Method for purifying lanosterol

Abstract

The invention discloses a purification method of lanosterol, which comprises the following steps: (a) acylating a lanosterol crude product to obtain an acylated crude product, (b) oxidizing the acylated crude product to obtain an oxidized crude product containing a compound shown as a formula III and a compound shown as a formula II, (c) carrying out separation treatment on the oxidized crude product containing the compound shown in the formula III and the compound shown in the formula II to respectively obtain the compound shown in the formula III and the compound shown in the formula II; and (d) converting the separated compound as shown in the formula III into lanosterol. The method is simple in process, convenient to operate, high in yield, high in purity, cheap and easily available in raw materials and suitable for industrialization; and the whole process is more economical and environment-friendly.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. 2023106722513 filed on June 7, 2023, and the entire contents of the above Chinese patent application are incorporated into this application by reference. Technical Field

[0003] The invention relates to a method for purifying high-purity lanosterol. Background Art

[0004] Lanosterol, also known as isocholesterol, is a white odorless powder with a molecular formula of C 30 H 50 O, melting point: ≥130℃, soluble in chloroform, ethanol, ether, and forms crystals in methanol and acetone. Lanosterol is an intermediate in the biosynthesis of sterols and an important raw material in the cosmetics, pharmaceutical and chemical industries. In addition, lanosterol has a variety of important biological activities and a wide range of pharmacological effects, such as anti-cancer, lowering blood pressure, treating leukemia, lowering blood sugar, and alleviating cataracts in experimental animals (rabbits and dogs). The separation and purification methods currently used in production and scientific research are mainly to directly extract lanosterol from animals and plants, including crystallization, complexation, saponification, molecular distillation, supercritical CO2 extraction, adsorption (column adsorption, high-pressure fluid adsorption), enzyme method, etc. Lanosterol sold on the market is basically about 60% crude product.

[0005] At present, there are many literature reports on the chemical purification process of crude lanosterol. A comprehensive literature analysis shows that the chemical purification processes of crude lanosterol reported in literature such as WO03040066A1 and WO03027133A1 can be summarized as follows, and their synthesis routes are as follows.

[0006] Route 1

[0007]

[0008] Physical purification methods for lanosterol mostly use solvent crystallization and column chromatography. Solvent crystallization is difficult to obtain a high-purity product even after multiple crystallizations; column chromatography uses a large amount of solvent, is difficult to recover the solvent, and is difficult to mass produce.

[0009] In the chemical purification method, route 1 has six chemical reactions. The second step requires column chromatography to remove compound 4, which is not conducive to large-scale industrial production. The third step reaction has large impurities and is easily formed isomer impurities, which also require column chromatography purification. When the applicant repeated the fifth step, it was found that it was difficult to repeat and the yield was low; route 2 and route 1 have similar problems and are difficult to industrialize.

[0010] Therefore, there is still a need in the art to develop a method for purifying lanosterol suitable for industrialization. Summary of the invention

[0011] In view of the problems existing in the prior art, the object of the present invention is to provide a method for purifying lanosterol, which has simple process, convenient operation, high yield, high purity, and the raw material (commercially available crude lanosterol containing about 30% dihydrolanosterol) is cheap and easy to obtain, thereby solving the problem that high-purity lanosterol cannot be obtained industrially; the whole process is more economical and environmentally friendly.

[0012] The present invention provides a method for purifying lanosterol, which comprises any one or more of the following steps (a) to (d):

[0013] (a) subjecting a crude lanosterol product containing dihydrolanosterol to an acylation treatment to obtain a crude acylated product containing a compound of formula I and a compound of formula II,

[0014]

[0015] Wherein, R1 is a hydroxyl protecting group, preferably a C1-C5 alkyl-C(O)-;

[0016] (b) subjecting the crude acylated product to an oxidation treatment, wherein the compound of formula I is oxidized to the compound of formula III, thereby obtaining a crude oxidation product containing the compound of formula III and the compound of formula II,

[0017]

[0018] (c) separating the crude oxidation product containing the compound of formula III and the compound of formula II to obtain the compound of formula III and the compound of formula II, respectively;

[0019] (d) converting the compound of formula III separated in step (c) into lanosterol.

[0020] In some embodiments, the purification method comprises steps (a) to (d).

[0021] In some embodiments, the step (d) comprises one or more steps of (d1) to (d3):

[0022] (d1) converting the compound of formula III into the compound of formula VI,

[0023]

[0024] R2 is a C1-C5 alkyl group;

[0025] (d2) converting the compound of formula VI into a compound of formula I,

[0026]

[0027] (d3) converting the compound of formula I into lanosterol,

[0028]

[0029] In some embodiments, step (d) includes (d1) to (d3).

[0030] In some embodiments, in step (a), acylation is carried out in a solvent using 4-dimethylaminopyridine as a catalyst and an acid anhydride or an acyl halide as an acylating agent. In some embodiments, the acylating agent is acetic anhydride or acetyl chloride.

[0031] In some embodiments, the solvent in step (a) is one or more of dichloromethane, acetone and tetrahydrofuran.

[0032] In some embodiments, in step (a), the acylation treatment is performed at 20-40°C.

[0033] In some embodiments, in step (b), the oxidation treatment using an oxidant is preferably carried out in a solvent.

[0034] In some embodiments, in step (b), the oxidant comprises one or more of hydrogen peroxide, m-chloroperbenzoic acid, peracetic acid and AD-Mix-β, preferably AD-Mix-β.

[0035] In some embodiments, in step (b), the solvent used in step (b) is a mixed solvent comprising water and an alcohol. In some embodiments, the mixed solvent comprises water and a C1-C4 alcohol. In some embodiments, the mixed solvent comprises water and one or more of n-butanol, ethanol and isopropanol.

[0036] In some embodiments, in step (b), a configuration stabilizer is also used. In some embodiments, the configuration stabilizer is methylsulfonamide.

[0037] In some embodiments, the amount of the oxidant used is 2-10 times the amount of the crude acylation product by mass.

[0038] In some embodiments, the amount of the configuration stabilizer is 0.1-1 times the amount of the crude acylation product by mass.

[0039] In some embodiments, in step (c), the separation treatment comprises mixing the crude oxidation product with methanol at 50-65° C. for a period of time, and then mixing at 20-40° C. for a period of time, followed by solid-liquid separation.

[0040] In some embodiments, in step (d1), the compound of formula III is converted into the compound of formula VI by condensing and dehydrating the compound in a solvent using trimethyl orthoformate, triethyl orthoformate or N,N-dimethylformamide dimethyl acetal as a protecting agent and pyridinium p-toluenesulfonate and / or p-toluenesulfonic acid as a catalyst.

[0041] In some embodiments, in step (d1), the condensation dehydration is carried out at 20-40°C.

[0042] In some embodiments, in step (d1), the amount of the protective agent is 1-15 times (by mass) of the compound of formula III.

[0043] In some embodiments, in step (d1), the amount of the catalyst used is 0.1-0.8 times (by mass) of the compound of formula III.

[0044] In some embodiments, in step (d1), the solvent is one or more of dichloromethane, ethyl acetate, and tetrahydrofuran, preferably dichloromethane.

[0045] In some embodiments, in step (d2), the compound of formula VI is converted into the compound of formula I by reacting in acetic anhydride at a temperature of 120°C to 150°C, preferably 140±5°C.

[0046] In some embodiments, in step (d3), the compound of formula I is converted into lanosterol in a base and an alcohol solvent. In some embodiments, the base is one or more of potassium hydroxide, sodium hydroxide and lithium hydroxide. In some embodiments, more preferably, the base is potassium hydroxide. In some embodiments, the solvent is one or more of methanol, ethanol and propanol.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects: the method has simple process, convenient operation, high yield, high purity, and cheap and readily available raw materials, which solves the problem that high-purity lanosterol cannot be obtained industrially; the entire process is more economical and environmentally friendly. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0049] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0050] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. 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.

[0051] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0052] The solvents used herein are commercially available. The following abbreviations are used herein:

[0053] DMAP: 4-dimethylaminopyridine

[0054] AD-Mix-β: AD-Mix-BETA, CAS No.: 148618-32-0

[0055] PPTS: Pyridinium p-toluenesulfonate

[0056] TLC: Thin layer chromatography

[0057] TBAF: Tetrabutylammonium fluoride

[0058] THF: Tetrahydrofuran

[0059] DCM: dichloromethane

[0060] DMF: N,N-dimethylformamide

[0061] eq: equivalent

[0062] Compounds are manually or The software names were used, and commercially available compounds were named using the supplier's catalog names.

[0063] Lanosterol raw material (crude product) (supplier is Shandong Junrui Co., Ltd. or Sibaiquan Chemical (Shanghai) Co., Ltd.)

[0064] In some embodiments, the purification route of lanosterol of the present invention is as follows:

[0065]

[0066] R1 is C1-C5 alkyl-C(=O)-, preferably acetyl, and R2 is C1-C5 alkyl, preferably methyl.

[0067] Example 1

[0068]

[0069] Acetylation

[0070] Weigh 10 g of crude lanosterol (containing about 60% lanosterol and 35% dihydrolanosterol) in a reaction flask, add 100 mL of dry dichloromethane, stir to dissolve, add DMAP (100 mg) and acetic anhydride (5 g), and stir at room temperature for 2 hours. After the reaction is completed, quench the reaction with 1 M hydrochloric acid and separate the liquids. The organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white solid, which is heated and pulped with 100 mL of methanol, cooled and filtered, and dried in vacuo to obtain 10.0 g of a white solid, i.e., the acetylated product 2, with a yield of 91.0%.

[0071] Oxidation treatment

[0072] The acetylated product 2 (10 g) was suspended in n-butanol / water (V=1 / 1) (200 mL), cooled to 0°C, and AD-Mix-β (30 g) and methylsulfonamide (2.5 g) were added. After the addition, the mixture was stirred at room temperature for 48 hours. After the reaction was completed, the mixture was cooled in an ice-water bath, 20 mL of saturated sodium sulfite solution was added to the reaction solution to quench the reaction, and the mixture was stirred for 1 hour; the mixture was extracted with 100 mL*2 of ethyl acetate, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white solid.

[0073] The prepared white solid was added to 100 mL of methanol, heated and stirred at 60°C for 2 hours, stirred at room temperature for 2 hours, and filtered. The filter cake was dried at 50°C to obtain compound 4, and the filtrate was concentrated under reduced pressure to obtain a white solid, i.e., 5.2 g of compound 3, with a yield of 81%.

[0074] Synthesis of Compound 5 from Compound 3

[0075] Compound 3 (5 g) was dissolved in 60 mL of dry dichloromethane, and triethyl orthoformate (12.5 g) and pyridinium p-toluenesulfonate (PPTS) (1.25 g) were added. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was filtered through 200-300 mesh silica gel and rinsed with an appropriate amount of DCM. The filtrate was concentrated under reduced pressure to obtain 5.2 g of white solid, namely compound 5, with a yield of 93.6%.

[0076] Synthesis of Compound 2 from Compound 5

[0077] Weigh compound 5 (10 g) into a reaction bottle, add acetic anhydride 120 mL, heat to 140 ° C for 3.5 hours, and the reaction is complete; cool to room temperature, pour the reaction solution into 200 mL of cold water, stir to precipitate a white solid, stir at room temperature for 15 minutes, and filter to obtain a white solid. The above solid is heated and slurried with methanol 100 mL, cooled and filtered, and vacuum dried to obtain 3.2 g of a white solid, namely compound 2, with a yield of 76.3%.

[0078] Synthesis of Compound 6 from Compound 2

[0079] Weigh potassium hydroxide (10 g) in anhydrous ethanol (200 mL), stir to dissolve; add compound 6 (10 g), heat to reflux 80°C, react for 3 hours, cool to room temperature, add 200 mL of purified water to the reaction solution, stir for 2 hours, precipitate a white solid, filter, recrystallize the obtained white solid with acetone, cool and crystallize to obtain 6.9 g of compound 6 (i.e., lanosterol), with a yield of 75.8% and a purity greater than 98%.

[0080] Structure confirmation:

[0081] APCI-MS, [M-1]=425, 409 is M-18+H (molecular dehydration peak).

[0082] 1 H NMR(500MHz, CDCl3)δ:5.08-5.11(m,1H),3.21-3.25(dd,J=4.55,4.5Hz,1H),1.99-2.05(m,5H),1.86-1.94(m,5H),1.71(s,3H),1.64-1 .69(m,1H),1.63(s,3H),1.04-1.60(m,12H),1.01(s,3H),1.00(s,3H),0.98(d,J=6.5Hz,3H),0.90(s,3H),0.81(s,3H),0.69(s,3H)ppm.

[0083] 13 C NMR(500MHz, CDCl3)δ:134.39,130.89,125.24,78.97,50.39,49.79,44.47,38.87,37.01,36.35,36.25,35.58,30 .89,30.84,28.19,27.95,27.84,26.49,25.71,24.91,24.24,20.99,19.12,18.62,18.25,17.62,15.73,15.40ppm.

[0084] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for purifying lanosterol, comprising any one or more of the following steps (a) to (d): (a) subjecting a crude lanosterol product containing dihydrolanosterol to an acylation treatment to obtain a crude acylated product containing a compound of formula I and a compound of formula II, in, R1 is a hydroxyl protecting group, preferably a C1-C5 alkyl-C(O)-; (b) subjecting the crude acylated product to an oxidation treatment, wherein the compound of formula I is oxidized to the compound of formula III, thereby obtaining a crude oxidation product containing the compound of formula III and the compound of formula II, (c) separating the crude oxidation product containing the compound of formula III and the compound of formula II to obtain the compound of formula III and the compound of formula II, respectively; (d) converting the compound of formula III separated in step (c) into lanosterol.

2. The method according to claim 1, wherein: The step (d) comprises one or more steps from (d1) to (d3): (d1) converting the compound of formula III into the compound of formula VI, R2 is a C1-C5 alkyl group; (d2) converting the compound of formula VI into a compound of formula I, (d3) converting the compound of formula I into lanosterol, 3. The method according to claim 1 or 2, characterized in that: In step (a), acylation is carried out in a solvent using 4-dimethylaminopyridine as a catalyst and an acid anhydride or an acyl halide as an acylating agent, preferably, the acylating agent is acetic anhydride or acetyl chloride; Preferably, the solvent in step (a) is one or more of dichloromethane, acetone and tetrahydrofuran; and / or Preferably, in step (a), the acylation treatment is carried out at 20-40°C.

4. The method according to any one of claims 1 to 3, characterized in that: In step (b), the oxidation treatment using an oxidant is preferably carried out in a solvent.

5. The method according to claim 4, characterized in that In step (b), the oxidant comprises one or more of hydrogen peroxide, m-chloroperbenzoic acid, peracetic acid and AD-Mix-β, preferably AD-Mix-β; In step (b), the solvent used in step (b) is a mixed solvent comprising water and alcohol, preferably, the mixed solvent comprises water and C1-C4 alcohol, more preferably, the mixed solvent comprises water and one or more of n-butanol, ethanol and isopropanol.

6. The method according to any one of claims 1 to 5, characterized in that: In step (b), a configuration stabilizer is also used, preferably, the configuration stabilizer is methylsulfonamide.

7. The method according to any one of claims 4 to 6, characterized in that: The amount of the oxidant used is 2-10 times the amount of the crude acylation product by mass; and / or The amount of the configuration stabilizer used is 0.1-1 times the amount of the crude acylation product by mass.

8. The method according to any one of claims 1 to 7, characterized in that: In step (c), the separation treatment comprises mixing the crude oxidation product with methanol at 50-65°C for a period of time, then mixing at 20-40°C for a period of time, and then performing solid-liquid separation.

9. The method according to any one of claims 2 to 8, characterized in that: In step (d1), the compound of formula III is condensed and dehydrated in a solvent using trimethyl orthoformate, triethyl orthoformate or N,N-dimethylformamide dimethyl acetal as a protective agent and pyridinium p-toluenesulfonate and / or p-toluenesulfonic acid as a catalyst to convert the compound into the compound of formula VI; Preferably, in step (d1), the condensation dehydration is carried out at 20-40° C.; and / or the amount of the protective agent is 1-15 times (by mass) of the compound of formula III, and / or the amount of the catalyst is 0.1-0.8 times (by mass) of the compound of formula III; and / or the solvent is one or more of dichloromethane, ethyl acetate, and tetrahydrofuran, preferably dichloromethane.

10. The method according to any one of claims 2 to 9, characterized in that: In step (d2), the compound of formula VI is reacted in acetic anhydride at a temperature of 120°C to 150°C, preferably 140±5°C, to convert it into a compound of formula I; and / or, in step (d3), the compound of formula I is converted into lanosterol in an alkali and alcohol solvent, Preferably, the base is one or more of potassium hydroxide, sodium hydroxide and lithium hydroxide, more preferably potassium hydroxide; Preferably, the solvent is one or more of methanol, ethanol and propanol.

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