A process for the preparation of 25-hydroxycholesterol
By using bis(hydroxyl) oxide as a starting material, first constructing a side chain carbon chain, then constructing an AB ring and performing a Grignard reaction, the problems of low yield and high cost in the preparation of 25-hydroxycholesterol in the prior art have been solved, realizing an efficient and low-cost preparation method with significantly improved yield and purity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGXI SHENTIAN BIOTECH CO LTD
- Filing Date
- 2023-12-11
- Publication Date
- 2026-07-03
AI Technical Summary
Existing methods for preparing 25-hydroxycholesterol suffer from problems such as long routes, low yields, high reagent toxicity, and expensive starting materials, making it difficult to achieve rapid, efficient, and low-cost synthesis.
Using bis(2-hydroxyol) oxide as the starting material, the carbon chain of the side chain was first constructed, then the AB ring was constructed, and the 25-hydroxyl group was constructed through Grignard reaction. Using KGP-06 as the raw material, the reaction temperature and conditions were controlled to carry out multi-step synthesis, and finally high-purity 25-hydroxycholesterol was obtained.
Achieving a yield of up to 97% and a purity of over 98%, this method provides a simple and efficient preparation method that reduces costs and environmental pollution risks.
Smart Images
Figure CN117700474B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of 25-hydroxycholesterol technology, and more specifically to a method for preparing 25-hydroxycholesterol. Background Technology
[0002] 25-Hydroxycholesterol possesses a wide range of pharmacological activities, such as antiviral, anti-inflammatory, anti-IR damage, and immunomodulatory effects in mammalian cells. It also plays a crucial role in lipid metabolism, particularly lipid biosynthesis and metabolism. Furthermore, 25-hydroxycholesterol is an important intermediate in the synthesis of calcitriol, thus holding significant research value. The current technical challenge lies in the modification of the side chain to introduce a 25-hydroxyl group. Currently, there are two main construction methods: one involves constructing the AB ring and side chain separately and then docking them via a reaction; the other involves modifying the cholesterol side chain to introduce a 25-hydroxyl group.
[0003] Patent CN112608361A discloses a method for synthesizing 25-hydroxycholesterol using bis(BA) as a starting material, the route of which is as follows:
[0004]
[0005] This route uses a large amount of lithium bromide and lithium carbonate for bromination, which is costly. In addition, the coupling of the brominated product with acrylate uses nickel chloride and zinc powder, which will generate a large amount of solid waste. At the same time, pyridine is used as a solvent, which also uses a large amount of methyl acrylate. Both have strong odors and are not environmentally friendly. Furthermore, due to the easy polymerization of methyl acrylate, the reaction is difficult to avoid.
[0006] Patent CN114315947A discloses a method for synthesizing 25-hydroxycholesterol using bis(BA) as a starting material, the route of which is as follows:
[0007]
[0008] The 3-trimethylsiloxy-3-methylbutylmagnesium bromide reagent used in this route requires a catalytic reaction with copper-lithium reagents and more than 10 equivalents of magnesium reagent for the reaction to proceed to completion. This results in low atom economy, and the high cost of 3-trimethylsiloxy-3-methylbutylmagnesium bromide and copper-lithium reagents leads to the high cost of 25-hydroxycholesterol.
[0009] Patent CN103626821A discloses a method for preparing 25-hydroxycholesterol using 24-dehydrocholesterol as a raw material, the route of which is as follows:
[0010]
[0011] This route synthesizes 2-hydroxycholesterol through reactions such as acylation, epoxidation, and ring-opening. However, the yield is low, the starting material 24-dehydrocholesterol is difficult to obtain, and high purity is required, resulting in high costs and preventing large-scale production. Furthermore, the process uses some heavy metal salts, causing significant pollution.
[0012] The current technical challenge lies in the modification of the side chain to introduce a hydroxyl group at position 25. There are currently two main construction methods: one is to construct the AB ring and side chain separately and then dock them through a reaction; the other is to modify the side chain of cholesterol to introduce a hydroxyl group at position 25.
[0013] Current reported preparation methods generally suffer from drawbacks such as long routes, low yields, high reagent toxicity, and expensive starting materials. Therefore, it is necessary to find a rapid, efficient, low-toxicity, and low-cost synthetic route. Summary of the Invention
[0014] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a method for preparing 25-hydroxycholesterol, solving the technical problems of long routes and low yields in existing methods for preparing 25-hydroxycholesterol.
[0015] To achieve the above-mentioned technical objectives, the present invention provides a method for preparing 25-hydroxycholesterol, comprising the following steps:
[0016] Tetrahydrofuran and KGP-06 were mixed and cooled to below 0°C under nitrogen protection. Then, methyl magnesium chloride solution was added dropwise, and the reaction temperature was controlled below 15°C. After the reaction was complete, hydrochloric acid solution was added to obtain the first reaction solution.
[0017] The first reaction solution was poured into acidic water, and the temperature was controlled below 40°C. Then it was concentrated, then water was added to cool it down to below 30°C, filtered, washed with water, and dried to obtain a solid. Then DCM and methanol were added to dissolve the solid, then it was concentrated, methanol was added to replace it to a viscous state, and then it was cooled to crystallize to obtain the 25-hydroxycholesterol.
[0018] The structural formula of KGP-06 is:
[0019]
[0020] Furthermore, the ratio of KGP-06 to the methyl magnesium chloride solution is 50g:(120-130)mL, and the concentration of the methyl magnesium chloride solution is 2.5-3mol / L.
[0021] Furthermore, the KGP-06 is prepared by the following steps:
[0022] Dichloromethane and methanol were mixed, then KGP-05 and Raney nickel were added, nitrogen gas was then introduced for purging, and the mixture was cooled to 0-5℃ and hydrogen gas was introduced to react and obtain KGP-06.
[0023] The structural formula of KGP-05 is as follows:
[0024]
[0025] Furthermore, the mass ratio of the KGP-05 to the Raney nickel is 100:(1-2).
[0026] Furthermore, the KGP-05 is prepared by the following steps:
[0027] Tetrahydrofuran, methanol, and sodium borohydride were mixed, followed by the addition of anhydrous lithium chloride. The mixture was then cooled to -5 to 0°C with stirring, and KGP-04 was added to continue the reaction. After the reaction was complete, a second reaction solution was obtained. This second reaction solution was slowly added to water, and then glacial acetic acid was added dropwise to adjust the pH to 4-5. The mixture was stirred to obtain KGP-05. The structural formula of KGP-04 is:
[0028]
[0029] Further, KGP-04 is prepared by the following steps: PTS is added to isopropyl acetate under stirring, followed by the addition of KGP-03, and then the temperature is raised to 80-85°C for reflux reaction. After the reaction is complete, the temperature is lowered to below 10°C, and triethylamine is added. Then the mixture is concentrated at 45-50°C, followed by the addition of methanol, and the mixture is further concentrated until the material becomes viscous. Finally, the temperature is lowered to 5-10°C and stirring is continued to obtain KGP-04.
[0030] The structural formula of the KGP-03 is as follows:
[0031]
[0032] Furthermore, the reflux reaction time is 3-4 hours.
[0033] Furthermore, the KGP-03 is prepared by the following steps:
[0034] KGP-02 and THF were mixed, then diethyl phosphonoacetate was added, followed by sodium methoxide. The mixture was stirred until the reaction was complete while maintaining the temperature below 40°C. Water and hydrochloric acid were then added to adjust the pH to 4-5 to obtain KGP-03.
[0035] The KGP-02 structure is as follows:
[0036]
[0037] Furthermore, the KGP-02 is prepared by the following steps:
[0038] (Methoxymethyl)triphenylphosphine chloride and DMSO were mixed, and then NaH was added. The reaction was carried out at a temperature below 30°C. KGP-01 was then added and the reaction was monitored by TLC at a temperature below 40°C. Water was then added, followed by hydrochloric acid to adjust the pH to 4-5. The aqueous layer was then extracted with dichloroisocyanuric acid. The organic phases were combined and concentrated to obtain an oil. Acetone was added to the oil and the temperature was raised to 40-45°C. Water was added dropwise, and the temperature was lowered to 5-10°C with stirring to obtain KGP-02.
[0039] The structural formula of the KGP-01 is:
[0040]
[0041] Furthermore, the mass ratio of (methoxymethyl)triphenylphosphine chloride to KGP-01 is 10:(5-6).
[0042] Compared with the prior art, the beneficial effects of the present invention include: the present invention mixes tetrahydrofuran and KGP-06 and cools it to below 0°C under nitrogen protection, then adds methyl magnesium chloride solution dropwise, controlling the reaction temperature to below 15°C; after the reaction is complete, hydrochloric acid solution is added to obtain a first reaction solution; the first reaction solution is poured into acid water, the temperature is controlled to below 40°C, then concentrated, then water is added to cool to below 30°C, filtered, washed with water, and dried to obtain a solid, then DCM and methanol are added to dissolve the solid, then concentrated, methanol is added to replace it to a viscous state, and then the mixture is cooled to crystallize to obtain the 25-hydroxycholesterol. The present invention uses KGP-06 as raw material to obtain 25-hydroxycholesterol through Grignard reaction with a yield as high as 97% and a purity greater than 98%. The preparation method is simple and has a high yield. Attached Figure Description
[0043] Figure 1 This is the HNMR spectrum of KGP-02 obtained in Example 1 of the present invention.
[0044] Figure 2 This is the CNMR spectrum of KGP-02 obtained in Example 1 of this invention.
[0045] Figure 3 This is the HNMR spectrum of KGP-03 obtained in Example 1 of the present invention.
[0046] Figure 4 This is the CNMR spectrum of KGP-03 obtained in Example 1 of this invention.
[0047] Figure 5 This is the HNMR spectrum of KGP-04 obtained in Example 1 of this invention.
[0048] Figure 6 This is the CNMR spectrum of KGP-04 obtained in Example 1 of this invention.
[0049] Figure 7 This is the HNMR spectrum of KGP-05 obtained in Example 1 of the present invention.
[0050] Figure 8 This is the CNMR spectrum of KGP-05 obtained in Example 1 of this invention.
[0051] Figure 9 This is the HPLC chromatogram of KGP-05 obtained in Example 1 of this invention.
[0052] Figure 10 This is the HNMR spectrum of KGP-07 obtained in Example 1 of this invention.
[0053] Figure 11 This is the CNMR spectrum of KGP-07 obtained in Example 1 of this invention.
[0054] Figure 12 This is the HPLC chromatogram of KGP-07 obtained in Example 1 of this invention. Detailed Implementation
[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0056] This specific embodiment provides a method for preparing 25-hydroxycholesterol, including the following steps:
[0057] (Methoxymethyl)triphenylphosphine chloride and DMSO were mixed, then NaH was added, and the reaction was carried out at a temperature below 30°C. KGP-01 was then added, and the reaction was monitored by TLC at a temperature below 40°C. Water was then added, followed by hydrochloric acid to adjust the pH to 4-5. The aqueous layer was then extracted with dichloroisocyanuric acid, and the organic phases were combined and concentrated to obtain an oily substance. Acetone was added to the oily substance, and the temperature was raised to 40-45°C. Water was then added dropwise, and the temperature was lowered to 5-10°C with stirring to obtain KGP-02. The mass ratio of (methoxymethyl)triphenylphosphine chloride to KGP-01 was 10:(5-6).
[0058] KGP-02 and THF were mixed, then diethyl phosphonoacetate was added, followed by sodium methoxide. The mixture was stirred until the reaction was complete while maintaining the temperature below 40°C. Water and hydrochloric acid were then added to adjust the pH to 4-5 to obtain KGP-03.
[0059] PTS was added to isopropyl acetate under stirring, followed by the addition of KGP-03. The mixture was then heated to 80-85°C and refluxed for 3-4 hours. After the reaction was complete, the temperature was lowered to below 10°C, and triethylamine was added. The mixture was then concentrated at 45-50°C, followed by the addition of methanol. The mixture was then concentrated until it became viscous, and the temperature was lowered to 5-10°C with stirring to obtain KGP-04.
[0060] Tetrahydrofuran, methanol and sodium borohydride were mixed, and then anhydrous lithium chloride was added. The mixture was then cooled to -5 to 0°C with stirring. KGP-04 was then added to continue the reaction. After the reaction was complete, a second reaction solution was obtained. The second reaction solution was slowly added to water, and then glacial acetic acid was added dropwise to adjust the pH to 4 to 5. The mixture was stirred to obtain KGP-05.
[0061] Dichloromethane and methanol were mixed, and then KGP-05 and Raney nickel were added, with the mass ratio of KGP-05 to Raney nickel being 100:(1-2). Nitrogen gas was then introduced for purging, and the mixture was cooled to 0-5°C and hydrogen gas was introduced to react and obtain KGP-06.
[0062] Tetrahydrofuran and KGP-06 were mixed and cooled to below 0°C under nitrogen protection. Then, methyl magnesium chloride solution was added dropwise, and the reaction temperature was controlled below 15°C. After the reaction was complete, hydrochloric acid solution was added to obtain the first reaction solution. The ratio of KGP-06 to methyl magnesium chloride solution was 50g:(120-130)mL, and the concentration of methyl magnesium chloride solution was 2.5-3mol / L.
[0063] The first reaction solution was poured into acidic water, and the temperature was controlled below 40°C. The solution was then concentrated, and water was added to cool it down to below 30°C. The solution was then filtered, washed with water, and dried to obtain a solid. DCM and methanol were then added to dissolve the solid. The solution was then concentrated, and methanol was added to displace it to a viscous state. The solution was then cooled to crystallize and obtain the 25-hydroxycholesterol (i.e., KGP-06).
[0064] The reaction mechanism is as follows:
[0065]
[0066] We adopted a different approach than previous strategies: using bis(oxo)ol oxide as the starting material, we first constructed the carbon chain of the side chain, and then constructed the AB ring with the carbon chain. After the AB ring was constructed, it was hydrogenated, and finally the 25-hydroxyl group was constructed by Grignard reaction to obtain 25-hydroxycholesterol.
[0067] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0068] Example 1
[0069] This embodiment proposes a method for preparing 25-hydroxycholesterol, including the following steps:
[0070] 100g of (methoxymethyl)triphenylphosphine chloride and 300ml of DMSO were added to a reaction flask. 2.5g of 60% NaH1 was added with stirring, maintaining the system temperature below 30℃. After the addition was complete, the mixture was stirred for 1 hour. Then, 50g of KGP-01 was added, maintaining the system temperature below 40℃. The reaction was monitored by TLC. The developing solvent was PE:EA = 4:1. After the reaction was complete, 4V of water was added to quench the reaction, and the pH was adjusted to 4 with hydrochloric acid. The aqueous layer was extracted with dichloroisocyanuric acid. The organic phases were combined, and the concentrated solvent was removed under reduced pressure at 40℃ to obtain an oily substance. 250ml of acetone was added to the oily substance, and the temperature was raised to 45℃. 350ml of water was slowly added dropwise. After the water addition was complete, the temperature was lowered to 5℃ and stirred for 30 minutes. The mixture was filtered, washed with water, and dried (at 50℃) to obtain the purified KGP-02, with a yield of 98.8%. Figure 1 and 2 The following are the proton and carbon spectra of KGP-02: 1 HNMR (400MHz, CDCl3) δ9.75(s,1H),5.72(s,1H),1.0-2.5(m,20H),1.19(s,3H),1.17–1.05(m,2H),1.03(m,3H),0.99–0.88(m,1H),0.77(s,3H).
[0071] 13 C NMR(101MHz, CDCl3)δ203.22(s),199.46(s),171.31(s),123.80(s),55.83(s),55.79(s),53.68(s),50.81(s),42.54(s),39.45(s),38 .55(s),35.67(s),35.57(s),33.95(s),32.85(s),31.94(s),31.51(s),28.37(s),24.08(s),20.96(s),19.95(s),17.36(s),11.95(s).
[0072]
[0073] At room temperature, 50g of KGP-02 and 300ml of THF were added to a reaction flask and stirred. 75g of diethyl methyl phosphonoacetate and 10g of sodium methoxide solid were then added. After the addition was complete, the system temperature was kept below 40℃, and the reaction was stirred until complete, which took about one hour. After the reaction was complete, 4V of water was added, and the pH was adjusted to 4 with an appropriate amount of hydrochloric acid. The THF was removed under reduced pressure at 40℃. After complete removal, an appropriate amount of water was added, the mixture was stirred, filtered, and dried (at 50℃) to obtain crude KGP-03 with a yield of 115%. Figure 3 and 4 The HNMR and CNMR spectra of KGP-03 are shown below:
[0074] 1 H NMR (400MHz, CDCl3) δ6.95(s,1H),5.55-6.04(m,2H),3.73(s,3H),2.33(m,5H),1.5-2.1(m,14H),1.18(m,6H),0.96(m,6H),0.72(s,3H).
[0075] 13 C NMR(101MHz, CDCl3)δ199.52(s),171.42(s),166.97(s),148.31(s),123.80 (s),122.23(s),55.77(s),55.52(s),57.32(s),51.37(s),42.48(s),39.43( s),38.99(s),38.57(s),35.68(s),35.61(s),35.60(s),33.91(s),32.89(s) ),31.98(s),28.16(s),24.14(s),20.98(s),18.92(s),17.37(s),11.96(s).
[0076]
[0077] Add 250 ml of isopropyl acetate to a clean, dry reaction flask. Add 0.5 g of PTS with stirring, and stir for 5 minutes. Then add 50 g of KGP-03. Heat to 85°C and reflux for 3 hours. After the reaction is complete, cool to below 10°C and slowly add 1 g of triethylamine. Concentrate under reduced pressure at 45°C to a small volume. Add 100 ml of methanol and continue to concentrate under reduced pressure until the material becomes viscous. Cool to 5°C and stir for 1 hour. Filter and dry at 55°C to obtain KGP-04, with a yield of 103%. Figure 5 and 6 The HNMR and CNMR spectra of KGP-04 are shown below:
[0078] 1H NMR (400MHz, CDCl3) δ6.96 (s, 1H), 5.82 (d, J = 15.6Hz, 1H), 5.69 (s, 1H), 5.39 (s, 1H), 3.73 (s ,3H),2.44(s,1H),2.29(s,1H),2.13(s,3H),2.07–1.04(m,19H),0.97(m,6H),0.72(s,3H).
[0079] 13 C NMR(101MHz, CDCl3)δ169.38(s),167.02(s),148.50(s),147.00(s),139.39(s),1 23.95(s),122.16(s),116.98(s),56.73(s),55.58(s),51.36(s),47.91(s),42.55 (s),39.56(s),39.07(s),35.66(s),34.89(s),33.77(s),31.82(s),31.75(s),28 .23(s),24.81(s),24.17(s),21.16(s),21.09(s),18.99(s),18.83(s),11.98(s).
[0080]
[0081] Under stirring, 200 ml of tetrahydrofuran, 200 ml of methanol, and 6.5 g of sodium borohydride were weighed and added to the reaction flask. Then, 3.5 g of anhydrous lithium chloride was weighed and added to the reaction flask. The mixture was stirred and cooled to -5°C. Then, 50 g of KGP-04 was added, and the mixture was kept at -5°C for 8 hours. After the reaction was complete, the second reaction solution was slowly added to 500 ml of water, and glacial acetic acid was slowly added dropwise to adjust the pH to 4. The mixture was stirred for 2 hours, filtered, and the filter cake was washed with drinking water until neutral. The filter cake was then dried to obtain wet KGP-05, with a yield of 88.9% and a purity of 96.5%. Figure 7 , 8 9 and 9 represent the HNMR, CNMR, and HPLC spectra of KGP-05, respectively, as follows:
[0082] 1H NMR (400MHz, CDCl3) δ6.96 (s, 1H), 5.82 (d, J = 15.6Hz, 1H), 5.35 (s, 1H), 3.73 (s, 3H), 3.52 (s, 1H), 2 .26(s,3H),1.96(s,3H),1.84(s,3H),1.74–1.07(m,14H),δ1.01(s,3H),0.95(m,3H),0.69(s,3H).
[0083] 13 C NMR(101MHz, CDCl3)δ167.06(s),148.55(s),140.81(s),122.14(s),121.59 (s),71.74(s),56.66(s),55.56(s),51.38(s),50.06(s),42.43(s),42.29( s),39.59(s),39.08(s),37.25(s),36.50(s),35.66(s),31.9(s),31.86(s) ,31.64(s),28.23(s),24.27(s),21.04(s),19.40(s),19.00(s),11.88(s).
[0084]
[0085] Add 200 ml of dichloromethane and 200 ml of methanol to a reaction flask. While stirring, add 50 g of KGP-05 and 0.5 g of Raney nickel. Purge the mixture with nitrogen three times. Cool to 0°C and then purge with hydrogen gas, maintaining the reaction temperature at 0°C for 8 hours. After the reaction is complete, stop the hydrogen purge, turn off the gas cylinder, purge with nitrogen, filter, carefully collect the filter cake for later use, and dry under vacuum to obtain a KGP-06 solution. Concentrate under reduced pressure at 40°C until viscous, then purge with 50 ml of methanol until viscous again. Cool to approximately 5°C to allow crystallization for 0.5 hours. Filter under vacuum, wash with a small amount of methanol, and dry the solid at 50°C to obtain KGP-06, with a yield of 99%.
[0086]
[0087] Add 250 ml of tetrahydrofuran to a clean, dry reaction flask, purge with nitrogen, add 50 g of KGP-06 solid and stir. Under nitrogen protection, cool to below 0°C. Once the reaction system temperature is below 0°C, add 120 ml of methyl magnesium chloride solution (3M) dropwise, maintaining the system temperature below 15°C. After the reaction is complete, add 400 ml of tap water and 50 ml of concentrated hydrochloric acid to a beaker, stir and cool. Slowly pour the first reaction solution into the acidic water, maintaining the internal temperature below 40°C. Concentrate under reduced pressure to remove tetrahydrofuran while maintaining the temperature below 50°C, cool and add water, measuring the pH to 4. Cool to below 30°C, filter, and wash with water. After drying the solid, add 250 ml of DCM and 250 ml of methanol to dissolve it, concentrate under reduced pressure at 40°C, purge with methanol until viscous, cool to approximately 5°C to crystallize for 1 hour, filter, and dry at 50°C to obtain KOH-07 (25-hydroxycholesterol), with a yield of 97% and a purity of 99.4%. Figure 10 , 11 12 and 12 are the HNMR, CNMR and HPLC spectra of KGP-07, respectively, as follows:
[0088] 1 H NMR (400MHz, DMSO) δ5.26(s,1H),4.58(s,1H),4.02(s,1H),,2.27–0.78(m,40H),0.65(s,3H).
[0089] 13 C NMR(101MHz,DMSO)δ141.74(s),120.88(s).,70.48(s),69.23(s),56.70(s),56.11(s),50.11(s),42.34(s),40.64(s),40. 43(s),40.23(s),40.02(s),39.81(s),39.39(s),36.54(s),31.96(s),29.89(s),29.71(s),19.62(s),19.04(s),12.14(s)( There are 6 carbon credits The signal peak overlaps with other signal peaks. ).
[0090]
[0091] Example 2
[0092] This embodiment proposes a method for preparing 25-hydroxycholesterol, including the following steps:
[0093] 100g of (methoxymethyl)triphenylphosphine chloride and 300ml of DMSO were added to a reaction flask. 2.5g of 60% NaH1 was added with stirring, maintaining the system temperature below 30℃. After the addition was complete, the mixture was stirred for 1 hour. Then, 60g of KGP-01 was added, maintaining the system temperature below 40℃. The reaction was monitored by TLC. The developing solvent was PE:EA = 4:1. After the reaction was complete, 4V of water was added to quench the reaction, and the pH was adjusted to 5 with hydrochloric acid. The aqueous layer was extracted with dichloroisocyanurate. The organic phases were combined, and the concentrated solvent was removed under reduced pressure at 40℃ to obtain an oily substance. 250ml of acetone was added to the oily substance, and the temperature was raised to 40℃. 350ml of water was slowly added dropwise. After the water addition was complete, the temperature was lowered to 10℃ and stirred for 30 minutes. The mixture was filtered, washed with water, and dried (at 50℃) to obtain pure KGP-02 with a yield greater than 98.2%.
[0094]
[0095] At room temperature, 50g of KGP-02 and 300ml of THF were added to a reaction flask and stirred. 75g of diethyl methyl phosphonoacetate and 10g of sodium methoxide solid were then added. After the addition was complete, the system temperature was kept below 40°C, and the mixture was stirred until the reaction was complete, which took about one hour. After the reaction was complete, 4V of water was added, and the pH was adjusted to 5 with an appropriate amount of hydrochloric acid. The THF was removed under reduced pressure at 40°C. After complete removal, an appropriate amount of water was added, the mixture was stirred, filtered, and dried (at 50°C) to obtain crude KGP-03 with a yield of 110%.
[0096]
[0097] Add 250 ml of isopropyl acetate to a clean, dry reaction flask. Add 0.5 g of PTS while stirring, and stir for 5 minutes. Then add 50 g of KGP-03. Heat to 80 °C and reflux for 3 hours. After the reaction is complete, cool to below 10 °C and slowly add 1 g of triethylamine. Concentrate under reduced pressure at 45 °C to a small volume. Add 100 ml of methanol and continue to concentrate under reduced pressure until the material becomes viscous. Cool to 5 °C and stir for 1 hour. Filter and dry at 50 °C to obtain KGP-04, with a yield of 104%.
[0098]
[0099] Under stirring, 200 ml of tetrahydrofuran, 200 ml of methanol, and 0.13 g of sodium borohydride were weighed and added to the reaction flask. Then, 3.5 g of anhydrous lithium chloride was weighed and added to the reaction flask. The mixture was stirred and cooled to -5°C. Then, 50 g of KGP-04 was added, and the mixture was kept at -5°C for 8 hours. After the reaction was complete, the second reaction solution was slowly added to 500 ml of water, and glacial acetic acid was slowly added dropwise to adjust the pH to 5. The mixture was stirred for 2 hours, filtered, and the filter cake was washed with drinking water until neutral. The filter cake was then dried to obtain wet KGP-05, with a yield of 88.3% and a purity of 94.7%.
[0100]
[0101] Add 200 ml of dichloromethane and 200 ml of methanol to a reaction flask. While stirring, add 50 g of KGP-05 and 1 g of Raney nickel. Purge the mixture three times with nitrogen. Cool to 0°C and then purge the system with hydrogen gas, maintaining the reaction temperature at 0°C for 8 hours. After the reaction is complete, stop the hydrogen purge, turn off the gas cylinder, purge with nitrogen, filter, carefully collect the filter cake for later use, and dry under vacuum to obtain a KGP-06 solution. Concentrate under reduced pressure at 40°C until viscous, then purge with 50 ml of methanol until viscous again. Cool to approximately 5°C to allow crystallization for 0.5 hours. Filter, wash with a small amount of methanol, and dry the solid at 50°C to obtain KGP-06, with a yield of 98.5%.
[0102]
[0103] Add 250 ml of tetrahydrofuran to a clean, dry reaction flask, purge with nitrogen, add 50 g of KGP-06 solid and stir. Under nitrogen protection, cool to below 0°C. Once the reaction system temperature is below 0°C, add 130 ml of methyl magnesium chloride solution (2.5 M) dropwise, maintaining the system temperature below 15°C. After the reaction is complete, add 400 ml of tap water and 50 ml of concentrated hydrochloric acid to a beaker, stir and cool. Slowly pour the first reaction solution into the acidic solution, maintaining the internal temperature below 40°C. Concentrate under reduced pressure to remove tetrahydrofuran while maintaining the temperature below 50°C, cool and add water, measuring the pH to 3. Cool to below 30°C, filter, and wash with water. After drying the solid, add 250 ml of DCM and 250 ml of methanol to dissolve it, concentrate under reduced pressure at 40°C, purge with methanol until viscous, cool to approximately 5°C to crystallize for 1 hour, filter, and dry at 50°C to obtain KOH-07 (25-hydroxycholesterol), with a yield of 96.4% and a purity of 98.3%.
[0104]
[0105] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for the preparation of 25-hydroxycholesterol, characterized in that, Includes the following steps: Tetrahydrofuran and KGP-06 were mixed and cooled to below 0°C under nitrogen protection. Then, methyl magnesium chloride solution was added dropwise, and the reaction temperature was controlled below 15°C. After the reaction was complete, the first reaction solution was obtained. The first reaction solution was poured into acidic water, and the temperature was controlled below 40°C. Then it was concentrated, then water was added to cool it down to below 30°C, filtered, washed with water, and dried to obtain a solid. Then DCM and methanol were added to dissolve the solid, then it was concentrated, methanol was added to replace it to a viscous state, and then it was cooled to crystallize to obtain the 25-hydroxycholesterol. The structural formula of KGP-06 is: ； The ratio of KGP-06 to the methyl magnesium chloride solution is 50g:(120-130)mL, and the concentration of the methyl magnesium chloride solution is 2.5-3mol / L.
2. The method for preparing 25-hydroxycholesterol according to claim 1, characterized in that, The KGP-06 is prepared by the following steps: Dichloromethane and methanol were mixed, then KGP-05 and Raney nickel were added, nitrogen gas was then introduced for purging, and the mixture was cooled to 0-5℃ and hydrogen gas was introduced to react and obtain KGP-06. The structural formula of KGP-05 is as follows: 。 3. The method for preparing 25-hydroxycholesterol according to claim 2, characterized in that, The mass ratio of KGP-05 to Raney nickel is 100:(1-2).
4. The method for preparing 25-hydroxycholesterol according to claim 2, characterized in that, The KGP-05 is prepared by the following steps: Tetrahydrofuran, methanol, and sodium borohydride were mixed, followed by the addition of anhydrous lithium chloride. The mixture was then cooled to -5 to 0°C with stirring, and KGP-04 was added to continue the reaction. After the reaction was complete, a second reaction solution was obtained. This second reaction solution was slowly added to water, and then glacial acetic acid was added dropwise to adjust the pH to 4-5. The mixture was stirred to obtain KGP-05. The structural formula of KGP-04 is: 。 5. The method for preparing 25-hydroxycholesterol according to claim 4, characterized in that, The KGP-04 is prepared by the following steps: PTS is added to isopropyl acetate under stirring, followed by the addition of KGP-03. The mixture is then heated to 80-85°C for reflux reaction. After the reaction is complete, the mixture is cooled to below 10°C, and triethylamine is added. The mixture is then concentrated at 45-50°C, followed by the addition of methanol. The mixture is then concentrated until the material becomes viscous. Finally, the mixture is cooled to 5-10°C and stirred to obtain KGP-04. The structural formula of the KGP-03 is as follows: 。 6. The method for preparing 25-hydroxycholesterol according to claim 5, characterized in that, The reflux reaction takes 3-4 hours.
7. The method for preparing 25-hydroxycholesterol according to claim 5, characterized in that, The KGP-03 is prepared by the following steps: KGP-02 and THF were mixed, then diethyl phosphonoacetate was added, followed by sodium methoxide. The mixture was stirred until the reaction was complete while maintaining the temperature below 40°C. Water and hydrochloric acid were then added to adjust the pH to 4-5 to obtain KGP-03. The KGP-02 structure is as follows: 。 8. The method for preparing 25-hydroxycholesterol according to claim 7, characterized in that, The KGP-02 is prepared by the following steps: (Methoxymethyl)triphenylphosphine chloride and DMSO were mixed, and then NaH was added. The reaction was carried out at a temperature below 30°C. KGP-01 was then added and the reaction was monitored by TLC at a temperature below 40°C. Water was then added, followed by hydrochloric acid to adjust the pH to 4-5. The aqueous layer was then extracted with dichloroisocyanuric acid. The organic phases were combined and concentrated to obtain an oil. Acetone was added to the oil and the temperature was raised to 40-45°C. Water was added dropwise, and the temperature was lowered to 5-10°C with stirring to obtain KGP-02. The structural formula of the KGP-01 is: 。 9. The method for preparing 25-hydroxycholesterol according to claim 8, characterized in that, The mass ratio of (methoxymethyl)triphenylphosphine chloride to KGP-01 is 10:(5-6).