CS269008B1 - Method of preparation of 4-tar d row 1t ha f e r A nu - Google Patents

Abstract

The method of preparation of 4-dehydrow!thaferin A tic, the liquid food containing a source of carbon and organic nitrogen is inoculated with Arthrobacter v1aplex /ATCC 6946/, then with the addition of 10 and 50 g of wilthaferlin A per 100 »1 pods and not cultured at a temperature of 28 and 30 °C for 30 ai 48 hours. The polished filtrate of the treated pod is extracted with acetic acid ester, the extract is evaporated to dryness, the obtained vapor is read! on a pinch of »1l1kagel, the eluate obtained was evaporated! to dryness and the product is crystallized from ethanol: chloroform pr! 4 °C. Flttrét of feraentated pods can also be extracted with chlorinated hydrocarbons*! with the number of 1 and 5 carbon atoms, preferably d1chloroethinoa

Description

The invention relates to a method for the preparation of a tablet of 4-dehydrowithaferin A inhibiting leukemia cells P388, which is prepared by transforming withaferin A into the microorganism Arthrobacter siaplex /ATCC 6946/.

Withaferin A is a steroid lactone that inhibits the growth of several tumors /Shohat ♦ t.al., Cancer Cheaother. Rep., JI,271,1977 ; Fuska et al., Heoplasaa 31 , 31,1984 /. The substance has been isolated from various plant species. Microorganism: Cunnighala elegans /HRRL 1393/ transforms withaferin A into 15 /3-hydroxywithaferin A /Rosazza at al., Steroids 31 ,676,1978/ and into 12 A-hydroxywithaferin A /Fuska et al.. Steroids 35,157,1982/. Salt metabolites of withaferin A have been prepared from withaferin A using Arthrobacter staple» and their structure has not been determined yet /Fuška et al., Folia microbiol., 30, 427,1985/.

The present solution describes a method for preparing 4-dehydrowithaferin A, which has higher antitumor activity than previously prepared withaferin A derivatives and withaferin A. The essence of our solution is that withaferin A (10-50 mg/lOOml) dissolved in a polar solvent, preferably in a diet of Iforaaeide, is added to a nutrient medium containing a source of carbon and organic nitrogen, inoculated with a culture of Arthrobacter siaplex /ATCC 6946/ and cultivated for 30-48 hours at a temperature of 28-30 °C with constant stirring and aeration. The filtrate of the fermented soil, which contains the new metabolite, is extracted with acetic acid ester, preferably ethyl acetate, and the obtained extract is evaporated to dryness after drying, purified on a silica gel plug, and the obtained eluate, after concentration at a temperature of up to 40 °C, is left at 4 °C, until pure 4-dehydrowithaferin A is obtained. Various chlorinated hydrocarbons with a carbon number of 1-5, preferably dichloroethane, can also be used for extracting the substance from the filtrate. For the recrystallization of the metabolite, ethyl acetate: n-heptane or ethyl acetate: ethanol are preferred solvents.

4-dehydrowithaferin A /1/ is a white crystalline substance, mp 276 - 277 °C, of the formula C _o H_,0,. In the system chloroform: acetone /4 : 2/ the substance gives after detection of p-anisaldehyde O j O o doo one icolor brown color with the value of R ^ 0.61. The values of the H-NMR spectra of 4-dehydrowithaferin A /1/ and withaferin A /2/, on the basis of which the substance was identified, are given in the following table:

Proton

Mu11 and lung and ta

2d .dd

4d

6s.br.

18s

6.21

6.94

3.77

3.23

0.71

6.87

3.43

0.74

19 and 1.41 1.40 21 d 1.01 1.02 22 ddd 4.43 4.45 23a dd 2.50 2/52 23b dd 2.20 2.20 27 ABq 4.36 4.30 28s and 2.04 2.04

SJ _2z3 = 10Hz; J _3z4 - 6Hz; J _20z21 = 6.5Hz; = 3Hz; J _22z23a = 13Hz; = ^{= 3H2} > ^J 23a,23b = ^18H2 >

4-dehydrowithaferin A inhibited the biochemical functions and growth of P388 leukemia cells at concentrations lower than 10 μg.ml more significantly than the original withaferin A and other derivatives prepared so far. The microorganism used transforms withaferin A very selectively and in a short period of cultivation into 4-dehydrowithaferin A.

The following examples describe the transformation of withaferin A into 4-dehydrowithaferin A and its isolation.

Example 1

Into 20 flat-bottomed boiling flasks with a volume of 500 ml, 100 ml of media was prepared, which had the following composition: glucose 2 g, corn extract /50 X suilny/ 3 g, yeast extract 5 g, tap water to 1000 ml, pH 6.5 -7.0. The media was sterilized for 20 minutes at 120 °C and after cooling, 15 ml of vegetative inoculum of Arthrobacter simplex /ATCC 6946/ was inoculated and cultivated on a rotary shaker at 220 rpm \ at a temperature of 28 °C. After 24 hours, 30 mg of withaferin A dissolved in 0.3 ml of dimethylformamide was added to each flask. The cultivation was terminated at 32 hours of cultivation, when the added substrate was completely transformed.

Example 2

2000 ml of the medium obtained after the transformation was centrifuged and then filtered to remove bacterial biomass. The obtained filtrate 1600 ml, pH 8.8 after adjusting the pH to 6.5, was extracted three times with 800 ml of ethyl acetate, with constant stirring, at a temperature of 20-24 °C for 15-20 minutes. The obtained extracts were combined, dried by adding anhydrous sodium sulfate and freed from the solvent under reduced pressure at a temperature of up to 40 °C. 190 mg of crude product was obtained, which was purified on a silica gel pad /60/120 mesh, column 4x35 cm/, eluent mixture of ethyl acetate » acetone /2:1/. The fractions containing 4-dehydrowithaferin A were combined, the solvents were removed, and the solid residue was recrystallized from ethanol:ethyl acetate to give 120 mg of pure crystalline material, which was identified as 4-dehydrowithaferin A.

Example 3

The biotransformation procedure was the same as that described in Example 1, but dichloromethane was used for metabolite extraction.

Claims (2)

SUBJECT OF THE INVENTION A method for preparing 4-dehydrowithafrin A, characterized in that a culture of Arthrobacter simplex is inoculated into a liquid medium containing a carbon source and organic nitrogen. CS 269008 81 3 /ATCC 6946/ and add 10 - 50 mg withaferin A per 100 ml of the soil, dissolved in a polar solvent, preferably in dimethy I formate, and the soil is allowed to cultivate with constant stirring and aeration. 30-48 hours, then the filtrate of the fermented soil is extracted with acetic acid esters, preferably ethyl acetate, the extract is evaporated to dryness at a temperature of up to 40 °C, the solid crude product is purified on a silica gel plug and after evaporation of the solvents, the substance is crystallized from a mixture of ethanol: chloroform or ethanol: ethyl acetate. 2. The preparation method according to item 1, characterized in that the filtrate of the fermented soil is extracted with chlorinated hydrocarbons with a carbon number of 1-5, preferably dichloromethane.