Method for preparing plant-derived cholesterol through chemical catalysis

Abstract

The invention discloses a method for preparing plant-derived cholesterol through chemical catalysis, which is characterized in that plant-derived BA is used as a raw material, chemical catalytic reduction is carried out through an intermediate compound 3, compared with an enzyme method, a large number of proper enzymes do not need to be screened, the cost is low, the stability is good, in addition, the influence of reaction sites on the Grignard addition yield is avoided through hydroxyl protection, and the yield of the plant-derived cholesterol is improved. Finished product isomer impurities can be reduced, the product yield is improved, and purification treatment is facilitated. The whole preparation route does not need an expensive catalyst, the reaction condition is mild, the specificity is high, the high-purity cholesterol can be obtained only through simple aftertreatment in each step, the method is suitable for industrial production, and the quality can meet the standards of medical injection and pharmaceutic adjuvants.

Description

Technical Field

[0001] The invention relates to the technical field of cholesterol synthesis, in particular to a method for preparing plant-derived cholesterol by chemical catalysis. Background Art

[0002] Cholesterol, also known as cholesterol, is a derivative of cyclopentane polyhydrophenanthrene. It was first discovered in gallstones and is the first steroid compound discovered by humans. Chemist Fischer named it "cholesterol". Cholesterol is the main component of mammalian cell membranes and can interact with phospholipids to regulate lipid membrane structure and dynamic properties. At the same time, cholesterol has important physiological functions in the human body and is a precursor for the synthesis of bile acids and steroid hormones. Cholesterol is an amphiphilic substance and can be used as a pharmaceutical excipient such as liposome membrane material, emulsifier, ointment base, etc., especially in liposome preparations. It plays an important role in improving the stability of preparations. Traditional cholesterol comes from animals, mainly from the saponification of brains and spinal tendons of pigs, cattle, and sheep, or lanolin, and is extracted with organic solvents. Since many diseases are now found to be transmitted to humans by animals, according to the latest guidelines for cell therapy and gene therapy, the use of human and animal-derived materials should be avoided as much as possible. Therefore, it is urgent to develop a plant-derived, safe, green, and high-purity cholesterol synthesis method.

[0003] The synthesis of cholesterol is reported mainly in the following ways:

[0004] 1. Chinese patent document CN105218610A discloses the synthesis of cholesterol with a total molar yield of 67% using stigmasterol degradation products as raw materials through 5 steps of reaction. However, when triethyl orthoformate is used in the first step of the patent to etherify the C-3 carbonyl group, the selectivity is poor and the side chain aldehyde group is easily reacted to form acetals, so the feasibility of this route is questionable.

[0005] 2. Chinese patent document CN105218609A discloses the synthesis of cholesterol with a total molar yield of 72% using pregnenolone as a raw material through a four-step reaction. This route uses a noble metal rhodium catalyst and a chiral phosphine ligand, which is expensive and not suitable for large-scale industrial production.

[0006] 3. Chinese patent document CN105237603A discloses the synthesis of cholesterol with a total molar yield of 68% using stigmasterol as a raw material through a five-step reaction. This route uses ozone in the synthesis process, which places higher requirements on monitoring the reaction and equipment, and the economy and safety are not good enough.

[0007] 4. Chinese patent document CN115678953A discloses the synthesis of cholesterol using BA as raw material through four steps of sulfonylation, Grignard addition, esterification and hydrolysis reduction. The reaction conditions are mild, the operability is strong, the specificity is strong, the isomer impurities of the reduction reaction can be effectively controlled, and the yield is high. However, there are multiple reaction sites in the second step of the reaction, the yield is low, and the impurities are difficult to remove, which is not suitable for industrial production.

[0008] 5. Chinese patent document CN113943336A discloses a method for synthesizing cholesterol using BA as a raw material, wherein the cholesterol is synthesized through the steps of oxidation, Wittig reaction, acetylation, reduction, and selective hydrogenation reduction. This route produces isomers in the second and fourth steps, which are difficult to separate. In addition, the last step uses a dangerous reagent, Raney nickel, and the degree of reaction of the selective hydrogenation reduction is difficult to control, which is not suitable for industrial production.

[0009] In summary, although the existing plant-derived cholesterol synthesis methods have their own advantages, they are complex processes, use a variety of toxic and hazardous reagents or dangerous processes, and are difficult to purify isomer impurities in the finished product. The yield and purity of the finished product are low, which makes it difficult to meet the requirements of large-scale industrial production. Therefore, it is urgent to develop a green, safe, and high-yield plant-derived cholesterol synthesis method. Summary of the invention

[0010] The present invention provides a method for preparing plant-derived cholesterol by chemical catalysis, so as to at least solve one of the many defects of the prior art, namely, high synthesis cost and the existence of isomers in the preparation process, which affects the purity and yield of the product.

[0011] In view of this, the scheme of the present invention is:

[0012] A method for preparing plant-derived cholesterol by chemical catalysis, comprising the following steps:

[0013] S1. Compound 3 undergoes reduction reaction under the action of a reducing agent, a catalyst and an organic base to generate compound 4;

[0014] S2. Compound 4 is protected by hydroxyl group under the action of organosilicon protecting agent to generate compound 5;

[0015] S3. Compound 5 undergoes a Grignard addition reaction under the action of a Grignard reagent to generate compound 6;

[0016] S4. Compound 6 undergoes a deprotection reaction under the action of a fluorination agent to generate compound 7, thereby obtaining plant-derived cholesterol;

[0017] The preparation method route is as follows:

[0018]

[0019] Further, in step S1:

[0020] The reducing agent is one or more selected from sodium borohydride, potassium borohydride, and lithium aluminum hydride, preferably sodium borohydride;

[0021] The catalyst is selected from one or more of calcium chloride, cerium chloride, and potassium chloride, preferably cerium chloride;

[0022] The organic base is selected from one or more of triethylamine, pyridine and imidazole, preferably pyridine;

[0023] The molar ratio of the compound 3 to the reducing agent, the catalyst and the organic base is 1:(1.5-4):(0.3-1):(1.5-3), preferably 1:2.2:0.3:2.5;

[0024] The reduction reaction temperature is -30 to 30°C, and the reaction time is 4 to 20 hours; preferably, the reaction is carried out at 0°C for 8 hours;

[0025] The solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, toluene and the like.

[0026] In the step S1, under the above conditions, the reduction reaction yield can reach 95%.

[0027] The post-treatment process of the reaction product in step S1 is as follows: adding purified water to the reaction product system, slowly adding acetic acid, stirring, filtering, rinsing with purified water, and collecting the filter cake. The filter cake is added to the reactor, anhydrous methanol is added, cooled to 0°C, and slurried for 1h. Filtering, rinsing the filter cake with 0°C anhydrous methanol, collecting the filter cake and vacuum drying can obtain compound 4 with a purity of more than 91%.

[0028] Further, in step S2:

[0029] The organosilicon protection agent is a chlorosilane, including but not limited to one or more of trimethylchlorosilane, tert-butyldimethylchlorosilane, triethoxychlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, preferably tert-butyldimethylchlorosilane;

[0030] The hydroxyl protection reaction also uses triethylamine, and the molar ratio of the compound 4 to the organosilicon protection reagent and triethylamine is 1:(1.2-2):(1.3-2), preferably 1:1.5:2;

[0031] The hydroxyl protection reaction temperature is 0 to 30°C, preferably 20°C;

[0032] The solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, N,N-dimethylformamide, N,N-dimethylacetamide, etc., preferably dichloromethane.

[0033] As described above, under the above conditions, the yield of the hydroxyl protection reaction in step S2 can reach 99%.

[0034] The post-treatment process of the reaction product in step S2 is as follows: adjust the pH to 7-8 with dilute hydrochloric acid, stand and separate the liquid, and remove the aqueous phase. Add purified water to the organic phase, stir, separate the aqueous phase, and repeat the above operation once. Add anhydrous sodium sulfate to the organic phase, stir and filter, collect the filtrate, and concentrate. Add anhydrous methanol, cool to 0°C, and beat for 1h. Filter, rinse with 0°C anhydrous methanol and collect the filter cake, and after vacuum drying, compound 5 with a purity of more than 98% can be obtained.

[0035] Further, in step S3:

[0036] The Grignard reaction is a reaction of compound 5 with isopropylmagnesium bromide and lithium tetrachlorocuprate in a molar ratio of 1:(2-8):(0.1-1); preferably, the molar ratio is 1:4:0.3.

[0037] Preferably, the Grignard reagent is prepared by reacting isopentyl bromide, magnesium powder and iodine in tetrahydrofuran; the molar ratio of isopentyl bromide, magnesium powder and iodine is 1:(1.1-1.5):(0.01-0.5), more preferably 1:1.2:0.03.

[0038] Preferably, the lithium tetrachlorocuprate solution is obtained by reacting a solution of copper chloride and lithium chloride in tetrahydrofuran, and the molar ratio of copper chloride to lithium chloride is 1:(1.5-3), more preferably 1:1.2:0.03.

[0039] Preferably, the Grignard addition reaction temperature is -30 to 25°C, and the reaction time is 3 to 20 hours; more preferably, the reaction temperature for dropwise addition of the Grignard reagent is -20°C, and after the dropwise addition is completed, the temperature is raised to 20°C and the reaction is continued for 8 hours.

[0040] Preferably, the Grignard addition reaction is carried out in an inert atmosphere.

[0041] Preferably, the solvent used in the Grignard addition reaction is selected from one or more of tetrahydrofuran, dimethyltetrahydrofuran, acetonitrile and the like.

[0042] As mentioned above, under the above conditions, the yield of the Grignard addition reaction in step S3 can reach 90%.

[0043] The post-treatment process of the reaction product in step S3 is as follows: take a sample point plate, after the raw material basically disappears, control the temperature at 0-20°C, add 5% hydrochloric acid to quench, precipitate a large amount of black solid, filter and decompress until there is basically no fraction. Add appropriate amount of water and DCM, stir and dissolve for 0.5-1 hour. Separate the liquid. The organic layer is concentrated under reduced pressure at 40-45°C until there is basically no fraction to obtain a light yellow oily solid, add appropriate amount of anhydrous methanol to the reactor, cool to 0°C, beat for 1h, filter to obtain a white solid, and dry to obtain compound 6 with a purity of more than 92%.

[0044] Further, in step S4:

[0045] The fluorination agent is selected from one or more of ferric chloride, tetrabutylammonium fluoride, and trifluoroacetic acid, preferably tetrabutylammonium fluoride;

[0046] The molar ratio of the compound 6 to the fluorination agent is 1:(2-15), preferably 1:4.

[0047] The solvent used in the reaction process is selected from one or more of tetrahydrofuran, dimethyltetrahydrofuran, acetonitrile, methanol and ethanol, preferably tetrahydrofuran.

[0048] As described above, under the above conditions, the reaction yield of step S4 can reach 99%.

[0049] The post-treatment process of the reaction product in step S4 is to distill the reaction product solution under reduced pressure until it is nearly dry, wash with water to precipitate a white solid, filter, and vacuum dry to obtain compound 7 with a purity of more than 99%.

[0050] Furthermore, the compound 3 uses BA as a raw material, first undergoes a sulfonylation reaction to obtain the compound 2, and the compound 2 is then obtained by an esterification reaction; the reaction process route is as follows:

[0051]

[0052] For the preparation of compound 2:

[0053] Preferably, the sulfonylation reaction process is that compound 1 (BA) is carried out under the action of sulfonyl chloride, 4-dimethylaminopyridine and triethylamine; the molar ratio of BA, sulfonyl chloride, 4-dimethylaminopyridine and triethylamine is 1: (1.1-2): (0.05-0.2): (1.2-3); preferably 1: 1.5: 0.15: 2;

[0054] The sulfonylation reaction temperature is 0 to 30°C, preferably 10°C;

[0055] The solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, toluene and the like.

[0056] Preferably, the sulfonyl chloride is selected from one or more of benzenesulfonic acid chloride, p-toluenesulfonic acid chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, and trifluoromethanesulfonate.

[0057] As described above, according to the above conditions, the yield of compound 2 prepared based on BA can reach 99%.

[0058] The post-treatment process of the product of the preparation process of compound 2 is as follows: cooling to room temperature, adding purified water, adjusting the pH value to neutral, standing for separation, collecting and concentrating the organic phase, adding anhydrous methanol to the concentrate, cooling to 0-5°C, beating for 2h, filtering, rinsing the filter cake with anhydrous methanol, collecting the filter cake, and vacuum drying to obtain compound 2 with a purity of more than 96%.

[0059] The process of preparing compound 3 from compound 2:

[0060] The reagents used in the esterification reaction include an esterification reagent and p-toluenesulfonic acid, and the molar ratio of the compound 2 to the esterification reagent and p-toluenesulfonic acid is 1:(5-20):(0.1-0.5), preferably 1:10:0.3;

[0061] The esterification agent is selected from one or more of isopropyl acetate, acetic anhydride acetyl chloride, and acetic anhydride, preferably isopropyl acetate;

[0062] The temperature of the esterification reaction is 0 to 50°C, preferably 40°C;

[0063] The solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, toluene, etc., preferably dichloromethane.

[0064] As described above, according to the above conditions, the yield of compound 3 prepared based on compound 2 can reach 97%.

[0065] The post-treatment process of the product of the preparation process of compound 3 is as follows: the reaction product system is concentrated, cooled to 0°C, anhydrous methanol is slowly added (violently exothermic), and slurrying is performed for 2 hours. Filtering, anhydrous methanol is eluted to filter cakes, and the filter cakes are collected and vacuum dried to obtain compound 3 with a purity of more than 95%.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] The preparation method of the present invention performs chemical catalytic reduction through the intermediate compound 3. Compared with the enzymatic method, it does not require a large amount of screening of suitable enzymes, has low cost and good stability. In addition, the influence of the reaction site on the Grignard addition yield is avoided by hydroxyl protection, which can reduce the isomeric impurities in the finished product, improve the product yield, and facilitate purification treatment.

[0068] The preparation method of the present invention can prepare cholesterol based on plant-derived BA as raw material. The overall preparation route does not require expensive catalysts, the reaction conditions are mild, and the specificity is strong. High-purity cholesterol can be obtained by simple post-treatment in each step. It is suitable for industrial production, and the quality can meet the standards of medical injections and pharmaceutical excipients. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the H NMR spectrum of compound 2 prepared in Example 1 of the present invention.

[0070] Figure 2 This is the H-NMR spectrum of the plant-derived cholesterol prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0071] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] Example 1

[0073] 1) Preparation of Compound 2

[0074] Add 200 mL of dichloromethane to the reactor and start stirring. Continue to add 2.4 g of DMAP, 50 g of BA and 46 ml of triethylamine, cool to about 10°C, and slowly add 45 g of p-toluenesulfonyl chloride dissolved in 50 ml of dichloromethane. Heat to 40°C, stir for 4.5 hours, monitor by TLC, and the reaction is complete. Cool to room temperature, add 250 ml of purified water, and add concentrated hydrochloric acid to adjust the pH value of the system to neutral. Let stand to separate the liquids, collect the organic phase, and concentrate the organic phase. Add 100 ml of anhydrous methanol to the concentrate, cool to 0-5°C, and beat for 2 hours. Filter, rinse the filter cake with 0°C anhydrous methanol, and collect the filter cake. Dry in a vacuum at 50°C to collect 72.5 g of compound 2, with a yield of 99% and a purity of 96.3%. The NMR hydrogen spectrum of the obtained compound 2 is as follows Figure 1 shown.

[0075] 2) Preparation of Compound 3

[0076] Add 180 ml of dichloromethane to the reactor and start stirring. Continue to add 60 g of compound 2, 6 g of p-toluenesulfonic acid and 120 ml of isopropyl acetate. Heat to 40 ° C and reflux, stir for 4-5 hours, and monitor by TLC. Concentrate the system, cool to 0 ° C, slowly add 100 mL of anhydrous methanol (violent heat release), and beat for 2 hours. Filter, rinse the filter cake with 10 g of anhydrous methanol at 0 ° C, and collect the filter cake. Dry in a vacuum at 50 ° C to collect 60 g of compound 3 with a yield of 92% and a purity of 95.4%.

[0077] 3) Preparation of Compound 4

[0078] Add tetrahydrofuran (120ml) and anhydrous methanol (120mL) to the reactor and start stirring. Continue to add 2.1g of anhydrous calcium chloride and 12mL of pyridine and stir until dissolved. Cool to -10~-15℃, add sodium borohydride in four batches (4.8g in total, 1.2g in each batch, with at least 10min interval between additions). Finally, add 30g of compound 3, naturally warm to room temperature, and stir overnight. Divide the above reaction system into two batches, add 300mL of purified water to each batch, and slowly add acetic acid (3mL) and stir for 30min. Filter, rinse with 10ml of purified water, and collect the filter cake. Combine the two batches of filter cakes and add them to the reactor, add 40ml of anhydrous methanol, cool to 0℃, and beat for 1h. Filter, rinse the filter cake with 0℃ anhydrous methanol, and collect the filter cake. Dry in vacuo at 50℃ to collect 25g of compound 4, with a yield of 90.6% and a purity of 91.2%.

[0079] 4) Preparation of Compound 5

[0080] Add 25 mL of dichloromethane to the reactor and start stirring. Continue to add 5 g of compound 4, 2 g of triethylamine and 3.15 g of TBSCl, stir overnight at room temperature, and monitor by TLC. Adjust the pH to 7-8 with 1 mol / L dilute hydrochloric acid, let stand to separate the liquid, and remove the aqueous phase. Add 10 ml of purified water to the organic phase, stir for 30 min, separate the aqueous phase, and repeat the above operation once. Add anhydrous sodium sulfate to the organic phase and stir for 30 min (observation of loose solids in the kettle is the drying end point). Filter, collect the filtrate, and concentrate to dryness. Add 10 mL of anhydrous methanol to the reactor, cool to 0 ° C, and beat for 1 hour. Filter, rinse the filter cake with 2 mL of anhydrous methanol at 0 ° C, and collect the filter cake. Dry in a vacuum at 50 ° C to collect 5.7 g of compound 5, with a yield of 95.1% and a purity of 98%.

[0081] 5) Preparation of Compound 6

[0082] a. Preparation of Grignard reagent: Add 2.11g magnesium powder, 0.1g iodine and dimethyltetrahydrofuran (40ml) into a three-necked flask, replace the gas with nitrogen three times, start stirring, and drop about bromoisopentane / THF (2ml) into the reaction solution. Wait for the reaction solution to change from brown to colorless (2-3 minutes). Start to drop the remaining bromoisopentane (12.08g) / THF (40ml) solution and raise the temperature to 45℃. After the dropwise addition, heat the Grignard reagent solution to 65℃ and react for 1h.

[0083] b. Preparation of lithium tetrachlorocuprate solution: Add copper chloride (0.456 g), lithium chloride (0.285 g), and THF (20 mL) to a 250 mL dry reaction bottle at room temperature, and replace with nitrogen three times. Control the temperature at 10-30°C and react for 1-2 hours until the solution becomes clear to obtain a lithium tetrachlorocuprate solution.

[0084] c. Cool the Grignard reagent solution to -20°C, add lithium tetrachlorocuprate solution, and then add a THF (60mL) solution of the intermediate compound 5 (6g). After the addition is complete, slowly heat up to room temperature and react for 3h. Take a sample point plate, the raw material basically disappears, control the temperature at 0-20°C, add 10mL 5% hydrochloric acid to quench, precipitate a large amount of black solid, filter and decompress until there is basically no fraction. Add 20mL water and 50mL DCM, stir and dissolve for 0.5 to 1 hour. Separate the liquid. The organic layer is concentrated under reduced pressure at 40-45°C until there is basically no fraction to obtain a light yellow oily solid. Add 10mL of anhydrous methanol to the reactor, cool to 0°C, beat for 1h, and filter to obtain 4.25g of white solid compound 6, with a yield of 85% and a purity of 92%.

[0085] 6) Add tetrahydrofuran and 4g of compound 6 to the reaction kettle, start stirring, add 10g of tetrabutylammonium fluoride, and stir for 1h. TLC monitors the completion of the reaction, and the solution is distilled to near dryness under reduced pressure. Add water (200ml) to wash the precipitated white solid, filter, and vacuum dry at 50℃ to obtain 2.96g of compound 7, with a yield of 96% and a purity of 99.3%. The product H NMR spectrum is as follows Figure 2 As shown, it was identified as cholesterol.

[0086] Example 2

[0087] 1) Preparation of Compound 2

[0088] Add 200 mL of dichloromethane to the reactor and start stirring. Continue to add 3 g DMAP, 50 g BA and 46 ml triethylamine, cool to about 10 ° C, dissolve 39 g of p-toluenesulfonyl chloride in 50 ml of dichloromethane and slowly add dropwise. Heat to 40 ° C, stir for 4.5 hours, monitor by TLC, and the reaction is complete. Cool to room temperature, add 250 ml of purified water, and add concentrated hydrochloric acid to adjust the pH value of the system to neutral. Let stand for separation, collect the organic phase, and concentrate the organic phase. Add 100 ml of anhydrous methanol to the concentrate, cool to 0-5 ° C, and beat for 2 hours. Filter, rinse the filter cake with 0 ° C anhydrous methanol, and collect the filter cake. Dry in a vacuum at 50 ° C to collect 71.3 g of compound 2, with a yield of 98.1% and a purity of 97.6%.

[0089] 2) Preparation of Compound 3

[0090] Add 180 ml of dichloromethane to the reactor and start stirring. Continue to add 60 g of compound 2, 7 g of p-toluenesulfonic acid and 90 ml of isopropenyl acetate. Heat to 40 ° C and reflux, stir for 4-5 hours, and monitor by TLC. Concentrate the system, cool to 0 ° C, slowly add 100 mL of anhydrous methanol (violent heat release), and beat for 2 hours. Filter, rinse the filter cake with 10 g of anhydrous methanol at 0 ° C, and collect the filter cake. Dry in a vacuum at 50 ° C to collect 61.g of compound 3 with a yield of 94.5% and a purity of 96.1%.

[0091] 3) Preparation of Compound 4

[0092] Add tetrahydrofuran (120ml) and anhydrous methanol (120mL) to the reactor and start stirring. Continue to add 2.3g of anhydrous calcium chloride and 14mL of pyridine and stir until dissolved. Cool to -10~-15℃, add sodium borohydride in four batches (6.4g in total, 1.6g in each batch, with at least 10min interval between additions). Finally, add 30g of compound 3, naturally warm to room temperature, and stir overnight. Divide the above reaction system into two batches, add 300mL of purified water to each batch, and slowly add acetic acid (3mL) and stir for 30min. Filter, rinse with 10ml of purified water, and collect the filter cake. Combine the two batches of filter cakes and add them to the reactor, add 40ml of anhydrous methanol, cool to 0℃, and beat for 1h. Filter, rinse the filter cake with 0℃ anhydrous methanol, and collect the filter cake. Dry in vacuo at 50℃ to collect 27.5g of compound 4, with a yield of 92.3% and a purity of 93.5%.

[0093] 4) Preparation of Compound 5

[0094] Add 25 mL of dichloromethane to the reactor and start stirring. Continue to add 5 g of compound 4, 1.5 g of triethylamine and 2.9 g of TBSCl, stir overnight at room temperature, and monitor by TLC. Adjust the pH to 7-8 with 1 mol / L dilute hydrochloric acid, let stand to separate the liquid, and remove the aqueous phase. Add 10 ml of purified water to the organic phase, stir for 30 min, separate the aqueous phase, and repeat the above operation once. Add anhydrous sodium sulfate to the organic phase and stir for 30 min (observation of loose solids in the kettle is the drying end point). Filter, collect the filtrate, and concentrate to dryness. Add 10 mL of anhydrous methanol to the reactor, cool to 0 ° C, and beat for 1 hour. Filter, rinse the filter cake with 2 mL of anhydrous methanol at 0 ° C, and collect the filter cake. Dry in a vacuum at 50 ° C to collect 5.84 g of compound 5, with a yield of 97.3% and a purity of 98.7%.

[0095] 5) Preparation of Compound 6

[0096] a. Preparation of Grignard reagent: Add 2.11g magnesium powder, 0.1g iodine and dimethyltetrahydrofuran (40ml) into a three-necked flask, replace the atmosphere with nitrogen three times, start stirring, and drop about bromoisopentane / THF (2ml) into the reaction solution. Wait for the reaction solution to change from brown to colorless (2-3 minutes). Start dropping the remaining bromoisopentane (8.5g) / THF (40ml) solution and raise the temperature to 45℃. After the dropwise addition, heat the Grignard reagent solution to 65℃ and react for 1h.

[0097] b. Preparation of lithium tetrachlorocuprate solution: Add copper chloride (0.456 g), lithium chloride (0.285 g), and THF (20 mL) to a 250 mL dry reaction bottle at room temperature, and replace with nitrogen three times. Control the temperature at 10-30°C and react for 1-2 hours until the solution becomes clear to obtain a lithium tetrachlorocuprate solution.

[0098] c. Cool the Grignard reagent solution to -20°C, add lithium tetrachlorocuprate solution, and then add a THF (60mL) solution of the intermediate compound 5 (6g). After the addition is complete, slowly heat up to room temperature and react for 3h. Take a sample point plate, the raw material basically disappears, control the temperature at 0-20°C, add 10mL 5% hydrochloric acid to quench, precipitate a large amount of black solid, filter and decompress until there is basically no fraction. Add 20mL water and 50mL DCM, stir and dissolve for 0.5 to 1 hour. Separate the liquid. The organic layer is concentrated under reduced pressure at 40-45°C until there is basically no fraction to obtain a light yellow oily solid. Add 10mL of anhydrous methanol to the reactor, cool to 0°C, beat for 1h, and filter to obtain 4.48g of white solid compound 6, with a yield of 89.5% and a purity of 91.8%.

[0099] 6) Preparation of Compound 7

[0100] Add tetrahydrofuran and 4g of compound 6 to the reaction kettle, start stirring, add 5g of tetrabutylammonium fluoride, and stir for 1h. TLC monitors the completion of the reaction, and the solution is distilled to near dryness under reduced pressure. Add water (200ml) to wash the precipitated white solid, filter it, and dry it in vacuo at 50℃ to obtain 3.02g of compound 7, with a yield of 97.6% and a purity of 99.5%.

[0101] Comparative Example 1

[0102] Compound 3 obtained in Example 1 was used to prepare Compound 4 by enzymatic reduction, specifically according to the steps of Example 6 of the specification of Chinese patent document CN115678953A. The yield of Compound 4 was 65.4%.

[0103] Comparative Example 2

[0104] Compound 7 was prepared by directly using compound 4 for Grignard addition reaction without hydroxyl protection. Compound 4 obtained in Example 1 was directly put into step 5) for Grignard addition reaction, and the reaction conditions were the same as those in Example 1. After the reaction, the post-treatment operation was the same as that in Example 1. The product yield was less than 50%, and there were many impurities. It was difficult to purify by conventional methods, and multiple column chromatography purifications were required, which was not suitable for industrial production.

[0105] It is not difficult to see from the above examples that, compared with Comparative Example 1, the chemical catalytic reduction method used in the example to prepare compound 4 has higher selectivity than the enzymatic catalytic reduction method used in CN115678953A, and can avoid reducing the content of isomers, thereby improving the yield. In addition, compared with Comparative Example 2, the Grignard addition reaction can avoid the influence of the reaction site on the Grignard addition yield after protecting the hydroxyl group of compound 4, thereby improving the product yield.

[0106] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing plant-derived cholesterol by chemical catalysis, characterized in that the steps include: S1. Compound 3 undergoes reduction reaction under the action of a reducing agent, a catalyst and an organic base to generate compound 4; S2. Compound 4 is protected by hydroxyl group under the action of organosilicon protecting agent to generate compound 5; S3. Compound 5 undergoes a Grignard addition reaction under the action of a Grignard reagent to generate compound 6; S4. Compound 6 undergoes a deprotection reaction under the action of a fluorination agent to generate compound 7, thereby obtaining plant-derived cholesterol; The preparation method route is as follows:

2. The method according to claim 1, characterized in that In step S1, the reducing agent is one or more selected from sodium borohydride, potassium borohydride, and lithium aluminum hydride; And / or, the catalyst is selected from one or more of calcium chloride, cerium chloride, and potassium chloride; And / or, the organic base is selected from one or more of triethylamine, pyridine and imidazole; and / or, the molar ratio of the compound 3 to the reducing agent, the catalyst, and the organic base is 1:(1.5-4):(0.3-1):(1.5-3); And / or, the reduction reaction temperature is -30 to 30°C, and the reaction time is 4 to 20 hours; And / or, the solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, toluene and the like.

3. The method according to claim 1, characterized in that In step S2, the organosilicon protecting agent is selected from one or more of trimethylsilyl chloride, tert-butyldimethylsilyl chloride, triethoxysilyl chloride, tert-butyldiphenylsilyl chloride, and triisopropylsilyl chloride; And / or, the hydroxyl protection reaction further uses triethylamine, and the molar ratio of the compound 4 to the organosilicon protection reagent and triethylamine is 1:(1.2-2):(1.3-2); And / or, the hydroxyl protection reaction temperature is 0 to 30°C; And / or, the solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, N,N-dimethylformamide, N,N-dimethylacetamide and the like.

4. The method according to claim 1, characterized in that: In step S3, the Grignard reaction is a reaction of compound 5 with isopropylmagnesium bromide and lithium tetrachlorocuprate in a molar ratio of 1:(2-8):(0.1-1).

5. The method according to claim 4, characterized in that The Grignard reagent is prepared by reacting isopentyl bromide, magnesium powder and iodine in tetrahydrofuran; the molar ratio of isopentyl bromide, magnesium powder and iodine is 1:(1.1-1.5):(0.01-0.5); And / or, the lithium tetrachlorocuprate solution is obtained by reacting a solution of copper chloride and lithium chloride in tetrahydrofuran, and the molar ratio of copper chloride to lithium chloride is 1:(1.5-3); And / or, the Grignard addition reaction temperature is -30 to 25°C, and the reaction time is 3 to 20 hours; And / or, the Grignard addition reaction is carried out in an inert atmosphere; And / or, the solvent used in the Grignard addition reaction is selected from one or more of tetrahydrofuran, dimethyltetrahydrofuran, acetonitrile and the like.

6. The method according to claim 1, characterized in that In step S4, the fluorination agent is selected from one or more of ferric chloride, tetrabutylammonium fluoride, and trifluoroacetic acid; and / or, the molar ratio of the compound 6 to the fluorination agent is 1:(2-15); And / or, the solvent used in the reaction process is selected from one or more of tetrahydrofuran, dimethyltetrahydrofuran, acetonitrile, methanol and ethanol.

7. The method according to claim 1, characterized in that The compound 3 uses BA as a raw material, first undergoes a sulfonylation reaction to obtain the compound 2, and then the compound 2 undergoes an esterification reaction to obtain the compound 2; the reaction process is as follows:

8. The method according to claim 7, characterized in that The preparation process of the compound 2 is carried out under the action of sulfonyl chloride, 4-dimethylaminopyridine and triethylamine; the molar ratio of BA, sulfonyl chloride, 4-dimethylaminopyridine and triethylamine is 1: (1.1-2): (0.05-0.2): (1.2-3); and / or, the sulfonylation reaction temperature is 0 to 30° C.; And / or, the solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, toluene and the like.

9. The method according to claim 8, characterized in that The sulfonyl chloride is selected from one or more of benzenesulfonic acid chloride, p-toluenesulfonic acid chloride, methanesulfonyl chloride, trifluoromethanesulfonic anhydride, and trifluoromethanesulfonate.

10. The method according to claim 7, characterized in that The reagents used in the esterification reaction include an esterification reagent and p-toluenesulfonic acid. The molar ratio of the compound 2 to the esterification reagent and p-toluenesulfonic acid is 1:(5-20):(0.1-0.5). The esterification reagent is selected from one or more of isopropyl acetate, acetic anhydride acetyl chloride, and acetic anhydride. The temperature of the esterification reaction is 0-50°C. And / or, the solvent used in the reaction process is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetonitrile, toluene and the like.

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