A method for the synthesis of 25-hydroxycholesterol

Abstract

The application discloses a synthesis method of 25-hydroxycholesterol, which takes plant source 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnorol or BA, as raw material, and synthesizes the 25-hydroxycholesterol through steps of oxidation, Wittig reaction, esterification, acetylation, reduction, selective hydrogenation reduction and Grignard reaction, and the total mole yield of seven-step reaction can reach 43.4%. The 25-hydroxycholesterol is synthesized by using the plant source BA as raw material, the raw material is cheap and easy to obtain, the operation is simple, the yield is high, the safety is high, and the risk of pathogenic bacteria and virus infection is avoided.

Description

Technical Field

[0001] This invention belongs to the field of organic chemical synthesis technology and relates to a method for synthesizing 25-hydroxy cholesterol. Specifically, it relates to a method for synthesizing 25-hydroxy cholesterol using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bisnoralcohol or BA, as a raw material. Background Technology

[0002] Calcitriol, also known as 1α,25-dihydroxyvitamin D3, is an important active metabolite of vitamin D3 in the body. It promotes the absorption of calcium and phosphorus and is mainly used clinically for postmenopausal and senile osteoporosis, chronic renal failure, and postoperative hypoparathyroidism. As a metabolite of vitamin D3, calcitriol is present in very small amounts in the body, making its isolation and purification difficult. Currently, it is mainly obtained through chemical synthesis in clinical practice. 25-hydroxycholesterol is a key intermediate in the synthesis of calcitriol and also possesses a wide range of biological activities, including anti-inflammatory, anti-tumor, and anti-Alzheimer's disease effects. Therefore, research on its synthetic route is of great value.

[0003] The main reported methods for the chemical synthesis of 25-hydroxycholesterol are as follows:

[0004] (1) Using 24-dehydrocholesterol as a starting material, 25-hydroxycholesterol was synthesized in a total molar yield of 55% through a three-step reaction (CN103626821 A, as shown in Scheme 1). The starting materials for this route are expensive and difficult to obtain.

[0005]

[0006] (2) Using dichlorol as a raw material, 25-hydroxycholesterol (CN114315947 A, as shown in Scheme 2) was synthesized in a total molar yield of 37.7% through a five-step reaction. This route utilizes lithium copper tetrachloride in the construction of the side chain, which is subject to harsh conditions and is not suitable for industrialization.

[0007]

[0008] (3) Using stigmasterol as a raw material, 25-hydroxycholesterol was synthesized in a total molar yield of 50.6% through a seven-step reaction (CN109021059A, as shown in Scheme 3). This route uses O3 in the synthesis process, which places high demands on reaction detection and equipment, making it unsuitable for industrialization.

[0009]

[0010] (4) Using stigmasterol as a starting material, 25-hydroxycholesterol was synthesized in a total molar yield of 26% through a seven-step reaction (US3822254, as shown in Scheme 4). This route uses hazardous reagents O3 and n-butyllithium in the synthesis process, which is difficult to operate and highly dangerous.

[0011]

[0012] Animal-derived 25-hydroxycholesterol carries the risk of infection by pathogenic bacteria and viruses. Currently reported chemical synthesis routes for 25-hydroxycholesterol suffer from drawbacks such as cumbersome operation, significant safety risks, and expensive catalysts. Therefore, it is necessary to develop a simple, safe, and low-cost synthetic route for 25-hydroxycholesterol based on plant-derived raw materials. Summary of the Invention

[0013] To address the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing 25-hydroxycholesterol from plant sources. This invention uses plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bis(2-hydroxy-3-one) or BA, as a raw material, and synthesizes the 25-hydroxycholesterol through steps including oxidation, Wittig reaction, esterification, acetylation, reduction, selective hydrogenation reduction, and Grignard reaction. This invention uses plant-derived BA to synthesize 25-hydroxycholesterol, which utilizes inexpensive and readily available raw materials, is simple to operate, has a high yield, and is highly safe, avoiding the risk of pathogenic bacteria and viral infections.

[0014] The raw material BA (bisnoralcohol) used in this invention comes from the fermentation of plant sterols, a byproduct of oil processing. It is a green raw material of plant origin, with an annual output of thousands of tons. It is inexpensive and can effectively avoid the risk of pathogenic bacteria and viral infection that may exist in animal-derived raw materials in the prior art.

[0015] In this synthesis method, the raw material BA includes, but is not limited to, obtaining it through bio-fermentation of phytosterols or through chemical synthesis.

[0016] This invention provides a method for synthesizing 25-hydroxycholesterol from plant-derived 21-hydroxy-20-methylpregn-4-en-3-one (BA). The method uses BA as a raw material and synthesizes the 25-hydroxycholesterol through steps including oxidation, Wittig reaction, esterification, acetylation, reduction, selective hydrogenation reduction, Grignard reaction, or oxidation, Wittig reaction, esterification, acetylation, reduction, Grignard reaction, and selective hydrogenation reduction.

[0017] The method includes the following steps:

[0018] Step (a): In the first solvent, BA represented by formula (1) is oxidized to obtain compound (2);

[0019] Step (b): In a second solvent, the compound of formula (2) undergoes a Wittig reaction to obtain the compound of formula (3);

[0020] Step (c): In a third solvent, the compound of formula (3) is esterified to obtain the compound of formula (4);

[0021] Step (d): In a fourth solvent, the compound of formula (4) is acetylated to obtain the compound of formula (5);

[0022] Step (e): In the fifth solvent, the compound of formula (5) is reduced to obtain the compound of formula (6);

[0023] Step (f): In the sixth solvent, the compound of formula (6) is selectively hydrogenated to obtain the compound of formula (7);

[0024] Step (g): In the seventh solvent, the compound of formula (7) is subjected to a Grignard reaction to give the compound of formula (9) 25-hydroxycholesterol;

[0025] Alternatively, the method may specifically include the following steps:

[0026] Step (a): In the first solvent, BA represented by formula (1) is oxidized to obtain compound (2);

[0027] Step (b): In a second solvent, the compound of formula (2) undergoes a Wittig reaction to obtain the compound of formula (3);

[0028] Step (c): In a third solvent, the compound of formula (3) is esterified to obtain the compound of formula (4);

[0029] Step (d): In a fourth solvent, the compound of formula (4) is acetylated to obtain the compound of formula (5);

[0030] Step (e): In the fifth solvent, the compound of formula (5) is reduced to obtain the compound of formula (6);

[0031] Step (h): In the seventh solvent, the compound of formula (6) undergoes a Grignard reaction to obtain the compound of formula (8);

[0032] Step (i): In the sixth solvent, the compound of formula (8) is selectively hydrogenated to obtain the compound of formula (9), 25-hydroxycholesterol.

[0033] The reaction process of the method is shown in route (A):

[0034]

[0035] Wherein, R is an alkyl group; preferably, it is a C1 to C20 alkyl group; more preferably, it is a C1 or C2 alkyl group.

[0036] In compounds (3) to (6) and (8), the ratio of the E configuration to the Z configuration of the double bond between C-22 and C-23 is 1:2 to 2:1 (via...). 1 H NMR integration determination); preferably, the ratio is 1:1.

[0037] In step (a) of the present invention, the oxidation reaction specifically involves the following: in the first solvent, the BA represented by formula (1) undergoes an oxidation reaction with 2,2,6,6-tetramethylpiperidine oxide (TEMPO), sodium bicarbonate, tetrabutylammonium bromide, and an oxidant to obtain the compound of formula (2).

[0038] Wherein, the molar ratio of BA, TEMPO, sodium bicarbonate, tetrabutylammonium bromide and oxidant shown in formula (1) is 1:(0~1):(0~20):(0~1):(1~5); preferably, it is 1:0.01:1.35:0.1:1.15.

[0039] The oxidation reaction is carried out under the action of an oxidant, which is selected from one or more of N-chlorosuccinimide (NCS), N-bromosuccinimide (NBS), 2-iodobenzoic acid (IBX), etc.; preferably, it is NCS.

[0040] The first solvent is selected from one or more of dichloromethane, tetrahydrofuran, toluene, dimethyl sulfoxide, and water; preferably, it is a mixed solvent of dichloromethane and water (volume ratio V / V = 5 / 2).

[0041] The oxidation reaction is carried out at a temperature of 0–30°C; preferably, it is carried out at 0°C.

[0042] The oxidation reaction takes 3 to 8 hours; preferably, it takes 6 hours.

[0043] In one specific embodiment, the synthesis steps of compound (2) include: dissolving BA as shown in formula (1) in a first solvent, and then adding TEMPO, sodium bicarbonate, tetrabutylammonium bromide and NCS to undergo an oxidation reaction to obtain compound (2).

[0044] In step (b) of the present invention, the Wittig reaction specifically involves the following: in the second solvent, the compound of formula (2), 2-carboxyethyltriphenylphosphine halide, and a base undergo a Wittig reaction to obtain the compound of formula (3).

[0045] Wherein, the molar ratio of the compound of formula (2), 2-carboxyethyltriphenylphosphine halide and base is 1:(1-4):(2-8); preferably, it is 1:2:4.

[0046] The second solvent is one or more of toluene, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, etc.; preferably, it is a mixed solvent of dimethyl sulfoxide and tetrahydrofuran (volume ratio V / V = 1 / 1).

[0047] The 2-carboxyethyltriphenylphosphine halide is selected from one or more of 2-carboxyethyltriphenylphosphine chloride, 2-carboxyethyltriphenylphosphine bromide, etc.; preferably, it is 2-carboxyethyltriphenylphosphine bromide.

[0048] The alkali is selected from one or more of sodium hydride, potassium tert-butoxide (t-BuOK), sodium tert-butoxide (t-BuONa), lithium bis(trimethylsilylamino)amine (LiHMDS), sodium hydroxide, potassium hydroxide, lithium diisopropylamino (LDA), etc.; preferably, it is potassium tert-butoxide.

[0049] The temperature of the Wittig reaction is -10 to 112°C; preferably, it is 25°C.

[0050] The Wittig reaction time is 0.5 to 9 hours; preferably, it is 2 hours.

[0051] In one specific embodiment, the synthesis steps of compound (3) include: dissolving compound (2) in a second solvent, then adding 2-carboxyethyltriphenylphosphine bromide and potassium tert-butoxide, and undergoing a Wittig reaction to obtain compound (3).

[0052] In step (c) of the present invention, the esterification reaction specifically involves the esterification reaction of the compound of formula (3) and the catalyst in the third solvent to obtain the compound of formula (4).

[0053] Wherein, the molar ratio of the compound of formula (3) to the catalyst is 1:(0.01~5); preferably, it is 1:0.1.

[0054] The catalyst is selected from one or more of thionyl chloride, concentrated sulfuric acid, and p-toluenesulfonic acid; preferably, it is concentrated sulfuric acid.

[0055] The third solvent is selected from one or more of methanol, ethanol, propanol, etc.; preferably, it is methanol.

[0056] The temperature of the esterification reaction is 20–100°C; preferably, it is 25°C.

[0057] The esterification reaction takes 0.5 to 20 hours, preferably 15 hours.

[0058] In one specific embodiment, the synthesis steps of compound (4) include: dissolving compound (3) in a third solvent, then adding concentrated sulfuric acid to undergo an esterification reaction to obtain compound (4).

[0059] In step (d) of the present invention, the acetylation reaction specifically involves the acetylation reaction of the compound of formula (4), acetyl chloride, and acetic anhydride in the fourth solvent to obtain the compound of formula (5).

[0060] Wherein, the molar ratio of the compound of formula (4), acetyl chloride, and acetic anhydride is 1:(0.5~62.5):(1~62.5); preferably, it is 1:25:24.

[0061] The fourth solvent is selected from one or more of acetic anhydride, acetyl chloride, ethyl acetate, dichloromethane, etc.; preferably, it is a mixed solvent of acetyl chloride and acetic anhydride.

[0062] The temperature of the acetylation reaction is 40–120°C; preferably, it is 70°C.

[0063] The acetylation reaction takes 1 to 20 hours. Preferably, it takes 5 hours.

[0064] In one specific embodiment, the synthesis steps of compound (5) include: adding acetyl chloride and acetic anhydride to compound (4) to undergo an acetylation reaction to obtain compound (5).

[0065] In step (e) of the present invention, the reduction reaction specifically involves adding a metal catalyst and a reducing agent to the fifth solvent to induce a reduction reaction and obtain the compound of formula (6).

[0066] Wherein, the molar ratio of the compound of formula (5), the metal catalyst and the reducing agent is 1:(0.1-5):(1-15); preferably, it is 1:0.1:4.

[0067] The fifth solvent is selected from one or more of tetrahydrofuran, methanol, ethanol, water, dichloromethane, 2-methyltetrahydrofuran, isopropanol, methyl tert-butyl ether, etc.; preferably, it is a mixed solvent of tetrahydrofuran and ethanol (volume ratio V / V = 2 / 1).

[0068] The reducing agent is selected from one or two of NaBH4, KBH4, etc.; preferably, it is NaBH4.

[0069] The metal catalyst is selected from one or more of the following: copper chloride, copper bromide, cuprous chloride, cuprous bromide, cuprous iodide, copper acetate, copper tartrate, copper glycinate, copper citrate, ferrous chloride, magnesium chloride, ferrous sulfate, nickel chloride, lithium chloride, aluminum chloride, manganese chloride, and calcium chloride; preferably, it is cuprous chloride.

[0070] The temperature of the reduction reaction is -10 to 50°C; preferably, it is 25°C.

[0071] The reduction reaction takes 0.1 to 20 hours; preferably, it takes 10 hours.

[0072] In one specific embodiment, the synthesis steps of compound (6) include: dissolving compound (5) in a fifth solvent, and undergoing a reduction reaction with a reducing agent under the action of a metal catalyst to obtain compound (6).

[0073] In step (f) of the present invention, the selective hydrogenation reduction reaction specifically involves the following: the compound of formula (6) undergoes a selective hydrogenation reduction reaction with a reducing agent in the sixth solvent under the action of a catalyst to obtain the compound of formula (7).

[0074] The reducing agent is H2.

[0075] The catalyst is Raney Ni.

[0076] Wherein, the mass ratio of the compound of formula (6) to the catalyst is 1:(0.05~5); preferably, it is 1:1.

[0077] The sixth solvent is selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, isopropanol, etc.; preferably, it is isopropanol.

[0078] The temperature of the selective hydrogenation reduction reaction is 0–60°C; preferably, it is 25°C.

[0079] The reducing agent H2 pressure in the selective hydrogenation reduction reaction is 1–20 atm; preferably, it is 1 atm.

[0080] The selective hydrogenation reduction reaction takes 1 to 10 hours; preferably, it takes 3 hours.

[0081] In one specific embodiment, the synthesis steps of compound (7) include: dissolving compound (6) in a sixth solvent, adding Raney Ni and H2 for substitution, and then undergoing a selective hydrogenation reduction reaction to obtain compound (7).

[0082] In step (g) of the present invention, the Grignard reaction specifically involves the following: the compound of formula (7) reacts with magnesium methyl halide in the seventh solvent to obtain the 25-hydroxycholesterol.

[0083] The process includes a purification step before obtaining 25-hydroxycholesterol, which may be one or more of column chromatography, recrystallization, or pulping.

[0084] Wherein, the molar ratio of the compound of formula (7) to magnesium methyl halide is 1:(3-12); preferably, it is 1:6.

[0085] The methyl magnesium halide is selected from one or more of methyl magnesium chloride, methyl magnesium bromide, and methyl magnesium iodide; preferably, it is methyl magnesium bromide.

[0086] The seventh solvent is selected from one or more of diethyl ether, 2-methyltetrahydrofuran, tetrahydrofuran, etc.; preferably, it is tetrahydrofuran.

[0087] The temperature of the Grignard reaction is 0–40°C; preferably, it is 25°C.

[0088] The Grignard reaction takes 1 to 15 hours; preferably, it takes 5 hours.

[0089] In one specific embodiment, the synthesis steps of 25-hydroxycholesterol include: dissolving the compound of formula (7) in a seventh solvent, adding methyl magnesium halide, and undergoing a Grignard reaction to obtain 25-hydroxycholesterol.

[0090] In step (h) of the present invention, the Grignard reaction specifically involves the Grignard reaction of the compound of formula (6) with magnesium methyl halide in the eighth solvent to obtain the compound of formula (8).

[0091] Wherein, the molar ratio of the compound of formula (6) and magnesium methyl halide is 1:(3-12); preferably, it is 1:6.

[0092] The methyl magnesium halide is selected from one or more of methyl magnesium chloride, methyl magnesium bromide, and methyl magnesium iodide; preferably, it is methyl magnesium bromide.

[0093] The eighth solvent is selected from one or more of diethyl ether, 2-methyltetrahydrofuran, tetrahydrofuran, etc.; preferably, it is tetrahydrofuran.

[0094] The temperature of the Grignard reaction is 0–40°C; preferably, it is 25°C.

[0095] The Grignard reaction takes 1 to 15 hours; preferably, it takes 5 hours.

[0096] In one specific embodiment, the synthesis steps of compound (8) include: dissolving compound (7) in an eighth solvent, adding methyl magnesium halide, and undergoing a Grignard reaction to obtain compound (8).

[0097] In step (i) of the present invention, the selective hydrogenation reduction reaction specifically involves the compound of formula (8) undergoing a selective hydrogenation reduction reaction with a reducing agent in the ninth solvent under the action of a catalyst to obtain the 25-hydroxycholesterol.

[0098] The reducing agent is H2.

[0099] The catalyst is Raney Ni.

[0100] Wherein, the mass ratio of the compound of formula (6) to the catalyst is 1:(0.05~5); preferably, it is 1:1.

[0101] The ninth solvent is selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, isopropanol, etc.; preferably, it is isopropanol.

[0102] The temperature of the selective hydrogenation reduction reaction is 0–60°C; preferably, it is 30°C.

[0103] The reducing agent H2 pressure in the selective hydrogenation reduction reaction is 1–20 atm; preferably, it is 1 atm.

[0104] The selective hydrogenation reduction reaction takes 1 to 10 hours; preferably, it takes 5 hours.

[0105] In one specific embodiment, the synthesis steps of 25-hydroxycholesterol include: dissolving the compound of formula (8) in a ninth solvent, adding Raney Ni and H2 for substitution, and then undergoing a selective hydrogenation reduction reaction to obtain 25-hydroxycholesterol.

[0106] The present invention also provides a compound having the structure shown in formula (9):

[0107]

[0108] The beneficial effects of this invention are as follows: This invention provides a new synthetic route for 25-hydroxycholesterol, avoiding the use of expensive reagents such as lithium copper tetrachloride, O3, and n-butyllithium, or reagents with high requirements for reaction conditions; the reaction operation is simple; and the reagents are inexpensive, with a high overall yield of up to 43.4%. Using plant-derived BA as a raw material ensures high safety and avoids the risk of infection by pathogenic bacteria and viruses. Detailed Implementation

[0109] The present invention will be further described in detail below with reference to specific embodiments. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0110] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0111] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0112] This invention discloses a method for synthesizing 25-hydroxycholesterol, using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bis(2-hydroxy-2-yl)-2-yl ...

[0113] In the following examples, the compound structures were determined using nuclear magnetic resonance and high-resolution mass spectrometry; reagents were mainly provided by Shanghai Guoyao Chemical Reagent Co., Ltd.; product purification was mainly performed by column chromatography; silica gel (200-300) was produced by Qingdao Ocean Chemical Plant.

[0114] Preparation of compound (2) in Example 1

[0115]

[0116] Dichloromethane (250 mL), BA (50 g, 151 mmol), TEMPO (469 mg, 3 mmol), sodium bicarbonate (17.6 g, 210 mmol), NCS (23.2 g, 173.7 mmol), tetrabutylammonium bromide (4.84 g, 15 mmol), and water (250 mL) were added sequentially to a flask and reacted at 0 °C for 6 h. After the reaction was complete as detected by TLC, sodium thiosulfate pentahydrate solution (2.64 g sodium thiosulfate pentahydrate / 50 mL water) was added and stirred at 5-10 °C for 20 min. The mixture was separated, and the aqueous phase was extracted with dichloromethane (200 mL × 2). The organic phases were combined, washed with 1% sodium hydroxide solution (200 mL), separated, and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure to obtain compound (2) (48.2 g white solid, 97% yield). 1HNMR(500MHz, CDCl3)δ9.55(d,J=3.2Hz,1H),5.71(d,J=1.9Hz,1H),2.45-2.39(m,1H),2.37( t,J=4.2Hz,1H),2.36-2.29(m,2H),2.28-2.24(m,1H),2.03-1.94(m,2H),1.91-1.79(m,2H), 1.72-1.63(m,2H),1.58-1.52(m,2H),1.51-1.46(m,1H),1.45-1.34(m,2H),1.26-1.20(m,1H ),1.17(s,3H),1.11(d,J=6.9Hz,3H),1.08-1.00(m,2H),0.96(d,J=4.2Hz,2H),0.75(s,3H). 13 C NMR (125MHz, CDCl3) δ204.84,199.48,171.14,123.88,55.15,53.73,50.94,49.42,42.99,39.29,38 .56,35.69,35.58,33.95,32.82,31.95,26.99,24.53,20.96,17.37,13.42,12.33.HRMS(ESI):calcd for C 22 H 32 NaO2[M+Na] + ,351.2295,found 351.2300.

[0117] Example 2 Preparation of compound (3)

[0118]

[0119] The present invention tested various reaction conditions (as shown in Table 1) and obtained the optimal Wittig reaction conditions, which resulted in the highest molar yield of target intermediate 3 (as shown in Table 1).

[0120] Table 1 shows the Wittig reaction of compounds of formula (2).

[0121]

[0122]

[0123] Note: a. The amount used is the molar equivalent; b. The molar yield of the compound obtained by column chromatography purification is calculated.

[0124] As shown in Table 1, the optimal reaction conditions were obtained by screening the type and amount of base, the amount of 2-carboxyethyltriphenylphosphine bromide, and the type of solvent. The optimal conditions were t-BuOK = 4 eq, 2-carboxyethyltriphenylphosphine bromide = 2 eq, and THF / DMSO (1 / 1, v / v) as solvent. The molar yield of compound 3 reached 83%.

[0125] Some of the embodiments are shown below:

[0126] Compound (2) (17.08 g, 52 mmol), 2-carboxyethyltriphenylphosphine bromide (43.19 g, 104 mmol), anhydrous DMSO (100 mL) and anhydrous THF (100 mL) were added to a flask and dissolved. t-BuOK (23.34 g, 208 mmol) was added under an ice-water bath. The mixture was protected with nitrogen and reacted at 25 °C for 5 h. After the reaction was complete as detected by TLC, the pH was adjusted to 10 with standard NaOH solution, and the mixture was extracted with EA (100 mL × 3). The organic phase was discarded. Under an ice-water bath, the pH of the aqueous phase was adjusted to 3-4 with 2N hydrochloric acid, and the mixture was extracted with EA (100 mL × 3). The organic phases were combined and washed successively with water (60 mL) and saturated sodium chloride solution (60 mL). The mixture was purified by column chromatography (PE:EA = 3:1) to obtain compounds (3E and 3Z, white solids, 16.6 g, molar yield 83%). The ratio of isomers 3E and 3Z is approximately 1:1 (via...). 1 H NMR integral determination). 1 HNMR(500MHz, CDCl3)δ5.73(s,2H),5.48-5.41(m,2H),5.41-5.35(m,2H),3.1 9-3.01(m,4H),2.46-2.21(m,10H),2.04-1.97(m,4H),1.86-1.79(m,2H),1.74 -1.63(m,4H),1.59-1.48(m,6H),1.48-1.39(m,2H),1.31-1.20(m,4H),1.19-1 .11(m,10H),1.03-0.95(m,10H),0.95-0.88(m,2H),0.75(s,3H),0.72(s,3H). 13CNMR(125MHz,CDCl3)δ199.98,177.44,171.89,141.36,140.20,123.83,123 .81,118.58,117.10,55.95,55.93,55.83,55.62,53.88,42.45,39.98,39.58 ,39.55,38.69,37.74,35.73,35.66,34.71,34.00,33.01,32.99,32.06,28.3 8,28.07,24.21,21.06,20.30,20.27,17.45,12.36,12.21.HRMS(ESI):calcd for C 25 H 36 NaO3[M+Na] + ,407.2557,found407.2566.

[0127] Compound (2) (1 g, 3.0 mmol), 2-carboxyethyltriphenylphosphine bromide (2.7 g, 6 mmol), and anhydrous DMSO (10 mL) were added to a flask and dissolved. t-BuOK (1.4 g, 12 mmol) was added under an ice-water bath. The mixture was protected with nitrogen and reacted at room temperature for 5 h. After the reaction was complete as detected by TLC, the pH was adjusted to 10 with standard NaOH solution, and the mixture was extracted with EA (20 mL × 3). The organic phase was discarded, and the pH of the aqueous phase was adjusted to 3-4 with 2N hydrochloric acid under an ice-water bath. The mixture was extracted with EA (20 mL × 3). The organic phases were combined and washed successively with water (20 mL) and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 3:1) to obtain compounds (3E and 3Z, white solids, 784 mg, molar yield 67%).

[0128] Compound (2) (1 g, 3.0 mmol), 2-carboxyethyltriphenylphosphine bromide (2.7 g, 6 mmol), anhydrous DMSO (5 mL) and anhydrous THF (5 mL) were added to a flask and dissolved. NaH (288 mg, 12 mmol) was added under ice-water bath and nitrogen protection was maintained. The reaction was carried out at room temperature for 5 h. After the reaction was completed by TLC, the pH was adjusted to 10 with standard NaOH solution and extracted with EA (20 mL × 3). The organic phase was discarded and the pH of the aqueous phase was adjusted to 3-4 with 2N hydrochloric acid under ice-water bath. EA (20 mL × 3) was extracted. The organic phases were combined and washed successively with water (20 mL) and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 3:1) to obtain compounds (3E and 3Z, white solids, 737 mg, molar yield 63%).

[0129] Note: In this invention, the E / Z configuration ratio of the intermediate compounds (4), (5), (6), and (8) obtained by esterification, acetylation, and reduction of compound (3) is 1:2 to 2:1. Since the cis-trans isomers of the double bond between C-22 and C-23 in compound (6) can all yield compound (9) 25-hydroxycholesterol after Raney nickel hydroreduction and Grignard reaction, the corresponding E / Z configuration ratios of the compounds are not indicated in the following examples.

[0130] Preparation of compound (4) in Example 3

[0131]

[0132] Compound (3) (5 g, 13 mmol), concentrated sulfuric acid (130 mg, 1.3 mmol), and anhydrous CH3OH (30 mL) were added to a flask and reacted at room temperature for 15 h. After the reaction was complete as detected by TLC, the mixture was concentrated under reduced pressure, water (60 mL) was added, and EA (20 mL × 3) was used for extraction. The organic phases were combined and washed successively with saturated sodium bicarbonate solution (30 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain compounds (4-1) (4-1E and 4-1Z, white solids, 5.18 g, molar yield 100%), which were used directly in the next step. HRMS(ESI):calcd for C 26 H 38 NaO3[M+Na]+,421.2713,found421.2720.

[0133]

[0134] Compound (3) (5 g, 13 mmol), concentrated sulfuric acid (130 mg, 1.3 mmol), and anhydrous ethanol (30 mL) were added to a flask and reacted at room temperature for 15 h. After the reaction was completed as detected by TLC, the mixture was concentrated under reduced pressure, water (60 mL) was added, and the mixture was extracted with EA (20 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (30 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain compounds (4-2) (4-2E and 4-2Z, white solids, 5.36 g, molar yield 100%), which were used directly in the next step.

[0135] Preparation of compound (5) in Example 4

[0136]

[0137] Compound (4-1) (2 g, 5 mmol), acetic anhydride (13 g, 120 mmol), and acetyl chloride (9.8 g, 125 mmol) were added to a flask and reacted at 70 °C for 9 h. After the reaction was almost complete as detected by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (PE:EA = 60:1) to give compounds (5-1E and 5-1Z, white solids, 1.94 g, molar yield 88%). The ratio of isomers 5-1E and 5-1Z was approximately 2:1 (using TLC). 1 H NMR integral determination). 1 H NMR (500MHz, CDCl3) δ5.68 (d, J = 2.3Hz, 1H), 5.44-5.33 (m, 3H), 3.69 (s, 1H), 3.67 (s, 2 H),3.16-2.97(m,2H),2.47-2.40(m,1H),2.16-2.08(m,6H),2.01-1.96(m,1H),1.87- 1.82(m,1H),1.67-1.61(m,3H),1.57-1.54(m,2H),1.45-1.42(m,1H),1.36-1.24(m,2 H),1.23-1.13(m,2H),1.13-1.05(m,2H),1.04-0.97(m,7H),0.74(s,1H),0.71(s,2H). 13 C NMR (125MHz, CDCl3) δ172.73,169.40,146.98,141.00,139.92,139.37,124.03,124.0 0,118.95,117.49,117.01,56.86,56.83,55.85,55.64,51.79,51.70,48.01,47.99,42 .45,39.91,39.65,39.61,37.85,34.91,34.62,33.78,33.13,31.84,31.74,28.35,28 .02,24.82,24.17,21.19,21.10,20.32,20.29,18.85,12.30,12.16.HRMS(ESI):calcd for C 28 H 40 NaO4[M+Na] + ,463.2819,found 463.2830.

[0138]

[0139] Compound (4-2) (5.36 g, 13 mmol), acetic anhydride (31.9 g, 312 mmol), and acetyl chloride (25.5 g, 325 mmol) were added to a flask and reacted at 70 °C for 5 h. After the reaction was complete as detected by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (PE:EA = 60:1) to give compounds (5-2E and 5-2Z, white solids, 5.14 g, molar yield 87%). The ratio of isomers 5-2E and 5-2Z was approximately 2:1 (using TLC). 1 H NMR integral determination). 1 H NMR (500MHz, CDCl3) δ5.68(d,J=2.3Hz,1H),5.43-5.32(m,3H),4.13(q,J=7.2Hz,2H),3.13-2.95(m,2H),2. 49-2.35(m,2H),2.16-2.09(m,5H),2.01-1.95(m,1H),1.86-1.83(m,1H),1.70-1.62(m,3H),1.57-1.53(m, 2H),1.44(dd,J=12.9,4.4Hz,1H),1.34-1.30(m,1H),1.30-1.25(m,3H),1.24(d,J=2.5Hz,1H),1.23-1.11( m,3H),1.09-1.05(m,2H),1.02(d,J=6.7Hz,1H),1.00-0.98(m,4H),0.98(s,1H),0.74(s,2H),0.71(s,1H). 13 C NMR (150MHz, CDCl3) δ172.38,169.70,147.30,141.20,140.12,139.69,124.35,124.32,119 .46,117.99,117.33,60.88,60.77,57.18,57.15,56.17,55.97,48.33,48.31,42.76,40.26 ,39.97,39.93,38.45,35.22,34.94,34.11,33.74,32.16,32.14,32.06,28.69,28.35,25.1 4,24.48,21.51,21.42,20.65,19.19,19.17,14.55,14.54,12.64,12.49.HRMS(ESI):calcd for C 29 H 42 NaO4[M+Na] + ,477.2975,found 477.2980.

[0140] Preparation of compound (6) in Example 5

[0141]

[0142] In the presence of a reducing agent, the ester group of compound (5-1) is reduced to a hydroxyl group; and the solvent used in this reduction reaction contains ethanol, which will cause an ester exchange reaction, converting the methyl ester group of compound (5-1) into an ethyl ester group, to generate compound (6-2).

[0143] The reduction reaction produces reduction byproduct isomers (10) and (6′-2). This invention tested various reaction conditions (as shown in Table 2) and obtained the optimal reaction conditions, which resulted in the highest molar yield of the target intermediate (6′-2) (as shown in Table 2).

[0144] Table 2 shows the reduction reactions of compounds of formula (5-1). a

[0145]

[0146]

[0147] a. Unless otherwise specified, the reduction reaction temperature is 25℃; b. The amount used is molar equivalent; c. The amount used is molar equivalent; d. The molar yields of compounds (6-2) and (10) were calculated by column chromatography purification; e. The separation yields of compounds 6-2 (3β-OH) and its isomer 6′-2 (3α-OH) were 90% and 5%, respectively; f. The reduction reaction temperature is 0℃; g. The reduction reaction temperature is 50℃.

[0148] As shown in Table 2, the optimal reduction reaction conditions were obtained by screening the solvent type, reducing agent type and amount, and metal catalyst type and amount. The optimal conditions were THF / EtOH (2:1, v / v) solvent, NaBH4 (4 eq) metal catalyst, CuCl (0.1 eq) compound (6-2) had the highest molar yield of 90%.

[0149] Some of the embodiments are shown below:

[0150] Compound (5-1) (2 g, 4.54 mmol), tetrahydrofuran (20 mL), ethanol (10 mL), and cuprous chloride (46 mg, 0.46 mmol) were added to a flask. Sodium borohydride (687 mg, 18.16 mmol) was slowly added under an ice-water bath, and the reaction was carried out at 25 °C for 10 h. After the reaction was completed as detected by TLC, water (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compounds (6-2) (6-2E and 6-2Z, white solids, 1.69 g, molar yield 90%). The ratio of isomers 6-2E and 6-2Z was approximately 2:1 (using TLC). 1 HNMR integral determination). 1 H NMR (500MHz, CDCl3) δ5.46-5.31(m,3H),4.14-4.09(m,2H),3.53-3.46(m, 1H),3.13-2.90(m,2H),2.42-2.03(m,3H),1.99-1.91(m,2H),1.84-1.79( m,3H),1.72-1.60(m,1H),1.54-1.42(m,6H),1.27-1.20(m,3H),1.19-1.0 3(m,4H),1.02-0.96(m,7H),0.95-0.86(m,1H),0.70(s,1H),0.67(s,2H). 13 C NMR (125MHz, CDCl3) δ172.68,141.26,141.15,140.17,121.95,121.93,119.43,117 .97,72.06,60.93,60.82,57.13,57.10,56.21,56.00,50.49,42.66,42.63,40.26, 40.03,39.99,38.47,37.62,36.86,34.95,33.77,32.23,32.22,31.97,28.70,28.3 6,24.60,21.41,20.68,20.66,19.75,14.57,14.55,12.55,12.40.HRMS(ESI):calcd for C 27 H 42 NaO3[M+Na] + ,437.3026,found437.3030.

[0151] Compound 6′-2 (6′-2E and 6′-2Z), white solid, 94 mg, molar yield 5%, isomers 6′-2E and 6′-2Z in a ratio of approximately 2:1 (via tracing). 1 H NMR integral judgment). 1 H NMR (500MHz, CDCl3) δ5.44-5.35(m,3H),4.19-4.09(m,2H),4.05-3.97(m,1H),3.15-2.93(m,2H),2.59-2.55(m,1H),2.14-1.92(m,4H),1. 76-1.60(m,5H),1.56-1.51(m,2H),1.48-1.40(m,3H),1.28-1.24(m, 4H),1.20-1.13(m,2H),1.08-0.99(m,9H),0.72(s,1H),0.69(s,2H). 13 C NMR (125MHz, CDCl3) δ172.31,140.93,138.55,123.98,119.10,117.62,67.11,60.56,60.45,56.77,55.83,55.63,50.37,42.31,39.93,39.87,3 9.62,38.14,37.35,34.61,33.43,33.23,31.95,31.83,28.91,28.34,28 .00,24.23,20.77,20.33,18.66,14.22,12.20,12.05.HRMS(ESI):calcd for C 27 H 42 NaO3[M+Na] + ,437.3026,found437.3030.

[0152] Compound 10 (white solid, 56 mg, molar yield 3%) 1H NMR (500MHz, CDCl3) δ5.42-5.19(m,3H),3.66-3.49(m,3H),2.50-2.33(m,1H),2.32- 2.27(m,1H),2.26-2.21(m,2H),2.13-1.93(m,3H),1.88-1.80(m,2H),1.71-1.63(m,1 H),1.59-1.42(m,7H),1.30-1.24(m,1H),1.23-1.10(m,2H),1.09-1.05(m,1H),1.05 -1.00(m,6H),0.99(d,J=6.6Hz,1H),0.94(d,J=5.1Hz,1H),0.72(s,1H),0.70(s,2H). 13 C NMR (125MHz, CDCl3) δ141.07,140.75,140.19,123.00,121.68,71.79,62.49,62.01,56.80,55.88,55.57,50.12,50.09,42.28,40.23,39.70 ,39.65,37.25,36.51,35.91,34.51,31.87,31.64,31.24,28.68,28.28,24.27,21.06,20.82,20.67,19.43,12.21,12.08.HRMS(ESI):calcd for C 25 H 40 NaO2[M+Na] + ,395.2921,found 395.2915.

[0153] Compound (5-1) (2 g, 4.54 mmol), tetrahydrofuran (20 mL), and ethanol (10 mL) were added to a flask. Sodium borohydride (687 mg, 18.16 mmol) was slowly added under an ice-water bath, and the reaction was carried out at 25 °C for 10 h. After the reaction was completed as detected by TLC, water (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compounds (6-2E and 6-2Z, white solids, 1.13 g, molar yield 60%).

[0154] Compound (5-1) (2 g, 4.54 mmol), tetrahydrofuran (20 mL), ethanol (10 mL), and cuprous bromide (65 mg, 0.46 mmol) were added to a flask. Sodium borohydride (687 mg, 18.16 mmol) was slowly added under an ice-water bath, and the reaction was carried out at 25 °C for 10 h. After the reaction was completed as detected by TLC, water (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compounds (6-2E and 6-2Z, white solids, 0.73 g, molar yield 39%).

[0155]

[0156] Compound (5-2) (2.06 g, 4.54 mmol), tetrahydrofuran (20 mL), ethanol (10 mL), and cuprous chloride (46 mg, 0.46 mmol) were added to a flask. Sodium borohydride (687 mg, 18.16 mmol) was slowly added under an ice-water bath, and the reaction was carried out at 25 °C for 10 h. After the reaction was completed as detected by TLC, water (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compounds (6-2E and 6-2Z, white solids, 1.67 g, molar yield 89%).

[0157]

[0158] Compound (5-1) (2 g, 4.54 mmol), tetrahydrofuran (20 mL), and methanol (10 mL) were added to a flask. Sodium borohydride (687 mg, 18.16 mmol) was slowly added under an ice-water bath, and the reaction was carried out at 25 °C for 10 h. After the reaction was completed as detected by TLC, water (15 mL) was added, and the mixture was extracted with EA (30 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 10:1) to obtain compound (6-1) (6-1E and 6-1Z, white solids, 0.62 g, molar yield 33%). The ratio of isomers 6-1E and 6-1Z was approximately 2:1 (using TLC). 1 HNMR integral determination). 1HNMR(500MHz, CDCl3)δ5.49-5.30(m,3H),3.67(d,J=6.4Hz,3H),3.55-3.48(m,1H),3.17-2.87(m,2H),2.39-2.05(m,3H),2.00-1.9 1(m,2H),1.87-1.80(m,2H),1.70-1.63(m,1H),1.55-1.42(m,6H),1.25-1.06(m,4H),1.03-0.88(m,9H),0.71(s,1H),0.68(s,2H). 13 C NMR (125MHz, CDCl3) δ172.78,141.04,140.76,139.95,121.67,121.65,118.90, 117.45,71.78,56.77,56.74,55.84,55.63,51.83,51.74,50.12,42.32,42.29, 39.92,39.68,39.64,37.86,37.26,36.51,34.63,33.13,31.88,31.86,31.65,2 8.35,28.03,24.27,21.06,20.33,20.29,19.41,12.20,12.05.HRMS(ESI):calcd for C 26 H 40 NaO3[M+Na] + ,423.2864,found 423.2870.

[0159] Example 6: Preparation of 25-hydroxycholesterol

[0160]

[0161] Compound (6-2) (2.07 g, 5 mmol) and isopropanol (20 mL) were added to a flask and dissolved. Then, activated Raney Ni (2 g, wet weight, activated according to standard procedure) was added, along with H2 (1 atm), and the reaction was carried out at room temperature for 3 h. After the reaction was confirmed by NMR, Raney Ni was removed by filtration through a diatomaceous earth filter. The filtrate was concentrated under reduced pressure to obtain compound (7-2), which was directly used in the next reaction step.

[0162] Compound of formula (7-2) and anhydrous THF (60 mL) were added to a flask. Methylmagnesium bromide (5 mL, 15 mmol) was slowly added under an ice-water bath. The reaction was carried out at room temperature for 5 h under nitrogen protection. After the reaction was completed as detected by TLC, the reaction was quenched by adding 2N HCl (20 mL) under an ice-water bath. Extraction was performed with EA (60 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The mixture was purified by column chromatography (PE:EA = 5:1) to obtain 25-hydroxycholesterol (white solid, 1.37 g, two-step yield 68%). 1 HNMR (600MHz, CDCl3) δ5.37-5.33(m,1H),3.55-3.49(m,1H),2.31-2.28(m,1H ),2.26-2.21(m,1H),2.04-1.95(m,2H),1.86-1.80(m,3H),1.60-1.48(m,5H), 1.48-1.43(m,3H),1.43-1.34(m,5H),1.27-1.23(m,2H),1.21(s,6H),1.18-1. 14(m,1H),1.11-1.03(m,4H),1.01(s,3H),0.93(d,J=6.5Hz,3H),0.68(s,3H). 13 C NMR (150MHz, CDCl3) δ140.81,121.73,71.83,71.15,56.79,56.10,50.15,44.46,42.37,42.34,39.81,37.28,36. 54,36.48,35.78,31.93,31.70,29.40,29.24,28.28,24.32,21.11,20.80,19.43,18.72,11.90.HRMS(ESI):calcd for C 27 H 46 NaO2[M+Na] + ,425.3391,found 425.3384.

[0163]

[0164] Compound (6-1) (2 g, 5 mmol) and isopropanol (20 mL) were added to a flask and dissolved. After dissolving, activated Raney Ni (2 g, wet weight, activated according to standard procedure) was added, along with H2 (1 atm), and the reaction was carried out at room temperature for 3 h. After the reaction was confirmed by NMR, Raney Ni was removed by filtration through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain compound (7-2), which was directly used in the next reaction.

[0165] Compound of formula (7-1) and anhydrous THF (60 mL) were added to a flask. Methylmagnesium bromide (5 mL, 15 mmol) was slowly added under an ice-water bath. The reaction was carried out at room temperature for 5 h under nitrogen protection. After the reaction was completed as detected by TLC, the reaction was quenched by adding 2N HCl (20 mL) under an ice-water bath. Extraction was performed with EA (60 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The mixture was purified by column chromatography (PE:EA = 5:1) to obtain 25-hydroxycholesterol (white solid, 1.32 g, two-step yield 66%). 1 HNMR (600MHz, CDCl3) δ5.37-5.33(m,1H),3.55-3.49(m,1H),2.31-2.28(m,1H ),2.26-2.21(m,1H),2.04-1.95(m,2H),1.86-1.80(m,3H),1.60-1.48(m,5H), 1.48-1.43(m,3H),1.43-1.34(m,5H),1.27-1.23(m,2H),1.21(s,6H),1.18-1. 14(m,1H),1.11-1.03(m,4H),1.01(s,3H),0.93(d,J=6.5Hz,3H),0.68(s,3H). 13 C NMR (150MHz, CDCl3) δ140.81,121.73,71.83,71.15,56.79,56.10,50.15,44.46,42.37,42.34,39.81,37.28,36. 54,36.48,35.78,31.93,31.70,29.40,29.24,28.28,24.32,21.11,20.80,19.43,18.72,11.90.HRMS(ESI):calcd for C 27 H 46 NaO2[M+Na] + ,425.3391,found 425.3384.

[0166] Preparation of compound (8) in Example 7

[0167]

[0168] Compound (6-2) (3.4 g, 8.2 mmol) and anhydrous THF (100 mL) were added to a flask. Methylmagnesium bromide (25 mL, 24.6 mmol) was slowly added under an ice-water bath. The reaction was carried out under nitrogen protection at room temperature for 5 h. After the reaction was detected by TLC, 2N HCl (5 mL) was added under an ice-water bath to quench the reaction. Water (80 mL) was added, and the mixture was extracted with EA (80 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 3:1) to obtain compound (8) (white solid, 2.14 g, yield 65%). The ratio of isomers 8E and 8Z was approximately 2:1 (after TLC analysis). 1 HNMR integral determination). 1 H NMR (500MHz, CDCl3) δ5.38-5.33(m,3H),3.54-3.50(m,1H),2.32-2.19(m,3H),2.13-2.08 (m,2H),1.20-1.94(m,2H),1.86-1.81(m,2H),1.71-1.63(m,2H),1.53-1.43(m,6H),1.26- 1.23(m,2H),1.21-1.19(m,6H),1.16-1.14(m,1H),1.10-1.05(m,2H),1.03(d,J=6.6Hz,3 H),1.01(d,J=2.6Hz,3H),0.98-0.96(m,1H),0.95-0.90(m,1H),0.71(s,1H),0.69(s,2H). 13 C NMR (125MHz, CDCl3) δ141.98,140.75,140.16,122.48,121.68,121.19,71. 78,70.95,70.54,56.79,56.06,55.62,50.09,46.84,42.29,41.66,40.37,3 9.65,37.25,36.51,34.31,31.87,31.65,29.16,29.05,28.84,28.17,24.33 ,24.27,21.06,20.75,20.67,19.44,19.42,12.22,12.09.HRMS(ESI):calcd for C 27 H 44 NaO2[M+Na] + ,423.3234,found423.3228.

[0169]

[0170] Compound (6-1) (3.28 g, 8.2 mmol) and anhydrous THF (100 mL) were added to a flask. Methylmagnesium bromide (25 mL, 24.6 mmol) was slowly added under an ice-water bath. The reaction was carried out at room temperature for 5 h under nitrogen protection. After the reaction was detected by TLC, 2N HCl (5 mL) was added under an ice-water bath to quench the reaction. Water (80 mL) was added, and the mixture was extracted with EA (80 mL × 3). The organic phases were combined and washed successively with saturated sodium bicarbonate solution (20 mL), water (20 mL), and saturated sodium chloride solution (20 mL). The mixture was purified by column chromatography (PE:EA = 3:1) to obtain compound (8) (white solid, 2.07 g, yield 63%). The ratio of isomers 8E and 8Z was approximately 2:1 (after TLC analysis). 1 HNMR integral determination). 1 H NMR (500MHz, CDCl3) δ5.38-5.33(m,3H),3.54-3.50(m,1H),2.32-2.19(m,3H),2.13-2.08 (m,2H),1.20-1.94(m,2H),1.86-1.81(m,2H),1.71-1.63(m,2H),1.53-1.43(m,6H),1.26- 1.23(m,2H),1.21-1.19(m,6H),1.16-1.14(m,1H),1.10-1.05(m,2H),1.03(d,J=6.6Hz,3 H),1.01(d,J=2.6Hz,3H),0.98-0.96(m,1H),0.95-0.90(m,1H),0.71(s,1H),0.69(s,2H). 13 C NMR (125MHz, CDCl3) δ141.98,140.75,140.16,122.48,121.68,121.19,71. 78,70.95,70.54,56.79,56.06,55.62,50.09,46.84,42.29,41.66,40.37,3 9.65,37.25,36.51,34.31,31.87,31.65,29.16,29.05,28.84,28.17,24.33 ,24.27,21.06,20.75,20.67,19.44,19.42,12.22,12.09.HRMS(ESI):calcd for C 27 H 44 NaO2[M+Na] + ,423.3234,found423.3228.

[0171] Example 8: Preparation of 25-hydroxycholesterol

[0172]

[0173] Compound of formula (8) (1.5 g, 3.7 mmol) and isopropanol (25 mL) were added to a flask and dissolved. Then, activated Raney Ni (1.5 g, wet weight, activated according to standard procedure) was added, along with H2 (1 atm), and the reaction was carried out at room temperature for 5 h. After the reaction was completed by TLC, Raney Ni was removed by filtration through a diatomaceous earth filter. The filtrate was concentrated under reduced pressure and subjected to column chromatography (PE:EA = 3:1) to obtain 25-hydroxycholesterol (white solid, 1.15 g, yield 76%). 1 H NMR (600MHz, CDCl3) δ5.37-5.33(m,1H),3.55-3.49(m,1H),2.31-2.28(m,1H) ,2.26-2.21(m,1H),2.04-1.95(m,2H),1.86-1.80(m,3H),1.60-1.48(m,5H),1 .48-1.43(m,3H),1.43-1.34(m,5H),1.27-1.23(m,2H),1.21(s,6H),1.18-1. 14(m,1H),1.11-1.03(m,4H),1.01(s,3H),0.93(d,J=6.5Hz,3H),0.68(s,3H). 13 C NMR (150MHz, CDCl3) δ140.81,121.73,71.83,71.15,56.79,56.10,50.15,44.46,42.37,42.34,39.81,37.28,36. 54,36.48,35.78,31.93,31.70,29.40,29.24,28.28,24.32,21.11,20.80,19.43,18.72,11.90.HRMS(ESI):calcd for C 27 H 46 NaO2[M+Na] + ,425.3391,found425.3384.

[0174] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0175] As used in this invention, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0176] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0177] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A method for synthesizing 25-hydroxycholesterol from plant-derived 21-hydroxy-20-methylpregn-4-en-3-one (BA), characterized in that, The method uses BA as a raw material and synthesizes the 25-hydroxycholesterol through oxidation, Wittig reaction, esterification, acetylation, reduction, selective hydrogenation reduction, and Grignard reaction, or through oxidation, Wittig reaction, esterification, acetylation, reduction, Grignard reaction, and selective hydrogenation reduction steps. The method includes the following steps: Step (a): In the first solvent, BA represented by formula (1) is oxidized to obtain compound (2); Step (b): In a second solvent, the compound of formula (2) undergoes a Wittig reaction to obtain the compound of formula (3); Step (c): In a third solvent, the compound of formula (3) is esterified to obtain the compound of formula (4); Step (d): In a fourth solvent, the compound of formula (4) is acetylated to obtain the compound of formula (5); Step (e): In the fifth solvent, the compound of formula (5) is reduced to obtain the compound of formula (6); Step (f): In the sixth solvent, the compound of formula (6) is selectively hydrogenated to obtain the compound of formula (7); Step (g): In the seventh solvent, the compound of formula (7) is subjected to a Grignard reaction to give the compound of formula (9) 25-hydroxycholesterol; Alternatively, the method may include the following steps: Step (a): In the first solvent, BA represented by formula (1) is oxidized to obtain compound (2); Step (b): In a second solvent, the compound of formula (2) undergoes a Wittig reaction to obtain the compound of formula (3); Step (c): In a third solvent, the compound of formula (3) is esterified to obtain the compound of formula (4); Step (d): In a fourth solvent, the compound of formula (4) is acetylated to obtain the compound of formula (5); Step (e): In the fifth solvent, the compound of formula (5) is reduced to obtain the compound of formula (6); Step (h): In the eighth solvent, the compound of formula (6) undergoes a Grignard reaction to obtain the compound of formula (8); Step (i): In the ninth solvent, the compound of formula (8) is selectively hydrogenated to obtain the compound of formula (9), 25-hydroxycholesterol. The reaction process of the method is shown in route (A): in, In the compounds (3) to (6) and (8), the ratio of the E configuration to the Z configuration of the double bond between C-22 and C-23 is determined by... 1 The HNMR integral is determined to be 1:2 to 2:1; R stands for alkyl group.

2. The method as described in claim 1, characterized in that, R is a C1 to C20 alkyl group.

3. The method as described in claim 1, characterized in that, In step (a), the oxidation reaction specifically involves the following: in the first solvent, BA represented by formula (1) undergoes an oxidation reaction with 2,2,6,6-tetramethylpiperidine oxide TEMPO, sodium bicarbonate, tetrabutylammonium bromide, and an oxidant to obtain the compound of formula (2). And / or, in step (b), the Wittig reaction specifically involves the reaction of the compound of formula (2), 2-carboxyethyltriphenylphosphine halide, and base in the second solvent to obtain the compound of formula (3); And / or, in step (c), the esterification reaction specifically involves the esterification reaction of the compound of formula (3) and the catalyst in the third solvent to obtain the compound of formula (4).

4. The method as described in claim 3, characterized in that, In step (a), the molar ratio of BA, TEMPO, sodium bicarbonate, tetrabutylammonium bromide, and oxidant shown in formula (1) is 1:(0-1):(0-20):(0-1):(1-5); and / or, the oxidant is selected from one or more of N-chlorosuccinimide NCS, N-bromosuccinimide NBS, and 2-iodobenzoic acid IBX; and / or, the first solvent is selected from one or more of dichloromethane, tetrahydrofuran, toluene, dimethyl sulfoxide, and water; and / or, the temperature of the oxidation reaction is 0-30°C; and / or, the time of the oxidation reaction is 3-8 h. And / or, in step (b), the molar ratio of the compound of formula (2), 2-carboxyethyltriphenylphosphine halide, and the base is 1:(1-4):(2-8); and / or, the second solvent is selected from one or more of toluene, dimethyl sulfoxide, tetrahydrofuran, and dichloromethane; and / or, the 2-carboxyethyltriphenylphosphine halide is selected from one or two of 2-carboxyethyltriphenylphosphine chloride and 2-carboxyethyltriphenylphosphine bromide; and / or, the base is selected from one or more of sodium hydride (NaH), potassium tert-butoxide (t-BuOK), sodium tert-butoxide (t-BuONa), lithium bis(trimethylsilylamino)amine (LiHMDS), sodium hydroxide, potassium hydroxide, and lithium diisopropylamino (LDA); and / or, the temperature of the Wittig reaction is -10 to 112°C; and / or, the time of the Wittig reaction is 0.5 to 9 h. And / or, in step (c), the molar ratio of the compound of formula (3) to the catalyst is 1:(0.01-5); and / or, the catalyst is selected from one or more of sulfoxide, concentrated sulfuric acid, and p-toluenesulfonic acid; and / or, the third solvent is selected from one or more of methanol, ethanol, and propanol; the temperature of the esterification reaction is 20-100°C; and / or, the time of the esterification reaction is 0.5-20 h.

5. The method as described in claim 1, characterized in that, In step (d), the acetylation reaction specifically involves the acetylation reaction of the compound of formula (4), acetyl chloride, and acetic anhydride in the fourth solvent to obtain the compound of formula (5). And / or, in step (e), the reduction reaction specifically involves: adding a metal catalyst and a reducing agent to the fifth solvent to reduce the compound of formula (5) to obtain the compound of formula (6); And / or, in step (f), the selective hydrogenation reduction reaction specifically involves the selective hydrogenation reduction reaction of the compound of formula (6) with a reducing agent in the sixth solvent under the action of a catalyst to obtain the compound of formula (7).

6. The method as described in claim 5, characterized in that, In step (d), the molar ratio of the compound of formula (4), acetyl chloride, and acetic anhydride is 1:(0.5-62.5):(1-62.5); and / or, the fourth solvent is selected from one or more of acetic anhydride, acetyl chloride, ethyl acetate, and dichloromethane; and / or, the temperature of the acetylation reaction is 40-120°C; and / or, the time of the acetylation reaction is 1-20 h. And / or, in step (e), the molar ratio of the compound of formula (5), the metal catalyst, and the reducing agent is 1:(0.1-5):(1-9); and / or, the fifth solvent is selected from one or more of tetrahydrofuran, methanol, ethanol, water, dichloromethane, 2-methyltetrahydrofuran, isopropanol, and methyl tert-butyl ether; and / or, the reducing agent is selected from one or two of sodium borohydride NaBH4 and potassium borohydride KBH4; and / or, the metal catalyst is selected from one or more of copper chloride, copper bromide, cuprous chloride, cuprous bromide, cuprous iodide, copper acetate, copper tartrate, copper glycinate, copper citrate, ferrous chloride, magnesium chloride, ferrous sulfate, nickel chloride, lithium chloride, aluminum chloride, manganese chloride, and calcium chloride; and / or, the temperature of the reduction reaction is -10 to 50°C; and / or, the time of the reduction reaction is 0.1 to 20 h. And / or, in step (f), the mass ratio of the compound of formula (6) to the catalyst is 1:(0.05-5); and / or, the reducing agent is H2; and / or, the catalyst is Raney Ni; and / or, the sixth solvent is selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, and isopropanol; and / or, the temperature of the selective hydrogenation reduction reaction is 0-60°C; and / or, the pressure of the reducing agent H2 in the selective hydrogenation reduction reaction is 1-20 atm; and / or, the time of the selective hydrogenation reduction reaction is 1-10 h.

7. The method as described in claim 1, characterized in that, In step (g), the Grignard reaction specifically involves the following: the compound of formula (7) reacts with magnesium methyl halide in the seventh solvent to obtain the 25-hydroxycholesterol; and / or, in step (h), the Grignard reaction specifically involves the following: the compound of formula (6) reacts with magnesium methyl halide in the eighth solvent to obtain the compound of formula (8). And / or, in step (i), the selective hydrogenation reduction reaction specifically involves the compound of formula (8) undergoing a selective hydrogenation reduction reaction with a reducing agent in the ninth solvent under the action of a catalyst to obtain the compound of formula (9), 25-hydroxycholesterol.

8. The method as described in claim 7, characterized in that, In step (g), the molar ratio of the compound of formula (7) to magnesium methyl halide is 1:(3-12); and / or, the magnesium methyl halide is selected from one or more of magnesium methyl chloride, magnesium methyl bromide, and magnesium methyl iodide; and / or, the seventh solvent is selected from one or more of diethyl ether, 2-methyltetrahydrofuran, and tetrahydrofuran; and / or, the Grignard reaction temperature is 0-40°C; and / or, the Grignard reaction time is 1-15 h; and / or, in step (h), the molar ratio of the compound of formula (6) to magnesium methyl halide is 1:(3-12); and / or, the magnesium methyl halide is selected from one or more of magnesium methyl chloride, magnesium methyl bromide, and magnesium methyl iodide; and / or, the eighth solvent is selected from one or more of diethyl ether, 2-methyltetrahydrofuran, and tetrahydrofuran; and / or, the Grignard reaction temperature is 0-40°C; and / or, the Grignard reaction time is 1-15 h.

9. The method as described in claim 7, characterized in that, In step (i), the mass ratio of the compound of formula (8) to the catalyst is 1:(0.05-5); and / or, the reducing agent is H2; and / or, the catalyst is Raney Ni; and / or, the ninth solvent is selected from one or more of 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, toluene, and isopropanol; and / or, the temperature of the selective hydrogenation reduction reaction is 0-60°C; and / or, the pressure of the reducing agent H2 in the selective hydrogenation reduction reaction is 1-20 atm; and / or, the time of the selective hydrogenation reduction reaction is 1-10 h.

10. A compound, characterized in that, The structure of the compound is shown in formula (10):

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