STEROIDAL COMPOUND, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

A steroidal compound, a preparation method therefor and an application thereof. On one hand, provided are a compound represented by formula I, a preparation method therefor, and an application thereof in a method for separating and purifying lanosterol; on the other hand, provided is the method for separating and purifying lanosterol. The method is simple to operate, stable in process, high in productivity and low in cost, and the obtained lanosterol has high purity and can meet medical application thereof.

Claims

1. A compound of formula I, ##STR00012## wherein X is ##STR00013## and R.sup.1, R.sup.2, and R.sup.3 are each independently C.sub.1-4 alkyl, C.sub.1-4 alkoxy, 5- to 9-membered heteroaryl, or C.sub.6-10 aryl, with the heteroatom in the 5-to 9-membered heteroaryl being selected from N, O, or S, and the number of the heteroatom in the 5-to 9-membered heteroaryl being 1, 2, or 3.

2. The compound according to claim 1, wherein X is TMS, TES, TBS, TBDPS, TIPS, DMIPS, TBDMS, or MDIPS.

3. A preparation method for the compound of formula I according to claim 1, comprising: reacting lanosterol with a hydroxyl-protecting agent to give the compound of formula I.

4. (canceled)

5. The preparation method according to claim 3, wherein the hydroxyl-protecting agent is selected from trimethylchlorosilane, triethylchlorosilane, tri-tert-butylchlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane and tert-butyldimethylsilyl trifluoromethanesulfonate; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted in the presence of an organic solvent; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted in the presence of a deacid reagent; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted at a temperature of 25-120 C.

6. A separation and purification method for the compound of formula I according to claim 1, comprising: separating and purifying a raw material A by column chromatography to give the compound of formula I, wherein the raw material A comprises the compound of formula I and a compound of formula I; ##STR00014##

7. The separation and purification method according to claim 6, further comprising: reacting a crude product of lanosterol with a hydroxyl-protecting agent to give the raw material A; ##STR00015##

8. The separation and purification method according to claim 7, wherein the hydroxyl-protecting agent is selected from trimethylchlorosilane, triethylchlorosilane, tri-tert-butylchlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane, and tert-butyldimethylsilyl trifluoromethanesulfonate; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted in the presence of an organic solvent; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted in the presence of a deacid reagent: or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted at a temperature of 25-120 C.

9. (canceled)

10. A separation and purification method, comprising: conducting hydroxyl deprotection to the compound of formula I according to claim 1 to give lanosterol; ##STR00016##

11. The separation and purification method according to claim 10, further comprising: separating and purifying a raw material A by column chromatography to give the compound of formula I, wherein the raw material A comprises the compound of formula I and a compound of formula I; ##STR00017## the hydroxyl deprotection is conducted in the presence of one or more of acetic acid, tetraalkylammonium fluoride, trifluoroacetic acid, or hydrochloric acid.

12. The separation and purification method according to claim 11, further comprising: reacting a crude product of lanosterol with a hydroxyl-protecting agent to give the raw material A; ##STR00018## or, the column chromatography is silica gel column chromatography; or, the hydroxyl deprotection is conducted in the presence of tetraalkylammonium fluoride.

13. A refinement method for a compound of formula I, comprising: recrystallizing a raw material A to give the compound of formula I, wherein the raw material A comprises the compound of formula I and a compound of formula I; ##STR00019## wherein X is as defined in claim 1, and the solvent for the recrystallization is selected from: 1) a mixture of ethyl acetate and isopropanol, or 2) isopropyl acetate.

14. The refinement method according to claim 13, wherein the mass percentage of the compound of formula I in the raw material A is 75% or greater; or, in the mixture of ethyl acetate and isopropanol, the volume ratio of ethyl acetate to isopropanol is 1:(0.5-10)

15. A composition Y, comprising a compound of formula I and a compound of formula I, ##STR00020## wherein X is ##STR00021## and R.sup.1, R.sup.2, and R.sup.3 are each independently C.sub.1-4 alkyl, C.sub.1-4 alkoxy, 5- to 9-membered heteroaryl, or C.sub.6-10 aryl, with the heteroatom in the 5-to 9-membered heteroaryl being selected from N, O, or S, and the number of the heteroatom in the 5- to 9-membered heteroaryl being 1, 2, or 3.

16. The composition Y according to claim 15, wherein X is TMS, TES, TBS, TBDPS, TIPS, DMIPS, TBDMS, or MDIPS; or, the mass percentage of the compound of formula I is 55%-95%.

17. The composition Y according to claim 16, wherein the mass percentage of the compound of formula I is 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%.

18. A method for purifying lanosterol, comprising: 1) reacting a crude product of lanosterol with a hydroxyl-protecting agent to give the raw material A according to claim 6, the raw material A comprising a compound of formula I and a compound of formula I; ##STR00022## 2) separating the raw material A by column chromatography to give the compound of formula I; and 3) conducting hydroxyl deprotection to the compound of formula I to give purified lanosterol; wherein the crude product of lanosterol comprises lanosterol and dihydrolanosterol; the hydroxyl-protecting agent is a silyl ether-based protecting agent.

19. The method according to claim 18, wherein the silyl ether-based protecting agent is selected from trimethylchlorosilane, triethylchlorosilane, tri-tert-butylchlorosilane, tert-butyldiphenylchlorosilane, triisopropylchlorosilane, dimethylisopropylchlorosilane, tert-butyldimethylchlorosilane, methyldiisopropylchlorosilane, triisopropylchlorosilane, and tert-butyldimethylsilyl trifluoromethanesulfonate; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted in the presence of an organic solvent; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted in the presence of a deacid reagent; or, the reaction of lanosterol with the hydroxyl-protecting agent is conducted at a temperature of 25-120 C.; or, the column chromatography is silica gel column chromatography; or, the hydroxyl deprotection is conducted in the presence of one or more of acetic acid, tetraalkylammonium fluoride, trifluoroacetic acid, or hydrochloric acid.

20-24. (canceled)

25. The separation and purification method according to claim 5, wherein the organic solvent is N,N-dimethylformamide or dichloromethane; or, the deacid reagent is an organic base or an inorganic base; or, the reaction of lanosterol and the silyl ether-based protecting agent is performed at a temperature of 60-85 C.

26. The separation and purification method according to claim 25, wherein the deacid reagent is pyridine, imidazole, diisopropylamine, triethylamine, triethanolamine, potassium carbonate, or sodium carbonate.

27. The separation and purification method according to claim 12, wherein the specification of the silica gel selected for the silica gel column chromatography is 100-200 mesh, 200-300 mesh, or 300-400 mesh.

Description

DETAILED DESCRIPTION

[0065] In order to make the objects, embodiments, and advantages of the present disclosure more apparent, the present disclosure is further described in detail with reference to the following examples. The specific examples described herein are merely illustrative of the present disclosure and do not constitute any limitation thereon. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to avoid unnecessary obscuration of concepts in the present disclosure. Such structures and techniques are also described in numerous publications.

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which the present disclosure belongs. For the purpose of illustrating the present disclosure, the following definitions will apply, and, where appropriate, terms used in the singular form will also include the plural form and vice versa.

[0067] As used herein, the terms a and an include plural references unless otherwise stated. For example, reference to a cell includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0068] The term about as used herein means a range of 20% of the value following it. In some embodiments, the term about means a range of 10% of the value following it. In some embodiments, the term about means a range of 5% of the value following it.

[0069] The solvents used herein are commercially available. The following abbreviations are used herein: [0070] TIPS: triisopropylsilane [0071] TIPSCl: triisopropylchlorosilane [0072] DMIPS: dimethylisopropylsilane [0073] DMIPSCl: dimethylisopropylchlorosilane [0074] TES: triethylsilane [0075] TESCl: triethylchlorosilane [0076] TMS: trimethylsilane [0077] TBDPS: tert-butyldiphenylsilane [0078] TBDPSCl: tert-butyldiphenylchlorosilane [0079] TBS: tert-butyldimethylsilane [0080] TBSCl: tert-butyldimethylchlorosilane [0081] TMSCl: trimethylchlorosilane [0082] TLC: thin-layer chromatography [0083] TBAF: tetrabutylammonium fluoride [0084] THF: tetrahydrofuran [0085] DCM: dichloromethane [0086] DMF: dimethylformamide [0087] eq: equivalent

[0088] Compounds are named either manually or by ChemDraw software, and supplier's catalog names are given for commercially available compounds.

[0089] Lanosterol material (crude) (supplied by Shandong Jun Rui Co., Ltd. or Spectrum Chemical Manufacturing Corp-China)

Examples

[0090] In the column chromatography, the conditions used to track the elution of the target intermediate in the eluent by thin-layer chromatography (TLC) were: petroleum ether: ethyl acetate=5:1, color development: phosphomolybdic acid.

[0091] Based on experience and nuclear magnetic resonance analysis, the TLC results of the collected eluate containing the target intermediate were divided into three types: those basically free of impurity spots (defined as high-purity products with a .sup.1H NMR characteristic peak integral ratio or HPLC purity of 85%-90%), those with impurity spots but significant improvement in purity (defined as relatively high-purity products with a .sup.1H NMR characteristic peak integral ratio or HPLC purity of 70%-85%), and those with impurity spots, no significant improvement in purity, and a higher concentration of the target intermediate in the eluate (defined as low-purity products with a .sup.1H NMR characteristic peak integral ratio or HPLC purity generally less than 70%). The improvement in purity was relative to the purity of the sample of the current column chromatography.

Example 1

1.1 Protection of Hydroxyl

[0092] 500 g of a commercial lanosterol material (about 60% lanosterol purity) was aliquoted into two replicates of 250 g each. Each replicate was subjected to the following processing.

[0093] At 15 C., imidazole (87.8 g, 1.29 mol) and TBSCl (132.5 g, 879 mmol) were added to a solution of a lanosterol material (250 g) in N,N-dimethylformamide (2500 mL). The mixture was stirred at 85 C. for 6 h. The mixture was then cooled to 15 C. and extracted with petroleum ether (25002 mL). The petroleum ether phase was washed sequentially with a saturated NaCl solution (2000 mL) and water (2000 mL.).

[0094] 348 g of intermediate 1a and 350 g of intermediate 1b were acquired.

[0095] According to the general knowledge in the art, the difference in purity between the intermediates 1a and 1b and the lanosterol material before and after the hydroxyl protection reaction is negligible.

1.2 Column Chromatography

[0096] 348 g of intermediate 1a was mixed with 1000 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 10 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a relatively high-purity product was collected, evaporated at a reduced pressure, and dried to give 125 g of intermediate 1c (66% yield).

[0097] 350 g of intermediate 1b was mixed with 1000 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 10 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a high-purity product was collected and dried to give 121 g of intermediate 1d (64% yield).

[0098] Intermediates 1c and 1d were combined and mixed with 740 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 7.4 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a high-purity product was collected, evaporated at a reduced pressure, and dried to give 179 g of intermediate le (47% yield).

[0099] 179 g of intermediate 1e was mixed with 540 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 5.4 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-hexane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a high-purity product was collected and dried to give 161 g of intermediate If (42% yield). Faint impurity spots were observed by the TLC monitoring of the intermediate If, and the .sup.1H NMR characteristic peak integral showed that TBS-lanosterol accounted for about 83% of the total sterols.

[0100] 161 g of intermediate If was mixed with 480 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 5.0 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-hexane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a high-purity product was collected, evaporated at a reduced pressure, and dried to give 147 g of intermediate 1 h. The overall yield of the 4 purifications was about 39%. Unremarkable impurity spots were observed in the TLC monitoring of the intermediate 1 h, and the .sup.1H NMR characteristic peak integral showed that TBS-lanosterol accounted for about 91% of the total sterols.

[0101] .sup.1H NMR (400 MHz, CDCl.sub.3) =5.07 (br t, J=7.2 Hz, 1H), 3.17 (dd, J=4.6, 11.2 Hz, 1H), 2.05-1.35 (m, 24H), 1.29 (br s, 5H), 1.18-0.97 (m, 5H), 0.95 (s, 3H), 0.90-0.88 (m, 4H), 0.84 (s, 9H), 0.74 (s, 3H), 0.66 (s, 3H), 0.00 (d, J=2.5 Hz, 7H) ppm.

1.3 Deprotection of Hydroxyl

[0102] 49.2 g of the intermediate 1h was dissolved in 250 mL of anhydrous tetrahydrofuran. At 15 C., 110 mL of a TBAF solution (1.0 M) was added, and the mixture was heated to reflux for 16 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was directly concentrated by rotary evaporation at a reduced pressure to give a residue. 600 mL of methanol was added to the residue, and the mixture was refluxed for 4 h to give a white suspension. The suspension was stirred at 60 C. for 24 h, and then cooled to 15 C. The mixture was filtered to give a filter cake. The filter cake was washed with 300 mL of methanol and then dried at a reduced pressure to give 38.3 g of final product (98.4% yield; 91% lanosterol purity by HPLC).

[0103] .sup.1H NMR (400 MHz, CDCl.sub.3) 5.12 (br t, J=7.15 Hz, 1H), 3.26 (dd, J=4.64, 11.42 Hz, 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=6.27 Hz, 3H), 0.90 (s, 3H), 0.83 (s, 3H), 0.71 (s, 3H) ppm.

Example 2

2.1 Protection of Hydroxyl

[0104] At 15 C., imidazole (87.8 g, 1.29 mol) and TESCl (132.5 g, 879 mmol) were added to a solution of a commercial lanosterol material (250 g, about 60% lanosterol purity) in N,N-dimethylformamide (2500 mL). The mixture was stirred at 80 C. for 3 h. The reaction was monitored by TLC. After the reaction was completed, the reaction mixture was cooled to 25 C., and 600 mL of methanol and 300 mL of water were added. The mixture was stirred and filtered to give a filter cake. The filter cake was washed with 300 mL of methanol, and then dried at a reduced pressure to give 341 g of intermediate 2a.

2.2 Column Chromatography

[0105] 341 g of intermediate 2a was mixed with 1000 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 10 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a high-purity product was collected, evaporated at a reduced pressure, and dried to give 168 g of intermediate 2b (88% yield) (faint impurity spots were still observed in TLC).

[0106] 168 g of intermediate 2b was mixed with 540 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm180 mm) with 5.4 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1).

[0107] The elution process was tracked by TLC. An eluate basically free of impurity spots was collected, evaporated at a reduced pressure, and dried to give 117 g of intermediate 2c (about 62% yield). The .sup.1H NMR characteristic peak integral showed that TES-lanosterol accounted for about 86% of the total sterols.

2.3 Deprotection of Hydroxyl

[0108] At 25 C., 27 g of intermediate 2c was dissolved in anhydrous THF (150 mL) and TBAF (55 mL, 0.055 mol). The mixture was incubated for 30 min for reaction. Then, the reaction mixture was warmed to 60 C., refluxed, and stirred for 4 h. The reaction completion was monitored by TLC. 80 mL of water and 160 mL of methanol were added, and the mixture was stirred for 1 h. A solid was precipitated. The mixture was filtered to give a filter cake. The filter cake was washed with a small amount of water and methanol, and then dried at a reduced pressure to give 18.5 g of the product (white solid, 87% yield; about 89% purity lanosterol by HPLC).

Example 3

3.1 Protection of Hydroxyl

[0109] At 15 C., imidazole (87.8 g, 1.29 mol) and TESCl (132.5 g, 879 mmol) were added to a solution of a commercial lanosterol material (250 g, about 60% lanosterol purity) in N,N-dimethylformamide (2500 mL). The mixture was stirred at 80 C. for 3 h. The reaction was monitored by TLC. After the reaction was completed, the reaction mixture was cooled to 25 C., and 600 mL of methanol and 300 mL of water were added. The mixture was stirred and filtered to give a filter cake. The filter cake was washed with 300 mL of methanol, and then dried at a reduced pressure to give 355 g of intermediate 3a.

3.2 Column Chromatography

[0110] 355 g of intermediate 3a was mixed with 1000 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 10 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC. Eluates containing a high-purity product and a relatively high-purity product were collected, evaporated at a reduced pressure, and dried to give 71 g of intermediate 3b with a purity of about 87%-89% and 96 g of intermediate 3c with a purity of about 70%-80%, respectively.

[0111] 96 g of intermediate 3c was mixed with 300 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm180 mm) with 3 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane and ethyl acetate (100:1). The elution process was tracked by TLC. An eluate containing a relatively high-purity product was collected, evaporated at a reduced pressure, and dried to give 44 g of intermediate 3d.

[0112] A total of 115 g of intermediates 3b and 3d (designated as intermediate 3e) was combined (61% yield). The .sup.1H NMR characteristic peak integral showed that TES-lanosterol accounted for about 90% of the total sterols

3.3 Deprotection of Hydroxyl

[0113] At 25 C., intermediate 3e (22 g, 0.04 mol) was dissolved in anhydrous THF (120 mL). TBAF (45 mL, 0.045 mol) was added to the reaction solution. The mixture was let stand for 30 min. Then, the reaction mixture was warmed to 60 C., refluxed, and stirred for 4 h. The reaction was monitored by TLC. After the reaction was completed, 60 mL of water and 120 mL of methanol were added, and the mixture was stirred for 1 h. A solid was precipitated. The solid was filtered to give a filter cake. The filter cake was washed with 60 mL of water and then washed with 100 mL of methanol. The mixture was filtered to give a filter cake. The filter cake was refluxed in 200 mL of methanol for 3 h, and then cooled to precipitate a solid. Again, the mixture was filtered to give 15.2 g of a white solid (88% yield; about 92% lanosterol purity by HPLC).

Example 4

4.1 Recrystallization

[0114] 50 g of the intermediate 3e obtained in Example 3 was added to a 750-mL mixture of ethyl acetate and isopropanol (volume ratio=1:1). The mixture was warmed to 90 C. and refluxed for 2 h until the solids were completely dissolved. The stirring was stopped and the mixture was slowly cooled. A solid was precipitated. The mixture was filtered to give a filter cake. The filter cake was washed and dried to give 40 g of intermediate 4a (80% yield). The .sup.1H NMR characteristic peak integral showed that TES-lanosterol accounted for about 93% of the total sterols.

[0115] .sup.1H NMR (400 MHz, CDCl) 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, 4.52 Hz, 1H), 5.12 (br t, J=7.03 Hz, 1H)

4.2 Deprotection

[0116] At 25 C., intermediate 4a (20 g, 0.037 mol) was dissolved in anhydrous THF (120 mL). TBAF (1 M, 1.5 eq) was added to the reaction solution. The reaction mixture was let stand for 30 min. The reaction mixture was warmed to 60 C., refluxed, and stirred for 4 h. The reaction completion was monitored by TLC. 60 ml of water and 120 mL of methanol were added, and the mixture was stirred for 1 h. A solid was precipitated. The solid was filtered to give a filter cake. The filter cake was washed sequentially with a small amount of water and methanol. The mixture was filtered to give a filter cake. The filter cake was refluxed for 3 h in 200 ml of methanol, and then cooled to precipitate a solid. The solid was filtered to give 13 g of a white solid. The yield was about 85%, and the lanosterol purity was >99% as measured by HPLC.

Example 5

5.1: Protection of Hydroxyl

[0117] At 15 C., imidazole (87.75 g, 1.29 mol) and TMSCl (95.47 g, 879 mmol) were added to a solution of a commercial lanosterol material (250 g, about 60% lanosterol purity) in N,N-dimethylformamide (2500 mL). The mixture was stirred at 70 C. for 5 h. After the reaction was completed, the reaction solution was cooled to 15 C. and extracted with petroleum ether (25002 mL). The petroleum ether phase was washed with a saturated NaCl solution (2000 mL) and water (2000 mL) to give 287 g of intermediate 5a.

5.2: Column Chromatography

[0118] 287 g of intermediate Sa was mixed with 850 g of silica gel of 100-200 mesh, and then subjected by column chromatography (1000 mm230 mm) with 8.5 kg of silica gel of 200-300 mesh. The column was eluted using a mixture of n-heptane, ethyl acetate, and aqueous ammonia (100:1:0.05). The elution process was tracked by TLC. An eluate was collected, evaporated at a reduced pressure, and dried to give 53 g of product 5b. The yield of TMS-lanosterol intermediate was about 30% with a purity of about 80% as determined by .sup.1H NMR. TLC showed faint impurity spots.

Example 6

Investigation of Protecting Agents and Conditions

[0119] At 15 C., imidazole (3.9 g) and triethylchlorosilane (1-2 eq.) were added to a solution of a commercial lanosterol material (10 g, 0.023 mol, about 60% purity) in DMF (100 mL). The reaction completion was monitored by TLC. The following post-treatments were performed for reactions with relatively complete conversion: The reaction mixture was cooled to room temperature and extracted with petroleum ether (752 mL). The petroleum ether phase obtained was washed with a saturated NaCl solution (75 mL) and water (750 mL), and then dried to give a corresponding crude product. The product was purified by column chromatography.

[0120] The solvent, protecting agent, temperature, and reaction time in the reaction described above were changed, and the corresponding test conditions and results are shown in the following table:

TABLE-US-00001 Hydroxyl- Column protecting Temperature, chromatography No. agent Solvent C. Time TLC results results 1 Benzoic acid DCM 60 24 h Irrecognizable impurity spots None 2 p-Nitrobenzoic acid DCM 60 24 h Irrecognizable impurity spots None 3 2-Picolinic acid DCM 60 24 h Irrecognizable impurity spots None 4 Acetic acid DCM 60 24 h Irrecognizable impurity spots None 5 Glycine DCM 60 24 h Incomplete reaction with None unstable resultant ester 6 TBSCl (1 eq) DMF 85 12 h Recognizable impurity spots None with a small amount of raw material 7 TBSCl (1.5 eq) DMF 85 6 h Complete conversion with About 37% yield recognizable impurity spots and about 86% purity 8 TBSCl (1.5 eq) DMF 120 4 h Complete conversion with About 34% yield recognizable impurity spots and about 86% purity TBSCl (1.5 eq) DMF 70 12 h Basically complete About 32% yield conversion with recognizable and about 85% impurity spots purity 10 TBSCl (1.5 eq) DMF 40 12 h Incomplete reaction with None recognizable impurity spots 11 TESCl (1.5 eq) DMF 80 4 h Complete conversion with About 58% yield recognizable impurity spots and about 87% purity 12 TESCl (1.1 eq) DMF 80 6 h Complete conversion with About 51% yield recognizable impurity spots and about 86% purity 13.sup.a TESCl (1.2 eq) DMF 80 4 h Complete conversion with About 59% yield recognizable impurity spots and about 87% purity Note: .sup.arepresents the following post-treatment procedures: the reaction system was cooled to room temperature; methanol (25 mL) and water (12.5 mL) were added; the mixture was stirred and filtered to give a filter cake; the filter cake was washed with a small amount of methanol, and then dried at a reduced pressure to give a corresponding crude product; the product was purified by column chromatography. In the post-treatment procedures, the product was directly precipitated and then filtered.

Example 7

[0121] 5.41 g of intermediate 2c purified by column chromatography obtained in Example 2 was tested with recrystallization conditions as shown in the following table, wherein the mass-volume ratio of the solute to the solvent was 1 g:10 mL.

TABLE-US-00002 No. Solvent (ratio) Other procedures Times Yield Purity 1 Ethyl The mixture was refluxed for 2 h 1 84% 91% acetate:isopropanol until complete dissolution, cooled (volume ratio = 1:1) to room temperature, and let stand overnight to precipitate a solid; the solid was filtered and dried at a reduced pressure 2 Ethyl The mixture was refluxed for 2 h 2 68% 91% acetate:isopropanol until complete dissolution, cooled (1:1) to room temperature, and let stand overnight to precipitate a solid; the solid was filtered and dried at a reduced pressure 3 Isopropyl acetate The mixture was refluxed for 2 h 1 80% 90% until complete dissolution, cooled to room temperature, and let stand overnight to precipitate a solid; the solid was filtered and dried at a reduced pressure 4 Isopropyl acetate The mixture was refluxed for 2 h 2 68% 90% until complete dissolution, cooled to room temperature, and let stand overnight to precipitate a solid; the solid was filtered and dried at a reduced pressure

[0122] The embodiments of the present disclosure are not limited to the specific examples described above, and any technical modifications made according to the embodiments of the present disclosure shall fall within the protection scope of the present disclosure.