Method for synthesizing high-purity plant-derived cholesterol

Abstract

The present invention discloses a method for synthesizing high-purity plant-derived cholesterol, which uses plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnoral alcohol or BA, as raw material, and can synthesize the above cholesterol through steps such as oxidation, Wittig reaction, acetylation, reduction, selective hydrogenation reduction, or can synthesize 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 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. At present, 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 preparing liposomes can significantly increase the drug-carrying capacity and stability of liposomes); 2. Cosmetic additives; 3. Raw materials for the production of liquid crystals; 4. Starting materials for the production of vitamin D3. At present, 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 five-step reaction with a total molar yield of 67% (CN105218610A, 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 reaction of the patent to etherify and protect the carbonyl group at the C-3 position, the selectivity was low, and the reaction of the aldehyde in the side chain was easily caused, 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, 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, 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 the raw material, cholesterol was synthesized through a five-step reaction with a total molar yield of 68% (CN105237603A, shown in Scheme 5). In this scheme, O3 was used in the synthesis process, which increases the requirements for reaction monitoring and equipment, making the economy and safety insufficient. [ka]

[0008] (6) Using stigmasterol as the raw material, cholesterol was synthesized through a four-step reaction with a total molar yield of 70% (CN106632565A, shown in Scheme 6). This scheme also uses O3, which increases the difficulty of the process and places high demands on reaction monitoring and equipment, making it less economical and safe. [ka]

[0009] 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. DISCLOSURE OF THEINVENTION

[0010] 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 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 raw material, and synthesizes the above cholesterol through steps such as oxidation, Wittig reaction, acetylation, reduction, selective hydrogenation reduction, or 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.

[0011] 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.

[0012] 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.

[0013] The two methods for synthesizing cholesterol using BA raw materials provided by the present invention include the following steps. Method 1: (a) oxidizing a 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) selectively reducing the compound of formula (5) by hydrogenolysis in a fifth solvent to obtain cholesterol. Method 2: (a) oxidizing a 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); (f) hydroxy-protecting the compound of formula (5) to obtain a compound of formula (6) in a sixth solvent; (g) selectively reducing the compound of formula (6) by hydrogenolysis in a seventh solvent to obtain a compound of formula (7); (h) A step of deprotecting or hydrolyzing the compound of formula (7) in an eighth solvent to obtain cholesterol.

[0014] 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 silicon 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] One or more selected from More preferably, R is an ethyl ester. [ka] Propyl ester [ka] [ka] Butyl ester 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 the following:

[0016] Note: In the compounds (3) to (6), the double bond between C-22 and C-23 has the E configuration as the main configuration and the Z configuration as the secondary configuration. The ratio of the E / Z configurations is 87 / 13 ( 1 1 H NMR), and this ratio does not change without subsequent purification steps.

[0017] 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).

[0018] 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.

[0019] 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).

[0020] 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).

[0021] Here, the temperature of the oxidation reaction is 0 to 30°C, and preferably 0°C.

[0022] Here, the time for the oxidation reaction is 3 to 8 hours, and preferably 6 hours.

[0023] 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).

[0024] In step (b) of the present invention, the Wittig reaction is specifically a reaction in which 1-halo-3-methylbutane and triphenylphosphine are added to a second solvent, refluxed, cooled, and suction filtered to obtain a quaternary phosphine salt, and then the quaternary phosphine salt and potassium tert-butoxide are added to the second solvent, and the compound of formula (2) is further added to perform a Wittig reaction to obtain a compound of formula (3).

[0025] Here, the molar ratio of the compound of formula (2), 1-halo-3-methylbutane, triphenylphosphine, and potassium tert-butoxide is 1:(1-4):(1-4):(1-4), and preferably 1:3:2:2.

[0026] Wherein, the second solvent is one or more of xylene, toluene, benzene, tetrahydrofuran, heptane, etc., and is preferably xylene.

[0027] Here, when synthesizing the quaternary phosphine salt, the solvent is preferably xylene.

[0028] Here, the 1-halo-3-methylbutane is one or two selected from 1-chloro-3-methylbutane, 1-bromo-3-methylbutane, etc., and is preferably 1-bromo-3-methylbutane.

[0029] Here, the temperature of the Wittig reaction is -10 to 145°C, and preferably 10°C.

[0030] Here, the time for the Wittig reaction is 0.5 to 24 hours, preferably 0.5 to 9 hours, and more preferably 1 hour.

[0031] 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).

[0032] 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.

[0033] Here, the base is one or more selected from pyridine, triethylamine, DIPEA, DMAP, diisopropylamine, etc., and is preferably diisopropylamine.

[0034] 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.

[0035] The temperature of the acetylation reaction is 40 to 110°C, and preferably 70°C.

[0036] Here, the acetylation reaction time is 1 to 10 hours, preferably 2 to 10 hours, and more preferably 6 hours.

[0037] Here, the acetylation reaction uses acetyl chloride and acetic anhydride as both reactants and solvents.

[0038] In a particular embodiment, the step of synthesizing the compound of formula (4) includes 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).

[0039] 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).

[0040] Here, the molar ratio of the compound of formula (4) to the reducing agent is 1:(1 to 25), and preferably 1:4.

[0041] 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).

[0042] Wherein, the reducing agent is one or more of NaBH4 and KBH4, etc., and is preferably NaBH4.

[0043] Here, the temperature of the reduction reaction is 0 to 50°C, and preferably 25°C.

[0044] Here, the reduction reaction time is 6 to 12 hours, and preferably 8 hours.

[0045] 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).

[0046] In step (e) of the present invention, the selective hydrogenation reduction reaction is specifically a reaction in which the compound of formula (5) is subjected to a selective hydrogenation reduction reaction with a reducing agent in a fifth solvent under the action of a catalyst to obtain cholesterol.

[0047] Wherein, before obtaining cholesterol, a purification step is further included, and the purification step is one or more of column chromatography, recrystallization, slurrying, etc. Here, the catalyst is Raney Ni. Here, the reducing agent is H2.

[0048] Here, the mass ratio of the compound of formula (5) to the catalyst Raney Ni is 1:(0.05 to 5), and preferably 1:1.

[0049] Here, the fifth solvent is one or more selected from isopropyl alcohol, dichloromethane, methanol, 2-methyltetrahydrofuran, tetrahydrofuran, ethanol, water, methyl tert-butyl ether, ethyl acetate, toluene, etc., and is preferably isopropyl alcohol.

[0050] Here, the temperature of the hydrogenation reduction reaction is 0 to 60°C, and preferably 30°C.

[0051] Here, the pressure of the reducing agent H2 in the hydrogenation reduction reaction is 1 to 20 atm, and preferably 1 atm.

[0052] Here, the time for the hydrogenation reduction reaction is 6 to 10 hours, and preferably 6 hours. In a particular embodiment, the step of synthesizing cholesterol includes dissolving the compound of formula (5) in a fifth solvent, adding Raney Ni, replacing with H2, and then selectively reducing the resulting mixture by hydrogenation to obtain cholesterol.

[0053] In step (f) of the present invention, when the hydroxy-protecting group R is an ester group, the hydroxy-protecting reaction is specifically a reaction in which the compound of formula (5) is reacted with a hydroxy-protecting reagent in a sixth solvent under the action of a base to obtain a compound of formula (6).

[0054] 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.

[0055] Here, the molar ratio of the compound of the formula (5), the hydroxy-protecting reagent, and the base is 1:(1-4):(0.05-5), and preferably 1:3:0.1.

[0056] Wherein, the sixth solvent is one or more of ethyl acetate, dichloromethane, chloroform, DMF, toluene, tetrahydrofuran, 2-methyltetrahydrofuran, etc., and is preferably ethyl acetate.

[0057] Here, the base is one or more selected from triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, etc., and is preferably DMAP.

[0058] Here, the reaction temperature is 0 to 50°C, and preferably 45°C.

[0059] Here, the reaction time is 2 to 24 hours, and preferably 4 hours.

[0060] In step (f) of the present invention, when the hydroxy-protecting group R is a silyl ether group, the hydroxy-protecting reaction is specifically a reaction of reacting the compound of formula (5) with a hydroxy-protecting reagent in a sixth solvent under the action of a base to obtain the compound of formula (6).

[0061] 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.

[0062] Here, the molar ratio of the compound of the formula (5), the hydroxy-protecting reagent, and the base is 1:(2-4):(4-8), and preferably 1:2.5:4.

[0063] Wherein, the sixth solvent is one or more of DMF, dichloromethane, chloroform, carbon tetrachloride, etc., and is preferably dichloromethane.

[0064] Here, the base is one or more selected from triethylamine, diisopropylethylamine, imidazole, pyridine, DMAP, etc., and is preferably imidazole.

[0065] Here, the reaction temperature is 0 to 50°C, and preferably 25°C.

[0066] Here, the reaction time is 2 to 24 hours, and preferably 12 hours.

[0067] In one particular embodiment, the step of synthesizing the compound of formula (6) comprises dissolving the compound of formula (5) in a sixth solvent and reacting with a hydroxy protecting reagent under the action of a base to obtain the compound of formula (6).

[0068] In step (g) 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 seventh 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 H2.

[0069] 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.

[0070] Here, the seventh solvent is one or more selected from 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, isopropyl alcohol, etc., and is preferably ethyl acetate.

[0071] Here, the temperature of the hydrogenation reduction reaction is 0 to 60°C, and preferably 30°C.

[0072] Here, the pressure of the reducing agent H2 in the hydrogenation reduction reaction is 1 to 20 atm, and preferably 1 atm.

[0073] Here, the time for the hydrogenation reduction reaction is 4 to 48 hours, preferably 4 to 30 hours, and more preferably 7 hours.

[0074] In a particular embodiment, the step of synthesizing the compound of formula (7) includes dissolving the compound of formula (6) in a seventh solvent, adding Raney Ni, replacing with H2, and then selectively reducing the solvent to obtain the compound of formula (7).

[0075] In step (h) of the present invention, when the hydroxy-protecting group R is an ester group, the hydrolysis reaction is specifically a reaction in which the compound of formula (7) is hydrolyzed in an eighth solvent under the action of a base to obtain cholesterol.

[0076] Here, the base is one or more selected from LiOH, KOH, NaOH, t-BuOK, K2CO3, etc., and is preferably K2CO3.

[0077] 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.

[0078] Here, the eighth solvent is one or two selected from methanol, ethanol, etc., and is preferably methanol.

[0079] The temperature of the hydrolysis reaction is 10 to 75°C, and preferably 65°C.

[0080] Here, the hydrolysis reaction time is 0.3 to 12 hours, and preferably 2 hours.

[0081] In step (h) of the present invention, when the hydroxy-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 eighth solvent under the action of a catalyst to obtain cholesterol.

[0082] Wherein, the catalyst is one or more selected from tetrabutylammonium fluoride (TBAF), tetrabutylammonium fluoride trihydrate (TBAF·3H2O), boron trifluoride etherate, acetic acid and hydrogen chloride in ethyl acetate solution, preferably TBAF·3H2O.

[0083] Here, the mass ratio of the compound of the formula (7) to the catalyst is 1:(1 to 6), and preferably 1:4.

[0084] Here, the eighth solvent is one or two selected from tetrahydrofuran, water, and the like, and is preferably tetrahydrofuran.

[0085] The temperature for the deprotection reaction is 10 to 75°C, and preferably 25°C.

[0086] Here, the time for the deprotection reaction is 2 to 48 hours, and preferably 24 hours. In a particular embodiment, the step of synthesizing cholesterol includes dissolving the compound of formula (7) in an eighth solvent, adding a base or a catalyst, and carrying out a deprotection or hydrolysis reaction to obtain cholesterol.

[0087] The present invention also provides 20 compounds, the structures of which are as shown in the following formulas: 6-2(E), 6-3(E), 6-5(E), 6-6(E), 6-2(Z), 6-3(Z), 6-5(Z), 6-6(Z), 6'-1(E), 6'-2(E), 6'-3(E), 6'-4(E), 6'-5(E), 6'-6(E), 6'-1(Z), 6'-2(Z), 6'-3(Z), 6'-4(Z), 6'-5(Z), 6'-6(Z). [ka]

[0088] The beneficial effects of the present invention include the following: In the cholesterol production method of the present invention, the commercialized raw material BA used is a plant-derived raw material, which avoids the risk of infection by pathogenic bacteria and viruses that may be present in animal-derived raw materials, 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]

[0089] [Figure 1] FIG. 1 shows a gas chromatogram of cholesterol obtained in Example 5 of the present invention. [Diagram 2] FIG. 2 shows the single crystal diffraction pattern of cholesterol. [Diagram 3] FIG. 3 shows the NMR hydrogen spectrum (CDCl3) of cholesterol. [Figure 4] FIG. 4 shows the NMR carbon spectrum (CDCl3) of cholesterol. [Diagram 5] FIG. 5 shows a gas chromatogram of the purified product of the compound of formula (7-1) obtained in Example 7 of the present invention. [Figure 6] FIG. 6 shows a gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-1) in Example 8 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] 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. 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 silica gel (200-300) was obtained from Qingdao Ocean Chemical Plant.

[0091] 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]

[0092] Note: In the compounds (3) to (6), the double bond between C-22 and C-23 has the E configuration as the main configuration and the Z configuration as the secondary configuration. The ratio of the E / Z configurations is 87 / 13 ( 1 1 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.

[0093] In the present invention, the compounds of the above formula (3), formula (4), formula (5), formula (5'), formula (6) and formula (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 ( 1 The 3β-OH / 3α-OH ratio in the resulting compounds (5) (3β-OH) and (5') (3α-OH) is about 92 / 8 (determined by H NMR). 1 H NMR), and this ratio does not change unless a subsequent 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.

[0094] Example 1 Preparation of the compound of formula (2) [ka] This example illustrates the preparation of compound of formula (2) under different experimental conditions.

[0095] (1) BA (50.00 g, 0.15 mol), TEMPO (235 mg, 1.50 mmol), dichloromethane (400 mL), sodium bicarbonate (17.60 g, 0.21 mol), NCS (23.10 g, 173.00 mmol), tetrabutylammonium bromide (4.84 g, 15 mmol), and water (160 mL) were added sequentially 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 (11.25 g sodium thiosulfate pentahydrate / 250 mL water) was added, stirred at 5-10°C for 30 minutes, separated, the aqueous phase was extracted with dichloromethane (300 mL x 2), the organic layers were combined, washed with 1% sodium hydroxide solution (300 mL), separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound of formula (2) (47.46 g, white solid, molar yield 94.9%). 1 H NMR(400MHz,CDCl3) δ 9.55(s,1H),5.71(s,1H),2.45-2.23(m,5H),1.99(t,J=13.7Hz,2H),1.91-1.78(m,2H),1.68(t,J=10.2Hz ,2H),1.43(m,5H),1.30-1.19(m,2H),1.17(s,3H),1.11(d,J=5.5Hz,3H),1.06-0.89(m,3H),0.75(s,3H). 13 C NMR(100MHz,CDCl3) δ 205.00,199.65,171.31,123.99,55.25,53.84,51.04,49.54,43.10,39.39,38.6 8,35.80,35.68,34.06,32.93,32.05,27.11,24.64,21.06,17.48,13.53,12.44. HRMS(ESI): calcd for C 22 H 32 NaO2[M+Na] + ,351.2295,found 351.2292.

[0096] (2) BA (50.00 g, 0.15 mol), TEMPO (236 mg, 1.5 mmol), dichloromethane (400 mL), sodium bicarbonate (17.6 g, 0.21 mol), NBS (35.6 g, 200 mmol), tetrabutylammonium bromide (4.84 g, 15 mmol), and water (320 mL) were added sequentially 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 (16 g sodium thiosulfate pentahydrate / 300 mL water) was added, stirred at 5-10°C for 20 minutes, separated, the aqueous phase was extracted with dichloromethane (300 mL x 2), the organic layers were combined, washed with 1% sodium hydroxide solution (200 mL), separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound of formula (2) (white solid 47.5 g, molar yield 95%). (3) The compound BA (50 g, 0.15 mol) of formula (1), IBX (84 g, 0.3 mmol), THF (350 mL), and DMSO (300 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 (500 mL) and water (300 mL). The organic phase was washed with 1% sodium hydroxide solution (300 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the compound of formula (2) (white solid 46.7 g, molar yield 93.4%).

[0097] Example 2 Preparation of compound of formula (3) [ka] This example illustrates the preparation of compound of formula (3) under different experimental conditions.

[0098] (1) Triphenylphosphine (5.25 g, 20 mmol), 1-bromo-3-methylbutane (6.04 g, 40 mmol), and 25 mL of toluene were added to a flask, and the mixture was heated under reflux for 72 hours. The mixture was then cooled to 25°C and suction filtered to obtain a quaternary phosphonium salt (white solid, 5.12 g, molar yield 62%).

[0099] In an ice bath, potassium tert-butoxide (1.39 g, 12.4 mmol) was added to the above quaternary phosphonium salt (5.12 g, 12.4 mmol) in toluene (25 mL) and stirred for 0.5 hours, after which the compound of formula (2) (2.63 g, 8 mmol) was added in batches and reacted at 10 ° C for 0.5 hours. After detecting the completion of the reaction by TLC, water (5 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 (30 mL × 2) and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to a mixed solution of ethanol / water, slurried at 25 ° C for 1 hour, and filtered to obtain the compound of formula (3) (3E and 3Z, 3E / 3Z ≒ 87 / 13, white solid 2.76 g, molar yield 90.2%). HRMS (ESI): calcd for C 27 H 42 NaO [M+Na] + ,405.3128,found 405.3136.

[0100] 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.

[0101] (2) Triphenylphosphine (5.25 g, 20 mmol), 1-bromo-3-methylbutane (4.53 g, 30 mmol), and 25 mL of xylene were added to a flask, and the mixture was heated under reflux for 48 hours. The mixture was then cooled to 25°C and suction filtered to obtain a quaternary phosphonium salt (white solid, 7.76 g, molar yield 93.94%).

[0102] In an ice bath, potassium tert-butoxide (2.11 g, 18.79 mmol) was added to the above quaternary phosphonium salt (7.76 g, 18.79 mmol) in xylene (25 mL) and stirred for 0.5 hours, after which the compound of formula (2) (4.92 g, 15 mmol) was added in batches and reacted at 5 ° C for 1 hour. After detecting the completion of the reaction by TLC, water (5 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 (40 mL × 2), and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to a mixed solution of ethanol / water, slurried at 25 ° C for 1 hour, and filtered to obtain the compound of formula (3) (3E and 3Z, white solid 5.38 g, molar yield 93.9%).

[0103] (3) Triphenylphosphine (5.25 g, 20 mmol), 1-chloro-3-methylbutane (3.2 g, 30 mmol), and 25 mL of xylene were added to a flask, and the mixture was heated under reflux for 72 hours. The mixture was then cooled to 25°C and suction filtered to obtain a quaternary phosphonium salt (white solid, 6.12 g, molar yield 83.04%).

[0104] In an ice bath, potassium tert-butoxide (1.86 g, 16.59 mmol) was added to the above quaternary phosphonium salt (6.12 g, 16.59 mmol) in xylene (25 mL) and stirred for 0.5 hours, after which the compound of formula (2) (4.58 g, 12 mmol) was added in batches and reacted at 10 ° C for 0.5 hours. After detecting the completion of the reaction by TLC, water (5 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 (30 mL × 2), and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to a mixed solution of ethanol / water, slurried at 25 ° C for 1 hour, and filtered to obtain the compound of formula (3) (3E and 3Z, white solid 4.04 g, molar yield 88.07%).

[0105] Example 3 Preparation of compound of formula (4) [ka] This example shows the preparation of compound of formula (4) under different experimental conditions.

[0106] (1) The compound of formula (3) (9.94 g, 26 mmol), acetic anhydride (63.71 g, 624 mmol), acetyl chloride (51.03 g, 650 mmol) and diisopropylamine (10.5 g, 103.8 mmol) were added to a flask, and the mixture was refluxed for 5 hours. After the completion of the reaction was detected by TLC, the mixture was cooled to 25° C., concentrated under reduced pressure, slightly cooled, and the reaction solution was added to ice water (150 mL), stirred for 15 minutes, filtered, and the water was removed. The resulting solid was added to methanol (20 mL) and pyridine (1 mL), slurried at 25° C. for 30 minutes, filtered, and the filter cake was washed with methanol (4 mL), suction filtered, and dried to obtain the compound of formula (4) (4E and 4Z, white solid 10.47 g, molar yield 94.97%).

[0107] (2) The compound of formula (3) (9.94 g, 26 mmol), acetic anhydride (39.46 g, 390 mmol), acetyl chloride (8.16 g, 104 mmol) and DIPEA (13.39 g, 103.6 mmol) were added to a flask, and the reaction was detected by TLC. The mixture was then cooled to 25° C., concentrated under reduced pressure, cooled slightly, and the reaction solution was added to ice water (150 mL), stirred for 15 minutes, filtered, and the water was removed. The resulting solid was added to methanol (20 mL) and pyridine (1 mL), slurried at 25° C. for 30 minutes, filtered, and the filter cake was washed with methanol (4 mL), suction filtered, and dried to obtain the compound of formula (4) (4E and 4Z, white solid 10.15 g, molar yield 92.07%).

[0108] (3) The compound of formula (3) (9.94 g, 26 mmol), acetic anhydride (63.71 g, 624 mmol) and acetyl chloride (51.03 g, 650 mmol) were added to a flask, and the completion of the reaction was detected by TLC. The mixture was then cooled to 25° C., concentrated under reduced pressure, and cooled slightly. The reaction solution was added to ice water (150 mL), stirred for 15 minutes, filtered, and the water was removed. The resulting solid was added to methanol (20 mL) and pyridine (1 mL), slurried at 25° C. for 30 minutes, filtered, and the filter cake was washed with methanol (4 mL), suction filtered, and dried to obtain the compound of formula (4) (4E and 4Z, white solid 9.8 g, molar yield 88.9%).

[0109] Example 4 Preparation of the crude product of the compound of formula (5) [ka] This example shows the preparation of the crude compound of formula (5) under different experimental conditions.

[0110] (1) The compound of formula (4) (8.48 g, 20 mmol) and a mixed solvent of tetrahydrofuran, ethanol and water (170 mL, V / V / V = 10 / 5 / 3) were added to a flask, sodium borohydride (3.03 g, 80 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, 6 M hydrochloric acid was added to adjust the pH of the reaction solution to 7-8, a large amount of solid was precipitated in the reaction solution, filtered, the filtrate was taken and the solvent was evaporated under reduced pressure, a large amount of solid was precipitated, dichloromethane (100 mL) and water (50 mL) were added for extraction, the liquid was separated, the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain a crude product of the compound of formula (5) (light yellow solid 7.53 g, molar yield 98.04%).

[0111] 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). 1According 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.

[0112] (2) The compound of formula (4) (8.48 g, 20 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 (1.51 g, 40 mmol) was added in batches and reacted at 25 ° C for 6 hours. After detecting 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 was precipitated in the reaction solution, filtered, the filtrate was taken and the solvent was evaporated under reduced pressure, a large amount of solid was precipitated, dichloromethane (100 mL) and water (50 mL) were added for extraction, the liquid was separated, the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain a crude product of the compound of formula (5) (light yellow solid 7.5 g, molar yield 97.9%).

[0113] (3) The compound of formula (4) (8.48 g, 20 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 (4.86 g, 90 mmol) was added in batches and reacted at 30 ° C for 8 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 was precipitated in the reaction solution, filtered, the filtrate was taken and the solvent was evaporated under reduced pressure, a large amount of solid was precipitated, dichloromethane (100 mL) and water (50 mL) were added for extraction, the liquid was separated, the organic phase was washed with water (100 mL), stirred, the layers were separated, and the solvent was evaporated under reduced pressure to obtain a crude product of the compound of formula (5) (light yellow solid 7.57 g, molar yield 98.6%).

[0114] 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. 1According to H NMR, compound 5(3β-OH) / 5′(3α-OH)≒85 / 15.

[0115] Example 5 Preparation of cholesterol by selective hydrogen reduction of the compound of formula (5) [ka] The crude product of the compound of formula (5) was purified by column chromatography (petroleum ether / ethyl acetate=40 / 1, v / v) to obtain compounds of formula (5) (5E and 5Z).

[0116] The purified compound of formula (5) (5E and 5Z, 3.84 g, 10 mmol), Raney Ni (3.9 g, wet weight), isopropyl alcohol (40 mL), and H2 (1 atm) were added to a flask and reacted at 30°C for 6 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 crude cholesterol, which was purified by recrystallization (ethanol / water) to obtain cholesterol (white solid 3.8 g, molar yield 98.9%, gas chromatography purity 95.54%, see Figure 1, CCDC 2099442, see Figure 2 for single crystal structure). mp: 147-149°C. 1 H NMR(600MHz,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=6.6Hz,3H),0.86(dd,J=6.6,2.8Hz,6H),0.68(s,3H). 13C NMR(150MHz,CDCl3) δ 140.77,121.73,71.82,56.78,56.17,50.14,42.33,42.32,39.80,39.53,37.27,36.52,36.20,35.80,3 1.93,31.92,31.68,28.25,28.03,24.31,23.84,22.84,22.58,21.10,19.41,18.73,11.87.HRMS(ESI): calcd for C 27 H 46 NaO [M+Na] + ,409.3441,found 409.3121.

[0117] Example 6 Preparation of the crude compound of formula (6) 1. Preparation of the crude product of the compound of formula (6-1) [ka] Ethyl acetate (76 mL) and the crude product of the compound of formula (5) (7.68 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 (100 mL) was added for extraction. The organic phase was washed with water and saturated saline, and concentrated under reduced pressure to obtain the crude product of the compound of formula (6-1) (white solid 8.18 g, molar yield 95.9%).

[0118] 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.

[0119] 2. Preparation of the crude product of the compound of formula (6-2) [ka] Ethyl acetate (50mL) and the crude product of the compound of formula (5) (7.68g, 20mmol) were added to a flask, stirred to dissolve transparently, DMAP (0.245g, 2mmol) and propionic anhydride (5.3g, 40mmol) were added, and the mixture was reacted at 50°C for 5 hours. After the completion of the reaction was detected by TLC, water (20mL) was added to quench the reaction, and ethyl acetate (100mL) was added to extract the product. The organic phase was washed with water and saturated saline, dried over anhydrous Na2SO4, 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 (6-2) (white solid 8.64g, molar yield 98.07%).

[0120] 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.

[0121] 3. Preparation of crude product of compound of formula (6-3) [ka] Ethyl acetate (50mL) and the crude product of the compound of formula (5) (7.68g, 20mmol) were added to a flask, stirred to dissolve transparently, DMAP (0.245g, 2mmol) and butyric anhydride (9.54g, 60mmol) were added, and the mixture was reacted at 55°C for 4 hours. After the completion of the reaction was detected by TLC, water (20mL) was added to quench the reaction, ethyl acetate (100mL) was added to extract, the organic phase was washed with water, further washed with saturated saline, dried over anhydrous Na2SO4, concentrated under reduced pressure, methanol / water was added, the mixture was slurried at 25°C for 5 hours, and the mixture was filtered by suction to obtain the crude product of the compound of formula (6-3) (white solid 8.82g, molar yield 97.03%).

[0122] 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.

[0123] 4. Preparation of crude compound of formula (6-4) [ka] Ethyl acetate (115 mL) and the crude product of the compound of formula (5) (7.68 g, 20 mmol) were added to a flask, and the mixture was stirred to dissolve transparently. DMAP (0.245 g, 2 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) (white solid 9.38 g, molar yield 95.9%).

[0124] 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.

[0125] 5. Preparation of crude compound of formula (6-5) [ka] Add DCM (50mL) and the crude product of the compound of formula (5) (7.68g, 20mmol) to a flask, stir to dissolve transparently, add triethylamine (2.64g, 26.14mmol) and DMAP (0.245g, 2mmol), add p-chlorobenzoyl chloride (6.8g, 39.2mmol) slowly at 0℃, react at 35℃ for 12 hours while protecting with N2, detect the completion of the reaction by TLC, add water (30mL) to quench the reaction, extract with DCM (80mL), wash the organic phase with saturated NaHCO3 aqueous solution, 2N diluted hydrochloric acid, water, saturated NaCl in sequence, concentrate under reduced pressure, add methanol / water, slurry at 25℃ for 5 hours, and suction filter to obtain the crude product of the compound of formula (6-5) (white solid 9.68g, molar yield 95.04%).

[0126] 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.

[0127] 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) (7.68 g, 20 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 and stirred for 10 minutes. The mixture was extracted and separated with DCM (80 mL). The organic phase was washed with water and saturated NaCl, concentrated under reduced pressure, methanol / water was added, and the mixture was slurried at 25 ° C for 3 hours. The mixture was then suction filtered to obtain the crude product of the compound of formula (6-6) (white solid 9.58 g, molar yield 96.09%). 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.

[0128] Example 7 Preparation of compound of formula (7) 1. Preparation of compound of formula (7-1) [ka] Ethyl acetate (75 mL) and the crude product of the compound of formula (6-1) (10 g, 23.54 mmol) were added to a flask, stirred to dissolve transparently, then Raney Ni (10 g, wet weight, activated according to the standard procedure) was added, and reacted at H2 (1 atm) and 35 ° C for 7 hours. After detecting the completion of the reaction by gas chromatography, Raney Ni was removed by diatomaceous earth filtration, and the filtrate was concentrated under reduced pressure to obtain the crude product of the compound of formula (7-1), which was directly used in the purification step. This 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 4 hours, and suction filtered to obtain the compound of formula (7-1) (white solid 8.4 g, molar yield 84.1%, gas chromatography purity 99.22%, Figure 5). 1 H NMR(600MHz,CDCl3) δ 5.40(d,J=4.9Hz,1H),4.68-4.57(m,1H),2.40-2.31(m,2H),2.05(s,3 H),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=6.5Hz,3H),0.89(dd,J=6.6,2.8Hz,6H),0.70(s,3H). 13 C NMR(151MHz,CDCl3) δ 170.55,139.67,122.66,73.99,56.69,56.14,50.04,42.32,39.74,39.53,38.13,37.00,36.60,36.19,3 5.80,31.91,31.87,28.24,28.02,27.78,24.29,23.83,22.83,22.57,21.45,21.03,19.32,18.72,11.86.

[0129] Note: The main components of the crude product of the compound of formula (7-1) were compound (7-1) and compound (7'-1), and the ratio thereof was compound (7-1) / (7'-1) ≒ 92 / 8.

[0130] 2. Preparation of Compound of Formula (7-2) [ka] Ethyl acetate (75mL) and the crude product of the compound of formula (6-2) (10.05g, 22.8mmol) were added to a flask, stirred to dissolve transparently, then Raney Ni (15g, wet weight, activated according to standard procedures) was added, and reacted at H2 (1atm) and 55°C for 7 hours. After detecting the completion of the reaction by gas chromatography, Raney Ni was removed by diatomaceous earth filtration, and 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. This 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 4 hours, and suction filtered to obtain the compound of formula (7-2) (white solid 8.17g, molar yield 81%, gas chromatography purity 98.69%). 1 H NMR(600MHz,CDCl3) δ 5.39(d,J=4.9Hz,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=6.5Hz,3H),0.89(dd,J=6.6,2.8Hz,6H),0.70(s,3H). 13 C NMR(151MHz,CDCl3) δ 173.96,139.75,122.59,56.70,56.14,50.03,42.32,39.74,39.53,38.16,37.01,36.61,36.19,35.80,3 1.91,31.87,28.24,28.02,27.94,27.81,24.29,23.83,22.83,22.57,21.04,19.33,18.72,11.86,9.19.

[0131] Note: The main components of the crude product of the compound of formula (7-2) were compound (7-2) and compound (7'-2), and the ratio thereof was compound (7-2) / (7'-2) ≒ 92 / 8.

[0132] 3. Preparation of Compound of Formula (7-3) [ka] Ethyl acetate (75mL) and the crude product of the compound of formula (6-3) (10g, 22mmol) were added to a flask, stirred to dissolve transparently, then Raney Ni (10g, wet weight, activated according to standard procedures) was added, and reacted at H2 (1atm) and 35°C for 10 hours. After detecting the completion of the reaction by gas chromatography, Raney Ni was removed by diatomaceous earth filtration, and 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. This 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 (7-3) (white solid 8.3g, molar yield 82.5%, gas chromatography purity 99.1%). 1 H NMR (600MHz, CDCl3) δ 5.39(d,J=4.9Hz,1H),4.74-4.58(m,1H),2.33(d,J=7.1Hz,2H),2.28(t,J=7. 4Hz,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=6.7,2.7Hz,6H),0.70(s,3H). 13 C NMR(151MHz,CDCl3) δ 173.14,139.74,122.59,73.68,56.70,56.14,50.03,42.32,39.74,39.53,38.18,37.01,36.60,36.19,35.8 0,31.91,31.87,28.24,28.02,27.83,24.29,23.84,22.83,22.57,21.04,19.33,18.72,18.56,13.65,11.86.

[0133] Note: The main components of the crude product of the compound of formula (7-3) were compound (7-3) and compound (7'-3), and the ratio thereof was compound (7-3) / (7'-3) ≒ 92 / 8.

[0134] 4. Preparation of Compound of Formula (7-4) [ka] Add tetrahydrofuran (35mL) and ethyl acetate (35mL) to a flask, add the crude product of the compound of formula (6-4) (10g, 20.46mmol), stir to dissolve transparently, then add Raney Ni (10g, wet weight, activated according to standard procedure), react for 24 hours at H2 (1atm) and 35℃, detect the completion of the reaction by gas chromatography, remove Raney Ni by diatomaceous earth filtration, concentrate the filtrate under reduced pressure, and obtain the crude product of the compound of formula (7-4) as a white solid, which was directly used in the purification step. Add this white solid to a mixed solution of methanol and ethyl acetate, heat to reflux until the solution becomes transparent, naturally cool to 15℃, stir for 4-5 hours, and suction filter to obtain the compound of formula (7-4) (white solid 8.33g, molar yield 82.96%, gas chromatography purity 98.96%). 1 H NMR(600MHz,CDCl3) δ 8.11-7.99(m,2H),7.59-7.51(m,1H),7.43(t,J=7.8Hz,2H),5.42(dd,J=5.1,2.1Hz,1H ),4.89-4.84(m,1H),2.47(d,J=8.0Hz,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,1 1H),1.04-0.99(m,3H),0.92(d,J=6.5Hz,3H),0.87(dd,J=6.6,2.8Hz,6H),0.69(s,3H). 13C NMR(151MHz,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.

[0135] Note: The main components of the crude product of the compound of formula (7-4) were compound (7-4) and compound (7'-4), and the ratio thereof was compound (7-4) / (7'-4) ≒ 92 / 8.

[0136] 5. Preparation of Compound of Formula (7-5) [ka] Add tetrahydrofuran (39mL) and ethyl acetate (39mL) to a flask, add the crude product of the compound of formula (6-5) (10g, 19.2mmol), stir to dissolve transparently, then add Raney Ni (15g, wet weight, activated according to standard procedure), react for 20 hours at H2 (1atm) and 30℃, detect the completion of the reaction by gas chromatography, remove Raney Ni by diatomaceous earth filtration, concentrate the filtrate under reduced pressure, and obtain the crude product of the compound of formula (7-5) as a white solid, which was directly used in the purification step. Add this white solid to a mixed solution of methanol and ethyl acetate, heat to reflux until the solution becomes transparent, naturally cool to 15℃, stir for 4 hours, and suction filter to obtain the compound of formula (7-5) (white solid 8.1g, molar yield 81%, gas chromatography purity 98.5%). 1H NMR(500MHz,CDCl3) δ 8.02(d,J=8.7Hz,2H),6.93(d,J=8.7Hz,2H),5.44(d,J=5.0Hz,1H),4.88-4.82(m,1H),3 .88(s,3H),2.47(d,J=7.9Hz,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=8.9Hz,3H),1.30-1.10(m,7H),1.09(s ,3H),1.07-0.99(m,3H),0.94(d,J=6.5Hz,3H),0.89(dd,J=6.6,2.4Hz,6H),0.71(s,3H). 13 C NMR(151MHz,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.

[0137] Note: The main components of the crude product of the compound of formula (7-5) were compound (7-5) and compound (7'-5), and the ratio thereof was compound (7-5) / (7'-5) ≒ 92 / 8.

[0138] 6. Preparation of Compound of Formula (7-6) [ka] Ethyl acetate (75mL) and the crude product of the compound of formula (6-6) (10g, 20.04mmol) were added to a flask, stirred to dissolve transparently, then Raney Ni (10g, wet weight, activated according to standard procedures) was added, and reacted at H2 (1atm) and 35°C for 15 hours. After detecting the completion of the reaction by gas chromatography, Raney Ni was removed by diatomaceous earth filtration, and 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. This 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 4 hours, and suction filtered to obtain the compound of formula (7-6) (white solid 8.25g, molar yield 82.5%, gas chromatography purity 98.3%). 1 H NMR(500MHz,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=6.4Hz,3H),0.91(s,9H),0.89(dd,J=6.6,2.4Hz,6H),0.69(s,3H),0.08(s,6H). 13 C NMR(151MHz,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.

[0139] Note: The main components of the crude product of the compound of formula (7-6) were compound (7-6) and compound (7'-6), and the ratio thereof was compound (7-6) / (7'-6) ≒ 92 / 8.

[0140] Example 8 Preparation of cholesterol by hydrolysis of the compound of formula (7) 1. Preparation of cholesterol by hydrolysis of the compound of formula (7-1) [ka] Methanol (43 mL) and K2CO3 (2.07 g, 15 mmol) were added to the flask, and after dissolving it transparently, under N2 protection, the compound of formula (7-1) (4.28 g, 10 mmol) was added, heated to 65 ° C and reacted for 2 hours, and after detecting the completion of the reaction by TLC, cooled to 25 ° C, 2 mol / L dilute hydrochloric acid was added to adjust the pH to 7 ~ 8, methanol was evaporated under reduced pressure, water (20 mL) was added, stirred at 25 ° C for 2 hours, suction filtered, water (20 mL) was added to the filter cake, stirred at 25 ° C for 2 hours, suction filtered, and dried to obtain purified cholesterol (white solid 3.80 g, molar yield 98.45%, gas chromatography purity 99.38%, Figure 6).

[0141] 2. Preparation of cholesterol by hydrolysis of the compound of formula (7-2) [ka] Methanol (50mL) and K2CO3 (2.07g, 15mmol) were added to the flask, and after dissolving it transparently, under N2 protection, the compound of formula (7-2) (4.4g, 10mmol) was added, heated to 65℃ and reacted for 2 hours, and after detecting the end of the reaction by TLC, cooled to 25℃, 2mol / L dilute hydrochloric acid was added to adjust the pH to 7-8, methanol was evaporated under reduced pressure, water (20mL) was added, stirred at 25℃ for 2 hours, suction filtered, water (20mL) was added to the filter cake, stirred at 25℃ for 2 hours, suction filtered, and dried to obtain purified cholesterol (white solid 3.81g, molar yield 98.7%, gas chromatography purity 99.0%).

[0142] 3. Preparation of cholesterol by hydrolysis of the compound of formula (7-3) [ka] Methanol (45mL) and K2CO3 (1.8g, 13mmol) were added to the flask, and after dissolving it transparently, under N2 protection, the compound of formula (7-3) (4.56g, 10mmol) was added, heated to 65℃ and reacted for 2 hours, and after detecting the end of the reaction by TLC, cooled to 25℃, 2mol / L dilute hydrochloric acid was added to adjust the pH to 7-8, methanol was evaporated under reduced pressure, water (20mL) was added, stirred at 25℃ for 2 hours, suction filtered, water (20mL) was added to the filter cake, stirred at 25℃ for 2 hours, suction filtered, and dried to obtain purified cholesterol (white solid 3.8g, molar yield 98.5%, gas chromatography purity 99.3%).

[0143] 4. Preparation of cholesterol by hydrolysis of the compound of formula (7-4) [ka] Methanol (50mL) and KOH (0.62g, 11mmol) were added to the flask, and after dissolving it transparently, under N2 protection, the compound of formula (7-4) (4.9g, 10mmol) was added, heated to 45℃ and reacted for 6 hours, and after detecting the end of the reaction by TLC, cooled to 25℃, 2mol / L dilute hydrochloric acid was added to adjust the pH to 5-6, methanol was evaporated under reduced pressure, water (20mL) was added, stirred at 25℃ for 2 hours, suction filtered, 80% ethanol (10ml) was added to the filter cake, stirred at 25℃ for 2 hours, suction filtered, and dried to obtain purified cholesterol (white solid 3.66g, molar yield 94.8%, gas chromatography purity 98.9%).

[0144] 5. Preparation of cholesterol by hydrolysis of the compound of formula (7-5) [ka] Methanol (60mL) and KOH (0.62g, 11mmol) were added to the flask, and after dissolving it transparently, under N2 protection, the compound of formula (7-5) (5.2g, 10mmol) was added, heated to 45℃ and reacted for 8 hours, and after detecting the end of the reaction by TLC, cooled to 25℃, 2mol / L dilute hydrochloric acid was added to adjust the pH to 5-6, methanol was evaporated under reduced pressure, water (20mL) was added, stirred at 25℃ for 2 hours, suction filtered, 80% ethanol (10ml) was added to the filter cake, stirred at 25℃ for 2 hours, suction filtered, and dried to obtain purified cholesterol (white solid 3.61g, molar yield 93.5%, gas chromatography purity 98.7%).

[0145] 6. Preparation of cholesterol by deprotection of compound of formula (7-6) [ka] A flask was charged with tetrahydrofuran (25 mL) and the compound of formula (7-6) (5.01 g, 10 mmol), and the mixture was dissolved in water. Under N2 protection, TBAF·3H2O (15.7 g, 50 mmol) was added and the mixture was reacted at 25°C for 24 hours. After the completion of the reaction was detected by TLC, the mixture was quenched by adding saturated ammonium chloride. The tetrahydrofuran was evaporated under reduced pressure, and dichloromethane (150 mL) and water (120 mL) were added for extraction and separation. The organic phase was washed with saturated aqueous NaHCO3, 2N dilute hydrochloric acid, water, and saturated NaCl, and concentrated under reduced pressure to obtain crude cholesterol. The filter cake was added with 80% ethanol (10 ml), slurried at 25°C for 2 hours, and filtered by suction to obtain purified cholesterol (white solid 3.68 g, molar yield 95.3%, gas chromatography purity 98.56%).

[0146] 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 shown in the following reaction formula 1. [ka]

[0147] 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.

[0148] 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 compound of formula (03') (shown in reaction formula 2) is obtained by post-treatment according to the method of patent document (CN105218610A), 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 to give compounds of (03') and (03''), but the compound of formula (03) described in patent document (CN105218610A) cannot be obtained. Apparently, the compounds of formula (03') and (03'') shown in reaction scheme 2 or scheme 3 cannot undergo the subsequent Wittig reaction. [ka]

[0149] 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 filter 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') (colorless oil 2.30g, molar yield 88%). 1 H NMR(600MHz,CDCl3) δ 5.23-5.19(m 1H),5.10(d,J=1.9Hz,1H),4.30(d,J=2.4Hz,1H),3.81-3.70(m,4H),3.6 1-3.55(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=7.0Hz ,3H),1.22-1.18(m,6H),0.99(d,J=6.7Hz,3H),0.96(s,3H),0.70(s,3H). 13 C NMR(150MHz,CDCl3) δ 154.52,141.06,118.03,106.07,99.04,64.60,63.35,62.14,56.48,51.88,48.33,42.59,40.39,39.6 2,35.18,33.86,31.92,31.86,27.72,25.55,24.42,21.17,18.96,15.49,15.37,14.68,11.94,11.89. HRMS(ESI): calcd for C 28 H 46 NaO3[M+Na] + ,453.3339,found 453.3328.

[0150] 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 filter 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') (colorless oil 2.32g, molar yield 95%). 1 H NMR(600MHz,CDCl3) δ 5.69(d,J=1.8Hz,1H),4.27(d,J=2.4Hz,1H),3.78-3.72(m,1H),3.58-3.53(m,1H),3.4 7-3.40(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,3 H),1.53-1.47(m,2H),1.41-1.27(m,3H),1.21-1.17(m,6H),1.16(d,J=4.1Hz,4H),1.13 -1.08(m,1H),1.04-0.98(m,2H),0.96(d,J=6.8Hz,3H),0.93-0.86(m,1H),0.69(s,3H). 13 C NMR(150MHz,CDCl3) δ 199.58,171.53,123.76,105.95,64.58,63.40,55.39,53.81,51.81,42.52,40.33,39.42,3 5.69,35.63,33.97,32.92,32.03,27.62,24.37,21.01,17.37,15.48,15.36,11.91,11.86.

[0151] Comparative Example 2 The second step Wittig reaction reported in the patent document (CN105218610A) is shown in the following reaction scheme 4. [ka]

[0152] In this patent document, toluene is used as a solvent, triphenylphosphine and 1-chloro-3-methylbutane are added and refluxed for 2 hours, potassium tert-butoxide and the compound of formula (03) are added and refluxed for 4 hours to obtain the compound of formula (04). Molar yield: 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 refluxes triphenylphosphine and 1-chloro-3-methylbutane for 2 hours. The reaction result is shown in Reaction Scheme 5, which shows that the compound of formula (3') is obtained, but the expected compound of formula (3) is not obtained. This shows that the method disclosed in patent document (CN105218610A) cannot be applied to the present invention. [ka]

[0153] 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 to react. 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. Then, the compound of formula (2) (500mg, 1.52mmol) was added, and the mixture was refluxed for 4 hours to react. After detecting the completion of the reaction by TLC, the reaction mixture was cooled to 25°C, and 2M HCl (4mL) was added to neutralize the solution. The solution was extracted with dichloromethane (50mL) and water (50mL). The organic phase was washed with water and saturated saline solution in that order, 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') (colorless oil, 516mg, molar yield 85%). 1H NMR(600MHz,CDCl3) δ 9.53(d,J=5.0Hz,1H),2.72-2.68(m,1H),2.39-2.25(m,6H),2.10(dd,J=14.4,4.8Hz,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=6.7Hz,3H),0.89(d,J=6.6Hz,6H),0.72(s,3H). 13 C NMR(150MHz,CDCl3) δ 205.71,198.54,163.30,133.32,55.49,54.31,51.82,48.85,42.21,39.05,38.94,38.40,35.35,3 5.16,33.95,32.12,29.71,28.43,27.31,26.49,23.74,23.21,22.54,20.74,17.80,13.60,12.97. HRMS(ESI): calcd for C 27 H 42 NaO2[M+Na] + ,421.3077,found 421.3075.

[0154] 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 to react. 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, followed by stirring for 0.5 hours in 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 to react. After detecting the completion of the reaction by TLC, the reaction mixture was cooled to 25°C, and 2M HCl (4mL) was added to neutralize the solution. The solution was extracted with dichloromethane (50mL) and water (50mL). The organic phase was washed with water and saturated saline solution in that order, 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') (colorless oil, 534mg, molar yield 88%).

[0155] Comparative Example 3 In the present invention, the ester group at the 3-position of the compound of formula (4) was reduced with NaBH4 to obtain the compound of formula (5), and then the compound of formula (5) was either directly subjected to selective hydrogenation reduction or was subjected to hydroxy protection, selective hydrogenation reduction, deprotection or hydrolysis to obtain cholesterol. In addition, an attempt was made to first perform selective hydrogenation reduction of the double bond of the side chain using Raney Ni / H2 as a reducing agent, and then reduce the ester group at the 3-position using NaBH4. As shown in reaction formula 6, the reaction results showed that the compounds of formulas (6-1) and (6'-1) were obtained, but the expected compound of formula (9) was not obtained. This shows that when Raney Ni / H2 is used as a reducing agent, the side chain cannot be selectively hydrogenated. Therefore, the reaction sequence of the present invention cannot be changed. [ka]

[0156] Experimental method: 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) were added to a flask and 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 isomeric compound of formula (6'-1) was 1:0.28.

[0157] 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]

[0158] In the present invention, the double bond of the side chain is selectively hydrogenated and reduced by using H2 as a reducing agent and Raney Ni as a catalyst, and the crude product of the compound of formula (7-1) obtained has a content of the compound of formula (8-4), which is an impurity and is an over-reduced product, of less than 0.7% ( 1 H NMR) and is easy to remove by purification. In addition, when the double bond of the side chain is selectively hydrogenated using H2 as a reducing agent and 10% Pd / C as a catalyst, the amount of the impurity compound of formula (8-4), which is an over-reduced product, in the crude product of the compound of formula (7-1) is more than 5% ( 1 H NMR), which was difficult to remove by purification, and the intended purpose could not be achieved. 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 reduction of side chains.

[0159] Experimental method: The compound of formula (6-1) (1.50 g, 3.56 mmol), 10% Pd / C (150 mg), and ethyl acetate (30 mL) were added to a flask, and the mixture was reacted at H2 (1 atm) and 30°C for 9 hours. After the completion of the reaction was detected by TLC, the Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure. 1 Judging from the H NMR spectrum, the content of the over-reduced impurity compound of formula (8-4) is more than 5%.

[0160] 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 is to use BA as a raw material and synthesize the cholesterol through oxidation, Wittig reaction, acetylation, reduction, selective hydrogenation reduction, or to synthesize the cholesterol through oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation reduction, deprotection, or hydrolysis; (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) selectively reducing the compound of formula (5) by hydrogenolysis in a fifth solvent to obtain cholesterol; Or, (f) hydroxy-protecting the compound of formula (5) to obtain a compound of formula (6) in a sixth solvent; (g) selectively reducing the compound of formula (6) by hydrogenolysis in a seventh solvent to obtain a compound of formula (7); (h) deprotecting or hydrolyzing the compound of formula (7) in an eighth solvent to obtain cholesterol; Including, The method for synthesizing cholesterol, wherein the reaction process of the method is as shown in scheme (A). 【Chemistry 64】 (wherein R is selected from an ester group and a silicon ether group).

2. The ester group is a C2-C10 linear ester group, an isobutyl ester 【Chemistry 65】 Isopentyl ester group 【Chemistry 66】 Phenyl ester group 【Chemistry 67】 and p-methoxyphenyl ester group 【Chemistry 68】 One or more selected from The silyl ether group is a trimethylsilyl ether group. 【Chemistry 69】 and tert-butyldimethylsilyl ether group 【Chemistry 70】 2. The method of claim 1, wherein the first and second halves of the first and second halves of the second and third ...

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 1-halo-3-methylbutane and triphenylphosphine are added to the second solvent, refluxed, cooled, and suction filtered to obtain a quaternary phosphine salt, and then the quaternary phosphine salt and potassium tert-butoxide are added to the second solvent, and the compound of formula (2) is further added, and the Wittig reaction is performed to obtain the compound of formula (3).

6. The method according to claim 5, wherein the molar ratio of the compound of formula (2), 1-halo-3-methylbutane, triphenylphosphine, and potassium tert-butoxide is 1:(1-4):(1-4):(1-4), and / or the second solvent is one or more selected from xylene, toluene, benzene, tetrahydrofuran, and heptane, and / or the 1-halo-3-methylbutane is one or two selected from 1-chloro-3-methylbutane and 1-bromo-3-methylbutane, and / or the temperature of the Wittig reaction is −10 to 145° C., and / or the time of the Wittig reaction is 0.5 to 24 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 acetylation reaction temperature is 40-110° C., and / or the acetylation reaction time 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 selective hydrogenation reduction reaction is specifically a reaction in which the compound of formula (5) is selectively hydrogenated and reduced with a reducing agent in the fifth solvent under the action of a catalyst to obtain cholesterol.

12. The reducing agent is H ２ and / or the catalyst is Raney Ni, and / or the mass ratio of the compound of formula (5) to the catalyst is 1:(0.05-5), and / or the fifth solvent is one or more selected from isopropyl alcohol, dichloromethane, methanol, 2-methyltetrahydrofuran, tetrahydrofuran, ethanol, water, methyl tert-butyl ether, ethyl acetate, and toluene, 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 time of the hydrogenation reduction reaction is 6 to 10 hours.

13. The method according to claim 1, characterized in that in step (f), the hydroxy protection reaction is specifically a reaction of reacting the compound of formula (5) with a hydroxy protection reagent in a sixth solvent under the action of a base to obtain a compound of formula (6).

14. when R is an ester group, the sixth solvent is one or more selected from ethyl acetate, dichloromethane, chloroform, DMF, toluene, tetrahydrofuran, and 2-methyltetrahydrofuran, and / or the base is one or two 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 of the hydroxyl protecting reaction is 2-24 hours, 14. The method of claim 13, wherein when R is a silyl ether group, the sixth 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; and / or 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 temperature of the hydroxy protecting reaction is 0-50° C.; and / or the time of the hydroxy protecting reaction is 2-24 hours.

15. The method according to claim 1, characterized in that in step (g), 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 seventh solvent under the action of a catalyst to obtain cholesterol.

16. 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 seventh 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.

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

18. The base is LiOH, KOH, NaOH, t-BuOK, and 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 eighth solvent is one or more selected from methanol and ethanol; and / or the hydrolysis reaction temperature is 10-75° C.; and / or the hydrolysis reaction time is 0.3-12 hours.

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

20. The catalyst is tetrabutylammonium fluoride (TBAF), tetrabutylammonium fluoride trihydrate (TBAF.3H) ２ and / or the mass ratio of the compound of formula (7) to the catalyst is 1:(1-6); and / or the eighth 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.

21. The compound has a structure represented by the formula 6-2(E), 6-3(E), 6-5(E), 6-6(E), 6-2(Z), 6-3(Z), 6-5(Z), 6-6(Z), 6'-1(E), 6'-2(E), 6'-3(E), 6'-4(E), 6'-5(E), 6'-6(E), 6'-1(Z), 6'-2(Z), 6'-3(Z), 6'-4(Z), 6'-5(Z), 6'-6(Z). 【Chemistry 71】

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