (20mg) was added under ice bath, and the cake was washed with water until the eluate became neutral, then drained and purified by column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain compound of formula (03') (2.30g colorless oil, molar yield: 88%). 1 H NMR (600 MHz, CDCl 3) δ 5.23 - 5.19 (m 1H), 5.10 (d, J = 1.9 Hz, 1H), 4.30 (d, J = 2.4 Hz, 1H), 3.81 - 3.70 (m, 4H), 3.61 - 3.55 (m, 1H), 3.49 - 3.41 (m, 2H), 2.31 - 2.25 (m, 1H), 2.18 - 1.96 (m, 4H), 1.85 - 1.78 (m, 2H), 1.71 - 1.52 (m, 7H), 1.43 - 1.33 (m, 4H), 1.29 (d, J = 7.0 Hz, 3H), 1.22 - 1.18 (m, 6H), 0.99 (d, J = 6.7 Hz, 3H), 0.96 (s, 3H), 0.70 (s, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 154.52, 141.06, 118.03, 106.07, 99.04, 64.60, 63.35, 62.14, 56.48, 51.88, 48.33, 42.59, 40.39, 39.62, 35.18, 33.86, 31.92, 31.86, 27.72, 25.55, 24.42, 21.17, 18.96, 15.49, 15.37, 14.68, 11.94, 11.89. HRMS(ESI): calcd for C 28 H 46 NaO 3 [M+Na] + ,453.3339, found 453.3328.

[0098] Experimental method: Ethanol (4ml), triethyl orthoformate (1ml), compound of formula (02) (2.00g, 70.20mmol) and p-toluenesulfonic acid (20mg, 0.12mmol) were added to a flask, and the flask was kept at 40℃ for 4 hours to react. The raw materials were completely reacted as detected by TLC. Sodium acetate (20mg) was added under ice bath, and the cake was washed with water until the eluate became neutral, then drained and purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound of formula (03'') (2.32g colorless oil, molar yield: 95%).1 H NMR (600 MHz, CDCl3) δ 5.69 (d, J = 1.8 Hz, 1H), 4.27 (d, J = 2.4 Hz, 1H), 3.78 - 3.72 (m, 1H), 3.58-3.53 (m, 1H), 3.47 - 3.40 (m, 2H), 2.42 - 2.21 (m, 4H), 2.01 - 1.97 (m, 2H), 1.84 - 1.77 (m, 2H), 1.69 - 1.58 (m, 3H), 1.53 - 1.47 (m, 2H), 1.41 - 1.27 (m, 3H), 1.21 - 1.17 (m, 6H), 1.16 (d, J = 4.1 Hz, 4H), 1.13 - 1.08 (m, 1H), 1.04 - 0.98 (m, 2H), 0.96 (d, J = 6.8 Hz, 3H), 0.93 - 0.86 (m, 1H), 0.69 (s, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 199.58, 171.53, 123.76, 105.95, 64.58, 63.40, 55.39, 53.81, 51.81, 42.52, 40.33, 39.42, 35.69, 35.63, 33.97, 32.92, 32.03, 27.62, 24.37, 21.01, 17.37, 15.48, 15.36, 11.91, 11.86.

[0099] Comparative Example 2 The second step Wittig reaction reported in the patent document (CN105218610A) is shown in the following reaction scheme 4. [ka]

[0100] In this patent document, toluene was used as a solvent, triphenylphosphine and 1-chloro-3-methylbutane were added and refluxed for 2 hours, potassium tert-butoxide and the compound of formula (03) were added and refluxed for 4 hours to obtain the compound of formula (04), with a molar yield of 90.29%. For example, the present invention adopts the method of patent document (CN105218610A) (Comparative Example 2, Reaction Scheme 4), and uses 1-chloro-3-methylbutane or 1-bromo-3-methylbutane to carry out the Wittig reaction of the compound of formula (2) of the present invention, and the reaction result is shown in Reaction Scheme 5, which shows that the compound of formula (3') was obtained, but the expected compound of formula (3) was not obtained. This shows that the method disclosed in patent document (CN105218610A) cannot be applied to the present invention. [ka]

[0101] Experimental method: Triphenylphosphine (797mg, 3.04mmol), 1-chloro-3-methylbutane (324mg, 3.04mmol), toluene (10mL) were added to a flask, and the mixture was refluxed for 2 hours. The mixture was then cooled to 25℃, and potassium tert-butoxide (307mg, 2.74.02mmol) was added in three portions under an ice bath, and the mixture was stirred for 0.5 hours under an ice bath. The compound of formula (2) (500mg, 1.52mmol) was then added, and the mixture was refluxed for 4 hours. After detecting the completion of the reaction by TLC, the reaction mixture was cooled to 25°C, and 2M HCl (4mL) was added to adjust the solution to neutrality. The mixture was extracted with dichloromethane (50mL) and water (50mL). The organic phase was washed with water and saturated saline solution in sequence, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE:EA=20:1) to obtain the compound of formula (3') (516mg of colorless oil, molar yield: 85%). 1 H NMR (600 MHz, CDCl 3) δ 9.53 (d, J = 5.0 Hz, 1H), 2.72 - 2.68 (m, 1H), 2.39 - 2.25 (m, 6H), 2.10 (dd, J = 14.4, 4.8 Hz, 1H), 1.94 - 1.86 (m, 3H), 1.68 - 1.53 (m, 8H), 1.43 - 1.30 (m, 4H), 1.25 (s, 3H), 1.15 (s, 3H), 1.04 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.6 Hz, 6H), 0.72 (s, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 205.71, 198.54, 163.30, 133.32, 55.49, 54.31, 51.82, 48.85, 42.21, 39.05, 38.94, 38.40, 35.35, 35.16, 33.95, 32.12, 29.71, 28.43, 27.31, 26.49, 23.74, 23.21, 22.54, 20.74, 17.80, 13.60, 12.97. HRMS(ESI): calcd for C 27 H 42 NaO 2 [M+Na] + ,421.3077, found 421.3075.

[0102] Triphenylphosphine (797 mg, 3.04 mmol), 1-bromo-3-methylbutane (459 mg, 3.04 mmol), and 10 mL of toluene were added to the flask, and the mixture was refluxed for 2 hours. The mixture was then cooled to 25°C, and potassium tert-butoxide (307 mg, 2.74 mmol) was added in batches under an ice bath, and the mixture was stirred for 0.5 hours under an ice bath. The compound of formula (2) (500 mg, 1.52 mmol) was then added, and the mixture was refluxed for 2.5 hours. After detecting the completion of the reaction by TLC, the reaction mixture was cooled to 25°C, and 2M HCl (4mL) was added to adjust the solution to neutrality. The mixture was extracted with dichloromethane (50mL) and water (50mL). The organic phase was washed with water and saturated saline solution in sequence, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE:EA=20:1) to obtain the compound of formula (3') (534mg of colorless oil, molar yield: 88%).

[0103] Comparative Example 3 In the present invention, the 3-position ester group of the compound of formula (4) is converted to NaBH 4 The compound of formula (5) was then reduced with Raney Ni / H to obtain the compound of formula (5), which was then subjected to hydroxy protection, selective hydrogenation reduction, deprotection or hydrolysis to obtain cholesterol. 2 Selective hydrogenation of the double bonds of the side chains was then carried out using NaBH 4 The reaction results, as shown in reaction scheme 6, gave the compounds of formula (6-1) and (6'-1), but did not give the expected compound of formula (9). This was due to the fact that the compound was reduced to 3-position by using Raney Ni / H 2 This shows that when is used as a reducing agent, the side chain cannot be selectively hydrogenated and reduced. Therefore, the reaction sequence of the present invention cannot be changed. [ka]

[0104] Experimental procedure: In a flask, add the compound of formula (4) (2.10 g, 17.2 mmol), Raney Ni (4.20 g, wet weight), isopropanol (30 mL), and H 2(1 atm), and reacted at 30°C for 11 hours. After detecting the completion of the reaction by TLC, Raney Ni was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a mixture of formula (6-1) and formula (6'-1). 1 Judging from the results of 1 H NMR, the ratio of the compound of formula (6-1) to the compound of isomeric formula (6'-1) was 1:0.28.

[0105] Comparative Example 4 In the present invention, the crude product of the compound of formula (6-1) is treated with Raney Ni / H 2 The crude product of the compound of formula (7-1) was synthesized by reduction with 2,4-dichloromethane (2,4-dichloromethane), as shown in the following reaction scheme 7. [ka]

[0106] In the present invention, the reducing agent is H 2 By using Raney Ni as a catalyst, the double bond of the side chain was selectively hydrogenated to obtain the crude product of the compound of formula (7-1). The content of the impurity compound of formula (8-4), which is an over-reduced product, was low (0.39%), and it was easy to remove it by purification. 2 When the double bond of the side chain was selectively hydrogenated using 10% Pd / C as a catalyst, the crude product of the compound of formula (7-1) contained the impurity compound of formula (8-4), which was an over-reduced product, at a level exceeding 3.4% (see FIG. 15), which was difficult to remove by purification and failed to achieve the intended purpose. This indicates that 10% Pd / C has a relatively high catalytic activity but low selectivity, and cannot replace Raney Ni as a catalyst for selective hydrogenation of the side chain.

[0107] Experimental method: Add the compound of formula (6-1) (1.50 g, 3.56 mmol), 10% Pd / C (150 mg), and ethyl acetate (30 mL) to a flask and hydrate with H 2The reaction was carried out at 1 atm and 30°C for 9 hours, and the completion of the reaction was detected by gas chromatography. After that, Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure. According to the gas chromatogram of the product, the content of the compound of formula (7-1) was 91.3%, the content of the compound of formula (8-4), which is an impurity that is an over-reduced product, was 3.4%, and the total content of intermediate impurities 1, 2, and 3 was 2%.

[0108] Comparative Example 5 In patent document CN113248557A, it is as shown in the following reaction scheme 8. The compound of formula (4) is reduced with sodium borohydride and purified by column chromatography to obtain the compound of formula (5) (3β-OH). The compound of formula (5) is subjected to selective hydrogenation with Raney Ni catalyst to obtain crude cholesterol. The crude cholesterol is then purified once by column chromatography or recrystallization to obtain purified cholesterol, with a gas chromatographic purity of 95-96%. [ka]

[0109] In the present invention, the cholesterol obtained by one-time recrystallization purification, deprotection or hydrolysis of the compound of formula (7) can reach a purity of 99.0% or more, and has high purity and good purification yield, and has important application value.

[0110] The protection of the present invention is not limited to the above embodiments. All modifications and advantages that can be imagined by those skilled in the art without departing from the spirit and scope of the concept of the present invention are included in the present invention and are protected by the appended claims.

Claims

1. A method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one BA as a raw material, comprising: The method uses BA as a raw material and synthesizes cholesterol through steps such as oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation reduction, deprotection, or hydrolysis. Specifically, the method includes: (a) oxidizing BA represented by formula (1) in a first solvent to obtain a compound of formula (2); (b) subjecting the compound of formula (2) to a Wittig reaction in a second solvent to obtain a compound of formula (3); (c) acetylating the compound of formula (3) in a third solvent to obtain a compound of formula (4); (d) reducing the compound of formula (4) in a fourth solvent to obtain a compound of formula (5); (e) subjecting the compound of formula (5) to a hydroxyl protection reaction in a fifth solvent to obtain a compound of formula (6); (f) selectively reducing the compound of formula (6) by hydrogenolysis in a sixth solvent to obtain a compound of formula (7); (g) deprotecting or hydrolyzing the compound of formula (7) in a seventh solvent to obtain cholesterol; Including, The method for synthesizing cholesterol, wherein the reaction process of the method is as shown in scheme (A). 【Chemistry 66】 (wherein R is selected from an ester group and a silyl ether group).

2. The ester group is a C2-C10 linear ester group, an isobutyl ester 【Chemistry 67】 Isopentyl ester group 【Chemistry 68】 Phenyl ester group 【Chemistry 69】 p-Methoxyphenyl ester group 【Chemistry 70】 one or more selected from The silyl ether group is a trimethylsilyl ether group. 【Chemistry 71】 and tert-butyldimethylsilyl ether group 【Chemical 72】 The method according to claim 1, characterized in that the one or two selected from the following are selected from the following:

3. The method according to claim 1, characterized in that in step (a), the oxidation reaction is specifically a reaction in which BA represented by formula (1) is oxidized with TEMPO, sodium bicarbonate, tetrabutylammonium bromide and an oxidizing agent in the first solvent to obtain a compound of formula (2).

4. The method according to claim 3, characterized in that the molar ratio of BA represented by the formula (1), TEMPO, sodium hydrogen carbonate, tetrabutylammonium bromide, and the oxidizing agent is 1:(0-1):(0-20):(0-1):(1-5), and / or the oxidizing agent is one or more selected from N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), and 2-iodoxybenzoic acid (IBX), and / or the first solvent is one or more selected from dichloromethane, tetrahydrofuran, toluene, dimethylsulfoxide, and water, and / or the temperature of the oxidation reaction is 0-30° C., and / or the time of the oxidation reaction is 3-8 hours.

5. The method according to claim 1, characterized in that in step (b), the Wittig reaction is specifically a reaction in which the compound of formula (2), 3,3-dimethylallyl halide, triphenylphosphine, and potassium tert-butoxide are subjected to a Wittig reaction in the second solvent to obtain the compound of formula (3).

6. The method according to claim 5, wherein the molar ratio of the compound of formula (2), 3,3-dimethylallyl halide, triphenylphosphine, and potassium tert-butoxide is 1:(1-4):(1-4):(1-4), and / or the second solvent is one or more selected from toluene, benzene, tetrahydrofuran, and heptane, and / or the 3,3-dimethylallyl halide is one or two selected from 3,3-dimethylallyl chloride and 3,3-dimethylallyl bromide, and / or the temperature of the Wittig reaction is −10 to 112° C., and / or the time of the Wittig reaction is 0.5 to 9 hours.

7. The method according to claim 1, characterized in that in step (c), the acetylation reaction is specifically a reaction in which the compound of formula (3), acetyl chloride, acetic anhydride, and a base are acetylated in a third solvent to obtain a compound of formula (4).

8. The method according to claim 7, characterized in that the molar ratio of the compound of formula (3), acetyl chloride, acetic anhydride and base is 1:(0.5-62.5):(1-62.5):(0-6), and / or the base is one or more selected from pyridine, triethylamine, DIPEA, DMAP and diisopropylamine, and / or the third solvent is one or more selected from acetic anhydride, acetyl chloride, ethyl acetate and dichloromethane, and / or the temperature of the acetylation reaction is 40-110° C., and / or the time of the acetylation reaction is 1-10 hours.

9. The method according to claim 1, characterized in that in step (d), the reduction reaction is specifically a reaction in which the compound of formula (4) is reduced with a reducing agent in the fourth solvent to obtain a compound of formula (5).

10. The molar ratio of the compound of formula (4) to the reducing agent is 1:(1-25); and / or the fourth solvent is one or more selected from tetrahydrofuran, ethanol, water, dichloromethane, 2-methyltetrahydrofuran, isopropyl alcohol, acetic acid, and methyl tert-butyl ether; and / or the reducing agent is NaBH ４ and K.B.H. ４ and / or the temperature of the reduction reaction is 0 to 50° C.; and / or the time of the reduction reaction is 6 to 12 hours.

11. The method according to claim 1, characterized in that in step (e), the hydroxyl protection reaction is specifically a reaction in which the compound of formula (5) is subjected to a condensation reaction with a hydroxyl protection reagent in the fifth solvent under the action of a base to obtain a compound of formula (6).

12. when R is an ester group, the fifth solvent is one or more selected from ethyl acetate, dichloromethane, chloroform, DMF, toluene, tetrahydrofuran and 2-methyltetrahydrofuran, and / or the base is one or more selected from triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, and / or the molar ratio of the compound of formula (5), the hydroxyl protecting reagent and the base is 1:(1-4):(0.05-5), and / or the temperature of the reaction is 0-50° C., and / or the time of the hydroxyl protecting reaction is 2-24 hours, 12. The method of claim 11, wherein when R is a silyl ether group, the fifth solvent is one or more of DMF, dichloromethane, chloroform, and carbon tetrachloride; and / or the base is one or more selected from triethylamine, diisopropylethylamine, imidazole, pyridine, and DMAP; the molar ratio of the compound of formula (5), the hydroxyl protecting reagent, and the base is 1:(2-4):(4-8); and / or the temperature of the reaction is 0-50° C.; and / or the time of the hydroxyl protecting reaction is 2-24 hours.

13. The method according to claim 1, characterized in that in step (f), the selective hydrogenation reduction reaction is specifically a reaction in which the compound of formula (6) is subjected to a selective hydrogenation reduction reaction with a reducing agent in the sixth solvent under the action of a catalyst to obtain a compound of formula (7).

14. The reducing agent is H ２ and / or the catalyst is Raney Ni, and / or the mass ratio of the compound of formula (6) to the catalyst is 1:(0.05-5), and / or the sixth solvent is one or more selected from 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, and isopropyl alcohol, and / or the temperature of the hydrogenation reduction reaction is 0-60° C., and / or the reducing agent H in the hydrogenation reduction reaction is ２ and / or the hydrogenation reduction reaction time is 4 to 48 hours.

15. The method according to claim 1, characterized in that, in step (g), when R is an ester group, the hydrolysis reaction is specifically a reaction of hydrolyzing the compound of formula (7) in a seventh solvent under the action of a base to obtain cholesterol.

16. The base is LiOH, KOH, NaOH, t-BuOK, K ２ CO ３ and / or the molar ratio of the compound of formula (7) to the base is 1:(0.5-2); and / or the seventh solvent is one or more selected from methanol and ethanol; and / or the temperature of the hydrolysis reaction is 10-75° C.; and / or the time of the hydrolysis reaction is 0.3-12 hours.

17. The method according to claim 1, characterized in that, in the case where R is a silyl ether group, in step (g), the deprotection reaction is specifically a reaction in which the compound of formula (7) is deprotected in the seventh solvent under the action of a catalyst to obtain cholesterol.

18. The catalyst is tetrabutylammonium fluoride (TBAF), tetrabutylammonium fluoride trihydrate (TBAF.3H) ２ and / or the molar ratio of the compound of formula (7) to the catalyst is 1:(1-6); and / or the seventh solvent is one or more selected from tetrahydrofuran and water; and / or the temperature of the deprotection reaction is 10-75° C.; and / or the time of the deprotection reaction is 2-48 hours.

19. The compound has a structure represented by the formula (6-2E), (6-3E), (6-4E), (6-5E), (6-6E), (6-2Z), (6-3Z), (6-4Z), (6-5Z), (6-6Z), (6'-2E), (6'-3E), (6'-4E), (6'-5E), (6'-6E), (6'-2Z), (6'-3Z), (6'-4Z), (6'-5Z), or (6'-6Z). 【Chemical 73】

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