Preparation of epothilones B lactam derivates

A technology of epothilone and lactam, which is applied in the field of producing epothilone B lactam derivatives, can solve the problems of lack of epothilone D, etc., and achieve the effect of high yield and short synthetic route

Inactive Publication Date: 2008-12-17
京山瑞生制药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] At present, there is a lack of a biosynthetic method for high-yield epothilone D, especially a practical technology suitable for the industrial scale production of epothilone D, and then using it as a starting material, using a simple semi-synthetic method to manufacture anticancer Drug epothilone B lactam derivative

Method used

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  • Preparation of epothilones B lactam derivates
  • Preparation of epothilones B lactam derivates
  • Preparation of epothilones B lactam derivates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Gene mutation and directional screening to obtain a mutant strain of S. cellulosus containing the inactivated epoK gene

[0036] The starting strain was Sorangium cellulosum ATCC15384 among the myxobacteria strains, which was obtained from the American Type Culture Collection.

[0037] The above strains were inoculated on a solid medium, and the components of the solid medium on the slant were: soybean peptone 8g / L, potato starch 20g / L, yeast extract 10g / L, anhydrous glucose 5g / L, CaCl 2 ·H 2 O 1g / L, MgSO 4 ·7H 2 O 1g / L, Fe-EDTA 8mg / L, agar powder 15g / L. After culturing at 30°C for 5 days, it was inoculated into the liquid fermentation medium. The composition of the liquid fermentation medium is: soybean peptone 10g / L, potato starch 20g / L, yeast extract 4g / L, anhydrous glucose 15g / L, CaCl 2 ·H 2 O 1g / L, MgSO 4 ·7H 2 O 1g / L, Fe-EDTA 8mg / L, resin XAD-1620ml / L. 30°C, 120 rpm, shaker for 4 days. The epoK inhibitor Mytyrapone was added to a final concentration of 5...

Embodiment 2

[0043] Fermentative production of epothilone D by mutant high-yielding strain

[0044] Continuous seed culture: 1ml of the frozen mutant high-yielding strain taken out of liquid nitrogen was inoculated into 10ml of G52 medium, and cultured at 30°C, 180 rpm, and 25mm displacement for 3 days. Add 5ml of seed culture to 45ml of G52 medium and grow for 3d. This 50ml culture was then added to 450ml G52 to grow for 3d. Every 3-4 days thereafter, 50 ml of the seed culture was inoculated into 450 ml of the G52 medium with a 10% inoculation amount to carry out continuous seed culture.

[0045] 20L intermediate seed culture: 18L G52 medium in a 30L fermenter, 2L of seed culture solution before inoculation, 30°C, 250 rpm, 0.5L air / L / min, and overpressure of 0.5bars for 3 days and 3 days ~4d, add 10ml of silicone antifoaming agent to prevent foam formation.

[0046] 250L fermentation culture:

[0047] 1B12 medium (potato starch Noredux A-150 (Blattmann, Waedenswil, Swizerland) 2%, def...

Embodiment 3

[0051] Synthesis of Epothilone B Lactam Derivatives Using Epothilone D as Starting Material

[0052] Epothilone D 129g (262.2mmol) was dissolved in 10.5L of anhydrous chloroform, m-CPBA 97.5g (390mmol) was added at -18°C, the reaction mixture was stirred at this temperature for 5h, and then diluted with 42L of dichloromethane, Add 45 L of saturated sodium bicarbonate to terminate the reaction. The layers were separated, the aqueous layer was extracted 3 times with dichloromethane, the combined organic phase was dried over magnesium sulfate, filtered and concentrated under reduced pressure, the residue was subjected to dynamic axial compression chromatography (silica gel, with n-hexane-ethyl acetate, 1:1 elution), to obtain epothilone B 108g (81%).

[0053] Epothilone B 101.2g (199.4mmol) and sodium azide 15.54g (240mmol) were suspended in THF-H 2 O (5:1) mixed solution 1.92L, degassed with nitrogen for 20min, then treated with a catalytic amount of triphenylphosphine palladi...

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Abstract

The invention relates to a method for the production of an epothilones B lactam derivative which is an anticancer drug. The method adopts a gene mutation method, utilizes an eopK inhibitor to promote the inactivation of cytochrome P450 oxidase which is the gene product of eopK, and directionally obtain epothilones C and epothilones D without the generation of epothilones A and epothilones B. The mutant strains are further treated with UV mutation breeding to obtain the high-yield strains of the epothilones D, and the strains are fermented to produce the epothilones D which is used as a starting material to synthesize the epothilones B lactam derivative by two-step reaction.

Description

field of invention [0001] The present invention relates to a production method of epothilone derivatives, in particular to a production method of epothilone D by fermentation of S. cellulosum in myxobacteria, and then using it as a starting material to produce it by a semi-synthetic method Methods of epothilone B lactam derivatives. Background technique [0002] Epothilones are a class of cytotoxic compounds secreted by myxobacterial cellulosicum with a 16-membered lactone macrocycle as the center body and thiazole ring ligands as side chains. New antineoplastic agents for tubulin polymerization and microtubule stabilization. Epothilones A and B were developed in the 1990s by He and his colleagues first found a myxobacteria Sorangiumcellulosum (Myxococcales) strain So ce90 from the soil on the banks of the Zambesi River in South Africa, and isolated a compound with anticancer activity from its culture. The structural formulas of epothilones A, B, C, and D are as follows:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16C12N1/20C12R1/01
Inventor 阎家麒
Owner 京山瑞生制药有限公司
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