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 relates to the field of organic chemical synthesis and concerns a method for synthesizing cholesterol using 21-hydroxy-20-methylpregna-4-en-3-one, also known as bisnoral alcohol or BA (Bisnoralcohol), of plant origin as a raw material. [Background technology]

[0002] Cholesterol is an essential and important substance for animal tissue cells, not only involved in the formation of cell membranes but also serving as a raw material for the synthesis of bile acids and steroid hormones in the body. Currently, commercially available cholesterol is mainly used for the following purposes: 1. Pharmaceutical excipients - liposome additives, for example, as an excipient for COVID-19 mRNA vaccines (adding 30-50% cholesterol when preparing liposomes can significantly increase the drug-carrying capacity and stability of the liposomes); 2. Cosmetic additives; 3. Raw material for liquid crystal manufacturing; 4. Starting material for vitamin D3 production. Currently, most commercially available cholesterol is derived from animal brainstem (brainstem cholesterol) or lanolin (lanolin cholesterol), both of which are animal-derived cholesterol. Studies have shown that animal-derived products are likely to contain animal pathogens and other harmful factors, and the safety of cholesterol has become a focus of attention, especially with the outbreaks of infectious diseases such as mad cow disease, swine streptococcus, and avian influenza. For example, cholesterol is an upstream raw material for vitamin D3 production, and European and American countries have long prohibited its use as a raw material due to the risk of disease. Since July 1, 2020, China has also restricted the use of brainstem cholesterol as a raw material. Therefore, in order to ensure people's health and safety, the development of plant-derived methods for synthesizing high-purity cholesterol has become an urgent necessity.

[0003] The following methods have been reported as the primary means of chemical synthesis of cholesterol: (1) Cholesterol was synthesized using diosgenin as a raw material in a 6-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 procedure, the high toxicity of the reagents used, and the high level of contamination. [ka]

[0004] (2) Cholesterol was synthesized in a total molar yield of 67% via a 5-step reaction using stigmasterol degradation products as raw materials (CN105218610A, shown in Scheme 2). However, the applicant of the present invention discovered through experiments that this scheme has the following problems: When triethyl orthoformate is used in the first step of the patent reaction to etherify and protect the carbonyl group at the C-3 position, the selectivity is low, and the reaction of the side chain aldehyde is likely to occur, resulting in the formation of an 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) Cholesterol was synthesized using pregnenolone as a starting material in 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 a raw material, cholesterol was synthesized with a total molar yield of 80% through a two-step reaction (shown in Scheme 4 of CN104961788A). However, in this scheme, the precious metal rhodium catalyst and chiral phosphine ligand used are expensive and not suitable for large-scale industrial production.

Chem.

[0007] (5) Using stigmasterol as a raw material, cholesterol was synthesized with a total molar yield of 68% through a five-step reaction (shown in Scheme 5 of CN105237603A). In this scheme, O3 is used in the synthesis process, which increases the requirements for reaction monitoring and equipment, and the economy and safety are not sufficient.

Chem.

[0008] (6) Using stigmasterol as a raw material, cholesterol was synthesized with a total molar yield of 70% through a four-step reaction (shown in Scheme 6 of CN106632565A). Since O3 is also used in this scheme, the process complexity increases, the requirements for reaction monitoring and equipment increase, and the economy and safety are not sufficient.

Chem.

[0009] Cholesterol derived from animals has a risk of being infected with diseases such as bovine spongiform encephalopathy, Streptococcus suis in pigs, and avian influenza. Among the reported chemical synthesis schemes of cholesterol, there are drawbacks such as complicated operations, high pollution 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 has important value.

Disclosure of the Invention

[0010] To overcome the shortcomings of existing technologies, 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-methylpregna-4-en-3-one, also known as bisnoral alcohol or BA, as a starting material and synthesizes the cholesterol through steps such as oxidation, Wittig reaction, acetylation, reduction, and selective hydrogenation / reduction, or through steps such as oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation / reduction, deprotection, or hydrolysis, and can achieve a purity of 99% or more. In the present invention, BA, as the starting material for synthesizing cholesterol, is safe and economical, the method for synthesizing cholesterol is easy to operate, has a high yield, high purity, is environmentally friendly, and is convenient for industrial production.

[0011] The raw material BA (bisnoralcohol) used in this invention is a plant-derived green raw material obtained by fermenting plant sterols remaining in the oil refining process. Currently, its annual production volume has reached approximately 1,000 tons, and it is inexpensive. Furthermore, it can effectively avoid the risks of pathogenic bacteria and viral infections that can be present in animal-derived cholesterol from 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 by chemical synthesis.

[0013] Two methods for synthesizing cholesterol using BA raw materials provided by the present invention include the following steps: Method 1: (a) A step of oxidizing BA represented by formula (1) in a first solvent to obtain the compound of formula (2); (b) In a second solvent, the compound of formula (2) is subjected to a Wittig reaction to obtain the compound of formula (3); (c) A step in which the compound of formula (3) is acetylated in a third solvent to obtain the compound of formula (4); (d) In a fourth solvent, the compound of formula (4) is reduced to obtain the compound of formula (5); (e) A step in which the compound of formula (5) is selectively hydrogenated and reduced in a fifth solvent to obtain cholesterol. Method 2: (a) A step of oxidizing BA represented by formula (1) in a first solvent to obtain the compound of formula (2); (b) In a second solvent, the compound of formula (2) is subjected to a Wittig reaction to obtain the compound of formula (3); (c) A step in which the compound of formula (3) is acetylated in a third solvent to obtain the compound of formula (4); (d) In a fourth solvent, the compound of formula (4) is reduced to obtain the compound of formula (5); (f) In a sixth solvent, protect the compound of formula (5) with hydroxyl to obtain the compound of formula (6); (g) A step in which the compound of formula (6) is selectively subjected to a hydrogenation-reduction reaction in a seventh solvent to obtain the compound of formula (7); (h) A step of obtaining cholesterol by deprotecting or hydrolyzing the compound of formula (7) in an eighth solvent.

[0014] The reaction process of the above method is shown in scheme (A). [ka]

[0015] Here, R is selected from an ester group and a silicon ether group. Preferably, R is a C2-C10 linear ester group, isobutyl ester [ka] isopentyl ester group [ka] Phenylen ester group [ka] p-methoxyphenyl ester group [ka] trimethylsilyl ether group [ka] and tert-butyldimethylsilyl ether group [ka] One or more selected from, For comfort, R is ethyl ester [ka] Propyl ester [ka] [ka] Butyl ester Isobutyl ester [ka] Isopentyl ester [ka] Phenylen ester [ka] p-methoxyphenyl ester [ka] trimethylsilyl ether group [ka] and tert-butyldimethylsilyl ether group [ka] It is one or more selected from the following.

[0016] Note: In compound formulas (3) to (6), the double bond between the C-22 and C-23 positions has a predominant E configuration and a secondary Z configuration, and the ratio of these two configurations is E / Z ≈ 87 / 13. 1 (Determined by 1H NMR), and this ratio does not change unless subsequent purification steps are performed.

[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 bicarbonate, tetrabutylammonium bromide, and an oxidizing agent in a first solvent to obtain a compound of formula (2).

[0018] Here, the molar ratio of BA, TEMPO, sodium bicarbonate, tetrabutylammonium bromide, and oxidizing agent represented by formula (1) 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, which is one or more selected from N-chlorosuccinimide NCS, N-bromosuccinimide NBS, and 2-iodoxybenzoic acid IBX, and is preferably N-chlorosuccinimide NCS.

[0020] Here, the first solvent is one or more selected from dichloromethane, tetrahydrofuran, toluene, dimethyl sulfoxide, and water, 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, preferably 0°C.

[0022] Here, the duration of the oxidation reaction is 3 to 8 hours, preferably 6 hours.

[0023] In one 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 filtered by suction 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 added and the Wittig reaction is carried out to obtain the 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] Here, the second solvent is one or more of xylene, toluene, benzene, tetrahydrofuran, and heptane, and is preferably xylene.

[0027] In this case, when synthesizing the quaternary phosphine salt, xylene is preferred as the solvent.

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

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

[0030] Here, the duration of 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, and diisopropylamine, and is preferably diisopropylamine.

[0034] Here, the third solvent is one or more of acetic anhydride, acetyl chloride, ethyl acetate, and dichloromethane, and is preferably a mixed solvent of acetyl chloride and acetic anhydride.

[0035] Here, the temperature of the acetylation reaction is 40 to 110°C, 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] In the acetylation reaction described above, acetyl chloride and acetic anhydride are used as both reactants and solvents.

[0038] In one particular embodiment, the synthesis step of the compound of formula (4) includes adding acetyl chloride, acetic anhydride, and a base to the compound of formula (3) and carrying out 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) and a reducing agent are reduced in a fourth solvent to obtain the compound of formula (5).

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

[0041] Here, the fourth solvent is one or more of the following: tetrahydrofuran, ethanol, water, dichloromethane, 2-methyltetrahydrofuran, isopropyl alcohol, acetic acid, and methyl tert-butyl ether, and is preferably a mixed solvent of tetrahydrofuran, ethanol, and water (volume ratio V / V / V = 10 / 5 / 3).

[0042] Here, the reducing agent is one or more of the following: NaBH4 and KBH4, and preferably NaBH4.

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

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

[0045] In one particular embodiment, the synthesis step of 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 selectively hydrogenated-reduce with a reducing agent in a fifth solvent under the action of a catalyst to obtain cholesterol.

[0047] Here, before obtaining cholesterol, a purification step is further included, the purification step being one or more of column chromatography, recrystallization, and slurrying. 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:(0.05~5), 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, and toluene, and is preferably isopropyl alcohol.

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

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

[0052] Here, the duration of the hydrogenation-reduction reaction is 6 to 10 hours, preferably 6 hours. In one particular embodiment, the cholesterol synthesis step includes dissolving the compound of formula (5) in a fifth solvent, adding Raney Ni, substituting with H2, and then subjecting it to a selective hydrogenation-reduction reaction to obtain cholesterol.

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

[0054] The aforementioned ester group is a C2-C10 linear ester group (ethyl ester). [ka] Propyl ester [ka] Butyl ester [ka] (etc.), isobutyl esters [ka] Isopentyl ester [ka] Phenylen ester [ka] and p-methoxyphenyl ester [ka] One or more ester groups are selected from among the following, and are preferably ethyl esters.

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

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

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

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

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

[0060] In step (f) of the present invention, if the hydroxyl protecting group R is a silyl ether group, the hydroxyl protection reaction is specifically a reaction in which the compound of formula (5) and a hydroxyl protecting reagent are reacted 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] One or more selected from the above, preferably tert-butyldimethylsilyl ether group [ka] That is the case.

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

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

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

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

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

[0067] In one particular embodiment, the synthesis step of the compound of formula (6) includes dissolving the compound of formula (5) in a sixth solvent and reacting it with a hydroxyl protecting agent 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 selectively hydrogenated-reduction with a reducing agent in a seventh 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.

[0069] Here, the mass ratio of the compound of formula (6) to the catalyst Raney Ni is 1:(0.05~5), preferably 1:1.

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

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

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

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

[0074] In one particular embodiment, the synthesis step of the compound of formula (7) includes dissolving the compound of formula (6) in a seventh solvent, adding Raney Ni, substituting with H2, and then subjecting it to a selective hydrogenation-reduction reaction to obtain the compound of formula (7).

[0075] In step (h) of the present invention, if the hydroxyl 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, and K2CO3, and is preferably K2CO3.

[0077] Here, the molar ratio of the compound in formula (7) to the base is 1:(0.5~2), preferably 1:1.3.

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

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

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

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

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

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

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

[0085] Here, the temperature of the deprotection reaction is 10 to 75°C, preferably 25°C.

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

[0087] The present invention also provides 20 compounds, the structures of which are 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 method for producing cholesterol of the present invention, the commercially available raw material BA used is a plant-derived raw material, thus avoiding the risk of infection by pathogenic bacteria and viruses that may be present in animal-derived raw materials, being inexpensive and readily available, and the resulting cholesterol is highly pure (over 99%), the synthesis process is simple, the yield is high, there are few side reactions, it is environmentally friendly, it is convenient for achieving industrial production of high-purity cholesterol, and it can solve the problems of existing cholesterol products, such as low safety, low purity, high synthesis costs, and being unenvironmentally unfriendly. [Brief explanation of the drawing]

[0089] [Figure 1] Figure 1 shows the gas chromatogram of cholesterol obtained in Example 5 of the present invention. [Figure 2] Figure 2 shows the single-crystal diffraction pattern of cholesterol. [Figure 3] Figure 3 shows the NMR hydrogen spectrum (CDCl3) of cholesterol. [Figure 4] Figure 4 shows the NMR carbon spectrum (CDCl3) of cholesterol. [Figure 5] Figure 5 shows the gas chromatogram of the purified product of the compound of formula (7-1) obtained in Example 7 of the present invention. [Figure 6] Figure 6 shows the gas chromatogram of cholesterol obtained by the hydrolysis reaction of the compound of formula (7-1) in Example 8 of the present invention. [Modes for carrying out the invention]

[0090] The present invention will be described in more detail based on the following specific examples and drawings. The processes, conditions, experimental methods, etc., for carrying out the present invention are common knowledge and prior art, except for those specifically mentioned below, and are not particularly limited to the present invention. In the following examples, the structure of the compounds was determined using nuclear magnetic resonance and high-resolution mass spectrometry, reagents were mainly supplied by Shanghai Guoyao Chemical Reagents Company, product purification was mainly carried out by slurrying and column chromatography, and silica gel (200-300) was obtained from Qingdao Marine Chemical Plant, etc.

[0091] The present invention provides a method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregna-4-en-3-one (BA) as a raw material, the reaction process of which is shown in scheme (A) above. Furthermore, the present invention further includes the generation of other by-products in the method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregna-4-en-3-one (BA) as a raw material, the specific reaction scheme of which is shown in (A'). [ka]

[0092] Note: In compound formulas (3) to (6), the double bond between the C-22 and C-23 positions has a predominant E configuration and a secondary Z configuration, and the ratio of these two configurations is E / Z ≈ 87 / 13. 1 (Determined by 1H NMR), and this ratio does not change unless subsequent purification is performed. The ratio of compound formula (5)(3β-OH) / (5')(3α-OH) is approximately 92 / 8( 1 (Determined by 1H NMR), and without subsequent purification, the ratios of compound formulas (6) / (6') and (7) / (7') remain unchanged.

[0093] In the present invention, the compounds of formulas (3), (4), (5), (5'), (6), and (6') above have a cis-trans configuration (E and Z configuration) in the D-ring side chain double bond (double bond at positions C-22 and C-23) introduced by the Wittig reaction, and the ratio of the two configurations is E / Z ≈ 87 / 13. 1 (Determined by 1H NMR), therefore, this ratio will not change unless a subsequent purification procedure is performed. Compound formulas (5)(3β-OH) and (5')(3α-OH) are obtained by reducing compound formula (4) with NaBH4 or KBH4, and the 3β-OH / 3α-OH ratio in the obtained compound formulas (5)(3β-OH) and (5')(3α-OH) is approximately 92 / 8. 1 (Determined by 1H NMR), and this ratio does not change unless a subsequent purification procedure is performed. Therefore, the compounds of formulas (3), (4), (5), (5'), (6), (6'), (7), and (7') are not single substances but mixtures.

[0094] Example 1: Preparation of the compound of formula (2) [ka] This example shows the results of preparing the compound of formula (2) under different experimental conditions.

[0095] (1) Add 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) to a flask in sequence, and react at 0 °C for 6 hours. After detecting the completion of the reaction by TLC, add a sodium thiosulfate pentahydrate solution (11.25 g sodium thiosulfate pentahydrate / 250 mL water), stir at 5 - 10 °C for 30 minutes, separate the layers, extract the aqueous phase with dichloromethane (300 mL × 2), combine the organic layers, wash with 1% sodium hydroxide solution (300 mL), separate the layers, dry the organic phase with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the compound of formula (2) (47.46 g, white solid, molar yield 94.9%). 1 H 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.

[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 reacted at 0°C for 6 hours. After detecting the end of the reaction by TLC, a sodium thiosulfate pentahydrate solution (16 g sodium thiosulfate pentahydrate / 300 mL water) was added, the mixture was stirred at 5-10°C for 20 minutes, and the liquid-liquid was separated. The aqueous phase was extracted with dichloromethane (300 mL x 2), the organic phases were combined, washed with 1% sodium hydroxide solution (200 mL), and separated again. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the compound of formula (2) (47.5 g white solid, molar yield 95%). (3) Compound BA of formula (1) (50 g, 0.15 mol), IBX (84 g, 0.3 mmol), THF (350 mL), and DMSO (300 mL) were sequentially added to a flask and reacted at 25°C for 5 hours. After detecting the end of the reaction by TLC, water was added, filtered by suction, and the filtrate was concentrated under reduced pressure. Dichloromethane (500 mL) and water (300 mL) were added for extraction. 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 the compound of formula (3) [ka] This example shows the results of preparing the 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, heated under reflux for 72 hours, then cooled to 25°C and filtered by suction to obtain a quaternary phosphonium salt (5.12 g white solid, molar yield 62%).

[0099] Under ice bath conditions, potassium tert-butoxide (1.39 g, 12.4 mmol) was added to a toluene (25 mL) solution of the above quaternary phosphonium salt (5.12 g, 12.4 mmol), and the mixture was stirred for 0.5 hours. Then, the compound of formula (2) (2.63 g, 8 mmol) was added in batches, and the mixture was reacted at 10°C for 0.5 hours. After detecting the end of the reaction by TLC, water (5 mL) was added to quench the reaction, and 2 M HCl was added to adjust the pH to 6-7. The organic phase was washed with water (30 mL x 2), and concentrated under reduced pressure to obtain a milky white solid. The solid was added to an ethanol / water mixture, slurryed at 25°C for 1 hour, and filtered to obtain the compound of formula (3) (3E and 3Z, 3E / 3Z ≈ 87 / 13, 2.76 g white solid, 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 intermediates obtained by acetylating and reducing the compound of formula (3), compounds of formula (4), (5), and (6), remains essentially unchanged. The double bond between the C-22 and C-23 positions has a predominant E configuration and a secondary Z configuration (3E / 3Z ≈ 87 / 13). The cis-trans isomers of the double bond between the C-22 and C-23 positions in compound (6) are reduced by Raney nickel, deprotected, or hydrolyzed to obtain the cholesterol product. Therefore, the E / Z configuration ratio of the corresponding compounds is not indicated in the later 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, heated under reflux for 48 hours, then cooled to 25°C and filtered by suction to obtain a quaternary phosphonium salt (7.76 g white solid, molar yield 93.94%).

[0102] Under ice bath conditions, potassium tert-butoxide (2.11 g, 18.79 mmol) was added to a xylene (25 mL) solution of the above quaternary phosphonium salt (7.76 g, 18.79 mmol), and the mixture was stirred for 0.5 hours. Then, the compound of formula (2) (4.92 g, 15 mmol) was added in batches, and the mixture was reacted at 5°C for 1 hour. After detecting the end of the reaction by TLC, the reaction was quenched with water (5 mL), and the pH was adjusted to 6-7 with 2 M HCl. The organic phase was washed with water (40 mL x 2), and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to an ethanol / water mixture, slurryed at 25°C for 1 hour, and filtered to obtain the compound of formula (3) (3E and 3Z, 5.38 g of white solid, molar yield 93.9%).

[0103] (3) Add triphenylphosphine (5.25 g, 20 mmol), 1-chloro-3-methylbutane (3.2 g, 30 mmol), and 25 mL of xylene to a flask, heat under reflux for 72 hours, then cool to 25°C and filter by suction to obtain a quaternary phosphonium salt (6.12 g white solid, molar yield 83.04%).

[0104] Under ice bath conditions, potassium tert-butoxide (1.86 g, 16.59 mmol) was added to a xylene (25 mL) solution of the above quaternary phosphonium salt (6.12 g, 16.59 mmol), and the mixture was stirred for 0.5 hours. Then, the compound of formula (2) (4.58 g, 12 mmol) was added in batches, and the mixture was reacted at 10°C for 0.5 hours. After detecting the end of the reaction by TLC, the reaction was quenched with water (5 mL), and the pH was adjusted to 6-7 with 2 M HCl. The organic phase was washed with water (30 mL x 2), and concentrated under reduced pressure to obtain a milky white solid. The above solid was added to an ethanol / water mixture, slurryed at 25°C for 1 hour, and filtered to obtain the compound of formula (3) (3E and 3Z, 4.04 g of white solid, molar yield 88.07%).

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

[0106] (1) Add 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) to a flask and reflux for 5 hours. After detecting the end of the reaction by TLC, cool to 25°C, concentrate under reduced pressure, cool slightly, add the reaction solution to ice water (150 mL), stir for 15 minutes, filter, drain the water, add the obtained solid to methanol (20 mL) and pyridine (1 mL), slurry at 25°C for 30 minutes, filter, wash the filter cake with methanol (4 mL), filter by suction, and dry to obtain the compound of formula (4) (4E and 4Z, 10.47 g white solid, molar yield 94.97%).

[0107] (2) Add 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) to a flask. After detecting the completion of the reaction by TLC, the mixture is cooled to 25°C, concentrated under reduced pressure, slightly cooled, and the reaction solution is added to ice water (150 mL). The mixture is stirred for 15 minutes, filtered, and the water is removed. The resulting solid is added to methanol (20 mL) and pyridine (1 mL), slurryed at 25°C for 30 minutes, filtered, the filter cake is washed with methanol (4 mL), filtered by suction, and dried to obtain the compound of formula (4) (4E and 4Z, 10.15 g of white solid, molar yield 92.07%).

[0108] (3) Add 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) to a flask. After detecting the completion of the reaction by TLC, the mixture is cooled to 25°C, concentrated under reduced pressure, slightly cooled, and the reaction solution is added to ice water (150 mL). The mixture is stirred for 15 minutes, filtered, and the water is removed. The resulting solid is added to methanol (20 mL) and pyridine (1 mL), slurryed at 25°C for 30 minutes, filtered, and the filter cake is washed with methanol (4 mL). The mixture is then filtered by suction and dried to obtain the compound of formula (4) (4E and 4Z, 9.8 g of white solid, molar yield 88.9%).

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

[0110] (1) Add the compound of formula (4) (8.48 g, 20 mmol), a mixed solvent of tetrahydrofuran, ethanol, and water (170 mL, V / V / V = 10 / 5 / 3) to a flask, add sodium borohydride (3.03 g, 80 mmol) in batches, and react at 30°C for 8 hours. After detecting the end of the reaction by TLC, add 6 M hydrochloric acid to adjust the pH of the reaction solution to 7-8, and a large amount of solid precipitates in the reaction solution. Filter the solution, take the filtrate and evaporate the solvent under reduced pressure, and a large amount of solid precipitates. Add dichloromethane (100 mL) and water (50 mL) to extract, separate the organic phase with water (100 mL), stir, separate the layers, and evaporate the solvent under reduced pressure to obtain the crude product of the compound of formula (5) (7.53 g pale yellow solid, 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 compound (5)(5E and 5Z, 3β-OH) and (5')(5'E and 5'Z, 3α-OH). 1According to 1H NMR analysis, the ratio of compound (5) / (5') was approximately 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 in this example, and therefore will not be mentioned hereafter.

[0112] (2) Add the compound of formula (4) (8.48 g, 20 mmol), a mixed solvent of dichloromethane, methanol, and water (95 mL, V / V / V = 18 / 9 / 1) to a flask, add sodium borohydride (1.51 g, 40 mmol) in batches, and react at 25°C for 6 hours. After detecting the end of the reaction by TLC, add 3 M hydrochloric acid to adjust the pH of the reaction solution to 7-8, and a large amount of solid precipitates in the reaction solution. Filter the solution, take the filtrate and evaporate the solvent under reduced pressure, and a large amount of solid precipitates. Add dichloromethane (100 mL) and water (50 mL) to extract, separate the organic phase with water (100 mL), stir, separate the layers, and evaporate the solvent under reduced pressure to obtain the crude product of the compound of formula (5) (7.5 g pale yellow solid, molar yield 97.9%).

[0113] (3) Add the compound of formula (4) (8.48 g, 20 mmol), a mixed solvent of tetrahydrofuran, ethanol, and water (200 mL, V / V / V = 10 / 5 / 3) to a flask, add potassium borohydride (4.86 g, 90 mmol) in batches, and react at 30°C for 8 hours. After detecting the end of the reaction by TLC, add 6 M hydrochloric acid to adjust the pH of the reaction solution to 7-8, and a large amount of solid precipitates in the reaction solution. Filter the solution, take the filtrate and evaporate the solvent under reduced pressure, and a large amount of solid precipitates. Add dichloromethane (100 mL) and water (50 mL) to extract, separate the organic phase with water (100 mL), stir, separate the layers, and evaporate the solvent under reduced pressure to obtain the crude product of the compound of formula (5) (7.57 g pale yellow solid, 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 compound (5)(5E and 5Z, 3β-OH) and (5')(5'E and 5'Z, 3α-OH), of which compound 5'(3α-OH) has a higher content. 1According to 1H NMR analysis, compound 5(3β-OH) / 5'(3α-OH) ≈ 85 / 15.

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

[0116] The compounds of formula (5) obtained by purification (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 detecting the end of the reaction by TLC, Raney Ni was filtered off, and the filtrate was concentrated under reduced pressure to obtain crude cholesterol. This 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 product of the compound of formula (6) 1. Preparation of the crude product of the compound of formula (6-1) [ka] Add ethyl acetate (76 mL) and the crude product of the compound of formula (5) (7.68 g, 20 mmol) to a flask, stir, and dissolve clearly. Add DMAP (0.245 g, 2 mmol) and acetic anhydride (6.13 g, 60 mmol), and react at 45°C for 4 hours. After detecting the end of the reaction by TLC, add water (20 mL) to quench the reaction, add ethyl acetate (100 mL) to extract, wash the organic phase with water and saturated brine, and concentrate under reduced pressure to obtain the crude product of the compound of formula (6-1) (8.18 g white solid, 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 their ratio was compound (6-1) / (6'-1) ≈ 92 / 8.

[0119] 2. Preparation of the crude product of the compound of formula (6-2) [ka] Add ethyl acetate (50 mL) and the crude product of the compound of formula (5) (7.68 g, 20 mmol) to a flask, stir, and dissolve clearly. Add DMAP (0.245 g, 2 mmol) and propionic anhydride (5.3 g, 40 mmol), and react at 50°C for 5 hours. After detecting the end of the reaction by TLC, add water (20 mL) to quench the reaction, add ethyl acetate (100 mL) to extract, wash the organic phase with water and saturated brine, dry over anhydrous Na2SO4, concentrate under reduced pressure, add methanol / water, and slurry at 25°C for 5 hours. Filter by suction filtration to obtain the crude product of the compound of formula (6-2) (8.64 g white solid, 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 their ratio was compound (6-2) / (6'-2) ≈ 92 / 8.

[0121] 3. Preparation of the crude product of the compound of formula (6-3) [ka] Add ethyl acetate (50 mL) and the crude product of the compound of formula (5) (7.68 g, 20 mmol) to a flask, stir, and dissolve clearly. Add DMAP (0.245 g, 2 mmol) and butyric anhydride (9.54 g, 60 mmol), and react at 55°C for 4 hours. After detecting the end of the reaction by TLC, add water (20 mL) to quench the reaction, add ethyl acetate (100 mL) to extract, wash the organic phase with water, then wash with saturated brine, dry over anhydrous Na2SO4, concentrate under reduced pressure, add methanol / water, and slurry at 25°C for 5 hours. Filter by suction to obtain the crude product of the compound of formula (6-3) (8.82 g white solid, 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 their ratio was compound (6-3) / (6'-3) ≈ 92 / 8.

[0123] 4. Preparation of the crude product of the compound of formula (6-4) [ka] In a flask, ethyl acetate (115 mL) and the crude product of the compound of formula (5) (7.68 g, 20 mmol) were added and stirred until clear. 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 detecting the end of the reaction by TLC, the mixture was concentrated under reduced pressure, methanol / water was added, and the mixture was slurryed at 25°C for 5 hours. The mixture was then filtered by suction to obtain the crude product of the compound of formula (6-4) (9.38 g white solid, 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 their ratio was compound (6-4) / (6'-4) ≈ 92 / 8.

[0125] 5. Preparation of the crude product of the compound of formula (6-5) [ka] Add DCM (50 mL) and the crude product of the compound of formula (5) (7.68 g, 20 mmol) to a flask, stir, and dissolve clearly. Add triethylamine (2.64 g, 26.14 mmol) and DMAP (0.245 g, 2 mmol), and slowly add p-chlorobenzoyl chloride (6.8 g, 39.2 mmol) dropwise at 0°C. React at 35°C for 12 hours while protecting with N2. After detecting the end of the reaction by TLC, add water (30 mL) to quench the reaction, extract with DCM (80 mL), wash the organic phase sequentially with saturated NaHCO3 aqueous solution, 2N dilute hydrochloric acid, water, and saturated NaCl, concentrate under reduced pressure, add methanol / water, slurry at 25°C for 5 hours, and filter by suction to obtain the crude product of the compound of formula (6-5) (9.68 g white solid, 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 their ratio was compound (6-5) / (6'-5) ≈ 92 / 8.

[0127] 6. Preparation of the crude product of the compound of formula (6-6) [ka] Add 100 mL of DCM and the crude product of the compound of formula (5) (7.68 g, 20 mmol) to a flask, stir until clear and dissolved, add 9.85 g of TBSCl and 65.35 mmol of imidazole (7.12 g, 104.56 mmol), react at 25°C for 12 hours, and after detecting the end of the reaction by TLC, add 100 mL of water and stir for 10 minutes, extract with 80 mL of DCM, separate the organic phase, wash with water and saturated NaCl, concentrate under reduced pressure, add methanol / water, slurry at 25°C for 3 hours, and filter by suction to obtain the crude product of the compound of formula (6-6) (9.58 g of white solid, molar yield 96.09%). Note: The main components of the crude product of the compound of formula (6-6) were compound (6-6)(6-6E and 6-6Z) and (6'-6)(6'-6E and 6'-6Z), and their ratio was compound (6-6) / (6'-6) ≈ 92 / 8.

[0128] Example 7 Preparation of the compound of formula (7) 1. Preparation of the compound of formula (7-1) [ka] 75 mL of ethyl acetate and the crude product of the compound of formula (6-1) (10 g, 23.54 mmol) were added to a flask and stirred until clear and dissolved. Then, 10 g of Raney Ni (wet weight, activated according to standard procedure) was added and the mixture was reacted at H2 (1 atm) at 35°C for 7 hours. After detecting the end of the reaction by gas chromatography, the 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 used directly in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became clear, allowed to cool naturally to 15°C, stirred for 4 hours, and then filtered by suction to obtain the compound of formula (7-1) (8.4 g of white solid, 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 their ratio was compound (7-1) / (7'-1) ≈ 92 / 8.

[0130] 2. Preparation of the compound of formula (7-2) [ka] In a flask, ethyl acetate (75 mL) and the crude product of the compound of formula (6-2) (10.05 g, 22.8 mmol) were added and stirred until clear and dissolved. Then, Raney Ni (15 g, wet weight, activated according to standard procedure) was added and the mixture was reacted at H2 (1 atm) at 55°C for 7 hours. After detecting the end of the reaction by gas chromatography, the 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 used directly in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became clear, allowed to cool naturally to 15°C, stirred for 4 hours, and then filtered by suction to obtain the compound of formula (7-2) (8.17 g white solid, 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 their ratio was compound (7-2) / (7'-2) ≈ 92 / 8.

[0132] 3. Preparation of the compound of formula (7-3) [ka] 75 mL of ethyl acetate and 10 g (22 mmol) of the crude product of the compound of formula (6-3) were added to a flask and stirred until clear and dissolved. Then, 10 g (wet weight, activated according to standard procedure) of Raney Ni was added and the mixture was reacted at 35°C in H2 (1 atm) for 10 hours. After detecting the end of the reaction by gas chromatography, the 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 used directly in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became clear, allowed to cool naturally to 15°C, stirred for 5 hours, and then filtered by suction to obtain the compound of formula (7-3) (8.3 g white solid, molar yield 82.5%, gas chromatography purity 99.1%). 1 1H 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 their ratio was compound (7-3) / (7'-3) ≈ 92 / 8.

[0134] 4. Preparation of the compound of formula (7-4) [ka] Tetrahydrofuran (35 mL), ethyl acetate (35 mL), and the crude product of the compound of formula (6-4) (10 g, 20.46 mmol) were added to a flask and stirred until clear and dissolved. Then, Raney Ni (10 g, wet weight, activated according to standard procedure) was added and the mixture was reacted at H2 (1 atm) at 35°C for 24 hours. After detecting the end of the reaction by gas chromatography, the 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 (7-4) as a white solid, which was used directly in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became clear, allowed to cool naturally to 15°C, stirred for 4-5 hours, and then filtered by suction to obtain the compound of formula (7-4) (8.33 g white solid, 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 their ratio was compound (7-4) / (7'-4) ≈ 92 / 8.

[0136] 5. Preparation of the compound of formula (7-5) [ka] Tetrahydrofuran (39 mL), ethyl acetate (39 mL), and the crude product of the compound of formula (6-5) (10 g, 19.2 mmol) were added to a flask and stirred until clear and dissolved. Then, Raney Ni (15 g, wet weight, activated according to standard procedure) was added and the mixture was reacted at H2 (1 atm) at 30°C for 20 hours. After detecting the end of the reaction by gas chromatography, the 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 (7-5) as a white solid, which was used directly in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became clear, allowed to cool naturally to 15°C, stirred for 4 hours, and then filtered by suction to obtain the compound of formula (7-5) (8.1 g white solid, 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 their ratio was compound (7-5) / (7'-5) ≈ 92 / 8.

[0138] 6. Preparation of the compound of formula (7-6) [ka] 75 mL of ethyl acetate and 10 g (20.04 mmol) of the crude product of the compound of formula (6-6) were added to a flask and stirred until clear and dissolved. Then, 10 g (wet weight, activated according to standard procedure) of Raney Ni was added and the mixture was reacted at 35°C under H2 (1 atm) for 15 hours. After detecting the end of the reaction by gas chromatography, the 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 used directly in the purification step. This white solid was added to a mixed solution of methanol and ethyl acetate, heated under reflux until the solution became clear, allowed to cool naturally to 15°C, stirred for 4 hours, and then filtered by suction to obtain the compound of formula (7-6) (8.25 g white solid, 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 their ratio was compound (7-6) / (7'-6) ≈ 92 / 8.

[0140] Example 8 Preparation of cholesterol by hydrolysis of compound (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 a flask and dissolved clearly. Under N2 protection, the compound of formula (7-1) (4.28 g, 10 mmol) was added, and the mixture was heated to 65°C and reacted for 2 hours. After detecting the end of the reaction 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. 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 filtered by suction, and water (20 mL) was added to the filter cake. The mixture was stirred at 25°C for 2 hours, filtered by suction, and dried to obtain purified cholesterol (3.80 g white solid, 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 (50 mL) and K2CO3 (2.07 g, 15 mmol) were added to a flask and dissolved clearly. Under N2 protection, the compound of formula (7-2) (4.4 g, 10 mmol) was added, and the mixture was heated to 65°C and reacted for 2 hours. After detecting the end of the reaction 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. 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 filtered by suction, and water (20 mL) was added to the filter cake. The mixture was stirred at 25°C for 2 hours, filtered by suction, and dried to obtain purified cholesterol (3.81 g white solid, 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 (45 mL) and K2CO3 (1.8 g, 13 mmol) were added to a flask and dissolved clearly. Under N2 protection, the compound of formula (7-3) (4.56 g, 10 mmol) was added, and the mixture was heated to 65°C and reacted for 2 hours. After detecting the end of the reaction 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. 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 filtered by suction, and water (20 mL) was added to the filter cake. The mixture was stirred at 25°C for 2 hours, filtered by suction, and dried to obtain purified cholesterol (3.8 g white solid, 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 (50 mL) and KOH (0.62 g, 11 mmol) were added to a flask and dissolved clearly. Under N2 protection, the compound of formula (7-4) (4.9 g, 10 mmol) was added, and the mixture was heated to 45°C and reacted for 6 hours. After detecting the end of the reaction 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. 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 filtered by suction, and 80% ethanol (10 mL) was added to the filter cake. The mixture was stirred at 25°C for 2 hours, filtered by suction, and dried to obtain purified cholesterol (3.66 g white solid, 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 (60 mL) and KOH (0.62 g, 11 mmol) were added to a flask and dissolved clearly. Under N2 protection, the compound of formula (7-5) (5.2 g, 10 mmol) was added and the mixture was heated to 45°C and reacted for 8 hours. After detecting the end of the reaction 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. 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 filtered by suction, and 80% ethanol (10 mL) was added to the filter cake. The mixture was stirred at 25°C for 2 hours, filtered by suction, and dried to obtain purified cholesterol (3.61 g white solid, molar yield 93.5%, gas chromatography purity 98.7%).

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

[0146] Comparative Example 1 As reported in the patent document (Background Art Scheme 2, CN105218610A), cholesterol was synthesized in a total molar yield of 67% through a five-step reaction using stigmasterol degradation products as a starting material. The reaction in the first step of the technical scheme in this patent document is shown in the following reaction formula 1. [ka]

[0147] In reaction formula 1 described in patent document CN105218610A, the compound of formula (02) after BA oxidation was used as a starting material, ethanol was used as a solvent, and under the action of p-toluenesulfonic acid and triethyl orthoformate, the mixture was heated to 40°C and held for 4 hours to obtain the compound of formula (03) (molar yield 97.50%).

[0148] According to the experimental method provided in the above-mentioned patent document, the present invention used ethanol as the solvent and the compound of formula (02) as the substrate, reacted at 40°C for 4 hours under the catalytic action of p-toluenesulfonic acid and triethyl orthoformate, and after TLC detected that the starting materials had reacted completely, post-treatment was performed according to the method of patent document (CN105218610A) to obtain the compound of formula (03') (shown in reaction formula 2), but this did not match the compound described in the said patent document. In the present invention, we also tried reducing the amount of triethyl orthoformate, but after TLC detected that the starting materials had reacted completely, the result of reaction formula 1 described in patent document (CN105218610A) was not obtained, and the result of reaction formula 3 below was 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, yielding compounds (03') and (03''), but not the compound of formula (03) described in Patent Document (CN105218610A). Clearly, the compounds of formula (03') and (03'') represented by reaction formula 2 or formula 3 cannot undergo the subsequent Wittig reaction. [ka]

[0149] Experimental method: Ethanol (4 ml), triethyl orthoformate (2 ml), 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. Complete reaction of the starting materials was detected by TLC. Sodium acetate (20 mg) was added under an ice bath, and the filtered cake was washed with water until the eluate was neutral. After draining, the mixture was purified by column chromatography (petroleum ether:ethyl acetate = 20:1) to obtain compound of formula (03') (2.30 g of colorless oily substance, 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 (4 ml), triethyl orthoformate (1 ml), 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. Complete reaction of the starting materials was detected by TLC. Sodium acetate (20 mg) was added under ice bath, and the filtered cake was washed with water until the eluate was neutral. After draining, the mixture was purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain compound of formula (03') (2.32 g of colorless oily substance, 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 of the Wittig reaction, reported in patent document (CN105218610A), is shown in the following reaction formula 4. [ka]

[0152] In this patent document, toluene was used as the solvent, triphenylphosphine and 1-chloro-3-methylbutane were added and refluxed for 2 hours, then potassium tert-butoxide and the compound of formula (03) were added and the reaction was carried out under reflux for 4 hours to obtain the compound of formula (04). Molar yield: 90.29%. For example, in the present invention, the method (Comparative Example 2, reaction formula 4) of patent document (CN105218610A) was adopted, and a Wittig reaction was carried out with the compound of formula (2) of the present invention using 1-chloro-3-methylbutane or 1-bromo-3-methylbutane, and triphenylphosphine and 1-chloro-3-methylbutane were refluxed for 2 hours. As shown in reaction formula 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]

[0153] 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 reacted under reflux for 2 hours. After cooling to 25°C, potassium tert-butoxide (307 mg, 2.74.02 mmol) was added in three portions under an ice bath, and the mixture was stirred under an ice bath for 0.5 hours. Then the compound of formula (2) (500 mg, 1.52 mmol) was added and reacted under reflux for 4 hours. After detecting the end of the reaction by TLC, the solution was cooled to 25°C, 2M HCl (4 mL) was added to neutralize it, and the solution was extracted with dichloromethane (50 mL) and water (50 mL). The organic phase was sequentially 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 colorless oil, 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 a flask and reacted under reflux for 2 hours. After cooling to 25°C, potassium tert-butoxide (307 mg, 2.74 mmol) was added in batches under an ice bath, and the mixture was stirred under an ice bath for 0.5 hours. Then the compound of formula (2) (500 mg, 1.52 mmol) was added and reacted under reflux for 2.5 hours. After detecting the end of the reaction by TLC, the solution was cooled to 25°C, 2M HCl (4 mL) was added to neutralize it, and the solution was extracted with dichloromethane (50 mL) and water (50 mL). The organic phase was sequentially 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 colorless oil, molar yield 88%).

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

[0156] Experimental method: Compound (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 detecting the end of the reaction by TLC, Raney Ni was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a mixture of formulas (6-1) and (6'-1). 1 Based on the 1H NMR results, the ratio of compound (6-1) to the isomer compound (6'-1) was 1:0.28.

[0157] Comparative Example 4 In this 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). This is as shown in the following reaction formula 7. [ka]

[0158] In this invention, by using H2 as a reducing agent and Raney Ni as a catalyst, the crude product of the compound of formula (7-1) obtained by selective hydrogenation reduction of the double bond of the side chain has an excess reduced impurity compound of formula (8-4) of less than 0.7%. 1 The concentration (determined by 1H NMR) is low and easily removed by purification. Furthermore, when selective hydrogenation reduction of the double bond of the side chain is performed using H2 as a reducing agent and 10% Pd / C as a catalyst, the crude product of the compound of formula (7-1) obtained contains an excess amount of the impurity compound of formula (8-4), which is the excess reduced form, exceeding 5%. 1 (As determined by 1H NMR) The amount of Pd was high and difficult to remove by purification, thus failing to achieve the intended objective. This indicates that 10% Pd / C has 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: 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 H2 (1 atm) and 30°C for 9 hours. After detecting the end of the reaction by TLC, Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure. 1 Judging from the 1H NMR spectrum, the content of the compound represented by formula (8-4), which is an excess reduction of the impurity, exceeds 5%.

[0160] The scope of protection of this invention is not limited to the embodiments described above. All modifications and advantages conceivable by those skilled in the art are included in this invention without departing from the spirit and scope of the invention, and the scope of protection is as defined by the appended claims.

Claims

1. A method for synthesizing cholesterol using plant-derived 21-hydroxy-20-methylpregna-4-en-3-one BA as a raw material, The above method involves using BA as a raw material and synthesizing the cholesterol via oxidation, Wittig reaction, acetylation, reduction, and selective hydrogenation, or synthesizing the cholesterol via oxidation, Wittig reaction, acetylation, reduction, hydroxyl protection, selective hydrogenation, deprotection, or hydrolysis. (a) A step of oxidizing BA represented by formula (1) in a first solvent to obtain the compound of formula (2); (b) The step of reacting the compound of formula (2) with the Wittig compound in a second solvent to obtain the compound of formula (3); (c) The step of acetylating the compound of formula (3) in a third solvent to obtain the compound of formula (4); (d) A step in which the compound of formula (4) is reduced in a fourth solvent to obtain the compound of formula (5); (e) A step of obtaining cholesterol by selectively hydrogenating and reducing the compound of formula (5) in a fifth solvent; Or, (f) In a sixth solvent, protect the hydroxyl group of the compound of formula (5) to obtain the compound of formula (6); (g) A step in which the compound of formula (6) is selectively subjected to a hydrogenation-reduction reaction in a seventh solvent to obtain the compound of formula (7); (h) A step of obtaining cholesterol by deprotecting or hydrolyzing the compound of formula (7) in an eighth solvent; Includes, In step (b), the Wittig reaction specifically involves adding 1-halo-3-methylbutane and triphenylphosphine to the second solvent, refluxing, cooling, and suction filtration to obtain a quaternary phosphine salt, then adding the quaternary phosphine salt and potassium tert-butoxide to the second solvent, and further adding the compound of formula (2) to undergo the Wittig reaction to obtain the compound of formula (3). Herein, the reaction process of the above method is as shown in scheme (A), which is a method for synthesizing cholesterol. 【Chemistry 64】 (Here, R-O is selected from an ester group and a silicon ether group.)

2. The ester group is a C2-C10 linear ester group, isobutyl ester 【Transformation 65】 isopentyl ester group 【Chemical Formula 66】 Phenylen ester group 【Transformation 67】 and p-methoxyphenyl ester group 【Transformation 68】 Selected from, The aforementioned silicon ether group is a trimethylsilyl ether group. 【Transformation 69】 and tert-butyldimethylsilyl ether group 【Transformation 70】 The method according to claim 1, characterized in that it is selected from among.

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, TEMPO, sodium bicarbonate, tetrabutylammonium bromide, and oxidizing agent represented by formula (1) 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 to 30°C, and / or the time of the oxidation reaction is 3 to 8 hours.

5. The method according to claim 1, characterized in that 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.

6. 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 the compound of formula (4).

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

8. 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) and a reducing agent are reduced in the fourth solvent to obtain the compound of formula (5).

9. The molar ratio of the compound of formula (4) to the reducing agent is 1:(1 to 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 KBH ４ The method according to 8, characterized in that it is one or two selected from, and / or the temperature of the reduction reaction is 0 to 50°C, and / or the duration of the reduction reaction is 6 to 12 hours.

10. 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-reduce with a reducing agent in the fifth solvent under the action of a catalyst to obtain cholesterol.

11. The reducing agent is H ２ The catalyst is selected from and / or Raney Ni, and / or the mass ratio of the compound of formula (5) to the catalyst is 1:(0.05 to 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 to 60°C, and / or the reducing agent H in the hydrogenation-reduction reaction ２ The method according to 10, characterized in that the pressure is 1 to 20 atm and / or the duration of the hydrogenation-reduction reaction is 6 to 10 hours.

12. The method according to claim 1, characterized in that, in step (f), the hydroxyl protection reaction is specifically a reaction in which the compound of formula (5) and a hydroxyl protecting agent are reacted in a sixth solvent under the action of a base to obtain the compound of formula (6).

13. When R-O 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 protection reagent and the base is 1:(1-4):(0.05-5), and / or the reaction temperature is 0-50°C, and / or the duration of the hydroxyl protection reaction is 2-24 hours. The method according to 12, characterized in that, when R-O is a silicon 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 hydroxyl protection reagent, and the base is 1:(2-4):(4-8), and / or the temperature of the hydroxyl protection reaction is 0-50°C, and / or the time of the hydroxyl protection reaction is 2-24 hours.

14. 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 selectively hydrogenated-reduce with a reducing agent in the seventh solvent under the action of a catalyst to obtain cholesterol.

15. The reducing agent is H ２ The catalyst is selected from and / or Raney Ni, and / or the mass ratio of the compound of formula (6) to the catalyst is 1:(0.05 to 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 to 60°C, and / or the reducing agent H in the hydrogenation-reduction reaction ２ The method according to 14, characterized in that the pressure is 1 to 20 atm and / or the duration of the hydrogenation-reduction reaction is 4 to 48 hours.

16. The method according to claim 1, characterized in that, when R-O is an ester group, in step (h), 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.

17. The aforementioned bases are LiOH, KOH, NaOH, t-BuOK, and K ２ CO ３ The method according to 16, characterized in that the eighth solvent is one or more selected from and / or the molar ratio of the compound of formula (7) to the base is 1:(0.5 to 2), and / or the eighth 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 hydrolysis reaction time is 0.3 to 12 hours.

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

19. The catalyst is one or more selected from tetrabutylammonium fluoride TBAF, tetrabutylammonium fluoride trihydrate TBAF·3 ２ O, boron trifluoride etherate, acetic acid, and an ethyl acetate solution of hydrogen chloride, and / or the mass ratio of the compound of the formula (7) to the catalyst is 1:(1-6), and / or the eighth solvent is one or two 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. The method according to claim 18, characterized in that.

20. The compound is characterized in that its structure is as shown by 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'-5(E), 6'-6(E), 6'-1(Z), 6'-2(Z), 6'-3(Z), 6'-5(Z), 6'-6(Z). 【Chemistry 71】

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