A synthesis process of 25-hydroxycholesterol

Abstract

The present invention relates to a synthesis process of 25-hydroxycholesterol, and belongs to the technical field of drug preparation. The present invention uses phytosterol as a raw material, and prepares the target product 25-hydroxycholesterol by steps such as ozonation, side chain extension, reduction and recrystallization. During the reaction process, the reaction conditions are precisely controlled to improve the selectivity of the reaction and reduce by-products; the efficient use of ozone is achieved by continuous flow technology, while the by-product generation of peroxide is reduced; the design of combining nitrogen replacement and sodium borohydride reduction is used to avoid excessive oxidation and by-product generation; and the yield and purity of the product are significantly improved by multiple recrystallizations and post-treatments.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation and relates to a synthesis process of 25-hydroxycholesterol. Background Art

[0002] 25-Hydroxycholesterol (25-Hydroxycholesterol) is an important oxysterol molecule widely distributed in human tissues and blood. As a key intermediate in cholesterol metabolism, it regulates intracellular cholesterol homeostasis. By binding to liver X receptors (LXRs), it participates in the regulation of cholesterol and lipid metabolism, as well as immune responses. Furthermore, 25-Hydroxycholesterol has demonstrated a significant role in antiviral immunity, inhibiting the replication of certain viruses (such as hepatitis B virus and Ebola virus), making it a hot topic in infectious disease research. Due to its unique effects in physiological regulation, anti-inflammatory and antiviral functions, and cancer treatment, 25-Hydroxycholesterol is widely used in drug development, biomedical research, and diagnostic reagent preparation, possessing enormous application prospects and market value.

[0003] At present, the preparation methods of 25-hydroxycholesterol are mainly divided into three methods: natural extraction, chemical synthesis and biotransformation. The natural extraction method is to separate 25-hydroxycholesterol from animal tissues (such as liver), but due to the limited source of raw materials and the complex purification process, its yield and economy are limited; the biotransformation method uses microorganisms or enzymes to hydroxylate cholesterol, which has the advantages of high selectivity and environmental friendliness, but is limited by the stability of biocatalysts and the harsh reaction conditions, and its industrial application still faces challenges. The chemical synthesis method has become the focus of current research because of its cheap and easy-to-obtain raw materials and high controllability of reaction conditions. In particular, the method of using plant sterols (such as stigmasterol) as the starting material and synthesizing it through steps such as ozonation reaction, side chain extension reaction and reduction reaction has the potential for simple operation and large-scale production.

[0004] The existing 25-hydroxycholesterol preparation technology still has some shortcomings in practical applications. The natural extraction method is limited by biological resources and is difficult to meet the needs of large-scale industrial production; although the biotransformation method is environmentally friendly, its production efficiency and economy need to be improved. Although the chemical synthesis method can achieve higher yields and industrial applications, there are still problems such as the generation of a large number of by-products, complex steps, and harsh operating conditions during the reaction process. In addition, the catalysts and solvents used in some reactions may pollute the environment, limiting its further application in green chemistry. Therefore, the development of a more efficient, economical, and environmentally friendly method for synthesizing 25-hydroxycholesterol to further improve the yield, simplify the operation, and reduce by-products is an important direction of current research and industrial development. Summary of the Invention

[0005] The present invention relates to a process for synthesizing 25-hydroxycholesterol, belonging to the technical field of pharmaceutical preparation. Using plant sterols as raw materials, the process prepares the target product, 25-hydroxycholesterol, through steps such as ozonation, side chain extension, reduction, and recrystallization. During the reaction, precise control of reaction conditions improves reaction selectivity and reduces byproducts. Continuous flow technology achieves efficient ozone utilization while reducing the generation of peroxide byproducts. A design combining nitrogen replacement and sodium borohydride reduction avoids over-oxidation and byproduct generation. Multiple recrystallizations and post-treatments significantly improve product purity.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A synthesis process for 25-hydroxycholesterol, the synthesis route is as follows:

[0008]

[0009] As a preferred technical solution of the present invention, the preparation method of the compound 2 is to sequentially add dichloromethane, compound 1 stigmasterol, 4-dimethylaminopyridine and triethylamine to a reaction flask, stir evenly, add p-toluenesulfonyl chloride, stir at 20-30°C for 12-20h, concentrate under reduced pressure, add deionized water to precipitate the solid, filter and dry it, and then dissolve it with methanol solvent, add activated carbon and stir for 30-60min, filter, add potassium acetate, and heat under reflux for 1-3h, concentrate under reduced pressure, add deionized water for recrystallization, and then recrystallize in a methanol-water-petroleum ether system, and then filter and dry to obtain compound 2; the mass ratio of dichloromethane, stigmasterol, 4-dimethylaminopyridine, triethylamine, p-toluenesulfonyl chloride, solvent methanol, activated carbon, and potassium acetate is 4-6:0.5-1.5:0.01-0.05:0.2-0.4:0.4-0.6:8-12:0.5-1.5:60-80.

[0010] As a preferred technical solution of the present invention, the preparation method of compound 3 is to sequentially add dichloromethane, methanol, compound 2 and sodium bicarbonate to a reaction flask, cool to -30 ° C, and use a continuous flow ozone reactor to pass 0.5-2.0wt% ozone oxygen mixed gas, the flow rate is controlled at 5-20mL / min, and then maintained for 20-50S. After nitrogen is replaced in the system, sodium borohydride is added, stirred at 20-30 ° C for 0.5-1.5h, and saturated ammonium chloride aqueous solution is added to quench the reaction. After the solvent is removed by distillation under reduced pressure, ethyl acetate is added to extract the aqueous phase, and the resulting organic phase is washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then petroleum ether is added to precipitate the solid, which is filtered and dried to obtain compound 3; the mass ratio of dichloromethane, methanol, compound 2, sodium bicarbonate, and sodium borohydride is 7-9:0.7-0.9:0.8-1.0:0.4-0.6:0.1-0.2.

[0011] As a preferred technical solution of the present invention, the preparation method of the compound 4 is to sequentially add dichloromethane, compound 3, 4-dimethylaminopyridine and triethylamine to a reaction flask, stir evenly, then add p-toluenesulfonyl chloride, stir at 20-30°C for 12-20h, and after the system is concentrated under reduced pressure, add deionized water to precipitate a large amount of solid, filter, and dry the filter cake to obtain compound 4; the mass ratio of dichloromethane, compound 3, 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 40-80:4-8:0.1-0.3:1-3:2-4.

[0012] As a preferred technical solution of the present invention, the preparation method of the compound 5 is to add tetrahydrofuran, compound 4, lithium bromide and lithium carbonate in a reaction bottle in sequence, stir at 50-70°C for 14-18h, concentrate under reduced pressure, add deionized water to slurry, and then filter and dry to obtain compound 5; the mass ratio of tetrahydrofuran, compound 4, lithium bromide and lithium carbonate is 30-50:6-10:2-4:0.5-0.7.

[0013] As a preferred technical solution of the present invention, the preparation method of the compound 6 is to sequentially add pyridine, zinc powder, nickel chloride hexahydrate and 10wt% boron trifluoride ether solution into a reaction flask, add methyl acrylate dropwise, stir at 40-60°C for 1-3h, cool to 20°C, add compound 5, stir at 25-35°C for 2-4h, add ethyl acetate to dilute, filter through diatomaceous earth, rinse the filter cake with ethyl acetate, add 10% hydrochloric acid to the filtrate to adjust the pH to 3, extract and separate the liquid, wash the organic phase with water to pH = 7, and then concentrate under reduced pressure to dryness, add methanol for pulping, filter, and dry the filter cake to obtain compound 6; the mass ratio of the pyridine, zinc powder, nickel chloride hexahydrate, 10wt% boron trifluoride ether solution, methyl acrylate, compound 5, and ethyl acetate is 30-40:6-8:8-10:0.5-1:15-17:7-8:15-25.

[0014] As a preferred technical solution of the present invention, the preparation method of the compound 7 is to add 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 in a reaction bottle in sequence, stir at 45-55 ° C for 2-4 hours, concentrate under reduced pressure, add pure water for pulping, filter, and dry to obtain compound 7; the mass ratio of the 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 is 20-24:10-12:0.1-0.5:4-6.

[0015] As a preferred technical solution of the present invention, the preparation method of the compound 8 is to add tetrahydrofuran and compound 7 to the reaction bottle in sequence and mix them evenly, cool to -10°C, add 3M methylmagnesium bromide solution dropwise, stir at 30-35°C for 0.5-1.5h, add saturated ammonium chloride aqueous solution to quench, concentrate under reduced pressure, add dichloromethane to extract the aqueous phase, and the resulting organic phase is washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure again, methanol is added to precipitate the solid, and the solid is dried by filtration to obtain compound 8; the mass ratio of the tetrahydrofuran, compound 7, methylmagnesium bromide solution, and saturated ammonium chloride aqueous solution is 30-50:3-5:10-20:5-10.

[0016] Beneficial effects of the present invention:

[0017] (1) In the conventional method, when synthesizing compound 2, the solid is precipitated by dissolving it in methanol and refluxing it. However, methanol cannot completely wash away the by-products generated in the reaction (such as excess p-toluenesulfonic acid). In the present invention, activated carbon is added for adsorption treatment before reflux to remove pigments and trace organic impurities. After the reflux treatment, a recrystallization step is introduced in a methanol-water-petroleum ether system to further improve the purity.

[0018] (2) During the ozonation process, there may be peroxidation reactions or side reactions. The present invention uses a continuous flow ozone reactor to precisely control the flow rate and residence time to ensure that the concentration of ozone is uniform throughout the system, avoid local peroxidation or side reactions, improve ozone utilization, reduce the generation of by-products, and improve operational safety. Ozone is a strong oxidant. If there is residual ozone or oxygen in the reaction system, it may be further oxidized after the target compound is generated, triggering side reactions, generating unwanted peroxides or other oxidation products, and reducing the yield of the target compound. By nitrogen replacement, ozone or oxygen can be completely removed from the reaction system, thereby avoiding excessive oxidation of reactants or intermediates. After nitrogen replacement, sodium borohydride quickly reduces the carbonyl compound to alcohol, avoiding side reactions caused by the instability of the carbonyl compound.

[0019] (3) In the synthesis of compound 6, methyl acrylate may generate a double addition by-product during the addition process. The present invention adjusts the addition rate of methyl acrylate and strictly controls the temperature to prevent unnecessary secondary addition, and adds a Lewis acid catalyst (10 wt% boron trifluoride etherate solution) to improve the reaction selectivity. DETAILED DESCRIPTION

[0020] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0021] Example 1

[0022] A synthesis process for 25-hydroxycholesterol, the synthesis route is as follows:

[0023]

[0024] The preparation method of the compound 2 is as follows: dichloromethane, compound 1 stigmasterol, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, stirred evenly, and then p-toluenesulfonyl chloride is added, stirred at 25° C. for 16 hours, concentrated under reduced pressure, deionized water is added to precipitate a solid, filtered and dried, and then dissolved with solvent methanol, activated carbon is added and stirred for 40 minutes, filtered, potassium acetate is added, heated under reflux for 2 hours, concentrated under reduced pressure, and then deionized water is added for recrystallization, and then recrystallization is performed in a methanol-water-petroleum ether system, and then filtered and dried to obtain compound 2; the mass ratio of the dichloromethane, stigmasterol, 4-dimethylaminopyridine, triethylamine, p-toluenesulfonyl chloride, solvent methanol, activated carbon, and potassium acetate is 5:1:0.03:0.3:0.5:10:1:70.

[0025] The preparation method of the compound 3 is as follows: dichloromethane, methanol, compound 2 and sodium bicarbonate are added to the reaction bottle in sequence, the temperature is lowered to -30 ° C, and a 1.0wt% ozone oxygen mixed gas is passed through a continuous flow ozone reactor, the flow rate is controlled at 10 mL / min, and the gas is vented for 60S and then maintained for 30S. After the nitrogen is replaced in the system, sodium borohydride is added, and the mixture is stirred at 25 ° C for 1 hour. After adding a saturated aqueous ammonium chloride solution to quench the reaction, the solvent is removed by distillation under reduced pressure, and then ethyl acetate is added to extract the aqueous phase. The obtained organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then petroleum ether is added to precipitate the solid, which is filtered and dried to obtain compound 3; the mass ratio of the dichloromethane, methanol, compound 2, sodium bicarbonate, and sodium borohydride is 8:0.8:0.9:0.5:0.15.

[0026] The preparation method of the compound 4 is as follows: dichloromethane, compound 3, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, and p-toluenesulfonyl chloride is added after stirring evenly. The mixture is stirred at 25° C. for 16 hours. After the system is concentrated under reduced pressure, deionized water is added to precipitate a large amount of solid, which is filtered and the filter cake is dried to obtain compound 4; the mass ratio of dichloromethane, compound 3, 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 60:6:0.2:2:3.

[0027] The preparation method of the compound 5 is as follows: tetrahydrofuran, compound 4, lithium bromide and lithium carbonate are added to a reaction bottle in sequence, stirred at 60°C for 16 hours, concentrated under reduced pressure, added with deionized water, slurried, and then filtered and dried to obtain compound 5; the mass ratio of tetrahydrofuran, compound 4, lithium bromide and lithium carbonate is 40:8:3:0.6.

[0028] The preparation method of the compound 6 is as follows: pyridine, zinc powder, nickel chloride hexahydrate and 10wt% boron trifluoride ether solution are added to a reaction flask in sequence, methyl acrylate is added dropwise, and the mixture is stirred at 50°C for 2h. After cooling to 20°C, compound 5 is added, and the mixture is stirred at 30°C for 3h. Ethyl acetate is added for dilution, and the mixture is filtered through diatomaceous earth. The filter cake is rinsed with ethyl acetate, 10% hydrochloric acid is added to the filtrate to adjust the pH to 3, and the liquid is extracted and separated. The organic phase is washed with water to pH = 7 and then concentrated to dryness under reduced pressure. Methanol is added for pulping, filtered, and the filter cake is dried to obtain compound 6; the mass ratio of the pyridine, zinc powder, nickel chloride hexahydrate, 10wt% boron trifluoride ether solution, methyl acrylate, compound 5, and ethyl acetate is 35:7:9:0.8:16:7.5:20.

[0029] The preparation method of compound 7 is to add 1,4-dioxane, deionized water, 98% concentrated sulfuric acid and compound 6 in a reaction bottle in sequence, stir at 50°C for 3 hours, concentrate under reduced pressure, add pure water for pulping, filter, and dry to obtain compound 7; the mass ratio of 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 is 22:11:0.3:5.

[0030] The preparation method of the compound 8 is as follows: tetrahydrofuran and compound 7 are added to a reaction flask in sequence and mixed evenly, the temperature is lowered to -10°C, a 3M methylmagnesium bromide solution is added dropwise, and the mixture is stirred at 32°C for 1 hour. A saturated aqueous ammonium chloride solution is added to quench the mixture, and the mixture is concentrated under reduced pressure. After that, dichloromethane is added to extract the aqueous phase. The obtained organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure again. Methanol is added to precipitate a solid, which is then filtered and dried to obtain compound 8. The mass ratio of the tetrahydrofuran, compound 7, methylmagnesium bromide solution, and saturated aqueous ammonium chloride is 40:4:15:8.

[0031] Example 2

[0032] A synthesis process for 25-hydroxycholesterol, the synthesis route is as follows:

[0033]

[0034] The preparation method of the compound 2 is as follows: dichloromethane, compound 1 stigmasterol, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, stirred evenly, and then p-toluenesulfonyl chloride is added, stirred at 20° C. for 12 hours, concentrated under reduced pressure, deionized water is added to precipitate a solid, filtered and dried, and then dissolved with solvent methanol, activated carbon is added and stirred for 30 minutes, filtered, potassium acetate is added, heated under reflux for 1 hour, concentrated under reduced pressure, and then deionized water is added for recrystallization, and then recrystallization is performed in a methanol-water-petroleum ether system, and then filtered and dried to obtain compound 2; the mass ratio of the dichloromethane, stigmasterol, 4-dimethylaminopyridine, triethylamine, p-toluenesulfonyl chloride, solvent methanol, activated carbon, and potassium acetate is 4:0.5:0.01:0.2:0.4:8:0.5:60.

[0035] The preparation method of the compound 3 is as follows: dichloromethane, methanol, compound 2 and sodium bicarbonate are added to a reaction flask in sequence, the temperature is lowered to -30°C, 0.5wt% ozone oxygen mixed gas is passed through a continuous flow ozone reactor, the flow rate is controlled at 5mL / min for 30S and then maintained for 20S, the system is replaced with nitrogen, sodium borohydride is added, and the mixture is stirred at 20°C for 0.5h. After adding a saturated aqueous ammonium chloride solution to quench the reaction, the solvent is removed by distillation under reduced pressure, and ethyl acetate is added to extract the aqueous phase. The resulting organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then petroleum ether is added to precipitate a solid, which is filtered and dried to obtain compound 3; the mass ratio of the dichloromethane, methanol, compound 2, sodium bicarbonate, and sodium borohydride is 7:0.7:0.:0.4:0.1.

[0036] The preparation method of the compound 4 is as follows: dichloromethane, compound 3, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, and p-toluenesulfonyl chloride is added after stirring evenly. The mixture is stirred at 20° C. for 12 hours. After the system is concentrated under reduced pressure, deionized water is added to precipitate a large amount of solid, which is filtered and the filter cake is dried to obtain compound 4; the mass ratio of dichloromethane, compound 3, 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 40:4:0.1:1:2.

[0037] The preparation method of the compound 5 is as follows: tetrahydrofuran, compound 4, lithium bromide and lithium carbonate are added to a reaction bottle in sequence, stirred at 50°C for 14 hours, concentrated under reduced pressure, added with deionized water, slurried, and then filtered and dried to obtain compound 5; the mass ratio of tetrahydrofuran, compound 4, lithium bromide and lithium carbonate is 30:6:2:0.5.

[0038] The preparation method of the compound 6 is as follows: pyridine, zinc powder, nickel chloride hexahydrate and 10wt% boron trifluoride ether solution are added to a reaction flask in sequence, methyl acrylate is added dropwise, stirred at 4°C for 1h, cooled to 20°C, compound 5 is added, stirred at 25°C for 2h, ethyl acetate is added for dilution, filtered through diatomaceous earth, the filter cake is rinsed with ethyl acetate, 10% hydrochloric acid is added to the filtrate to adjust the pH to 3, extraction and separation are carried out, the organic phase is washed with water to pH = 7, and then concentrated to dryness under reduced pressure, methanol is added for pulping, filtered, and the filter cake is dried to obtain compound 6; the mass ratio of the pyridine, zinc powder, nickel chloride hexahydrate, 10wt% boron trifluoride ether solution, methyl acrylate, compound 5, and ethyl acetate is 30:6:8:0.5:15:7:15.

[0039] The preparation method of compound 7 is to add 1,4-dioxane, deionized water, 98% concentrated sulfuric acid and compound 6 in a reaction bottle in sequence, stir at 45°C for 2 hours, concentrate under reduced pressure, add pure water for pulping, filter, and dry to obtain compound 7; the mass ratio of 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 is 20:10:0.1:4.

[0040] The preparation method of the compound 8 is as follows: tetrahydrofuran and compound 7 are added to a reaction flask in sequence and mixed evenly, the mixture is cooled to -10°C, a 3M methylmagnesium bromide solution is added dropwise, and the mixture is stirred at 30°C for 0.5h. A saturated aqueous ammonium chloride solution is added to quench the mixture, and the mixture is concentrated under reduced pressure. After that, dichloromethane is added to extract the aqueous phase. The obtained organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure again. Methanol is added to precipitate a solid, which is then filtered and dried to obtain compound 8; the mass ratio of the tetrahydrofuran, compound 7, methylmagnesium bromide solution, and saturated aqueous ammonium chloride is 30:3:10:5.

[0041] Example 3

[0042] A synthesis process for 25-hydroxycholesterol, the synthesis route is as follows:

[0043]

[0044] The preparation method of the compound 2 is as follows: dichloromethane, compound 1 stigmasterol, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, stirred evenly, and then p-toluenesulfonyl chloride is added, stirred at 30°C for 20 hours, concentrated under reduced pressure, deionized water is added to precipitate a solid, filtered and dried, and then dissolved with solvent methanol, activated carbon is added and stirred for 60 minutes, filtered, potassium acetate is added, heated under reflux for 3 hours, concentrated under reduced pressure, and then deionized water is added for recrystallization, and then recrystallization is performed in a methanol-water-petroleum ether system, and then filtered and dried to obtain compound 2; the mass ratio of the dichloromethane, stigmasterol, 4-dimethylaminopyridine, triethylamine, p-toluenesulfonyl chloride, solvent methanol, activated carbon, and potassium acetate is 6:1.5:0.05:0.4:0.6:12:1.5:80.

[0045] The preparation method of the compound 3 is as follows: dichloromethane, methanol, compound 2 and sodium bicarbonate are added to a reaction flask in sequence, the temperature is lowered to -30°C, 2.0wt% ozone oxygen mixed gas is passed through a continuous flow ozone reactor, the flow rate is controlled at 20mL / min, the ventilation is maintained for 120S and then 50S, the system is replaced with nitrogen, sodium borohydride is added, and the mixture is stirred at 30°C for 1.5h. After adding a saturated aqueous ammonium chloride solution to quench the reaction, the solvent is removed by distillation under reduced pressure, and ethyl acetate is added to extract the aqueous phase. The resulting organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then petroleum ether is added to precipitate a solid, which is filtered and dried to obtain compound 3; the mass ratio of the dichloromethane, methanol, compound 2, sodium bicarbonate, and sodium borohydride is 9:0.9:1.0:0.6:0.2.

[0046] The preparation method of the compound 4 is as follows: dichloromethane, compound 3, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, and p-toluenesulfonyl chloride is added after stirring evenly. The mixture is stirred at 30° C. for 20 hours. After the system is concentrated under reduced pressure, deionized water is added to precipitate a large amount of solid, which is filtered and the filter cake is dried to obtain compound 4; the mass ratio of the dichloromethane, compound 3, 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 80:8:0.3:3:4.

[0047] The preparation method of the compound 5 is as follows: tetrahydrofuran, compound 4, lithium bromide and lithium carbonate are added to a reaction bottle in sequence, stirred at 70°C for 18 hours, concentrated under reduced pressure, added with deionized water, slurried, and then filtered and dried to obtain compound 5; the mass ratio of tetrahydrofuran, compound 4, lithium bromide and lithium carbonate is 50:10:4:0.7.

[0048] The preparation method of the compound 6 is as follows: pyridine, zinc powder, nickel chloride hexahydrate and 10wt% boron trifluoride ether solution are added to a reaction flask in sequence, methyl acrylate is added dropwise, and the mixture is stirred at 60°C for 3h. After cooling to 20°C, compound 5 is added and stirred at 35°C for 4h. Ethyl acetate is added for dilution, and the mixture is filtered through diatomaceous earth. The filter cake is rinsed with ethyl acetate, 10% hydrochloric acid is added to the filtrate to adjust the pH to 3, and the liquid is extracted and separated. The organic phase is washed with water to pH = 7 and then concentrated to dryness under reduced pressure. Methanol is added for pulping, filtered, and the filter cake is dried to obtain compound 6; the mass ratio of the pyridine, zinc powder, nickel chloride hexahydrate, 10wt% boron trifluoride ether solution, methyl acrylate, compound 5, and ethyl acetate is 40:8:10:1:17:8:25.

[0049] The preparation method of compound 7 is to add 1,4-dioxane, deionized water, 98% concentrated sulfuric acid and compound 6 in a reaction bottle in sequence, stir at 55°C for 4 hours, concentrate under reduced pressure, add pure water for pulping, filter, and dry to obtain compound 7; the mass ratio of 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 is 24:12:0.5:6.

[0050] The preparation method of the compound 8 is as follows: tetrahydrofuran and compound 7 are added to a reaction flask in sequence and mixed evenly, the mixture is cooled to -10°C, a 3M methylmagnesium bromide solution is added dropwise, and the mixture is stirred at 35°C for 1.5 hours. A saturated aqueous ammonium chloride solution is added to quench the mixture, and the mixture is concentrated under reduced pressure. After that, dichloromethane is added to extract the aqueous phase. The obtained organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure again. Methanol is added to precipitate a solid, which is then filtered and dried to obtain compound 8; the mass ratio of the tetrahydrofuran, compound 7, methylmagnesium bromide solution, and saturated aqueous ammonium chloride is 50:5:20:10.

[0051] Example 4

[0052] A synthesis process for 25-hydroxycholesterol, the synthesis route is as follows:

[0053]

[0054] The preparation method of the compound 2 is as follows: dichloromethane, compound 1 stigmasterol, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, stirred evenly, and then p-toluenesulfonyl chloride is added, stirred at 20°C for 20 hours, concentrated under reduced pressure, deionized water is added to precipitate a solid, filtered and dried, and then dissolved with solvent methanol, activated carbon is added and stirred for 30 minutes, filtered, potassium acetate is added, heated under reflux for 3 hours, concentrated under reduced pressure, recrystallized with deionized water, and then recrystallized in a methanol-water-petroleum ether system, and then filtered and dried to obtain compound 2; the mass ratio of the dichloromethane, stigmasterol, 4-dimethylaminopyridine, triethylamine, p-toluenesulfonyl chloride, solvent methanol, activated carbon, and potassium acetate is 4:0.5:0.05:0.4:0.4:8:1.5:80.

[0055] The preparation method of the compound 3 is as follows: dichloromethane, methanol, compound 2 and sodium bicarbonate are added to the reaction bottle in sequence, the temperature is lowered to -30 ° C, and a 0.5wt% ozone oxygen mixed gas is passed through a continuous flow ozone reactor, the flow rate is controlled at 5 mL / min, and the ventilation is maintained for 50 seconds after 120 seconds. After the nitrogen is replaced in the system, sodium borohydride is added, and the mixture is stirred at 20 ° C for 0.5 hours. After adding a saturated ammonium chloride aqueous solution to quench the reaction, the solvent is removed by distillation under reduced pressure, and then ethyl acetate is added to extract the aqueous phase. The obtained organic phase is washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then petroleum ether is added to precipitate the solid, which is filtered and dried to obtain compound 3; the mass ratio of the dichloromethane, methanol, compound 2, sodium bicarbonate, and sodium borohydride is 7:0.7:1.0:0.6:0.2.

[0056] The preparation method of the compound 4 is as follows: dichloromethane, compound 3, 4-dimethylaminopyridine and triethylamine are added to a reaction flask in sequence, and p-toluenesulfonyl chloride is added after stirring evenly. The mixture is stirred at 20° C. for 20 hours. After the system is concentrated under reduced pressure, deionized water is added to precipitate a large amount of solid, which is filtered and the filter cake is dried to obtain compound 4; the mass ratio of the dichloromethane, compound 3, 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 80:8:0.1:1:2.

[0057] The preparation method of the compound 5 is as follows: tetrahydrofuran, compound 4, lithium bromide and lithium carbonate are added to a reaction bottle in sequence, stirred at 70°C for 14 hours, concentrated under reduced pressure, added with deionized water, slurried, and then filtered and dried to obtain compound 5; the mass ratio of tetrahydrofuran, compound 4, lithium bromide and lithium carbonate is 30:6:4:0.7.

[0058] The preparation method of the compound 6 is as follows: pyridine, zinc powder, nickel chloride hexahydrate and 10wt% boron trifluoride ether solution are added to a reaction flask in sequence, methyl acrylate is added dropwise, stirred at 60°C for 1h, cooled to 20°C, compound 5 is added, stirred at 25°C for 4h, ethyl acetate is added for dilution, filtered through diatomaceous earth, the filter cake is rinsed with ethyl acetate, 10% hydrochloric acid is added to the filtrate to adjust the pH to 3, extraction and separation are performed, the organic phase is washed with water to pH = 7, and then concentrated to dryness under reduced pressure, methanol is added for pulping, filtered, and the filter cake is dried to obtain compound 6; the mass ratio of the pyridine, zinc powder, nickel chloride hexahydrate, 10wt% boron trifluoride ether solution, methyl acrylate, compound 5, and ethyl acetate is 30:6:10:1:15:7:25.

[0059] The preparation method of compound 7 is to add 1,4-dioxane, deionized water, 98% concentrated sulfuric acid and compound 6 in a reaction bottle in sequence, stir at 55°C for 2 hours, concentrate under reduced pressure, add pure water for pulping, filter, and dry to obtain compound 7; the mass ratio of 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 is 20:10:0.5:6.

[0060] The preparation method of the compound 8 is as follows: tetrahydrofuran and compound 7 are added to a reaction flask in sequence and mixed evenly, the mixture is cooled to -10°C, a 3M methylmagnesium bromide solution is added dropwise, the mixture is stirred at 30°C for 1.5 hours, a saturated aqueous ammonium chloride solution is added to quench, and the mixture is concentrated under reduced pressure. After that, dichloromethane is added to extract the aqueous phase, and the obtained organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure again. Methanol is added to precipitate a solid, which is then filtered and dried to obtain compound 8; the mass ratio of the tetrahydrofuran, compound 7, methylmagnesium bromide solution, and saturated aqueous ammonium chloride is 50:3:20:5.

[0061] Performance Testing

[0062] The yield is calculated based on the chemical equation of the reaction and the mass (or molar number) of the starting material to calculate the theoretical product mass, and then the actual product mass is measured. The calculation formula is:

[0063]

[0064]

[0065] The test results show that the present invention improves the selectivity of the reaction and reduces by-products by precisely controlling the reaction conditions; achieves efficient utilization of ozone while reducing the generation of peroxide by-products through continuous flow technology; avoids excessive oxidation and by-product generation through a design that combines nitrogen replacement and sodium borohydride reduction; and improves the product yield through multiple recrystallizations and post-treatments.

[0066] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any indirect modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A process for synthesizing 25-hydroxycholesterol, characterized in that: The synthetic route is as follows:

2. A process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of the compound 2 is as follows: dichloromethane, compound 1 stigmasterol, 4-dimethylaminopyridine and triethylamine are added to a reaction bottle in sequence, and p-toluenesulfonyl chloride is added after stirring evenly, and the mixture is stirred at 20-30° C. for 12-20 hours. After reduced pressure concentration, deionized water is added to precipitate a solid, and the solid is dissolved in solvent methanol after filtering and drying, and activated carbon is added and stirred for 30-60 minutes. After filtering, potassium acetate is added and heated to reflux for reaction for 1-3 hours, and after reduced pressure concentration, deionized water is added for recrystallization, and then recrystallization is performed in a methanol-water-petroleum ether system, and then filtering and drying is performed to obtain compound 2; the mass ratio of dichloromethane, stigmasterol, 4-dimethylaminopyridine, triethylamine, p-toluenesulfonyl chloride, solvent methanol, activated carbon, and potassium acetate is 4-6:0.5-1.5:0.01-0.05:0.2-0.4:0.4-0.6:8-12:0.5-1.5:60-80.

3. A process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of the compound 3 is as follows: dichloromethane, methanol, compound 2 and sodium bicarbonate are added to a reaction bottle in sequence, the temperature is lowered to -30°C, 0.5-2.0wt% ozone oxygen mixed gas is passed through a continuous flow ozone reactor, the flow rate is controlled at 5-20mL / min, aeration is maintained for 30-120S and then maintained for 20-50S, sodium borohydride is added after the system replaces nitrogen, and the mixture is stirred at 20-30°C for 0.5-1.5h, saturated ammonium chloride aqueous solution is added to quench the reaction, and the solvent is removed by reduced pressure distillation, and ethyl acetate is added to extract the aqueous phase, the obtained organic phase is washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then petroleum ether is added to precipitate solids, which are filtered and dried to obtain compound 3; the mass ratio of dichloromethane, methanol, compound 2, sodium bicarbonate, and sodium borohydride is 7-9:0.7-0.9:0.8-1.0:0.4-0.6:0.1-0.

2.

4. A process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of the compound 4 is as follows: dichloromethane, compound 3, 4-dimethylaminopyridine and triethylamine are added to a reaction bottle in sequence, p-toluenesulfonyl chloride is added after stirring evenly, and the mixture is stirred at 20-30° C. for 12-20 hours. After the system is concentrated under reduced pressure, deionized water is added to precipitate a solid, and the solid is filtered and the filter cake is dried to obtain compound 4; the mass ratio of the dichloromethane, compound 3, 4-dimethylaminopyridine, triethylamine and p-toluenesulfonyl chloride is 40-80:4-8:0.1-0.3:1-3:2-4.

5. A process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of the compound 5 is to add tetrahydrofuran, compound 4, lithium bromide and lithium carbonate in a reaction bottle in sequence, stir at 50-70°C for 14-18h, concentrate under reduced pressure, add deionized water for pulping, and then filter and dry to obtain compound 5; the mass ratio of tetrahydrofuran, compound 4, lithium bromide and lithium carbonate is 30-50:6-10:2-4:0.5-0.

7.

6. A process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of the compound 6 is as follows: pyridine, zinc powder, nickel chloride hexahydrate and 10wt% boron trifluoride ether solution are added to a reaction bottle in sequence, methyl acrylate is added dropwise, and the mixture is stirred at 40-60°C for 1-3h. After cooling to 20°C, compound 5 is added, and the mixture is stirred at 25-35°C for 2-4h. Ethyl acetate is added to dilute the mixture, and the mixture is filtered through diatomaceous earth. The filter cake is rinsed with ethyl acetate, 10% hydrochloric acid is added to the filtrate to adjust the pH to 3, and the extraction is performed. The organic phase is washed with water until the pH is 7, and then concentrated to dryness under reduced pressure. Methanol is added for pulping, and the mixture is filtered. The filter cake is dried to obtain compound 6. The mass ratio of pyridine, zinc powder, nickel chloride hexahydrate, 10wt% boron trifluoride ether solution, methyl acrylate, compound 5 and ethyl acetate is 30-40:6-8:8-10:0.5-1:15-17:7-8:15-25.

7. A process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of compound 7 is to add 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 in a reaction bottle in sequence, stir at 45-55° C. for 2-4 hours, concentrate under reduced pressure, add pure water for pulping, filter and dry to obtain compound 7; the mass ratio of 1,4-dioxane, deionized water, concentrated sulfuric acid and compound 6 is 20-24:10-12:0.1-0.5:4-6.

8. The process for synthesizing 25-hydroxycholesterol according to claim 1, characterized in that: The preparation method of the compound 8 is as follows: tetrahydrofuran and compound 7 are added to a reaction bottle in sequence and mixed evenly, the mixture is cooled to -10°C, a 3M methylmagnesium bromide solution is added dropwise, the mixture is stirred at 30-35°C for 0.5-1.5h, a saturated aqueous ammonium chloride solution is added to quench, the mixture is concentrated under reduced pressure, dichloromethane is added to extract the aqueous phase, the obtained organic phase is washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure again, methanol is added to precipitate a solid, and the solid is filtered and dried to obtain compound 8; the mass ratio of the tetrahydrofuran, compound 7, methylmagnesium bromide solution, and saturated aqueous ammonium chloride solution is 30-50:3-5:10-20:5-10.

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