Steroid compounds, methods for preparing them, and their use

Abstract

The present invention provides a steroid compound, its preparation method and its use in a method for separating and purifying lanosterol, and a method for separating and purifying lanosterol, which is simple to operate, has a stable process, has a high productivity, is low in cost, and the purity of the obtained lanosterol is high, and can meet its medical uses.

Description

[Technical Field]

[0001] This application claims priority to Chinese patent application 2021110137424, filed on 31 August 2021. This application incorporates the full text of the aforementioned Chinese patent application.

[0002] This invention relates to the field of separation and purification of organic compounds, and more specifically to steroid compounds, methods for preparing them, and their use. [Background technology]

[0003] Lanosterol belongs to the tetracyclic triterpenoid group and is an intermediate in cholesterol biosynthesis. Currently, lanosterol is mainly obtained by isolation and extraction from crude lanosterol. However, crude lanosterol is a mixture of tetracyclic triterpenoids separated from lanolin by crystallization, and generally contains about 60% lanosterol (CAS number: 79-63-0), about 30% dihydromanosterol (CAS number: 79-62-9), and other impurities such as cholesterol (CAS number: 57-88-5). Of these, lanosterol and dihydrolanosterol are very similar in polarity, making their separation extremely difficult.

[0004] CN101691391 reports the preparation of lanosterol with a separation purity of 97% by high-performance liquid chromatography, but the amount prepared is only 250 mg, making it costly and difficult to produce on a large scale commercially. Currently, there are no purification processes for the large-scale production and preparation of lanosterol with a purity of over 90%, making it difficult to meet the needs for medical applications.

[0005] Furthermore, separating lanosterol from other impurity compounds is extremely difficult, and it is virtually impossible to completely separate lanosterol by column chromatography or recrystallization. For example, one study found that after recrystallizing lanosterol mixed with other impurities, the proportion of impurities in the precipitated lanosterol crystals did not change significantly from that before crystallization.

[0006] Therefore, in order to better study lanosterol and use it in the medical field, there is an urgent need in the market for methods to isolate and purify lanosterol. [Overview of the project]

[0007] This invention provides a steroid compound, a method for preparing the same, and its use to solve one of the above-mentioned technical problems in the prior art. Furthermore, this invention provides a method for purifying lanosterol that can separate and purify lanosterol from crude lanosterol. This method is easy to operate, has a stable process, high production capacity, low cost, and the resulting lanosterol has high purity and can be used for various pharmaceutical applications.

[0008] One aspect of the present invention provides a compound having a structure represented by formula I. JPEG0007857399000001.jpg48170JPEG0007857399000002.jpg40170

[0009] According to some embodiments of the present invention, X is TMS, TES, TBS, TBDPS, TIPS, DMIPS, TBDMS, or MDIPS.

[0010] Another aspect of the present invention provides a method for preparing a compound of formula I, comprising the step of reacting lanosterol with a hydroxyprotectant to obtain a compound of formula I.

[0011] According to some embodiments of the present invention, the hydroxyl protective agent is a silyl ether protective agent. According to some embodiments of the present invention, the hydroxyl protective agent is selected from trimethylchlorosilane, triethylchlorosilane, tert-butyltrichlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane, and tert-butyldimethylsilyl trifluoromethanesulfonate.

[0012] According to some embodiments of the present invention, the reaction between lanosterol and the hydroxyprotectant can be carried out in the presence of an organic solvent. According to some other embodiments of the present invention, the organic solvent is N,N-dimethylformamide or dichloromethane.

[0013] According to some embodiments of the present invention, the reaction between lanosterol and the hydroxyprotectant is carried out in the presence of an acid binder. According to some other embodiments of the present invention, the acid binder is an organic base or an inorganic base. According to some further embodiments of the present invention, the acid binder is pyridine, imidazole, diisopropylamine, triethylamine, triethanolamine, potassium carbonate, or sodium carbonate.

[0014] According to some embodiments of the present invention, the reaction between the lanosterol and the hydroxyprotectant can be carried out at a temperature of 25 to 120°C. According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxyprotectant can be carried out at a temperature of 60 to 85°C.

[0015] Another aspect of the present invention provides a method for separating and purifying a compound of formula I, comprising the step of separating and purifying a starting material A containing a compound of formula I and a compound of formula I' by column chromatography to obtain a compound of formula I. JPEG0007857399000003.jpg54170

[0016] According to some embodiments of the present invention, in raw material A, the mass percentage of compound I is 45% to 85%. According to some embodiments of the present invention, in raw material A, the mass percentage of compound I is 55% to 70%. According to some embodiments of the present invention, in raw material A, the mass percentage of compound I is 60% to 70%.

[0017] According to some embodiments of the present invention, the column chromatography is silica gel column chromatography.

[0018] According to some embodiments of the present invention, the specifications of the silica gel selected for the silica gel column chromatography are 100-200 mesh, 200-300 mesh, or 300-400 mesh.

[0019] According to some embodiments of the present invention, the eluent used in the silica gel column chromatography is one or more of petroleum ether, n-heptane, n-hexane, dichloromethane, and ethyl acetate. According to some embodiments of the present invention, aqueous ammonia is added to the eluent used in the silica gel column chromatography. According to some embodiments of the present invention, the eluent used in the silica gel column chromatography may be a mixture of n-heptane and ethyl acetate, a mixture of n-hexane and ethyl acetate, or a mixture of n-heptane, ethyl acetate and aqueous ammonia. According to some embodiments of the present invention, the eluent used in the silica gel column chromatography may be a mixture of n-heptane and ethyl acetate, where the volume ratio of n-heptane to ethyl acetate is (90-100):(10-0), for example 100:1. According to some embodiments of the present invention, the eluent used in the silica gel column chromatography may be a mixture of n-hexane and ethyl acetate, where the volume ratio of n-hexane to ethyl acetate is (90-100):(10-0), for example 100:1.

[0020] According to some embodiments of the present invention, after the raw material A is mixed with silica gel of 100-200 mesh, the above silica gel column chromatography is carried out.

[0021] According to some embodiments of the present invention, the silica gel column chromatography can be carried out one or more times, for example, 1, 2, 3, 4 or 5 times.

[0022] According to some embodiments of the present invention, the method for separating and purifying the compound of formula I further comprises a step of reacting crude lanosterol with a hydroxy protecting agent to obtain the raw material A. [[ID=S9]] JPEG0007857399000004.jpg59170

[0023] According to some embodiments of the present invention, the hydroxy protecting agent is a silyl ether protecting agent; for example, trimethylchlorosilane, triethylchlorosilane, tert-butyltrichlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane, and tert-butyldimethylsilyl trifluoromethanesulfonate.

[0024] According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxy protecting agent is carried out in the presence of an organic solvent. According to some embodiments of the present invention, the organic solvent is N,N-dimethylformamide or dichloromethane.

[0025] According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxy protecting agent is carried out in the presence of an acid binder. According to some embodiments of the present invention, the acid binder is an organic base or an inorganic base. According to some embodiments of the present invention, the acid binder is pyridine, imidazole, diisopropylamine, triethylamine, triethanolamine, potassium carbonate, or sodium carbonate.

[0026] According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxy protecting agent is carried out at a temperature of 25 to 120 °C. According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxy protecting agent is carried out at a temperature of 60 to 85 °C.

[0027] Another aspect of the present invention provides the use of the compound of formula I in the separation and purification of lanosterol. According to some embodiments of the present invention, the use includes a step of hydroxy-deprotecting the compound of formula I to obtain lanosterol. JPEG0007857399000005.jpg38170

[0028] According to some embodiments of the present invention, the hydroxy-deprotection is carried out in the presence of one or more of acetic acid, tetraalkylammonium fluoride, trifluoroacetic acid, or hydrochloric acid. According to some embodiments of the present invention, the hydroxy-deprotection is carried out in the presence of tetrabutylammonium fluoride.

[0029] According to some embodiments of the present invention, the use further includes a step of separating and purifying raw material A containing the compound of formula I and the compound of formula I' by column chromatography to obtain the compound of formula I. JPEG0007857399000006.jpg57170

[0030] JPEG0007857399000007.jpg38170

[0031] According to some embodiments of the present invention, X is TMS, TES, TBS, TBDPS, TIPS, DMIPS, TBDMS or MDIPS.

[0032] According to some embodiments of the present invention, in raw material A, the mass percentage of compound I is 45% to 85%. According to some embodiments of the present invention, in raw material A, the mass percentage of compound I is 55% to 70%. According to some embodiments of the present invention, in raw material A, the mass percentage of compound I is 60% to 70%.

[0033] According to some embodiments of the present invention, the column chromatography is silica gel column chromatography.

[0034] According to some embodiments of the present invention, the specifications of the silica gel selected for the silica gel column chromatography are 100-200 mesh, 200-300 mesh, or 300-400 mesh.

[0035] According to some embodiments of the present invention, the eluent used in the silica gel column chromatography is one or more of petroleum ether, n-heptane, n-hexane, dichloromethane, and ethyl acetate. According to some embodiments of the present invention, aqueous ammonia is added to the eluent used in the silica gel column chromatography. According to some embodiments of the present invention, the eluent used in the silica gel column chromatography may be a mixture of n-heptane and ethyl acetate, a mixture of n-hexane and ethyl acetate, or a mixture of n-heptane, ethyl acetate and aqueous ammonia. According to some embodiments of the present invention, the eluent used in the silica gel column chromatography may be a mixture of n-heptane and ethyl acetate, where the volume ratio of n-heptane to ethyl acetate is (90-100):(10-0), for example 100:1. According to some embodiments of the present invention, the eluent used in the silica gel column chromatography may be a mixture of n-hexane and ethyl acetate, where the volume ratio of n-hexane to ethyl acetate is (90-100):(10-0), for example 100:1.

[0036] According to some embodiments of the present invention, the raw material A is mixed with silica gel of 100 to 200 mesh, and then the silica gel column chromatography described above is performed.

[0037] According to some embodiments of the present invention, the silica gel column chromatography can be performed once or multiple times, for example, 1, 2, 3, 4, or 5 times.

[0038] According to some embodiments of the present invention, a method for separating and purifying the compound of formula I further includes the step of reacting crude lanosterol with a hydroxyprotecting agent to obtain the starting material A. JPEG0007857399000008.jpg64170

[0039] According to some embodiments of the present invention, the hydroxyl protective agent is a silyl ether protective agent; for example, selected from trimethylchlorosilane, triethylchlorosilane, tert-butyltrichlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane, and tert-butyldimethylsilyl trifluoromethanesulfonate.

[0040] According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxyprotectant is carried out in the presence of an organic solvent. According to some embodiments of the present invention, the organic solvent is N,N-dimethylformamide or dichloromethane.

[0041] According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxyprotectant is carried out in the presence of an acid binder. According to some embodiments of the present invention, the acid binder is an organic base or an inorganic base. According to some embodiments of the present invention, the acid binder is pyridine, imidazole, diisopropylamine, triethylamine, triethanolamine, potassium carbonate, or sodium carbonate.

[0042] According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxyprotectant is carried out at a temperature of 25 to 120°C. According to some embodiments of the present invention, the reaction between the crude lanosterol and the hydroxyprotectant is carried out at a temperature of 60 to 85°C.

[0043] Another aspect of the present invention provides a method for purifying a compound of formula I, comprising the step of obtaining the compound of formula I by recrystallizing a raw material A containing the compound of formula I and the compound of formula I'. JPEG0007857399000009.jpg62170

[0044] Here, the definition of X is as described above, and the solvent for the recrystallization is, 1) A mixture of ethyl acetate and isopropyl alcohol, or 2) Isopropyl acetate, selected from the above.

[0045] According to some embodiments of the present invention, in the mixture of ethyl acetate and isopropyl alcohol, the volume ratio of ethyl acetate to isopropyl alcohol is 1:(0.5~10), preferably 1:(1~10), for example 1:(0.5~2).

[0046] According to some embodiments of the present invention, in raw material A, the mass percentage of the compound of formula I is 75% or more, preferably 85% or more, and more preferably 90% or more.

[0047] According to some embodiments of the present invention, the recrystallization involves first dissolving the raw material A in the solvent, raising the temperature until it is dissolved, and then cooling it to room temperature.

[0048] Another aspect of the present invention provides a composition Y comprising a compound of formula I and a compound of formula I'. JPEG0007857399000010.jpg49170

[0049] JPEG0007857399000011.jpg40170

[0050] According to some embodiments of the present invention, X is TMS, TES, TBS, TBDPS, TIPS, DMIPS, TBDMS, or MDIPS.

[0051] According to some embodiments of the present invention, in composition Y, the mass percentage of the compound of formula I is 55% to 95%, for example, 56%, 57%, 58%, 59%, 60%, 61%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%.

[0052] Another aspect of the present invention is, 1) A step of reacting crude lanosterol with a hydroxyl protective agent to obtain starting material A containing compound I and compound I'; 2) A step of separating the raw material A by column chromatography to obtain the compound of formula I; and 3) A step of obtaining purified lanosterol by hydroxy deprotecting the compound of formula I; This invention provides a method for purifying lanosterol containing [the specified substance]. Here, the crude lanosterol includes lanosterol and dihydrolanosterol. The aforementioned hydroxy protective agent is a silyl ether protective agent.

[0053] According to some embodiments of the present invention, the silyl ether protective agent is selected from trimethylchlorosilane, triethylchlorosilane, tert-butyltrichlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane, and tert-butyldimethylsilyl trifluoromethanesulfonate.

[0054] In the method for purifying lanosterol described above, the raw material A, the compound of formula I, and the reaction conditions and operations for each step are as described in one of the schemes above.

[0055] This invention aims to purify crude lanosterol (raw material) by first protecting the crude lanosterol with a hydroxysilyl ether. By utilizing the difference in polarity between the hydroxysilyl ether-protected lanosterol and the hydroxysilyl ether-protected dihydrolanosterol, the two can be separated by silica gel column chromatography to obtain pure hydroxysilyl ether-protected lanosterol. Then, the silyl ether protecting group is removed to obtain pure lanosterol. Furthermore, this invention has found that hydroxysilyl ether-protected lanosterol possesses good crystallization properties, and by recrystallizing before removing the protecting group, some impurities can be retained in the mother liquor. Purified lanosterol can then be obtained by removing the hydroxy protecting group. The resulting lanosterol can meet not only the commercial needs of large-scale preparation but also the needs of pharmaceutical use. Numerous experiments have demonstrated that hydroxy protecting groups of other non-silyl ether groups, such as alkyl ethers, carboxylic acid esters, and amino acid esters, cannot separate lanosterol from dihydrolanosterol.

[0056] Examples are provided below to aid in understanding the present invention. However, it should be understood that these examples are used solely to illustrate the present invention and do not constitute any limitation. The actual scope of protection of the present invention is described in the claims. It should be understood that any modifications and changes are possible without departing from the spirit of the invention. [Modes for carrying out the invention]

[0057] To make the object, technical solution, and advantages of the present invention clearer and easier to understand, the present invention will be described in more detail below in relation to examples. The specific examples described herein are used solely to illustrate the present invention and are not intended to limit it. Furthermore, in order to avoid unnecessarily confusing the concepts of the present invention, descriptions of well-known structures and techniques will be omitted in the following description. Such structures and techniques are also described in many publications.

[0058] Unless otherwise defined, all technical and scientific terms used in this invention have the same meanings as those commonly used in the art to which this invention pertains. For the purposes of interpreting this specification, the following definitions apply, with singular terms also having plural forms where applicable, and vice versa.

[0059] As used herein, the expressions “one” and “one” include multiple referents unless the context clearly indicates otherwise. For example, a reference to “one cell” includes multiple such cells and equivalents known to those skilled in the art.

[0060] As used herein, the term “approximately” means a range of ±20% of the following number. In some embodiments, the term “approximately” means a range of ±10% of the following number. In some embodiments, the term “approximately” means a range of ±5% of the following number.

[0061] The solvents used herein are commercially available. The following abbreviations are used herein: TIPS: Triisopropylsilane TIPSCl: Triisopropylchlorosilane DMIPS: Dimethylisopropylsilane DMIPSCl: Dimethylisopropylchlorosilane TES: Triethylsilane TESCl: Triethylchlorosilane TMS: Trimethylsilane TBDPS: tert-butyldiphenylsilane TBDPSCl:tert-butyldiphenylchlorosilane TBS: tert-butyldimethylsilane TBSCl:tert-butyldimethylchlorosilane TMSCl: Trimethylchlorosilane TLC: Thin-layer chromatography TBAF: Tetrabutylammonium fluoride THF: Tetrahydrofuran DCM: Dichloromethane DMF: Dimethylformamide eq: equivalent Compounds were named manually or using the software ChemDraw®, and commercially available compounds were named using the supplier's catalog name.

[0062] Lanosterol raw material (crude product) (Suppliers are Shandong Junrui Medical Technology Co., Ltd. or Spectrum China Ltd.)

[0063] <Examples> In column chromatography, the elution of the target intermediate in the eluent was tracked by thin-layer chromatography (TLC), using petroleum ether:ethyl acetate in a 5:1 ratio, with phosphomolybdic acid used for color development.

[0064] Based on experience, we classified the TLC results of collected eluates containing target intermediates into three categories, in combination with nuclear magnetic analysis: those with virtually no impurity spots (defined as high-purity products), 1 The integral ratio of the 1H NMR characteristic peak or the HPLC purity is between approximately 85 and 90%), and although there are impurity spots, there is a significant improvement in purity (defined as a product with relatively high purity). 1The integral ratio of the 1H NMR characteristic peak or the HPLC purity is between approximately 70-85%), there are impurity spots but no significant improvement in purity is observed, and the concentration of the target intermediate in the eluent is relatively high (defined as a low-purity product). 1 The integration ratio of the 1H NMR characteristic peak or HPLC purity is generally <70%), where the improvement in purity is relative to the purity of the sample obtained by column chromatography at that point.

[0065] <Example 1> 1.1 Hydroxyprotection 500g of commercially available lanosterol raw material (lanosterol purity is approximately 60%) was divided into two equal parts of 250g each, and each was subjected to the following processing.

[0066] At 15°C, 250 g of lanosterol raw material was prepared in an N,N-dimethylformamide solution (2500 mL), to which imidazole (87.8 g, 1.29 mol) and TBSCl (132.5 g, 879 mmol) were added, and the mixture was stirred at 85°C for 6 hours. Next, the mixture was cooled to 15°C, and petroleum ether (2500 × 2 mL) was added for extraction. The petroleum ether phase was then sequentially washed with saturated NaCl solution (2000 mL) and water (2000 mL).

[0067] 348 g of intermediate product 1a and 350 g of intermediate product 1b were obtained, respectively.

[0068] According to common knowledge in this field, the difference in purity between the intermediate products 1a and 1b before and after the hydroxy protection reaction and the lanosterol starting material is negligible.

[0069] 1.2 Column chromatography 348 g of intermediate product 1a was mixed with 1000 g of 100-200 mesh silica gel, and chromatography was performed using a 10 kg 200-300 mesh silica gel column (1000 mm × 230 mm). Elution was performed with a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate of the relatively high-purity product was collected, dried by vacuum distillation, and 125 g of intermediate product 1c was obtained, with a yield of approximately 66%.

[0070] 350 g of intermediate product 1b was mixed with 1000 g of 100-200 mesh silica gel, and column chromatography was performed using 10 kg of 200-300 mesh silica gel (1000 mm × 230 mm). Elution was performed with a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate of the high-purity product was collected, dried, and 121 g of intermediate product 1d was obtained, with a yield of approximately 64%.

[0071] Intermediate products 1c and 1d were combined and mixed with 740 g of 100-200 mesh silica gel. Chromatography was performed using a 7.4 kg 200-300 mesh silica gel column (1000 mm × 230 mm), and elution was performed with a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate of the high-purity product was collected. After drying by vacuum distillation, 179 g of intermediate product 1e was obtained, with a yield of approximately 47%.

[0072] 179 g of intermediate product 1e was mixed with 540 g of 100-200 mesh silica gel, and chromatography was performed on a 5.4 kg 200-300 mesh silica gel column (1000 mm × 230 mm). Elution was performed with a mixture of n-hexane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate of the high-purity product was collected, dried, and 161 g of intermediate product 1f was obtained, with a yield of approximately 42%. When intermediate product 1f was monitored by TLC, the presence of faint impurity spots was still observed. 1 Integration of 1H NMR characteristic peaks showed that TBS-lanosterol accounts for approximately 83% of the total sterols.

[0073] 161 g of intermediate product 1f was mixed with 480 g of 100-200 mesh silica gel, and chromatography was performed on a 5.0 kg 200-300 mesh silica gel column (1000 mm × 230 mm). Elution was performed with a mixture of n-hexane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate of the high-purity product was collected, dried by vacuum distillation, and 147 g of intermediate product 1h was obtained. The total yield of the four purifications was approximately 39%. Monitoring of intermediate product 1h by TLC showed no clear impurity spots. 1 Integration of 1H NMR characteristic peaks showed that TBS-lanosterol accounts for approximately 91% of the total sterol content.

[0074] 1 H NMR(400MHz, CDCl3)δ=5.07(br t,J=7.2Hz,1H),3.17(dd,J=4.6,11.2Hz,1H),2.05‐1.35(m,24H),1.29(br ppm.

[0075] 1.3 Hydroxy deprotection 49.2 g of the intermediate product was taken and dissolved in 250 mL of anhydrous tetrahydrofuran. 110 mL of TBAF solution (1.0 M) was added at 15°C, and the mixture was heated under reflux for 16 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was directly spun-dried under reduced pressure to obtain the residue. 600 mL of methanol was added to the residue, and the mixture was refluxed for 4 hours to obtain a white suspension. The mixture was stirred at 60°C for 24 hours, then cooled to 15°C, filtered, and the cake was washed with 300 mL of methanol and dried by suction to obtain 38.3 g of the final product. The yield was approximately 98.4%, and the purity of lanosterol measured by HPLC was approximately 91%.

[0076] 1H NMR(400MHz,CDCl3)δ=5.12(br t,J=7.15Hz,1H),3.26(dd,J=4.64,11.42Hz,1H),1.73-2.13(m,10H),1.71(s,3H),1.64-1.68(m,1H),1.63(s,3H) ,1.05‐1.59(m,12H),1.02(s,3H),1.00(s,3H),0.93(d,J=6.27Hz,3H),0.90(s,3H),0.83(s,3H),0.71(s,3H)ppm.

[0077] <Example 2> 2.1 Hydroxyprotection At 15°C, imidazole (87.8 g, 1.29 mol) and TESCl (132.5 g, 879 mmol) were added to a 2500 mL N,N-dimethylformamide solution of commercially available lanosterol starting material (250 g, lanosterol purity approximately 60%), and the mixture was stirred at 80°C for 3 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to 25°C, 600 mL of methanol and 300 mL of water were added, the mixture was stirred, and the solution was filtered. The cake was washed with 300 mL of methanol, and the cake was dried by suction to obtain 341 g of intermediate product 2a.

[0078] 2.2 Column chromatography 341 g of intermediate product 2a was mixed with 1000 g of 100-200 mesh silica gel, and chromatography was performed on a 10 kg 200-300 mesh silica gel column (1000 mm × 230 mm). Elution was performed with a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate of the high-purity product was collected, dried by vacuum distillation, and 168 g of intermediate product 2b was obtained (the presence of faint impurity spots was still observed on TLC), with a yield of approximately 88%.

[0079] 168 g of intermediate product 2b was mixed with 540 g of 100-200 mesh silica gel, and chromatography was performed using a 5.4 kg 200-300 mesh silica gel column (1000 mm × 180 mm). Elution was performed with a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC, and the eluate, which was essentially free of impurity spots, was collected. After drying by vacuum distillation, 117 g of intermediate product 2c was obtained, with a yield of approximately 62%. 1 Integration of 1H NMR characteristic peaks showed that TES-lanosterol accounts for approximately 86% of the total sterol content.

[0080] 2.3 Hydroxy deprotection At 25°C, 27 g of intermediate product 2c was weighed and dissolved in dry THF (150 mL) and TBAF (55 mL, 0.055 mol), and the mixture was reacted for 30 minutes. Next, the temperature was raised to 60°C, refluxed, and stirred for 4 hours. The reaction was monitored by TLC until completion. 80 mL of water and 160 mL of methanol were added, and the mixture was stirred for 1 hour, at which point a solid precipitate formed. The mixture was filtered, the cake was washed with a small amount of water and methanol, and dried by suction to obtain 18.5 g of a white solid final product. The yield was 87%, and the purity of lanosterol, as measured by HPLC, was approximately 89%.

[0081] <Example 3> 3.1 Hydroxyprotection At 15°C, imidazole (87.8 g, 1.29 mol) and TESCl (132.5 g, 879 mmol) were added to a 2500 mL N,N-dimethylformamide solution of commercially available lanosterol starting material (250 g, lanosterol purity approximately 60%), and the mixture was stirred at 80°C for 3 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to 25°C, 600 mL of methanol and 300 mL of water were added, the mixture was stirred, filtered, and the cake was dried by suction to obtain 355 g of intermediate product 3a.

[0082] 3.2 Column chromatography 355 g of the intermediate product 3a was mixed with 1000 g of silica gel of 100 - 200 mesh, and chromatography was performed on a silica gel column (1000 mm × 230 mm) of 10 kg of 200 - 300 mesh. Elution was carried out with a mixture of n - heptane and ethyl acetate (100:1). The elution process was monitored by TLC, and the eluates of high - purity products and products with relatively high purity were collected respectively, dried by vacuum distillation, and then 71 g of intermediate product 3b with a purity of about 87 - 89% and 96 g of intermediate product 3c with a purity of about 70 - 80% were obtained respectively.

[0083] 96 g of the intermediate product 3c was mixed again with 300 g of silica gel of 100 - 200 mesh, and chromatography was performed on a silica gel column (1000 mm × 180 mm) of 3 kg of 200 - 300 mesh. Elution was carried out with a mixture of n - heptane and ethyl acetate (100:1). The elution process was monitored by TLC, and the eluate with relatively high purity was collected, dried by vacuum distillation, and then 44 g of intermediate product 3d was obtained.

[0084] The intermediate products 3b and 3d were combined (named intermediate product 3e), with a total of 115 g and a yield of 61%. 1 By the integration of the H NMR characteristic peaks, it was shown that TES - lanosterol accounted for about 90% of the total sterol content.

[0085] 3.3 Hydroxy Deprotection At 25°C, 22 g (0.04 mol) of the intermediate product 3e was dissolved in dry THF (120 mL), TBAF (45 mL, 0.045 mol) was added to the reaction solution, and it was held for 30 minutes. Next, the temperature was raised to 60°C, refluxed, and stirred for 4 hours. The reaction was monitored by TLC. After the reaction was completed, 60 mL of water and 120 mL of methanol were added and stirred for 1 hour, and a solid precipitated. It was filtered, the cake was washed with 60 mL of water, then with 100 mL of methanol, filtered to obtain a cake, refluxed with 200 mL of methanol for 3 hours, cooled to precipitate, and filtered again to obtain 15.2 g of a white solid. The yield was about 88%, and the purity of lanosterol measured by HPLC was about 92%.

[0086] <Example 4> 4.1 Recrystallization 50 g of intermediate product 3e obtained in Example 3 was taken and added to 750 ml of a mixture of ethyl acetate and isopropyl alcohol (volume ratio 1:1). The mixture was heated to 90°C and refluxed for 2 hours to dissolve and clarify. After stopping the stirring, the mixture was slowly cooled, and a solid precipitated. The solid was filtered to obtain a cake, which was washed and dried to obtain 40 g of intermediate product 4a, with a yield of 80%. 1 Integration of 1H NMR characteristic peaks showed that TES-lanosterol accounts for approximately 93% of the total sterol content.

[0087] 1 H NMR(400MHz, CDCl3)δ=0.56-0.66(m,6H),0.70-0.91(m,9H),0.92-1.07(m,19H) ,1.12-1.77(m,21H),1.82-2.12(m,7H),3.25(dd,J=11.29,4.52Hz,1H),5.12(br t,J=7.03Hz,1H).

[0088] 4.2 Deprotection At 25°C, intermediate product 4a (20 g, 0.037 mol) was dissolved in dry THF (120 mL). TBAF (1 M, 1.5 eq) was added to the reaction solution and held for 30 minutes. The temperature was raised to 60°C, refluxed, and stirred for 4 hours. The reaction was monitored by TLC until completion. 60 mL of water and 120 mL of methanol were added, and the mixture was stirred for 1 hour, after which a solid precipitated. The mixture was filtered, and the cake was sequentially washed with small amounts of water and methanol, filtered, and the cake was obtained. 200 mL of methanol was added to the cake, refluxed for 3 hours, cooled and precipitated, and filtered to obtain 13 g of a white solid. The yield was approximately 85%, and the purity of lanosterol, as measured by HPLC, was >99%.

[0089] <Example 5> 5.1: Hydroxyprotection At 15°C, imidazole (87.75 g, 1.29 mol) and TMSCl (95.47 g, 879 mmol) were added to a 2500 mL N,N-dimethylformamide solution of commercially available lanosterol raw material (250 g, lanosterol purity approximately 60%), and the mixture was stirred at 70°C for 5 hours. After the reaction was complete, the reaction solution was cooled to 15°C, and petroleum ether (2500 × 2 mL) was added for extraction. The petroleum ether phase was washed with saturated NaCl solution (2000 mL) and water (2000 mL), respectively, to obtain 287 g of intermediate product 5a.

[0090] 5.2: Column chromatography 287 g of intermediate product 5a was mixed with 850 g of 100-200 mesh silica gel, and chromatography was performed on an 8.5 kg 200-300 mesh silica gel column (1000 mm × 230 mm). Elution was performed with a mixture of n-heptane, ethyl acetate, and aqueous ammonia (100:1:0.05). After tracking and observation by TLC, the eluate was collected, dried by vacuum distillation, and 53 g of product 5b was obtained. The yield of the TMS-lanosterol intermediate was approximately 30%. 1 The purity measured by 1H NMR was approximately 80%, and TLC showed the presence of faint impurity spots.

[0091] <Example 6> Experiment: Consideration of Protective Agents and Experimental Conditions At 15°C, 100 mL of a DMF solution of commercially available lanosterol starting material (10 g, 0.023 mol, approximately 60% purity) was mixed with imidazole (3.9 g) and triethylchlorosilane (1-2 eq). The reaction was monitored by TLC, and if the conversion was relatively clean, the following workup procedure was performed. The mixture was cooled to room temperature, and petroleum ether (75 × 2 mL) was added for extraction. The petroleum ether phase was washed with saturated NaCl solution (75 mL) and water (750 mL), respectively, and dried to obtain the corresponding crude products, which were then purified by column chromatography.

[0092] The table below shows the conditions and results obtained by varying the solvent, protective reagent, temperature, and reaction time in the above reaction. JPEG0007857399000012.jpg159170

[0093] Note: a. The post-processing procedure is as follows: Cool to room temperature, add methanol (25 mL) and water (12.5 mL), stir, filter, wash the cake with a small amount of methanol, and suction dry the cake to obtain the corresponding crude product, which is then purified by column. Here, by employing this post-processing procedure, the product can be precipitated directly and filtered.

[0094] <Example 7> 5.41 g of the intermediate product 2c purified by column in Example 2 was taken, and the recrystallization conditions shown in the table below, with a solute-to-solvent mass-volume ratio of 1 g:10 mL, were investigated. JPEG0007857399000013.jpg60170

[0095] The technical solutions of the present invention are not limited to the specific embodiments described above, and all technical modifications made based on the technical solutions of the present invention are within the scope of protection of the present invention.

Claims

1. A compound having the structure represented by formula I. (Here, X is triethylsilyl.)

2. Compounds having the structure represented by formula I and compounds having the structure represented by formula I' are (Here, X is triethylsilyl) A method for separating and purifying a compound of formula I according to claim 1, characterized by comprising the step of separating and purifying a raw material A containing a compound of formula I and a compound of formula I' by column chromatography to obtain a compound of formula I.

3. A method for separating and purifying a compound of formula I according to claim 2, further comprising the step of reacting crude lanosterol with a hydroxyl protective agent to obtain the raw material A, wherein the hydroxyl protective agent is triethylchlorosilane.

4. A method for purifying a compound of formula I, comprising the step of obtaining the compound of formula I by recrystallizing a raw material A containing the compound of formula I and the compound of formula I'. (Here, the definition of X is as described in claim 1, and the solvent for recrystallization is selected from 1) a mixture of ethyl acetate and isopropyl alcohol, or 2) isopropyl acetate.)

5. A purification method according to claim 4, wherein any one of the following conditions is met. (1) The mass percentage of the compound of formula I in raw material A is 90% or more. (2) In a mixture of ethyl acetate and isopropyl alcohol, the volume ratio of ethyl acetate to isopropyl alcohol is 1:(0.5 to 2).

6. 1) A step of reacting crude lanosterol with a hydroxyl protective agent to obtain a starting material A containing the compound of formula I and the compound of formula I' described in claim 2; 2) A step of separating the raw material A by column chromatography to obtain the compound of formula I; and 3) The step of hydroxy-deprotecting the compound of formula I to obtain purified lanosterol; Here, the crude lanosterol comprises lanosterol and dihydrolanosterol; The hydroxyprotective agent is triethylchlorosilane. A method for purifying lanosterol characterized by the following features.

7. The separation and purification method according to claim 3, wherein the reaction between the crude lanosterol and the hydroxyprotecting agent satisfies any one of the following conditions. (1) The reaction between crude lanosterol and a hydroxyprotectant is carried out in the presence of an organic solvent, the organic solvent being N,N-dimethylformamide or dichloromethane. (2) The reaction between crude lanosterol and a hydroxyprotectant is carried out in the presence of an acid binder, the acid binder being pyridine, imidazole, diisopropylamine, triethylamine, triethanolamine, potassium carbonate, or sodium carbonate. (3) The reaction between crude lanosterol and the hydroxyprotectant is carried out at a temperature of 60–85°C.

8. The method according to claim 6, wherein the reaction between the crude lanosterol and the hydroxyprotectant satisfies any one of the following conditions. (1) The reaction between crude lanosterol and a hydroxyprotectant is carried out in the presence of an organic solvent, the organic solvent being N,N-dimethylformamide or dichloromethane. (2) The reaction between crude lanosterol and a hydroxyprotectant is carried out in the presence of an acid binder, the acid binder being pyridine, imidazole, diisopropylamine, triethylamine, triethanolamine, potassium carbonate, or sodium carbonate. (3) The reaction between crude lanosterol and the hydroxyprotectant is carried out at a temperature of 60–85°C.

9. The method according to claim 6, wherein the column chromatography is silica gel column chromatography, and the silica gel column chromatography satisfies any one of the following conditions. (1) The specifications of the silica gel selected for the silica gel column chromatography are 100-200 mesh, 200-300 mesh, or 300-400 mesh. (2) The eluent used in the silica gel column chromatography is one or more of petroleum ether, n-heptane, n-hexane, dichloromethane, and ethyl acetate. (3) Ammonia water is added to the eluent used in the silica gel column chromatography.

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