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A method for separating and purifying epothilone by high-speed countercurrent chromatography

A high-speed counter-current chromatography, epothilone technology, applied in the field of high-speed counter-current chromatography separation and purification of epothilone, can solve problems such as no high-speed counter-current chromatography epothilone, to avoid sample loss, high purity, separation and purification The effect of mild process conditions

Active Publication Date: 2015-12-02
FUJIAN INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The description of using myxobacteria to produce epothilones and further separating and extracting epothilones can be found in WO93 / 10121, WO99 / 42602, etc., but in the existing literature, there is no high-speed countercurrent chromatography for the separation of epothilones A / B domestic and foreign reports

Method used

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  • A method for separating and purifying epothilone by high-speed countercurrent chromatography
  • A method for separating and purifying epothilone by high-speed countercurrent chromatography
  • A method for separating and purifying epothilone by high-speed countercurrent chromatography

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Prepare a solvent system, wherein the volume ratio of n-hexane:ethyl acetate:methanol:water is 1:1:1:1, a total of 1500ml is miscible in a separatory funnel, shake it up and let it stand for stratification, and take the upper phase as the stationary phase , the lower phase is the mobile phase, after ultrasonic degassing, first pump the stationary phase into the separation column, set the temperature of the constant temperature circulator to 20°C, then turn on the host of the high-speed countercurrent chromatograph, adjust the speed of the host to 850rpm, and the mobile phase to 1.5ml / min flow rate pumped into the separation column, until the entire stationary phase - mobile phase system to establish a dynamic equilibrium, balance 10min.

[0050] The retention rate ρ of this solvent system is 78.8% (ρ=(V 总 -V 出 ) / V 总 ×100%, where V 总 : Total volume of the separation column pipeline (ml), V 出 : volume of stationary phase pushed out by mobile phase during equilibration ...

Embodiment 2

[0053] Prepare a solvent system, wherein the volume ratio of n-hexane:ethyl acetate:methanol:water is 1.2:1.2:1:1, a total of 1500ml is miscible in a separatory funnel, shake it up and let it stand for stratification, and take the upper phase as the stationary phase , the lower phase is the mobile phase. After ultrasonic degassing, first pump the stationary phase into the entire separation column, set the temperature of the constant temperature circulator to 20°C, and then turn on the main engine of the high-speed countercurrent chromatograph, adjust the main engine speed to 850rpm, and the mobile phase to Pump at a flow rate of 1.5ml / min into the separation cartridge until the entire stationary phase-mobile phase system establishes a dynamic equilibrium for 10 minutes.

[0054] The retention rate p of this solvent system when calculating equilibrium is 80.5% (p=(V 总 -V 出 ) / V 总 ×100%, where V 总 : Total volume of the separation column pipeline (ml), V 出 : volume of stationa...

Embodiment 3

[0057] Prepare a solvent system, wherein the volume ratio of n-hexane: ethyl acetate: methanol: water is 1:0.8:0.8:1, a total of 1500ml is miscible in a separatory funnel, shake it up and let it stand for stratification, and take the upper phase as the stationary phase , the lower phase is the mobile phase. After ultrasonic degassing, first pump the stationary phase into the entire separation column, set the temperature of the constant temperature circulator to 20°C, and then turn on the main engine of the high-speed countercurrent chromatograph, adjust the main engine speed to 850rpm, and the mobile phase to Pump at a flow rate of 1.5ml / min into the separation cartridge until the entire stationary phase-mobile phase system establishes a dynamic equilibrium for 10 minutes.

[0058] The retention rate p of this solvent system when calculating balance is 82.5% (p=(V 总 -V 出 ) / V 总 ×100%, where V 总 : total volume of the column tubing (ml), V 出 : volume of stationary phase pushe...

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Abstract

The invention provides a method for separating and purifying epothilone by high-speed counter-current chromatography, which comprises the following steps: preparing solvent systems for constituting a stationary phase and a mobile phase of a high-speed counter-current chromatograph; fully filling the stationary phase in the high-speed counter-current chromatograph, setting the temperature of a constant temperature circulator, regulating the rotation speed of the host of the high-speed counter-current chromatograph, and finally, pumping in the mobile phase until the whole system establishes a dynamic balance; dissolving an epothilone crude extract in the mobile phase, and introducing the sample by an introduction valve; and collecting the target product according to an ultraviolet detector chromatograph or high performance liquid chromatograph. The invention has the advantages of high separation degree for the epothilone crude extract, and high purity of the purified target product, is suitable for automatic production, and has very high economic benefit.

Description

【Technical field】 [0001] The invention relates to a method for separating and purifying epothilone by high-speed countercurrent chromatography. 【Background technique】 [0002] Epothilone (epothilone, ) is currently clinically following paclitaxel (paclitaxel, ) after the most successful anticancer chemotherapy drugs. In 1993, Hofle et al. reported finding a strain of Sorangiumcellulosum Myxococcales from the soil of the Zambesi River in South Africa, and isolated a new 16-membered macrolide compound from its culture. Bleomycin A and B. A schematic diagram of epothilones obtained is described by Hofle et al. in WO93 / 10121. Epothilone A and B have the following structural formula: [0003] [0004] In 1995, Bollag et al. found that epothilone has the property of promoting tubulin polymerization similar to paclitaxel (Bollag, D.M., Cancer Res 55, 2325-2333, 1995). In an example of therapeutic use, International Application WO99 / 43320 describes various modes of admini...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D493/04
Inventor 杨煌建张祝兰唐文力任林英庄鸿陈宏孙菲郑卫
Owner FUJIAN INST OF MICROBIOLOGY
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