CN102219764A - Method for separating and purifying paclitaxel industrially - Google Patents

Abstract

The invention discloses an industrialized method for efficiently separating and purifying paclitaxel, which belongs to the field of compound separation and purification. The method uses column chromatography to separate paclitaxel, and any three or four of C1-10 alkanes, chlorinated hydrocarbons, C1-4 alcohols, saturated alkane esters, and alkane ketones are combined with amine compounds. The combination was used as the mobile phase, and ordinary 200-300 mesh silica gel was used as the stationary phase, and paclitaxel was separated and purified by normal phase column chromatography. The mixed solvent mobile phase of the multi-component system increases the separation degree of difficult-to-separate substance pairs, and solves the phenomenon that paclitaxel and cephalomannine cannot be effectively separated in previous separation methods. After separation and purification by column chromatography, the purity of the raw material paclitaxel can be increased to over 98%. According to the condition of the raw material, the total yield can be as high as 90% after only one or two elutions. The method uses common solvents and silica gel, and has the advantages of low cost, simple operation, large sample loading, and easy industrial production.

Description

A kind of method for industrial separation and purification of paclitaxel

technical field

The invention relates to a separation and purification method of paclitaxel, in particular to an industrialized high-efficiency separation and purification method of paclitaxel and cephalomannine, which belongs to the field of compound separation and purification.

Background technique

Paclitaxel is a new type of anticancer drug isolated from Taxus genus with a taxane diterpene skeleton. It is also the only drug known to promote microtubule polymerization and stabilize the polymerized microtubules. It has a strong inhibitory effect on most solid tumors, but basically has no effect on normal cells, especially for advanced ovarian cancer, breast cancer, non-small cell lung cancer and Kaposi's sarcoma, with definite curative effect and less side effects. With the further research, the anti-cancer effect of paclitaxel drugs has been more and more widely used, and has now become a first-line anti-cancer drug and a broad-spectrum anti-tumor drug.

Taxus plants, which are the main source of paclitaxel, are rare species and are listed as national first-class protected plants in my country. The paclitaxel content in yew bark is extremely low, accounting for about 0.01%, which is far from meeting the market demand for paclitaxel. In the past 20 years, the preparation methods of paclitaxel have been exploratoryly reported from multiple disciplines and perspectives. There are paclitaxel cell culture, fungal fermentation, chemical semi-synthesis and total synthesis methods. No matter what method is used to prepare paclitaxel, it is necessary to separate and purify paclitaxel from paclitaxel analogues. The common paclitaxel analogs mainly include cephalomannine, baccatin III, 10-deacetyl paclitaxel, 7-epi-paclitaxel, paclitaxel C and so on. Due to the similar structure and properties, it is very difficult to separate paclitaxel and its analogues. In addition, as a biological macromolecular substance, paclitaxel is easily degraded or isomerized to generate other taxanes under the influence of environmental conditions such as temperature, organic solvent, acid, and alkali. For example, paclitaxel will be degraded to baccatin III or isomerized to 7-epi-paclitaxel under strong acidic or weak alkaline conditions; paclitaxel will also undergo degradation reaction at higher temperature to generate corresponding small molecular substances. As a drug in clinical use, paclitaxel has high requirements for its purity in practical applications. The resource of paclitaxel is precious and the price remains high. The purity and yield of separation become the key factors restricting the cost of paclitaxel for pharmaceutical use. Based on the above aspects, the development of more efficient and cheap industrial separation and purification technology and new production separation and purification technology has become the focus of paclitaxel research.

At present, the commonly used methods for separation and purification of paclitaxel and paclitaxel analogues mainly include column chromatography, thin layer chromatography, precipitation method, micellar electrokinetic chromatography, membrane separation method, resin adsorption separation method, chemical reaction method and so on. However, column chromatography is the only method suitable for industrialized large-scale production, and other methods are limited to laboratory research. Column chromatography is divided into two methods: normal phase column method and reversed phase column method.

There are many patent applications related to the separation and purification of paclitaxel: such as Chinese patent CN 1377882A "a method for jointly preparing high-purity paclitaxel, cephalomannine, and 10-deacetylbaccatin III"; Chinese patent CN 1611496A "a A method for preparing high-purity paclitaxel by high-pressure liquid chromatography"; Chinese patent CN 101391989A "a method for preparing polyhydroxytaxane and paclitaxel".

The deficiency of existing paclitaxel separation and purification method mainly lies in: the method for various separation paclitaxel at present still can't separate paclitaxel and cephalomannine effectively; As far as the method is concerned, the normal phase method and the reverse phase method have their own defects. Normal phase column chromatography often needs to introduce an oxidation reaction to increase the separation degree of cephalomannine and paclitaxel by changing the chemical structure of cephalomannine. This not only changes the chemical structure of the separated object, but also requires multiple Pass through the column and recrystallize several times for purification. The cumbersome operation steps not only reduce the yield of the target paclitaxel and increase the production cost, but also only one product of paclitaxel can be obtained as a result of separation, and the cephalomannine after the structure change can only be discarded as waste, which wastes resources and pollutes the environment . Although reversed-phase column chromatography does not need to change the chemical structure of the separated object, in practical application, this method needs to use a special reversed-phase column silica gel, and its market price is as high as 500,000 yuan/kg. In industrial production, at least 50 kg of reversed-phase column silica gel will be used to separate 1 kg of paclitaxel, and the cost of reversed-phase column silica gel alone will be 25 million yuan, which greatly increases the production cost of separating paclitaxel and limits its industrial scale. mass production.

Contents of the invention

The object of the present invention is to provide a paclitaxel separation and purification method with high separation efficiency, low cost and easy industrial operation.

In order to achieve the purpose of the present invention, normal phase column chromatography is used to separate and purify the crude product of paclitaxel by selecting a multi-component (quaternary or pentadic) system mixed solvent as the mobile phase. The mobile phase is a combination of any three or four of C1-10 alkanes, chlorinated hydrocarbons, C1-4 alcohols, saturated alkane esters, alkane ketones and amine compounds.

The amine solvent is selected from diethylamine or triethylamine; the C1-10 alkane solvent is selected from normal hexane, normal pentane or cyclohexane; the chlorinated hydrocarbon solvent is selected from dichloromethane, chloroform or tetrachloromethane Chloromethane; the C1-4 alcohol solvent is selected from methanol or ethanol; the saturated alkane ester solvent is selected from ethyl acetate; the alkane ketone solvent is selected from acetone or isopropanone. The amount of amine solvent accounts for 8% to 16% of the mobile phase volume.

Usually, paclitaxel with a purity of 93% to 95% accounts for 80% to 90% after one or two column elutions; paclitaxel with a purity of 30% to 90% after one or two column elutions accounts for 5% to 15%. ; Paclitaxel with a purity lower than 30% after one or two elutions from the chromatographic column accounts for 1% to 3%; the sample is recrystallized once to obtain the target paclitaxel.

In practical applications, the higher the content of the raw material paclitaxel, the less the number of elution times required for the chromatographic column. When using this method to separate paclitaxel, if the paclitaxel content of the raw material is higher than 70%, then only one column elution is required to obtain the ideal separation effect; if the paclitaxel content of the raw material is 50% to 70%, two column washes The ideal separation effect can be obtained.

The innovation of the present invention lies in the selection of mobile phase of the multi-element (quaternary or quinary) system, which saves the chemical reaction steps such as bromination method in the separation and purification process, avoids the change of the chemical structure in the separation object, and reduces the concentration of raw material samples. More than 80% of paclitaxel can be separated by only passing through the column once. At the same time, as a result of the separation, two substances, paclitaxel and cephalomannine, were obtained with high purity. In addition to direct utilization of cephalomannine itself as a high value-added raw material drug (150,000 yuan/kg), it can also be made into docetaxel or paclitaxel to obtain more economic benefits.

The innovation point of the present invention is that the mobile phase selects the combination of any three or four of C1-10 alkanes, chlorinated hydrocarbons, C1-4 alcohols, saturated alkane esters, alkane ketones and amine compounds; The phase is ordinary 200-300 mesh normal-phase column silica gel at 50 yuan/Kg. The materials used in the separation and purification process are simple, easy-to-obtain and low-cost materials, which are easy to carry out industrial production and reduce production costs. Compared with the currently commonly used reversed-phase column separation paclitaxel method, the paclitaxel and cephalomannine can be effectively separated, the number of column passes is small, the separation efficiency is high, the production cost is low, and industrial production can be carried out on a large scale.

The innovation point of the present invention lies in the application of amine reagents, which adjusts the pH value of the mobile phase, so that the separation process is carried out under alkaline conditions. At this time, the separated substances all exist in molecular form, which reduces the adsorption on silica gel, and improves The separation degree of paclitaxel and cephalomannine for difficult-to-separate substances has been improved, and the problem that paclitaxel and cephalomannine cannot be effectively separated by various separation methods in the past has been well solved.

The innovative point of the present invention is that in the eluent of the chromatographic column, the liquid containing cephalomannine flows out first, and the liquid containing paclitaxel flows out later. This is related to the use of multiple (quaternary or pentadic) system mobile phases and amine reagents, and the alkaline separation environment makes cephalomannine elute before paclitaxel.

Adopting the method of the present invention can effectively separate paclitaxel and cephalomannine, the effect of separation and purification is good, and the purity of the crude paclitaxel product can be easily increased to more than 98%. Up to 90%. Compared with the existing separation method of paclitaxel, the method has fewer column elution times, simple and safe operation, high separation efficiency, low cost, large loading amount, and easy industrialized large-scale production.

Detailed ways

For a better description of the present invention, the specific implementation is as follows:

Example 1

A sample with a paclitaxel content of 70% was taken as a raw material, and the weight ratio of the raw material to silica gel was 2:100. The raw material was dissolved in dichloromethane for later use. The mobile phase of the quaternary system is n-hexane: dichloromethane: methanol: triethylamine volume ratio = 3:6:2:2, 200-300 mesh silica gel as the stationary phase. The wet method is used to load the column. After the sample is loaded, the column volume is flushed with dichloromethane solvent and then separated with the prepared mobile phase. After 1.4 column volumes of the mobile phase were flushed, paclitaxel C began to flow out, followed by cephalomannine, and after 0.3 column volumes, single paclitaxel flowed out, and paclitaxel could be completely separated after 2 column volumes , Paclitaxel with a purity of more than 90% after one elution accounted for 85%. The paclitaxel effluent with a purity of 30% to 90% was combined and eluted twice. (The separation process can be monitored by TLC or HPLC) Combine the effluents of paclitaxel with a purity of more than 90% in the two eluents, and concentrate them in vacuum at 45°C. : 6 volume ratio for recrystallization, a large amount of white precipitates precipitated after stirring for 10 minutes, paclitaxel can be completely precipitated after stirring for 20 minutes. After suction filtration and vacuum drying, the pure paclitaxel with a purity of 98.6% was obtained, and the total yield of paclitaxel reached 97.6%.

Example 2

A sample with a paclitaxel content of 60% was taken as a raw material, and the weight ratio of the raw material to silica gel was 1.5:100. The raw material was dissolved in dichloromethane for later use. The mobile phase of the five-component system is n-hexane: dichloromethane: acetone: triethylamine: ethyl acetate volume ratio = 6:3:1:1:1.5, and 200-300 mesh silica gel is used as the stationary phase. The wet method is used to load the column. After the sample is loaded, the column volume is flushed with dichloromethane solvent and then separated with the prepared mobile phase. After flushing the mobile phase for 1.7 column volumes, paclitaxel C began to flow out, followed by cephalomannine, and after 0.5 column volumes, single paclitaxel flowed out, and paclitaxel could be completely separated after 2 column volumes , Paclitaxel with a purity of more than 90% after one elution accounted for 75%. The paclitaxel effluent with a purity of 30% to 90% was combined and eluted twice. (The separation process can be monitored by TLC or HPLC.) The paclitaxel effluent with a purity of more than 90% in the two eluents can be combined. (The separation process can be monitored by TLC or HPLC.) The paclitaxel-containing effluent is vacuum concentrated at 45°C, concentrated The product was recrystallized at room temperature (25°C) with acetone: n-hexane = 1: 6 volume ratio. After stirring for 10 minutes, a large amount of white precipitates precipitated. After stirring for 20 minutes, paclitaxel could be completely precipitated. After suction filtration and vacuum drying, the pure paclitaxel with a purity of 98.5% was obtained, and the total yield of paclitaxel reached 94.6%.

Example 3

A sample with a paclitaxel content of 50% was taken as a raw material, and the weight ratio of the raw material to silica gel was 2:100. The raw materials were dissolved in chloroform for later use. The mobile phase of the five-component system is n-hexane: chloroform: acetone: diethylamine: ethyl acetate volume ratio = 4:3:1:1:1.5, and 200-300 mesh silica gel is used as the stationary phase. The wet method is used to load the column. After loading the sample, rinse the column volume with chloroform solvent for 2 column volumes and then separate with the prepared mobile phase. After the mobile phase was flushed for 2 column volumes, paclitaxel C began to flow out, followed by cephalomannine, and after 0.5 column volumes, single paclitaxel flowed out, and paclitaxel could be completely separated after 2.5 column volumes , paclitaxel with a purity of more than 90% after one elution accounted for 66.7%. The paclitaxel effluent with a purity of 30% to 90% was combined and eluted twice. (The separation process can be monitored by TLC or HPLC) The paclitaxel-containing effluent was concentrated in vacuo at 45°C, and the concentrate was recrystallized at room temperature (25°C) with acetone:n-hexane=1:6 volume ratio, and stirred for 10 minutes A large amount of white precipitates were precipitated, and paclitaxel could be completely precipitated after stirring for 20 minutes. After suction filtration and vacuum drying, pure paclitaxel with a purity of 98.3% was obtained, and the total yield of paclitaxel was 90.7%.

Claims (3)

1. the method for an industrial separation purification of paclitaxel, adopt normal phase column chromatography isolation of taxol, it is characterized in that moving phase is the combination of any three kinds or four kinds and aminated compounds in C1-10 alkanes, chlorinated hydrocarbons, C1-4 alcohols, saturated alkane ester class, the alkane ketone compounds.

2. in the separating and purifying taxol method as claimed in claim 1, it is characterized in that aminated compounds is selected from diethylamine or triethylamine; The C1-10 alkane derivative is selected from normal hexane, Skellysolve A or hexanaphthene; The chlorinated hydrocarbons compound is selected from methylene dichloride, trichloromethane or tetrachloromethane; The C1-4 alcohol compound is selected from methyl alcohol or ethanol; The saturated alkane ester compound is selected from ethyl acetate; The alkane ketone compounds is selected from acetone or isopropyl acetone.

3. in the separating and purifying taxol method as claimed in claim 1 or 2, it is characterized in that the consumption of aminated compounds accounts for 8%～16% of mobile phase volume.