阅读说明：本技术 一种多西他赛的分离纯化方法 (Separation and purification method of docetaxel ) 是由 陶冶 王希 郁明钧 于 2021-08-27 设计创作，主要内容包括：本发明提供了一种多西他赛的分离纯化方法。本发明的多西他赛的分离纯化方法,包括如下步骤：1)将多西他赛粗品进行溶解、过滤,得到多西他赛溶液；2)将步骤1)得到的多西他赛溶液上样到装有UniSil微球的层析柱中进行层析,采用有机溶剂作为流动相对多西他赛溶液进行洗脱；3)分段收集经步骤2)层析、洗脱后的目的峰值的多西他赛溶液,对符合要求的组份液进行汇总,得到纯化的多西他赛。本发明的分离纯化方法用于多西他赛的深度精纯,仅需一步层析纯化即可满足总纯度>99.5％的要求,纯化收率高而稳定,同时本发明分离方法简单方便,可用于规模化生产,大大降低生产成本。(The invention provides a separation and purification method of docetaxel. The method for separating and purifying docetaxel comprises the following steps: 1) dissolving and filtering a docetaxel crude product to obtain a docetaxel solution; 2) loading the docetaxel solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the docetaxel solution by using an organic solvent as a mobile phase; 3) collecting docetaxel solution of the target peak value after chromatography and elution in the step 2) in sections, and collecting component solution meeting the requirements to obtain purified docetaxel. The separation and purification method is used for deep fine purification of docetaxel, can meet the requirement that the total purity is more than 99.5% only by one-step chromatographic purification, has high and stable purification yield, is simple and convenient, can be used for large-scale production, and greatly reduces the production cost.)

1. A separation and purification method of docetaxel, which is characterized by comprising the following steps:

1) dissolving and filtering a docetaxel crude product to obtain a docetaxel solution;

2) loading the docetaxel solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the docetaxel solution by using an organic solvent as a mobile phase, wherein the organic solvent is a mixed solution of dichloromethane and ethyl acetate in a volume ratio of (8-10) to 1;

3) collecting docetaxel solution of the target peak value after chromatography and elution in the step 2) in sections, and collecting component solution meeting the requirements to obtain purified docetaxel.

2. The separation and purification method according to claim 1, wherein the UniSil microspheres are normal phase silica gel UniSil microspheres in step 2).

3. The separation and purification method according to claim 1 or 2, wherein in step 1), the crude docetaxel is dissolved in an organic phase;

preferably, the organic phase is dichloromethane or ethyl acetate.

4. The separation and purification method according to claim 3, wherein in step 1), the docetaxel purity of the crude docetaxel is 95-98%, and the concentration of the docetaxel solution is 40-60 mg/mL;

preferably, in step 2), the amount of the mobile phase is 20-30 column volumes, based on 1 column volume of the packing filled in the chromatography column.

5. The separation and purification method according to any one of claims 1 to 4, wherein the specific process of elution is as follows: and (3) adopting dichloromethane and ethyl acetate with the volume ratio of (8-10):1 as a flow phase to elute the docetaxel solution, wherein the elution time is 200-260min, and the flow rate of the mobile phase is 50-70 mL/min.

6. The separation and purification method according to any one of claims 1 to 5, wherein the step 2) further comprises a step of pre-column treatment of the chromatography column before loading.

7. The separation and purification method according to claim 6, wherein the specific process of the pre-column treatment is as follows: removing impurities from the chromatographic column by using ethyl acetate, and balancing the chromatographic column after removing the impurities by using a mixed solution of dichloromethane and ethyl acetate with a volume ratio of (8-10):1 as a flow.

8. The separation and purification method according to any one of claims 1 to 7, wherein in step 1), the filtration is performed with a filter membrane having a pore size of 0.3 to 0.5 μm.

9. The separation and purification method according to any one of claims 1 to 8, wherein the separation and purification method comprises the steps of:

1) dissolving docetaxel crude product with a purity of 95-98% by using an organic phase, and filtering by using a filter membrane with a pore size of 0.3-0.5 mu m to obtain a docetaxel solution with a concentration of 40-60 mg/mL;

2) removing impurities from a chromatographic column filled with normal-phase silica gel UniSil microspheres by using ethyl acetate, balancing the chromatographic column after the impurities are removed by using a mixed solution of dichloromethane and ethyl acetate with a volume ratio of (8-10):1 as a flow phase, loading the docetaxel solution obtained in the step 1) into the chromatographic column subjected to the balancing treatment for chromatography, eluting the docetaxel solution by using dichloromethane and ethyl acetate with a volume ratio of (8-10):1 as a flow phase, wherein the elution time is 200-260min, and the flow rate of the mobile phase is 50-70 mL/min;

3) collecting docetaxel solution of the target peak value after chromatography and elution in the step 2) in sections, and collecting component solution meeting the requirements to obtain purified docetaxel.

10. Docetaxel obtained by the separation and purification process according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of drug purification, relates to a separation and purification method of docetaxel, and particularly relates to a high-efficiency separation and purification method of docetaxel.

Background

Docetaxel (Docetaxel), a Docetaxel (Docetaxel), acts like Paclitaxel (PTX), and is an M-phase cycle-specific drug that promotes the polymerization of tubules into stable microtubules and inhibits their aggregation, thereby significantly reducing the number of tubules and destroying the microtubule network. In vitro experiments show that the compound has cytotoxic effect on various mouse and human tumor cell strains, and the antitumor spectrum is wider than that of PTX.

Docetaxel is a paclitaxel derivative synthesized in the process of modifying the structure of paclitaxel, and has good bioavailability and small toxic and side effects. Docetaxel is a taxoid antineoplastic agent that acts as an antineoplastic agent by interfering with the microtubule network necessary for cell mitosis and interphase cell function. Docetaxel can bind to free tubulin, promote the assembly of tubulin into stable microtubules, and inhibit its depolymerization, resulting in the generation of microtubule bundles with loss of normal function and the fixation of microtubules, thereby inhibiting the mitosis of cells. The association of docetaxel with microtubules did not change the number of strands. This is in contrast to most spindle-toxic drugs used clinically.

Docetaxel has the following structural formula:

CN110305080A discloses a method for purifying docetaxel. The method comprises the following steps: (1) dissolving a docetaxel crude product by using ethyl acetate, filtering by using a polytetrafluoroethylene microporous membrane, adding dichloromethane and acetic acid into the obtained filtrate, adding alkane while stirring, crystallizing and filtering to obtain a docetaxel primary purification product; (2) and dissolving the first purified product of the docetaxel by using the ethyl acetate, the dichloromethane and the acetic acid, adding the alkane, and crystallizing, filtering and drying in vacuum to obtain a second purified product of the docetaxel. The purity of the docetaxel secondary purification product obtained by the purification method reaches more than 99.5%, and the method has the advantages of high recovery rate, simple operation and easy industrial production. However, the purification method needs two times of purification, and has the disadvantages of complicated process and high cost.

CN109836401A discloses a method for purifying docetaxel, which comprises precipitating docetaxel solid in a mixed solution of dichloromethane and toluene. The purification method of the invention greatly reduces the content of each single impurity in the docetaxel, introduces few new impurities, improves the product purity, has high yield, is very suitable for industrial mass production, and the obtained product meets the requirements of preparations and can be directly used for preparing docetaxel injection. However, the purity and yield of the purification process are to be further improved.

Therefore, it is necessary to provide a method for separating and purifying docetaxel with high purity and high yield.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a separation and purification method of docetaxel, which can meet the requirement of purity of more than 99.5% only by one-step chromatographic purification and has high and stable purification yield.

In order to achieve the purpose, the invention adopts the following technical scheme:

a separation and purification method of docetaxel comprises the following steps:

1) dissolving and filtering a docetaxel crude product to obtain a docetaxel solution;

2) loading the docetaxel solution obtained in the step 1) into a chromatographic column filled with UniSil microspheres for chromatography, and eluting the docetaxel solution by using an organic solvent as a mobile phase, wherein the organic solvent is a mixed solution of dichloromethane and ethyl acetate in a volume ratio of (8-10) to 1;

3) collecting docetaxel solution of the target peak value after chromatography and elution in the step 2) in sections, and collecting component solution meeting the requirements to obtain purified docetaxel.

The method for separating and purifying docetaxel adopts a chromatographic column filled with UniSil microspheres for chromatography, can meet the requirement of purity of more than 99.5% by only one-step chromatographic purification, has the purification yield of more than 95%, is stable, is simple and convenient to operate, can repeatedly utilize a used stationary phase, saves a small amount of used mobile phase, and greatly reduces the cost.

In the step 2), the UniSil microspheres are normal phase silica gel UniSil microspheres.

In step 2), the volume ratio of the dichloromethane to the ethyl acetate is (8-10):1, for example, 8:1, 9:1 or 10:1, preferably 9: 1.

In the step 1), the purity of docetaxel in the docetaxel crude product is 95-98%, such as 95%, 96%, 97% or 98%; the docetaxel solution has a concentration of 40 to 60mg/mL, for example, 40mg/mL, 41mg/mL, 42mg/mL, 43mg/mL, 44mg/mL, 45mg/mL, 46mg/mL, 47mg/mL, 48mg/mL, 49mg/mL, 50mg/mL, 51mg/mL, 52mg/mL, 53mg/mL, 54mg/mL, 55mg/mL, 56mg/mL, 57mg/mL, 58mg/mL, 59mg/mL, or 60mg/mL, etc.

In the step 1), the docetaxel crude product is dissolved by adopting an organic phase.

Preferably, the organic phase is dichloromethane or ethyl acetate.

Preferably, in step 2), the amount of the mobile phase is 20-30 column volumes, based on 1 column volume of the packing filled in the chromatography column. Wherein, the volume of the filler in the invention is 490mL, namely the volume of one column is 490 mL.

The specific process of elution is as follows: adopting dichloromethane and ethyl acetate with volume ratio of (8-10):1 as flow phase to perform elution to docetaxel solution, wherein the elution time is 200-260min, such as 200min, 210min, 220min, 230min, 240min, 250, 260min and the like; the flow rate of the mobile phase is 50-70mL/min, such as 50mL/min, 51mL/min, 52mL/min, 53mL/min, 54mL/min, 55mL/min, 56mL/min, 57mL/min, 58mL/min, 59mL/min, 60mL/min, 61mL/min, 62mL/min, 63mL/min, 64mL/min, 65mL/min, 66mL/min, 67mL/min, 68mL/min, 69mL/min, or 70mL/min, and the like.

Step 2) before loading, the method also comprises a step of carrying out column pretreatment on the chromatographic column.

The specific process of the column pretreatment is as follows: removing impurities from the chromatographic column by using ethyl acetate, and balancing the chromatographic column after removing the impurities by using a mixed solution of dichloromethane and ethyl acetate with a volume ratio of (8-10):1 as a flow.

In step 1), the filtration is performed by using a filter membrane with a pore size of 0.3-0.5 μm, for example, the pore size of the filter membrane is 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm or 0.5 μm.

As a preferred embodiment of the present invention, the method for separating and purifying docetaxel comprises the following steps:

1) dissolving docetaxel crude product with a purity of 95-98% by using an organic phase, and filtering by using a filter membrane with a pore size of 0.3-0.5 mu m to obtain a docetaxel solution with a concentration of 40-60 mg/mL;

2) removing impurities from a chromatographic column filled with normal-phase silica gel UniSil microspheres by using ethyl acetate, balancing the chromatographic column after the impurities are removed by using a mixed solution of dichloromethane and ethyl acetate with a volume ratio of (8-10):1 as a flow phase, loading the docetaxel solution obtained in the step 1) into the chromatographic column subjected to the balancing treatment for chromatography, eluting the docetaxel solution by using dichloromethane and ethyl acetate with a volume ratio of (8-10):1 as a flow phase, wherein the elution time is 200-260min, and the flow rate of the mobile phase is 50-70 mL/min;

3) collecting docetaxel solution of the target peak value after chromatography and elution in the step 2) in sections, and collecting component solution meeting the requirements to obtain purified docetaxel.

The second objective of the present invention is to provide docetaxel obtained by the separation and purification method described in the first objective.

Compared with the prior art, the invention has the beneficial effects that:

the method for separating and purifying docetaxel has the advantages of high purity, high yield, stability, simple and convenient operation, reusable stationary phase, less mobile phase and greatly reduced cost. Specifically, the requirement of purity of more than 99.5 percent can be met only by one-step chromatographic purification, and the purification yield is more than 95 percent.

Drawings

FIG. 1 is a scanning electron micrograph of UniSil normal phase silica gel used in example 1 of the present invention;

fig. 2 is a high performance liquid chromatography analysis of docetaxel before purification in example 1 of the present invention;

fig. 3 is a high performance liquid chromatography analysis of docetaxel purified in example 1 of the present invention.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached figures 1-3.

Unless otherwise specified, various starting materials of the present invention are commercially available or prepared according to conventional methods in the art.

Example 1

1) Taking a docetaxel crude product with the purity of 97%, adding dichloromethane to dissolve the docetaxel crude product to obtain a docetaxel solution with the concentration of 50 mg/mL; after the solution is clarified, filtering the solution by using a filter membrane with the aperture of 0.45 mu m, and collecting filtrate for later use;

2) a chromatographic column with the size of 50 multiplied by 250mm and UniSil normal phase silica gel (produced by Suzhou nano-micro technology Co., Ltd.) are adopted as the chromatographic column packing, and the volume of the packed column is 490 mL; pre-column pretreatment is carried out on a chromatographic column, ethyl acetate is used for removing impurities, and then mobile phase with volume ratio of dichloromethane to ethyl acetate being 9:1 is used for balancing; then, sampling, wherein the sampling liquid is 1) the filtrate; then, a mixed solution with the volume ratio of dichloromethane to ethyl acetate being 9:1 is used as a mobile phase for elution for 240min, and the flow rate is controlled at 60 mL/min;

3) and collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 99.62% and the yield of 96.6%.

Wherein, fig. 1 is a scanning electron micrograph of UniSil normal phase silica gel used in example 1, and fig. 2 is a hplc analysis of docetaxel before purification, which shows that certain impurities are present. Fig. 3 shows the high performance liquid chromatography analysis of purified docetaxel, which shows very few impurities and very small peaks.

Example 2

1) Taking a docetaxel crude product with the purity of 97%, adding dichloromethane to dissolve the docetaxel crude product to obtain a docetaxel solution with the concentration of 50 mg/mL; after the solution is clarified, filtering the solution by using a filter membrane with the aperture of 0.45 mu m, and collecting filtrate for later use;

2) a chromatographic column with the size of 50 multiplied by 250mm and UniSil normal phase silica gel (produced by Suzhou nano-micro technology Co., Ltd.) are adopted as the chromatographic column packing, and the volume of the packed column is 490 mL; the chromatographic column is pretreated before the column, impurities are removed by ethyl acetate, and then the chromatographic column is equilibrated by a mobile phase with the volume ratio of dichloromethane to ethyl acetate being 8.5: 1.5. Then eluting for 240min by adopting a mobile phase with the volume ratio of dichloromethane to ethyl acetate being 8.5:1.5, and controlling the flow rate at 60 ml/min;

3) and collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 99.56 percent and the yield of 95.7 percent.

Example 3

This example differs from example 1 in that the organic solution in which the docetaxel was dissolved was ethyl acetate, and the rest was the same as example 1.

And collecting the solution with the target peak value in a segmented manner, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 99.51% and the yield of 95.2%.

Comparative example 1

This comparative example differs from example 1 in that the packing phase in the column is conventional Partisil high purity amorphous silica gel, otherwise the same as in example 1.

And collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 99.21% and the yield of 90.5%.

Comparative example 2

The comparative example is a conventional method for purifying docetaxel, and a repeated crystallization and impurity removal method is directly adopted.

The purity of the finished product docetaxel is 98.22%.

Comparative example 3

This comparative example is different from example 1 in that methylene chloride was used as a mobile phase for the equilibration treatment and elution treatment, and the rest was the same as example 1.

And collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 98.54 percent and the yield of 90.1 percent.

Comparative example 4

This comparative example is different from example 1 in that the mobile phase used in the equilibration and elution treatments was ethyl acetate, and the rest was the same as example 1.

And collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 98.12% and the yield of 98.5%.

Comparative example 5

This example is different from example 1 in that the mobile phases used for the equilibration and elution are dichloromethane and ethyl acetate in a volume ratio of 5:1, and the rest are the same as example 1.

And collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 98.68% and the yield of 97.6%.

Comparative example 6

This comparative example differs from example 1 in that the mobile phases used for the equilibration and elution treatments were dichloromethane and ethyl acetate in a volume ratio of 15:1, and the rest were the same as in example 1.

And collecting the solution of the target peak value in sections, summarizing component solutions meeting the requirements, and analyzing by high performance liquid chromatography to obtain the docetaxel in the eluent with the purity of 99.12 percent and the yield of 93.4 percent.

The method is used for deeply and finely purifying docetaxel, can meet the requirement that the purity of the docetaxel is more than 99.5% only by one-step chromatographic purification, has the purification yield of more than 95% and is stable, and meanwhile, the separation and purification method is simple and convenient, can be used for large-scale production, and greatly reduces the production cost.

The present invention is illustrated by the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, i.e. it is not meant to imply that the present invention must rely on the above-mentioned detailed process equipment and process flow to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.