SU896070A1 - Method of preparing steroid - Google Patents

Description

This invention relates to the medical industry and relates to the preparation of drug steroids. A known method for producing a steroid is by microbiological hydrolization of the steroid ester, followed by isolation of the target product fll. However, the known method provides a slight yield of the target product (40%). The purpose of the invention is to increase the output and steroid. This object is achieved in that the method is carried out for preparing steroid way microbiological steroid ester hydrolysis followed by isolation of the product left tse and hydrolysis is carried out using a strain of Arthrobacter cftreus IBPM B-51 Arthrobacter citreus IBPM B Brevibacterium luteum 185 B 134 B Brevibacterium cltreum IBSM 131 sludge Corunebacterium insidlosum IBPM B-13 Microbiological transformation is carried out by any known method (growing culture or washed cells), the steroid is applied in an organic solvent or in a microcrystalline state. Example 1. The culture of Arthrobacter citeum B-51 support on corn-glucose agar,%: glucose 1; corn extract 1; agaragar 3; tap water up to I l; pH 6.8-7.2. For the growth of the microorganism, 750 ml Erlenmeira flasks are used, containing 150 ml of medium of similar composition, but without agar-agar. For seeding from scythes, wash out 7.5 ml of sterile tap water. 5.0 ml of a bacterial suspension containing 2.0 g / l of cells was added to a flask with 150 ml of medium. Biomass is measured nephelometrically, followed by weight of dry biomass. The culture is grown for 24-26 hours on a rocking chair of l80 o b / min at up to

a biomass of 2-3 g / l, then a steroid — cortisone 21-acetate (300 mg) in ethanol solution (10 ml) is introduced.

The final steroid concentration is 2 g / L. After 48 hours of transformation, the process ends. The results of the chromatographic analysis of the culture fluid show that -. the content of the hydrolyzed steroid, cortisone, is 95%.

Similarly, growing cells of the following strains are used to hydrolyze cortisone acetate (2 g / l). The results of the analyzes are given in: table.I, table 1

Arthrobacter citreus B-t85 Brevibacterium citreum B-131 Brevibacterium luteum B-134 Corynebacterium insidiosum B-137 Example 2. Culture Arthrobacter citreus B-51 is grown analogously to example I. After cultivation, the cells are separated by centrifugation at BOOC, resuspened for 20 min, and the material is resuspended in a wedge of saline. to the required concentration (8 g / l) and used for transformation. The transformation was carried out under conditions similar to bleaching in 750 ml Erlenmeyre flasks containing 150 ml of the reaction mixture. Microcrystalline steroid-2-cortisone acetic acid (particle size 5-30 //) is introduced to a final concentration of 100 g / l. After 10–12 days of transformation, it ends, the culture fluid is filtered on a funnel with a glass porous bottom (filter No. 1) with one paper filter and two cloth filters. The filter cake was transferred to a flask and boiled for 30 minutes with a 30-fold excess of isopropanol. The operation is repeated three times, decanting the solution and adding a new portion of the solvent. The combined isopropanol solution of cortisone is treated with 0.3 g of activated carbon at room temperature (20 h). The carbon is filtered off with a No. 4 glass porous filter and the filtrate is evaporated to a volume of 10-15 ml. The concentrated solution is cooled and incubated for 20 hours at 0 ° C. The precipitate (cortisone) is filtered off, washed with cooled isoprop98 98 95 95 NOL and dried in a vacuum desiccator to constant weight. Of 4.85 g of cortisone acetate, 3.6 g of cortisone is obtained (82.0% of the theoretical). T. pl. 219-221e, E 450 (in methanol). The admixture of cortisone acetate is 1% and the 20 / -oxy derivative of cortisone is followed by the amount. 0.15 g of cortisone is further isolated from the mother liquor by extraction with ethyl acetate and subsequent recrystallization of the final product from isopropanol. 214-215s, - / cm methanol). The overall yield of cortisone is 85.5% of the theoretical amount. EXAMPLE 3 A culture of Arthrobacter citreus IBFM B-51 was grown as in Example 1. For transformation, cells separated from the medium, resuspended in tap water, were used in an amount of 3 g / l. The steroid microcrystalline 21-prednisone acetate (particle size 530 / ") is carried away at a concentration of 10 g / l. (The transformation is carried out in conditions similar to cultivation. After 48 h of transformation, the culture liquid (total volume 200 ml:: 100 ml of enzyme mixture + 100 ml distilled water for washing) is extracted with ethyl acetate 4 times in 200 ml. The solution is extracted with ethyl acetate 4 times in 200 ml. The solution is filtered through a filter paper and evaporated on a rotary evaporator.

up to the volume of 5-7 ml. The concentrated prednisone solution is cooled and kept for 20 h, washed with chilled isopropanol, dried in a vacuum desiccator to constant weight. From 0.95 g of prednisone acetate, 0.70 g of prednisone is obtained (82% of the theoretical amount), m.p. 224-220 C, 440 (in methanol).

The impurity of prednisone acetate is 1% and 20 (-oxy derivative prednisone is traces.

0.02 g is additionally separated from the mother liquor by extraction with ethyl acetate and recrystallization of the crude product from isopropanol. prednisone with T. pl. 220-222 s. Overall

the output of prednisone, 0.72 g (85% of theoretical amount).

In addition to cortisone acetate and prednisone acetate, Arthobacter cltreus culture IBPM B-51 transforms other steroid esters (Table 2). Under the growth conditions described in Example 2, washed cells are used for transformation at

biomass of 2 g / l and steroid concentration of 1 g / l. As follows from the chromatographic analysis, the yield of the transformation products is 95-100%. The results of the hydrolysis of various.

Steroids esters culture Arthrobacter citreus IBFM-51 (according to chromatographic analysis of the system benzene-acetone 3: 1, silufol) are given in table. 2

Tabpitsa2

Claims (1)

Claim A method of obtaining a steroid by microbiological hydrolysis of a steroid ester with subsequent isolation of the target product, characterized in that, with the aim of higher. VNIIPI Order 11631/10 ... _________ Branch of PPP Patent, Uzhhorod, st. Project, 4