A method for purifying 7-dehydrocholesterol intermediate

Abstract

The present invention provides a method for purifying a 7-dehydrocholesterol intermediate, using 7-dehydrocholesterol acetate impurities as a template, based on a low eutectic solvent as a functional monomer, preparing a molecular imprinting polymer, and then using the molecular imprinting polymer to separate 7-dehydrocholesterol acetate impurities from a crude 7-dehydrocholesterol acetate product to obtain high-purity 7-dehydrocholesterol acetate. The method is easy to operate, the process is stable and reliable, and the impurities can be effectively removed to obtain 7-dehydrocholesterol acetate with an impurity content of less than 0.10% and a purity of more than 97.0%.

Description

Technical Field

[0001] The invention belongs to the technical field of purification, and in particular relates to a method for purifying a 7-dehydrocholesterol intermediate. Background Art

[0002] Vitamin D3, also known as cholecalciferol, is a drug with anti-rickets activity and is widely considered to be an important substance that regulates calcium and phosphorus metabolism. Before the discovery of vitamin D3, osteomalacia (also known as rickets) was one of the important diseases that troubled human health. Later, people gradually discovered that more sun exposure can play a role in treating osteomalacia to a certain extent, which also indicates that certain substances in the human body can be converted into effective anti-rickets active substances after light exposure. By the early 1930s, scientists Waddel and Windaus finally confirmed that the active component of anti-rickets in the human body was cholecalciferol generated by 7-dehydrocholesterol (having a structure shown in Formula I) after light exposure, and named it vitamin D3.

[0003]

[0004] The chemical synthesis of vitamin D3 can be divided into full synthesis and semi-synthesis. The semi-synthesis is to first synthesize 7-dehydrocholesterol, and then convert it into vitamin D3 through photolysis and thermal isomerization reactions. At present, there are two main production processes for vitamin D3. One is the bromination / debromination method, which is accompanied by many by-products such as bromide and isomers; the other method is the redox method, which uses NF-grade cholesterol as raw material, and successively synthesizes 7-dehydrocholesterol through esterification, catalytic oxidation, hydrazone formation, and dehydrazone reaction, and then uses a photochemical synthesis process to perform light reaction, thermal isomerization reaction and other steps on 7-dehydrocholesterol to generate vitamin D3.

[0005] In the process of synthesizing 7-dehydrocholesterol by redox method, the most important step is the dehydrazone reaction of 7-toluenesulfonylhydrazone-cholesterol acetate to generate 7-dehydrocholesterol acetate. The dehydrazone reaction will generate an impurity that is difficult to detect and remove, and has a structure shown in Formula II:

[0006]

[0007] At present, there is no literature describing the removal method of this impurity. 7-dehydrocholesterol is an important starting material for the production of vitamin D3, and the impurity content and purity will directly affect the quality of vitamin D3. Therefore, it is of great significance to study the purification method of this impurity and obtain 7-dehydrocholesterol with high purity and low impurity content. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a method for purifying a 7-dehydrocholesterol intermediate, which can effectively remove impurities and obtain 7-dehydrocholesterol acetate with high purity.

[0009] The present invention provides a method for purifying a 7-dehydrocholesterol intermediate, comprising the following steps:

[0010] In an inert atmosphere, a template, a low eutectic solvent, a cross-linking agent and an initiator are mixed to carry out a polymerization reaction, the template is eluted, and the mixture is dried to obtain a molecularly imprinted polymer;

[0011] The template has a structure of Formula II:

[0012]

[0013] The crude 7-dehydrocholesterol acetate is mixed with an alcohol solvent or a ketone solvent, stirred until dissolved, and then the molecular imprinted polymer material is added, adsorbed, filtered, and the filter cake is eluted to obtain a regenerated molecular imprinted polymer material; the crude 7-dehydrocholesterol acetate includes impurities shown in formula II;

[0014] The regenerated molecular imprinted polymer material is added to the solution for adsorption, and the cycle is repeated to obtain 7-dehydrocholesterol acetate.

[0015] In some embodiments of the present invention, the low eutectic solvent serves as a functional monomer; the template and the functional monomer are bonded together by covalent bonds. The low eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor;

[0016] The hydrogen bond acceptor is selected from one or more of carboxylic acids, polyacids, polyols, sugars, urea, amides and polyphenols; the polyacid is selected from citric acid and / or caffeic acid; the polyol is selected from glycerol and / or ethylene glycol; the sugar is selected from glucose;

[0017] The hydrogen bond donor is selected from one or more of quaternary ammonium salts and zwitterions; the quaternary ammonium salt is selected from choline chloride and / or tetrabutylammonium bromide; and the zwitterion is selected from betaine.

[0018] In the present invention, the low eutectic solvent is prepared by heating a two-component or three-component mixture of a hydrogen bond donor and a hydrogen bond acceptor in different molar ratios.

[0019] After the polymerization reaction is completed, the present invention grinds and sieves the polymerization product to obtain a uniform granular material. The present invention dissolves the granular material in a mixed solvent and uses Soxhlet extraction to elute the template molecule; after the template molecule is eluted, a binding site that can interact with the target molecule through non-covalent interaction is formed in the imprinting cavity of the imprinted polymer. The mixed solvent is selected from an ethanol-acetic acid mixed solvent with a volume ratio of 9 to 20:1; or a methanol-acetic acid mixed solvent with a volume ratio of 9 to 20:1. The elution time is 22 to 26 hours.

[0020] After the template molecules are eluted, they are dried at room temperature to produce water, thereby obtaining a molecularly imprinted polymer with a certain cavity.

[0021] The invention mixes crude 7-dehydrocholesterol acetate with an alcohol solvent or a ketone solvent, stirs until the mixture is dissolved, then adds the molecular imprinting polymer, performs adsorption, filtration, and elution of the filter cake to obtain the regenerated molecular imprinting polymer.

[0022] In the present invention, the crude 7-dehydrocholesterol acetate includes an impurity shown in Formula II; the carbon spectrum and hydrogen spectrum of the impurity of Formula II are as follows: Figure 1 and Figure 2 shown.

[0023] In the present invention, the mass ratio of the crude 7-dehydrocholesterol acetate to the alcohol solvent or ketone solvent is 1 g: (10-20) mL. The present invention uses a mixed solvent to elute the filter cake; the mixed solvent is selected from an ethanol-acetic acid mixed solvent with a volume ratio of 9-20:1; or a methanol-acetic acid mixed solvent with a volume ratio of 9-20:1.

[0024] The present invention adds the regenerated molecular imprinting polymer material to a solution for adsorption, and then cycles to obtain 7-dehydrocholesterol acetate. In some embodiments, the adsorption-desorption cycle is 4 to 8 times. The obtained solution is concentrated to dryness to obtain high-purity 7-dehydrocholesterol acetate.

[0025] The present invention provides a method for purifying a 7-dehydrocholesterol intermediate, using 7-dehydrocholesterol acetate impurities as a template and a low eutectic solvent as a functional monomer to prepare a molecular imprinting polymer, which is then used to separate 7-dehydrocholesterol acetate impurities from a crude 7-dehydrocholesterol acetate product to obtain high-purity 7-dehydrocholesterol acetate. The method is easy to operate, has a stable and reliable process, a high yield, and a low cost. The method can effectively remove the impurities to obtain 7-dehydrocholesterol acetate with an impurity content of less than 0.10% and a purity of more than 97.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The carbon spectrum of the compound of formula II;

[0027] Figure 2 and Figure 3 All of them are hydrogen spectra of compounds with structure of formula II;

[0028] Figure 4 Characterization of the molecular imprinted polymer material in Example 1 of the present invention, wherein A is the infrared spectra of DES and DES-MIPs, B is the thermogravimetric curve of DES-MIPs, C is the SEM image of DES-MIPs, D is the SEM image of DES-NIPs, and E is the EDX image of DES-MIPs;

[0029] Figure 5 This is the spectrum of the crude product of 7-dehydrocholesterol acetate in Example 1 of the present invention;

[0030] Figure 6 is the spectrum of 7-dehydrocholesterol acetate in Example 1 of the present invention;

[0031] Figure 7 This is the spectrum of the crude product of 7-dehydrocholesterol acetate in Example 2 of the present invention;

[0032] Figure 8 is the spectrum of 7-dehydrocholesterol acetate in Example 2 of the present invention;

[0033] Fig. 9 This is the spectrum of the crude product of 7-dehydrocholesterol acetate in Example 3 of the present invention;

[0034] Fig.10 This is the spectrum of 7-dehydrocholesterol acetate in Example 3 of the present invention. DETAILED DESCRIPTION

[0035] In order to further illustrate the present invention, a method for purifying a 7-dehydrocholesterol intermediate provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] 2 g of caffeic acid, 12 g of choline chloride, and 6 g of formic acid were weighed and added into a 50 mL round-bottom flask, and stirred continuously in an oil bath at 90 °C until a uniform dark brown liquid was formed, and then cooled to room temperature for the preparation of 7-dehydrocholesterol acetate impurity molecular imprinting polymer.

[0038] 200 mg of 7-dehydrocholesterol acetate impurity (having the structure of formula II) was accurately weighed and dissolved in 50 mL of chloroform, followed by the addition of 10 mL of a low eutectic solvent and prepolymerization at room temperature for 12 h. After the reaction was completed, 10 mL of crosslinker ethylene glycol dimethacrylate (EGDMA) and 200 mg of initiator azobisisobutyronitrile (AIBN) were added, and then the mixture was bubbled with nitrogen for 5 min, and a three-necked flask was sealed under nitrogen protection, and then the reaction vessel was placed in a water bath for 6 h at 60 ° C and 18 h at 70 ° C. After the polymerization reaction was completed, in order to obtain small particles, the polymer was ground and sieved and repeatedly washed in methanol. The resulting product was eluted with the template molecule by Soxhlet extraction using an ethanol-acetic acid (9:1, v / v) mixture solution. After elution for 24 h, the product was dried at room temperature to obtain 7-dehydrocholesterol acetate impurity molecular imprinting polymers (DES-MIPs) with a certain cavity, which were dried for use. Meanwhile, non-molecularly imprinted polymers (DES-NIPs) were prepared by the same procedure without adding 7-dehydrocholesterol acetate impurity.

[0039] Add 19.6g of 7-toluenesulfonylhydrazone-cholesterol acetate and 196ml of toluene into a single-necked bottle, stir until dissolved, then add 1.3g of lithium hydride, heat to reflux for 2 hours, cool to 0-10°C, add 196ml of water dropwise, extract and separate, collect the organic layer, and concentrate the organic layer to dryness at 60°C to obtain 11.6g of crude 7-dehydrocholesterol acetate.

[0040] 11.6g crude 7-dehydrocholesterol acetate (HPLC purity of 78.22%, impurity content: 20.64%) was dissolved in 232ml ethanol, and then 5.0g 7-dehydrocholesterol acetate impurity molecular imprinting polymer was added, and stirred at 25°C for 12h. After the stirring was completed and the first round of adsorption was completed, the filter cake obtained was the 7-dehydrocholesterol acetate impurity molecular imprinting polymer material adsorbing impurities, and the 7-dehydrocholesterol acetate impurity molecular imprinting polymer material adsorbing impurities was washed with a mixed solvent of ethanol-acetic acid (9:1v / v), and dried to obtain a regenerated 7-dehydrocholesterol acetate impurity molecular imprinting polymer. The regenerated 7-dehydrocholesterol acetate impurity molecular imprinting polymer was added to the solution for the next adsorption cycle. After 6 adsorption-desorption cycles, the 7-dehydrocholesterol acetate impurity content in the solution was determined to be less than 0.1%. The adsorption-completed solution was concentrated to dryness under reduced pressure at 40-45°C to obtain 9.3g 7-dehydrocholesterol acetate. The purity detected by HPLC was 98.46%, and the impurity content was 0%.

[0041] Embodiment 2:

[0042] 2 g of caffeic acid, 10 g of choline chloride, and 10 g of formic acid were weighed and added into a 50 mL round-bottom flask, and stirred continuously in an oil bath at 90 °C until a uniform dark brown liquid was formed, and then cooled to room temperature for the preparation of 7-dehydrocholesterol acetate impurity molecular imprinting polymer.

[0043] Accurately weigh 200 mg of 7-dehydrocholesterol acetate impurity and dissolve it in 50 mL of chloroform, then add 12 mL of low eutectic solvent, and prepolymerize it at room temperature for 12 hours. After the reaction is completed, add 20 mL of cross-linking agent ethylene glycol dimethacrylate (EGDMA) and 200 mg of initiator azobisisobutyronitrile (AIBN), then bubble the mixture with nitrogen for 5 minutes, seal the three-necked bottle under nitrogen protection, and then place the reaction vessel in a water bath for 6 hours at 60 ° C and 18 hours at 70 ° C. After the polymerization reaction is completed, in order to obtain small particles, the polymer is ground and sieved, and repeatedly washed in methanol. The resulting product is eluted with the template molecule by Soxhlet extraction using a mixed solvent of ethanol-acetic acid (9:1, v / v). After elution for 24 hours, the product is dried at room temperature to obtain a 7-dehydrocholesterol acetate impurity molecular imprinting polymer with a certain cavity, which is dried for use.

[0044] Add 19.0g of 7-toluenesulfonylhydrazone-cholesterol acetate and 190ml of toluene into a single-necked bottle, stir until dissolved, then add 1.2g of lithium hydride, heat to reflux for 2 hours, cool to 0-10°C, add 190ml of water dropwise, extract and separate, collect the organic layer, and concentrate the organic layer to dryness at 60°C to obtain 11g of crude 7-dehydrocholesterol acetate.

[0045] 11.0g crude 7-dehydrocholesterol acetate (HPLC purity of 81.51%, impurity content: 17.19%) was dissolved in 220ml methanol, and then 7.0g 7-dehydrocholesterol acetate impurity molecular imprinting polymer was added, and stirred at 25°C for 12h. After the stirring was completed and the first round of adsorption was completed, the filter cake obtained was the 7-dehydrocholesterol acetate impurity molecular imprinting polymer material adsorbing impurities, and the 7-dehydrocholesterol acetate impurity molecular imprinting polymer material adsorbing impurities was washed with a mixed solvent of methanol-acetic acid (9:1v / v), and dried to obtain a regenerated 7-dehydrocholesterol acetate impurity molecular imprinting polymer. The regenerated 7-dehydrocholesterol acetate impurity molecular imprinting polymer was added to the solution for the next adsorption cycle. After 6 adsorption-desorption cycles, the 7-dehydrocholesterol acetate impurity content in the solution was determined to be less than 0.1%. The adsorption-completed solution was concentrated to dryness under reduced pressure at 40-45°C to obtain 9.0g 7-dehydrocholesterol acetate. The purity detected by HPLC was 98.32%, and the impurity content was 0%.

[0046] Example 3

[0047] 1.5 g of caffeic acid, 9 g of choline chloride, and 9 g of formic acid were weighed and added into a 50 mL round-bottom flask, and stirred continuously in an oil bath at 90 °C until a uniform dark brown liquid was formed, and then cooled to room temperature for the preparation of 7-dehydrocholesterol acetate impurity molecular imprinting polymer.

[0048] Accurately weigh 200 mg of 7-dehydrocholesterol acetate impurity and dissolve it in 50 mL of chloroform, then add 15 mL of low eutectic solvent, and prepolymerize it at room temperature for 12 hours. After the reaction is completed, add 4 mL of cross-linking agent ethylene glycol dimethacrylate (EGDMA) and 200 mg of initiator azobisisobutyronitrile (AIBN), then bubble the mixture with nitrogen for 5 minutes, seal the three-necked bottle under nitrogen protection, and then place the reaction vessel in a water bath for 6 hours at 60 ° C and 18 hours at 70 ° C. After the polymerization reaction is completed, in order to obtain small particles, the polymer is ground and sieved, and repeatedly washed in methanol. The resulting product is eluted with the template molecule by Soxhlet extraction using a mixed solvent of ethanol-acetic acid (9:1, v / v). After elution for 24 hours, the product is dried at room temperature to obtain a 7-dehydrocholesterol acetate impurity molecular imprinting polymer with a certain cavity, which is dried for use.

[0049] Add 19.6g of 7-toluenesulfonylhydrazone-cholesterol acetate and 196ml of toluene into a single-necked bottle, stir until dissolved, then add 1.3g of lithium hydride, heat to reflux for 2 hours, cool to 0-10°C, add 196ml of water dropwise, extract and separate, collect the organic layer, and concentrate the organic layer to dryness at 60°C to obtain 11.8g of crude 7-dehydrocholesterol acetate.

[0050] 11.8g crude 7-dehydrocholesterol acetate (HPLC purity of 84.38%, impurity content: 13.45%) was dissolved in 236ml acetone, and then 6.0g 7-dehydrocholesterol acetate impurity molecular imprinting polymer was added, and stirred at 25℃ for 12h. After the stirring was completed and the first round of adsorption was completed, the filter cake obtained was the 7-dehydrocholesterol acetate impurity molecular imprinting polymer material adsorbing impurities, and the 7-dehydrocholesterol acetate impurity molecular imprinting polymer material adsorbing impurities was washed with a mixed solvent of ethanol-acetic acid (9:1v / v), and dried to obtain a regenerated 7-dehydrocholesterol acetate impurity molecular imprinting polymer. The regenerated 7-dehydrocholesterol acetate impurity molecular imprinting polymer was added to the solution for the next adsorption cycle. After 5 adsorption-desorption cycles, the 7-dehydrocholesterol acetate impurity content in the solution was determined to be less than 0.1%. The adsorption-completed solution was concentrated to dryness under reduced pressure at 40-45℃ to obtain 10.0g 7-dehydrocholesterol acetate. The purity detected by HPLC was 97.97%, and the impurity content was 0%.

[0051] It can be seen from the above embodiments that the purification method provided by the present invention is easy to operate, has a stable and reliable process, a high yield, and a low cost; the method can effectively remove the impurity to obtain 7-dehydrocholesterol acetate with an impurity content of less than 0.10% and a purity greater than 97.0%.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for purifying a 7-dehydrocholesterol intermediate, comprising the following steps: In an inert atmosphere, a template, a low eutectic solvent, a cross-linking agent and an initiator are mixed to carry out a polymerization reaction, the template molecules are eluted, and the mixture is dried to obtain a molecularly imprinted polymer; The template has a structure of Formula II: The crude 7-dehydrocholesterol acetate is mixed with an alcohol solvent or a ketone solvent, stirred until dissolved, and then the molecular imprinted polymer is added, adsorbed, filtered, and the filter cake is eluted to obtain a regenerated molecular imprinted polymer; the crude 7-dehydrocholesterol acetate includes impurities shown in formula II; adding the regenerated molecular imprinting polymer material to the solution for adsorption, and performing a cycle to obtain 7-dehydrocholesterol acetate; The deep eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor; The hydrogen bond donor is selected from polyacids and / or polyols; the polyacid is selected from citric acid and / or caffeic acid; the polyol is selected from glycerol and / or ethylene glycol; The hydrogen bond acceptor is selected from formic acid and / or a quaternary ammonium salt; the quaternary ammonium salt is selected from choline chloride and / or tetrabutylammonium bromide.

2. The purification method according to claim 1, characterized in that The cross-linking agent is selected from ethylene glycol dimethacrylate and / or zein; The initiator is selected from one or more of azobisisobutyronitrile, azobisisoheptanenitrile, acyl peroxides, ester peroxides and hydroperoxides.

3. The purification method according to claim 1, characterized in that The alcohol solvent is selected from C1-C4 alcohol solvents; the ketone solvent is selected from acetone or butanone.

4. The purification method according to claim 1, characterized in that The elution template molecule and the elution filter cake are independently selected from a mixed solvent; the mixed solvent is selected from an ethanol-acetic acid mixed solvent with a volume ratio of 9 to 20:1; or a methanol-acetic acid mixed solvent with a volume ratio of 9 to 20:

1.

5. The purification method according to claim 1, characterized in that The volume ratio of the mass of the crude 7-dehydrocholesterol acetate to the alcohol solvent or ketone solvent is 1 g: (10-20) mL.

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