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 not only plays a role in the formation of cell membranes, but also serves as a raw material for the synthesis of bile acids and steroid hormones in the body. Currently, commercial cholesterol is primarily used for the following purposes: 1. Pharmaceutical excipients - liposome additives, such as excipients for novel coronavirus mRNA vaccines (adding 30-50% cholesterol during liposome production can significantly increase the drug-loading capacity and stability of liposomes); 2. Cosmetic additives; 3. Raw material for liquid crystal production; and 4. Starting material for vitamin D3 production. Most commercially available cholesterol is derived from animal brainstem (brainstem cholesterol) or lanolin (lanolin cholesterol), both of which are animal-derived cholesterol. Research has revealed that animal-derived products are likely to contain animal pathogens and other harmful factors. The safety of cholesterol has come under scrutiny, particularly following the outbreaks of infectious diseases such as mad cow disease, Streptococcus suis, and avian influenza. For example, cholesterol is an upstream raw material for vitamin D3 production, and Europe and the United States have long banned the use of brainstem cholesterol as a raw material due to the risk of epidemics. China also restricted the use of brainstem cholesterol as a raw material from July 1, 2020. Therefore, to ensure people's health and safety, it is urgent to develop a method for synthesizing high-purity cholesterol from plants.

[0003] The chemical synthesis of cholesterol has been reported to mainly involve the following methods: (1) Using diosgenin as a raw material, cholesterol was synthesized through a six-step reaction 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 cost of the raw materials, the complicated procedures, and the high toxicity and polluting nature of the reagents used. [ka]

[0004] (2) Using stigmasterol degradation products as raw materials, cholesterol was synthesized through a five-step reaction with a total molar yield of 67% (CN105218610A, as shown in Scheme 2). However, the applicant of the present invention discovered through experiments that this scheme had the following problems: When triethyl orthoformate was used in the first step of the reaction to etherify the carbonyl group at the C-3 position, the selectivity was low, which easily led to the reaction of the aldehyde in the side chain, resulting in the formation of an acetal (see "Comparative Example 1" in the description 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 requires expensive noble metal rhodium catalysts and chiral phosphine ligands, making it unsuitable for large-scale industrial production. [ka]

[0006] (4) Using pregnenolone as a raw material, cholesterol was synthesized through a two-step reaction with an overall molar yield of 80% (CN104961788A, as shown in Scheme 4). This scheme also requires expensive noble metal rhodium catalysts and chiral phosphine ligands, making it unsuitable 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, O3 was used in the synthesis process, which increased the demands on reaction monitoring and equipment, making it less economical and safer. [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). This scheme also uses O3, which increases the process difficulty and increases the demands on reaction monitoring and equipment, resulting in insufficient economics 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 process used in this scheme involves column chromatography, which is not suitable for industrial production. Furthermore, the purity of the cholesterol obtained in this scheme is low (95%–96%), making it 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, Streptococcus suis, and avian influenza. Previously reported chemical synthesis schemes for cholesterol have drawbacks, including complicated procedures, high contamination levels, expensive catalysts, and low product purity. Therefore, the development of a safer and more efficient method for synthesizing high-purity cholesterol using plant-derived raw materials is of great value. Summary of the Invention

[0011] To address the shortcomings of existing technologies, the present invention provides a method for synthesizing high-purity plant-derived cholesterol. The present invention uses plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnoral alcohol or BA, as a starting material. The cholesterol is synthesized through steps such as oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation reduction, deprotection, and hydrolysis, resulting in a purity of 99% or more. The BA used as the starting material for cholesterol synthesis in the present invention is safe and economical, and the method for synthesizing cholesterol is simple, has high yields, 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. Its current annual production volume has reached 1,000 tons, its price is low, and it can fully avoid the risk of pathogenic bacterial and viral infections that can be present in animal-derived cholesterol in 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: (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 hydrogenating the compound of formula (6) 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] wherein R is selected from an ester group and a silyl ether group; Preferably, R is a C2-C10 straight chain ester group, 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 one or more selected from More preferably, R is an ethyl ester. [ka] Propyl ester [ka] Butyl ester [ka] Isobutyl ester [ka] Isopentyl ester [ka] Phenyl ester [ka] p-Methoxyphenyl ester [ka] Trimethylsilyl ether group [ka] and tert-butyldimethylsilyl ether group [ka] One or more selected from:

[0016] Note: In the compound formulas (3) to (6), the double bond between the C-22 and C-23 positions is predominantly E-configuration and predominantly Z-configuration, with the ratio of E / Z being approximately 87 / 13 ( 1 1 H NMR), and this ratio does not change without subsequent purification procedures. 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 bicarbonate, 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 preferably N-chlorosuccinimide (NCS).

[0019] Here, the first solvent is one or more selected from dichloromethane, tetrahydrofuran, toluene, dimethyl sulfoxide, 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, preferably 6 hours.

[0020] In a particular embodiment, the synthesis step of the compound of formula (2) comprises dissolving the BA of formula (1) in a first solvent, followed by 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., and 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, 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 preferably diisopropylamine. Here, the third solvent is one or more of acetic anhydride, acetyl chloride, ethyl acetate, dichloromethane, etc., and preferably a mixed solvent of acetyl chloride and acetic anhydride. The temperature for the acetylation reaction is 40 to 110°C, and preferably 70°C. 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, thereby obtaining 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), 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 one or more of NaBH4 and KBH4, etc., and is preferably NaBH4. The temperature of the reduction reaction is 0 to 50°C, and preferably 25°C. The reduction reaction time is 6 to 12 hours, preferably 8 hours.

[0029] In one particular embodiment, the step of synthesizing the compound of formula (5) comprises 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 (e.g., ethyl ester [ka] Propyl ester [ka] Butyl ester [ka] etc.), isobutyl ester [ka] Isopentyl ester [ka] Phenyl ester [ka] and p-methoxyphenyl ester [ka] and the like, preferably an ethyl ester.

[0032] Here, the molar ratio of the compound of formula (5), the protecting reagent for the ester group, and the base is 1:(1 to 4):(0.05 to 5), 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 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, preferably 45°C. Here, the reaction time is 2 to 24 hours, 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 the compound of formula (5) is reacted with a protecting reagent for the 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] is.

[0035] Here, the molar ratio of the compound of formula (5), the protecting reagent for the silyl ether group, and the base is 1:(2 to 4):(4 to 8), preferably 1:2.5:4. wherein the fifth solvent is one or more of DMF, dichloromethane, chloroform, carbon tetrachloride, etc., and 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. The reaction time is 2 to 24 hours, 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 it 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 the compound of formula (7). Here, the catalyst is Raney Ni. Here, the reducing agent is H2. Here, the mass ratio of the compound of formula (6) to the catalyst Raney Ni is 1:(0.05 to 5), 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 pressure of the reducing agent H2 in the hydrogenation reduction reaction 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 a particular embodiment, the step of synthesizing cholesterol includes dissolving the compound of formula (6) in a sixth solvent, adding Raney Ni, replacing with H, and then selectively reducing by hydrogenation to obtain the 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 one or more selected from LiOH, KOH, NaOH, t-BuOK, K2CO3, etc., and is preferably K2CO3. Here, the molar ratio of the compound of the formula (7) to the base is 1:(0.5 to 2), 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. 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 one or more selected from tetrabutylammonium fluoride (TBAF), tetrabutylammonium fluoride trihydrate (TBAF·3H2O), boron trifluoride etherate, and a solution of acetic acid and hydrogen chloride in ethyl acetate, and is preferably TBAF·3H2O. Here, the molar ratio of the compound of the formula (7) to the catalyst is 1:(1 to 6), 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, preferably 25°C. The deprotection reaction time is 2 to 48 hours, preferably 24 hours.

[0041] In one 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 performing 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), (6'-6Z). [ka]

[0043] The beneficial effects of the present invention include the following: In the method for producing high-purity cholesterol of the present invention, the commercial raw material BA used is a plant-derived raw material, thereby avoiding the risk of infection by pathogenic bacteria and viruses that may be present in animal-derived raw materials, and is inexpensive and easily available. In addition, the obtained cholesterol is highly pure (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 for achieving industrial production of high-purity cholesterol. This solves the problems of existing cholesterol products, such as low safety, low purity, high synthesis costs, and environmental unfriendliness. [Brief explanation 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. [Figure 2]FIG. 2 shows a gas chromatogram of the purified compound of formula (7-1) obtained in Example 6 of the present invention. [Figure 3] FIG. 3 shows a gas chromatogram of the purified 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. [Figure 5] FIG. 5 shows a gas chromatogram of the purified 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 the gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-3). [Figure 12] FIG. 12 shows the gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-4). [Figure 13] FIG. 13 shows a gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-5). [Figure 14] FIG. 14 shows the 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) under a 10% Pd / C catalyst. DETAILED DESCRIPTION OF THE INVENTION

[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 content specifically described below, and the content thereof is 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 Guoyang Chemical Reagent Company, the purification of the products was mainly carried out by slurrying and column chromatography, and 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 starting material, and the reaction process is as shown in Scheme (A) above. Note that the method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one (BA) as a starting material further includes producing other by-products, and the specific reaction scheme is as shown in Scheme (A'). [ka]

[0048] wherein R is selected from an ester group and a silyl ether group; Note: In the compound formulas (3) to (6), the double bond between the C-22 and C-23 positions is predominantly E-configuration and predominantly Z-configuration, with the ratio of E / Z being approximately 87 / 13 ( 1 The ratio of compound (5) (3β-OH) / (5') (3α-OH) is approximately 92 / 8 (determined by H NMR). 1 1 H NMR), and the ratios of compounds of formula (6) / (6') and (7) / (7') do not change unless a subsequent purification procedure is performed.

[0049] In the present invention, the compounds of formula (3), formula (4), formula (5), formula (5'), formula (6), and formula (6') have cis-trans (E and Z) configurations of the D-ring side chain double bonds (double bonds at C-22 and C-23) introduced by the Wittig reaction, with the ratio of the two configurations being E / Z ≈ 87 / 13, and this ratio will not change without subsequent purification procedures. Compounds of formula (5) (3β-OH) and (5') (3α-OH) can be obtained by reducing compound of formula (4) with NaBH4 or KBH4. The 3β-OH / 3α-OH ratio in the resulting compounds of formula (5) (3β-OH) and (5') (3α-OH) is approximately 92 / 8, and this ratio will not change without subsequent purification procedures. 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] This example demonstrates the preparation of compound of formula (2) under different experimental conditions.

[0051] (1) BA (24.79 g, 0.075 mol), TEMPO (118 mg, 0.750 mmol), dichloromethane (200 mL), sodium bicarbonate (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 reacted 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 to 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%). 1H NMR (400 MHz, CDCl3) 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, CDCl3) 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 NaO2 [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 added to a flask in this order, 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 to 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 of formula (1) BA (24.79 g, 75 mmol), IBX (42 g, 150 mmol), THF (250 mL), and DMSO (200 mL) were sequentially added to a flask and reacted at 25° C. for 5 hours. After detecting the completion of the reaction by TLC, water was added, the mixture was suction filtered, and the filtrate was concentrated under reduced pressure. Dichloromethane (400 mL) and water (300 mL) were added for extraction. 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 a white solid, molar yield: 93.5%).

[0054] Example 2 Preparation of the compound of formula (3) [ka] This example demonstrates the preparation of compound of formula (3) under different experimental conditions.

[0055] (1) Triphenylphosphine (23.97 g, 91.40 mmol), 3,3-dimethylallylbromide (13.62 g, 91.40 mmol), and 230 mL of toluene were added to a flask and stirred at 80 °C for 30 minutes. After heating and refluxing for 2 hours, the mixture was cooled to 25 °C. In an ice bath, potassium tert-butoxide (10.26 g, 91.40 mmol) was added and stirred for 0.5 hours. Then, the compound of formula (2) (23.09 g, 70.30 mmol) was added in batches and reacted at 10 °C for 0.5 hours. After detecting the completion of the reaction by TLC, water (23 mL) was added to quench the reaction. 2 M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (120 mL × 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 a 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) remains essentially unchanged, and the double bond between the C-22 and C-23 positions is predominantly E-configuration and predominantly Z-configuration (3E / 3Z ≒ 87 / 13). The cis-trans isomers of the double bond between the C-22 and C-23 positions in the compound of formula (6) are reduced with Raney nickel and then 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) A flask was charged with triphenylphosphine (23.97 g, 91.40 mmol), 3,3-dimethylallylbromide (13.62 g, 91.40 mmol), and 230 mL of n-heptane. The mixture was heated to reflux for 3 hours, then cooled to 25 °C. In an ice bath, potassium tert-butoxide (10.26 g, 91.40 mmol) was added and stirred for 0.5 hours. Then, the compound of formula (2) (23.00 g, 70.07 mmol) 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, quenched by adding water (23 mL), and the pH was adjusted to 6-7 by adding 2 M HCl. The organic phase was washed with water (120 mL × 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 a white solid, molar yield: 91.94%).

[0058] (3) Triphenylphosphine (23.97 g, 91.40 mmol), 3,3-dimethylallylbromide (13.62 g, 91.40 mmol), and 230 mL of toluene were added to a flask and stirred at 80 °C for 30 minutes. After heating and refluxing for 2 hours, the mixture was cooled to 25 °C, and potassium tert-butoxide (11.27 g, 100.5 mmol) was added in an ice bath and stirred for 40 minutes. Then, the compound of formula (2) (23.09 g, 70.30 mmol) was added in batches and reacted at 10 °C for 2 hours. After detecting the completion of the reaction by TLC, water (23 mL) was added to quench the reaction. 2 M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (120 mL × 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 a white solid, molar yield: 92.5%).

[0059] (4) A flask was charged with triphenylphosphine (23.97 g, 91.40 mmol), 3,3-dimethylallylbromide (13.62 g, 91.40 mmol), and 230 mL of toluene. The mixture was refluxed for 2 hours, cooled to 25°C, and potassium tert-butoxide (9.23 g, 82.26 mmol) was added in an ice bath. The mixture was stirred for 0.5 hours, and then the compound of formula (2) (15.00 g, 45.70 mmol) was added. The mixture was refluxed for 2.5 hours. After completion of the reaction was confirmed by TLC, the mixture was cooled to 25°C and quenched with water (23 mL). The pH was adjusted to 6-7 with 2 M HCl. The organic phase was washed with water (100 mL × 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 a white solid, molar yield: 96.20%).

[0060] (5) A flask was charged with triphenylphosphine (23.97 g, 91.40 mmol), 3,3-dimethylallyl chloride (9.56 g, 91.40 mmol), and 230 mL of toluene. The mixture was refluxed for 10 hours, cooled to 25°C, and potassium tert-butoxide (9.23 g, 82.26 mmol) was added in an ice bath. The mixture was stirred for 0.5 hours, and then the compound of formula (2) (7.5 g, 22.85 mmol) was added and refluxed for 4.5 hours. After completion of the reaction was confirmed by TLC, the mixture was cooled to 25°C and quenched by adding water (23 mL). The pH was adjusted to 6-7 by adding 2 M HCl. The organic phase was washed with water (100 mL × 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 a white solid, molar yield: 72.04%).

[0061] Example 3 Preparation of the compound of formula (4) [ka] This example demonstrates the preparation of compound of formula (4) under different experimental conditions.

[0062] (1) The compound of formula (3) (15 g, 39.44 mmol), acetic anhydride (16.09 g, 157.76 mmol) and acetyl chloride (3.08 g, 39.44 mmol) were added to a flask and reacted at 70 ° C for 6 hours. After the completion of the reaction was detected by TLC, the mixture was cooled to 25 ° C, concentrated under reduced pressure, slightly cooled, pyridine (2 g) and methanol (75 mL) were added, stirred to disperse, concentrated under reduced pressure, slightly cooled, methanol (75 mL) 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 (24 mL), suction filtered and dried to obtain the compound of formula (4) (4E and 4Z, 15.50 g of white solid, molar yield: 93.03%). HRMS (ESI): calcd for C 29 H 42 NaO2 [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 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, and slightly cooled. The reaction solution was added to ice water (200 mL), stirred for 15 minutes, filtered, and the water was drained off. 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 a 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 slightly cooled. The reaction solution was added to ice water (300 mL), stirred for 15 minutes, filtered, and the water was drained off. The resulting solid was added to methanol (60 mL) and pyridine (3 mL), and the mixture was 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 compounds of formula (4) (4E and 4Z, 14.99 g of a white solid, molar yield: 89.97%).

[0065] (4) A flask was charged with 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), 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, and slightly cooled. The reaction solution was added to ice water (300 mL), stirred for 15 minutes, filtered, and the water was drained off. The resulting solid was added to methanol (60 mL) and pyridine (3 mL), and the mixture was 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 compounds 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 demonstrates the preparation of crude compound of formula (5) under different experimental conditions.

[0067] (1) Compound (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. The reaction was allowed to proceed at 30°C for 8 hours. After completion of the reaction was confirmed by TLC, the pH of the reaction solution was adjusted to 7-8 with 6 M hydrochloric acid. A large amount of solid precipitated in the reaction solution. The solution was filtered, and the filtrate was removed and the solvent was evaporated under reduced pressure. A large amount of solid precipitated in the reaction solution. The solution was extracted with dichloromethane (100 mL) and water (50 mL), and the organic phase was washed with water (100 mL). The mixture was stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain the crude product of compound (5) (8.71 g, pale yellow solid, molar yield: 98.09%), which was used directly in the next step of the 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 essentially the same as that in this example, and therefore will not be shown hereinafter.

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

[0070] (3) Compound (4) (9.80 g, 23.21 mmol) and a mixed solvent of dichloromethane, methanol, and water (95 mL, V / V / V = 18 / 9 / 1) were added to a flask, and sodium borohydride (1.76 g, 46.44 mmol) was added in batches. The reaction was allowed to proceed at 25 °C for 6 hours. After confirming the completion of the reaction by TLC, 3 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. The reaction solution was filtered, and the filtrate was removed and the solvent was evaporated under reduced pressure. A large amount of solid precipitated in the reaction solution. The solution was extracted with dichloromethane (100 mL) and water (50 mL). The organic phase was washed with water (100 mL), stirred, and the layers were separated. The solvent was evaporated under reduced pressure to obtain the crude product of compound (5) (8.77 g, pale yellow solid, molar yield: 98.76%), which was directly used in the next step of the reaction.

[0071] (4) Compound (4) (9.80 g, 23.21 mmol) and a mixed solvent of tetrahydrofuran, ethanol, and water (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. The reaction was allowed to proceed at 30 °C for 8 hours. After the completion of the reaction was confirmed 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. The solution was filtered, and the filtrate was removed and the solvent was evaporated under reduced pressure. A large amount of solid precipitated in the reaction solution. The solution was extracted with dichloromethane (100 mL) and water (50 mL). The organic phase was washed with water (100 mL), stirred, and the layers were separated. The solvent was evaporated under reduced pressure to obtain the crude product of compound (5) (8.80 g, pale yellow solid, molar yield: 99.1%), which was directly used in the next step of the 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, the ratio of compound 5(3β-OH) / 5′(3α-OH) was 85 / 15.

[0072] Example 5 Preparation of crude product of compound of formula (6) 1. Preparation of crude 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 stirred to dissolve the compound. DMAP (0.245 g, 2 mmol) and acetic anhydride (6.13 g, 60 mmol) were added and the reaction was carried out 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. Ethyl acetate (76 mL) was added for extraction. The organic phase was washed with water and saturated brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give the crude product of the compound of formula (6-1) (8.3 g of a white solid, molar yield: 97.8%).

[0073] Note: The main components of the crude product of compound (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 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 stirred to dissolve it transparently. DMAP (0.159 g, 1.3 mmol) and propionic anhydride (5.07 g, 39 mmol) were added and the reaction was carried out 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 brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, added methanol / water, slurried at 25 °C for 5 hours, and filtered by 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 compound (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 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 stirred to dissolve it transparently. DMAP (0.159 g, 1.3 mmol) and butyric anhydride (6.2 g, 39 mmol) were added and the reaction was carried out 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 brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, added methanol / water, slurried at 25 °C for 5 hours, and filtered by 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 compound (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 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 stirred to dissolve it transparently. DMAP (0.488 g, 4 mmol) and benzoic anhydride (13.5 g, 60 mmol) were added and 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 compound (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 compound of formula (6-5) [ka] A flask was charged with DCM (50 mL) and the crude product of the compound of formula (5) (5 g, 13.07 mmol), and the mixture was stirred to dissolve the compound. Triethylamine (2.64 g, 26.14 mmol) and DMAP (0.318 g, 2.61 mmol) were added, and p-chlorobenzoyl chloride (3.4 g, 19.6 mmol) was slowly added dropwise at 0 °C. The mixture was then reacted at 35 °C for 12 hours under N protection. After the completion of the reaction was confirmed by TLC, water (30 mL) was added to quench the reaction. The organic phase was washed successively with saturated aqueous NaHCO3, 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 by 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 compound (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 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), and the mixture was stirred to dissolve the compound until it was 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 confirmed by TLC, water (100 mL) was added, the mixture was stirred for 10 minutes, and the layers were separated. The organic phase was washed with water and saturated NaCl, concentrated under reduced pressure, and methanol / water was added. The mixture was slurried at 25°C for 3 hours and then suction filtered to obtain the crude product of the compound of formula (6-6) (12.43 g of a white solid, molar yield: 95%). Note: The main components of the crude product of compound (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) and the crude product of formula (6-1) (5 g, 11.77 mmol). The mixture was stirred to dissolve the compound, followed by the addition of Raney Ni (5 g, wet weight, activated according to standard procedures) and the reaction mixture was stirred at 35 °C under 1 atm H2 for 7 hours. The reaction was monitored for completion by gas chromatography. The Raney Ni was removed by diatomaceous earth filtration, and the filtrate was concentrated under reduced pressure to give the crude product of formula (7-1) (white solid, GC purity: 91.25%, see Figure 1). This was used directly for purification. The white solid was added to a mixture of methanol and ethyl acetate and refluxed until the solution became clear. The mixture was allowed to cool to 15 °C, stirred for 5 hours, and then filtered with suction to give the compound of formula (7-1) (4.2 g, white solid, molar yield: 84%, GC purity: 99.40%, see Figure 2). 1 H NMR (600 MHz, CDCl3) 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). 13C NMR (151 MHz, CDCl3) 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 of compounds (7-1) / (7'-1) was approximately 92 / 8.

[0080] 2. Preparation of Compound of Formula (7-2) [ka] Ethyl acetate (75 mL) and the crude product of formula (6-2) (5 g, 11.4 mmol) were added to a flask and stirred to dissolve the compound. Raney Ni (7.5 g, wet weight, activated according to standard procedures) was then added and the reaction mixture was stirred at 55°C under 1 atm H2 for 7 hours. The reaction was monitored for completion by gas chromatography. The Raney Ni was removed by filtration with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give the crude product of formula (7-2) as a white solid, which was used directly for purification. This white solid was added to a mixture of methanol and ethyl acetate and heated to reflux until the solution became clear. The mixture was allowed to cool to 15°C, stirred for 4 hours, and then filtered with suction to give formula (7-2) (4.0 g of a white solid, molar yield: 80%, gas chromatography purity: 98.35%, see Figure 3). 1H NMR (600 MHz, CDCl3) 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, CDCl3) 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 of compounds (7-2) / (7'-2) was approximately 92 / 8.

[0081] 3. Preparation of Compound of Formula (7-3) [ka] A flask was charged with ethyl acetate (75 mL) and the crude product of formula (6-3) (5 g, 11 mmol). The mixture was stirred to form a clear solution. Then, Raney Ni (5 g, wet weight, activated according to standard procedures) was added and the reaction mixture was stirred at 35 °C under 1 atm H2 for 10 hours. The reaction was monitored for completion by gas chromatography. The Raney Ni was removed by filtration with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give the crude product of formula (7-3) as a white solid, which was used directly for purification. This white solid was added to a mixture of methanol and ethyl acetate, heated to reflux until the solution became clear, allowed to cool to 15 °C, stirred for 5 hours, and then filtered with suction to give formula (7-3) (4.1 g, white solid, molar yield: 82%, gas chromatography purity: 99.5%, see Figure 4). 1 H NMR (600 MHz, CDCl3) 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, CDCl3) 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 of compounds (7-3) / (7'-3) was approximately 92 / 8.

[0082] 4. Preparation of Compound of Formula (7-4) [ka] A flask was charged with tetrahydrofuran (35 mL) and ethyl acetate (35 mL), and the crude product of formula (6-4) (4.87 g, 10 mmol) was dissolved by stirring. Raney Ni (4.87 g, wet weight, activated according to standard procedures) was then added and reacted at 35 °C under H2 (1 atm) for 24 h. The reaction was monitored for completion by gas chromatography. After the Raney Ni was removed by filtration with diatomaceous earth, the filtrate was concentrated under reduced pressure to give the crude product of formula (7-4) as a white solid, which was used directly for purification. This white solid was added to a mixture of methanol and ethyl acetate, heated to reflux until the solution became clear, allowed to cool to 15 °C, stirred for 4–5 h, and then filtered with suction to give formula (7-4) (4.1 g, white solid, molar yield: 84.2%, gas chromatography purity: 98.08%, see Figure 5). 1 H NMR (600 MHz, CDCl3) 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). 13C NMR (151 MHz, CDCl3) 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 approximately 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 formula (6-5) (5.16 g, 10 mmol) and stirred to dissolve. Then, Raney Ni (10 g, wet weight, activated according to standard procedures) was added and reacted at 30 °C under 1 atm H2 for 24 hours. The reaction was monitored for completion by gas chromatography. The Raney Ni was removed by filtration with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give the crude product of formula (7-5) as a white solid, which was used directly for purification. This white solid was added to a mixture of methanol and ethyl acetate, heated to reflux until the solution became clear, allowed to cool to 15 °C, stirred for 4 hours, and then filtered with suction to give formula (7-5) (4.23 g, white solid, molar yield: 82%, gas chromatography purity: 97.96%, see Figure 6). 1H NMR (500 MHz, CDCl3) 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, CDCl3) 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 of compounds (7-5) / (7'-5) was approximately 92 / 8.

[0084] 6. Preparation of Compound of Formula (7-6) [ka] A flask was charged with ethyl acetate (75 mL) and the crude product of formula (6-6) (5 g, 10 mmol). The mixture was stirred to form a clear solution. Then, Raney Ni (5 g, wet weight, activated according to standard procedures) was added and the reaction mixture was stirred at 35 °C under 1 atm H2 for 12 hours. The reaction was monitored for completion by gas chromatography. The Raney Ni was removed by filtration with diatomaceous earth, and the filtrate was concentrated under reduced pressure to give the crude product of formula (7-6) as a white solid, which was used directly for purification. This white solid was added to a mixture of methanol and ethyl acetate, heated to reflux until the solution became clear, allowed to cool to 15 °C, stirred for 4 hours, and then filtered with suction to give formula (7-6) (4.05 g of a white solid, molar yield: 81%, gas chromatography purity: 98.57%, see Figure 7). 1 H NMR (500 MHz, CDCl3) 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). 13 C NMR (151 MHz, CDCl3) 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 of compounds (7-6) / (7'-6) was approximately 92 / 8.

[0085] Example 7 Production of cholesterol 1. Preparation of cholesterol by hydrolysis of the compound of formula (7-1) [ka] Methanol (40 mL) and K2CO3 (1.28 g, 12.1 mmol) were added to a flask and dissolved to a clear solution. Under N2 protection, compound (7-1) (4 g, 9.33 mmol) was added and heated to 65 °C for 2 hours. After completion of the reaction was confirmed by TLC, the mixture was cooled to 25 °C, and 2 mol / L dilute hydrochloric acid was added to adjust the pH to 7-8. The methanol was evaporated under reduced pressure, water (20 mL) was added, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered. The cake was added to water (20 mL), stirred at 25 °C for 2 hours, suction filtered, and dried to obtain purified cholesterol (CCDC 2099442, single crystal structure shown in Figure 8, 3.57 g white solid, molar yield: 99.1%, gas chromatography purity: 99.10%, see Figure 9). mp: 147-149 °C. 1 H NMR (600 MHz, CDCl3) 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). 13C NMR (150 MHz, CDCl3) 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] Methanol (40 mL) and K2CO3 (1.57 g, 11.4 mmol) were added to a flask and dissolved to a clear solution. Then, under N2 protection, compound (7-2) (4 g, 9 mmol) was added and heated to 65 °C for 2 hours. After completion of the reaction was confirmed by TLC, the mixture was cooled to 25 °C, and 2 mol / L dilute hydrochloric acid was added to adjust the pH to 7-8. The methanol was evaporated under reduced pressure, water (20 mL) was added, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered. The cake was added to water (20 mL), stirred at 25 °C for 2 hours, suction filtered, and dried to obtain purified cholesterol (3.46 g of a 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] Methanol (40 mL) and K2CO3 (1.57 g, 11.4 mmol) were added to a flask and dissolved to a clear solution. Then, under N2 protection, compound (7-3) (4 g, 8.76 mmol) was added and heated to 65 °C for 2 hours. After completion of the reaction was confirmed by TLC, the mixture was cooled to 25 °C, and 2 mol / L dilute hydrochloric acid was added to adjust the pH to 7-8. The methanol was evaporated under reduced pressure, water (20 mL) was added, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered. The cake was added to water (20 mL), stirred at 25 °C for 2 hours, suction filtered, and dried to obtain purified cholesterol (3.33 g of a white solid, molar yield: 98.5%, gas chromatography purity: 99.41%, see Figure 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 a flask and dissolved to a clear solution. Under N2 protection, the compound of formula (7-4) (2.45 g, 5 mmol) was added and reacted at 45 °C for 6 hours. After completion of the reaction was confirmed by TLC, the mixture was cooled to 25 °C, and 2 mol / L dilute hydrochloric acid was added to adjust the pH to 5-6. The methanol was evaporated under reduced pressure, water (20 mL) was added, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered. 80% ethanol (10 mL) was added to the cake, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered and dried to obtain purified cholesterol (1.83 g of a white solid, molar yield: 94.8%, gas chromatography purity: 99.44%, see Figure 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 a flask and dissolved to a clear solution. Under N2 protection, the compound of formula (7-5) (2.6 g, 5 mmol) was added and reacted at 45 °C for 8 hours. After the completion of the reaction was confirmed by TLC, the mixture was cooled to 25 °C, and 2 mol / L dilute hydrochloric acid was added to adjust the pH to 5-6. The methanol was evaporated under reduced pressure, water (20 mL) was added, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered. 80% ethanol (10 mL) was added to the cake, and the mixture was stirred at 25 °C for 2 hours. The mixture was then suction filtered and dried to obtain purified cholesterol (1.8 g of a white solid, molar yield: 93.3%, gas chromatography purity: 98.69%, see Figure 13).

[0090] 6. Preparation of cholesterol by deprotection of the compound of formula (7-6) [ka] Tetrahydrofuran (25 mL) and the compound of formula (7-6) (2.5 g, 5 mmol) were added to a flask and dissolved to form a clear solution. TBAF·3H2O (7.88 g, 25 mmol) was added under N2 protection and the reaction mixture was allowed to react at 25 °C for 24 hours. After completion of the reaction was confirmed by TLC, the mixture was quenched with saturated ammonium chloride. The tetrahydrofuran was evaporated under reduced pressure, and the mixture was extracted with dichloromethane (70 mL) and water (50 mL). The organic phase was washed sequentially with saturated aqueous NaHCO3, 2 N dilute hydrochloric acid, water, and saturated NaCl, and concentrated under reduced pressure to obtain crude cholesterol. The cake was added to 80% ethanol (10 mL), slurried at 25 °C for 3 hours, and then suction filtered to obtain purified cholesterol (1.84 g of a white solid, molar yield: 95.3%, GC purity: 98.6%, see Figure 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 formulas (8-1), (8-2), (8-3) and (8-4). [ka]

[0092] In the gas chromatogram of the crude product 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 a single recrystallization, and its purity increased from 91.25% to 99.40% (see Figure 2 for the gas chromatogram). The 3α-OR by-product was essentially eliminated, and the corresponding impurity contents decreased, with retention times of 9.168 min (0.19%), 9.382 min (0.12%), 9.576 min (0.05%), and 10.366 min (0.17%), respectively. The molar yield of this recrystallization procedure was 84%. The gas chromatographic purity of cholesterol obtained by alkaline hydrolysis was 99.10% (see Figure 9), which was essentially consistent with the purity of the compound of formula (7-1).

[0094] Comparative Example 1 As reported in a patent document (Scheme 2 of the Background Art, CN105218610A), cholesterol was synthesized using a stigmasterol degradation product as a raw material 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 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 used ethanol as a solvent and the compound of formula (02) as a substrate, and reacted it under the catalysis of p-toluenesulfonic acid and triethyl orthoformate at 40°C for 4 hours. After the complete reaction of the raw materials was detected by TLC, the workup was carried out according to the method of patent document (CN105218610A). A compound of formula (03') (shown in Reaction Scheme 2) was obtained, but it did not match the compound described in the patent document. In the present invention, an attempt was made to reduce the amount of triethyl orthoformate, but after the complete reaction of the raw materials was detected by TLC, the result of Reaction Scheme 1 described in patent document (CN105218610A) was not obtained, and instead the result of Reaction Scheme 3 below was obtained. This indicates that when the carbonyl group at position 3 of 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 compounds of formula (03') and (03'') shown in Reaction Scheme 2 or 3, but not the compound of formula (03) described in patent document (CN105218610A). Apparently, the compounds of formula (03') and (03'') shown in Reaction Scheme 2 or 3 cannot undergo the subsequent Wittig reaction. [ka]

[0097] Experimental method: Ethanol (4 ml), triethyl orthoformate (2 ml), the compound of formula (02) (2.00 g, 70.20 mmol), and p-toluenesulfonic acid (20 mg, 0.12 mmol) were added to a flask and reacted at 40°C for 4 hours. TLC confirmed that the raw materials had completely reacted. Sodium acetate (20 mg) was added in an ice bath, and the cake was washed with water until the eluate became neutral. The cake was then drained and purified by column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the compound of formula (03') (2.30 g, colorless oil, molar yield: 88%). 1H NMR (600 MHz, CDCl3) 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, CDCl3) 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; 28 H 46 NaO3 [M+Na] + ,453.3339, found 453.3328.

[0098] Experimental method: Ethanol (4 ml), triethyl orthoformate (1 ml), the compound of formula (02) (2.00 g, 70.20 mmol), and p-toluenesulfonic acid (20 mg, 0.12 mmol) were added to a flask and reacted at 40°C for 4 hours. TLC confirmed that the raw materials had completely reacted. Sodium acetate (20 mg) was added in an ice bath, and the cake was washed with water until the eluate became neutral. The cake was then drained and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain the compound of formula (03'') (2.32 g, 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, CDCl3) 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, the Wittig reaction, reported in the patent document (CN105218610A) is shown in the following reaction scheme 4. [ka]

[0100] In this patent, toluene was used as a solvent, triphenylphosphine and 1-chloro-3-methylbutane were added, and the mixture was refluxed for 2 hours. Potassium tert-butoxide and the compound of formula (03) were then 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 employs the method of patent document (CN105218610A) (Comparative Example 2, Reaction Scheme 4) to perform a Wittig reaction on the compound of formula (2) of the present invention using 1-chloro-3-methylbutane or 1-bromo-3-methylbutane. As shown in Reaction Scheme 5, the compound of formula (3') was obtained, but the expected compound of formula (3) was not obtained. This indicates that the method disclosed in patent document (CN105218610A) is not applicable to the present invention. [ka]

[0101] Experimental method: Triphenylphosphine (797 mg, 3.04 mmol), 1-chloro-3-methylbutane (324 mg, 3.04 mmol), and toluene (10 mL) were added to a flask and refluxed for 2 hours. The mixture was then cooled to 25°C, and potassium tert-butoxide (307 mg, 2.744.02 mmol) was added in three portions in an ice bath. The mixture was stirred for 0.5 hours in an ice bath, and then the compound of formula (2) (500 mg, 1.52 mmol) was added and refluxed for 4 hours. After detecting the completion of the reaction by TLC, the mixture was cooled to 25°C, and 2M HCl (4 mL) was added to adjust the solution to neutrality. The mixture was extracted with dichloromethane (50 mL) and water (50 mL). The organic phase was washed with water and saturated brine, 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') (516 mg of a colorless oil, molar yield: 85%). 1H NMR (600 MHz, CDCl3) 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, CDCl3) 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 NaO2[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 in an ice bath. The mixture was stirred for 0.5 hours in an ice bath, and then the compound of formula (2) (500 mg, 1.52 mmol) 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 2M HCl (4 mL) was added to adjust the solution to neutrality. The mixture was extracted with dichloromethane (50 mL) and water (50 mL). The organic phase was washed with water and saturated brine, 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') (534 mg of a colorless oil, molar yield: 88%).

[0103] Comparative Example 3 In the present invention, the 3-position ester group of compound (4) was reduced with NaBH4 to obtain compound (5), which was then subjected to hydroxy protection, selective hydrogenation reduction, deprotection, or hydrolysis to obtain cholesterol. In another study, we first performed selective hydrogenation reduction of the double bond in the side chain using Raney Ni / H2 as a reducing agent, followed by reduction of the 3-position ester group using NaBH4. As shown in Reaction Scheme 6, the compounds of formulas (6-1) and (6'-1) were obtained, but the expected compound of formula (9) was not. This indicates that selective hydrogenation reduction of the side chain is not possible when Raney Ni / H2 is used as a reducing agent. Therefore, the reaction sequence of the present invention cannot be changed. [ka]

[0104] Experimental method: A flask was charged with the compound of formula (4) (2.10 g, 17.2 mmol), Raney Ni (4.20 g, wet weight), isopropanol (30 mL), and H2 (1 atm), and the mixture was reacted at 30°C for 11 hours. After the completion of the reaction was detected by TLC, the 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 isomer 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) was reduced with Raney Ni / H2 to synthesize the crude product of the compound of formula (7-1), as shown in the following reaction scheme 7. [ka]

[0106] In the present invention, the side chain double bond was selectively hydrogenated using H2 as the reducing agent and Raney Ni as the catalyst. The crude product of the compound of formula (7-1) obtained by hydrogenation contained a low content (0.39%) of the over-reduced impurity compound of formula (8-4), which was easy to remove by purification. Furthermore, when the side chain double bond was selectively hydrogenated using H2 as the reducing agent and 10% Pd / C as the catalyst, the crude product of the compound of formula (7-1) contained the over-reduced impurity compound of formula (8-4), at a content exceeding 3.4% (see Figure 15), which was difficult to remove by purification and failed to achieve the intended purpose. This indicates that 10% Pd / C has relatively high catalytic activity but low selectivity, and therefore cannot replace Raney Ni as a catalyst for selective side chain hydrogenation.

[0107] Experimental method: Compound (6-1) (1.50 g, 3.56 mmol), 10% Pd / C (150 mg), and ethyl acetate (30 mL) were added to a flask and reacted at 30°C under H2 (1 atm) for 9 hours. After completion of the reaction was confirmed by gas chromatography, the Pd / C was removed by filtration and the filtrate was concentrated under reduced pressure. The gas chromatogram of the product indicated that the content of compound (7-1) was 91.3%, the content of the over-reduced impurity compound (8-4) 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) was reduced with sodium borohydride and purified by column chromatography to obtain the compound of formula (5) (3β-OH). The compound of formula (5) was subjected to selective hydrogenation with a Raney Ni catalyst to obtain crude cholesterol. The crude cholesterol was 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 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, making it of great application value.

[0110] The protection content of the present invention is not limited to the above examples. All modifications and advantages that can be conceived 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 covered by the scope of the accompanying 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, and specifically includes 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) subjecting the compound of formula (5) to a hydroxyl protection reaction in a fifth solvent to obtain a compound of formula (6); (f) selectively hydrogenating the compound of formula (6) 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). 【Hua 66】 (wherein R—O 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 【Chemical 67】 isopentyl ester group 【Chemistry 68】 Phenyl ester group 【Chemical Formula 69】 p-methoxyphenyl ester group 【Chemistry 70】 is selected from The silyl ether group is a trimethylsilyl ether group. 【Chemical 71】 and tert-butyldimethylsilyl ether group 【Chemical 72】 2. The method of claim 1, wherein the compound is selected from the group consisting of:

3. 2. The method according to claim 1, wherein 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, wherein the molar ratio of BA represented by the formula (1), TEMPO, sodium bicarbonate, 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, dimethyl sulfoxide, 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, wherein 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, wherein 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 the compound of formula (4).

8. The method according to claim 7, wherein 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. 2. The method according to claim 1, wherein 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) and a hydroxy protecting reagent are subjected to a condensation reaction in the fifth solvent under the action of a base to obtain a compound of formula (6).

12. when R—O 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, and 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 reaction temperature is 0-50° C., and / or the time for the hydroxyl protecting reaction is 2-24 hours, 12. The method of claim 11, wherein when R—O 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 hydroxy-protecting reagent, and the base is 1:(2-4):(4-8), and / or the reaction temperature is 0-50° C., and / or the hydroxy-protecting reaction time is 2-24 hours.

13. 2. The method according to claim 1, wherein 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, wherein, when R—O is an ester group, in step (g), 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 may be 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 to 2), and / or the seventh solvent is one or two selected from methanol and ethanol, and / or the temperature of the hydrolysis reaction is 10 to 75°C, and / or the time of the hydrolysis reaction is 0.3 to 12 hours.

17. The method according to claim 1, wherein, when R—O 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 18. The method of claim 17, wherein the seventh solvent is one or more selected from the group consisting of tetrahydrofuran and water, boron trifluoride etherate, acetic acid, and hydrogen chloride in ethyl acetate, and / or the molar ratio of the compound of formula (7) to the catalyst is 1:(1 to 6), and / or the seventh solvent is one or two selected from tetrahydrofuran and water, and / or the temperature of the deprotection reaction is 10 to 75°C, and / or the time of the deprotection reaction is 2 to 48 hours.

19. The structure of the compound is as shown in 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), (6'-6Z). 【Chemical 73】

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