JPH07501689A - Method for producing 14α-hydroxy-4-androstene-3,17-dione - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for producing 14α-hydroxy-4-androstene-3,17-dione The present invention relates to carpularia lunata ((:urvularia 1unata) by fermenting 4-androstene-3,17-dione using cultured bacteria. Method for producing 14α-hydroxy-4-androstene-3,17-dione , 4-androstene-3,17-dione was suspended in water, and the obtained 14α-hydroxy-4-andros, characterized by fermenting @/R liquid The present invention relates to a method for producing ten-3,17-dione.

In Soviet Patent No. 801517 (see C, A, 110゜133697u) 4-androstene-3,17-dio 14α-hydroxy-4-androstene-3,1 by fermenting A method for producing 7-dione is described. However, the patent specification does not include the Which public strain depository does the organism come from and under what number? Since there are no instructions included, it is not possible to retry the method described. Applicable According to the technical means in the specification, 4-androstene-3゜17-dione is converted into methanol. 0.5-1.5 g/l of substrate dissolved in dimethylformamide or dimethylformamide at 40% After ~66 hours of fermentation, the 14α-hydroxy was apparently severely contaminated. 35-70% of -4-androstene-3,17-dione crude product is obtained.

(This is evident from the work-up method, i.e. the fermentation batch is chloroformed. The chloroform phase is concentrated and the residue is boiled with hexane.

Therefore, all the products obtained in this way are extractable and in hexane Must contain insoluble substances. The product is contained in a fermentation patch With all steroids as well as corresponding calbularia-containing substances and vehicle components ).

In contrast, the method according to the invention provides more than 5 g of 4-andro per liter. Stene-3,17-dione can be reproduced within about 12 hours and 14α-hydroxy- It can be converted to 4-androstene-3,17-dione. the processed product The yield is about 60% pure product.

The method according to the present invention is based on the known microbiological method using cultured microorganisms of Calburial Inata. It is carried out under fermentation conditions that are also used for substrate conversion.

The liquid under aeration in a suitable medium under culture conditions normally used for the microorganism. Grow cultured bacteria. Next, the cultured bacteria were treated with 4-androstene-3,17-dione. Water-based! Add liquid 15 and ferment until maximum substrate conversion is reached. 4-Andro Aqueous suspensions of sten-3,17-dione can be prepared in a simple manner. For example, the substrate is mixed with 2 to 5 times the amount of water while adding a nonionic surfactant. The substrate is ground in a ball mill until the substrate has a particle size of approximately 1-5 μm. Appropriate Surfactants include, for example, ethylene oxide adducts of polyglycols or fatty acid esters. It is. Suitable surfactants include, for example, commercially available wetting agents such as Tegin ( R), Tween (R) or 5pan (R).

After fermentation, the cultured bacteria is post-treated using a conventional method. The extract was concentrated and the resulting residue was chromatographed. Purification by tography and/or crystallization.

Pharmacology of the obtained 14α-hydroxy-4-androstene-3,17-dione The subsequent treatments for steroids that act as (J, Amer, Chew, Sac, 91, 1969.1228 onwards) (Reference: European Patent Application Publication No. 0300062, etc.).

The invention will now be explained in detail with reference to the following examples.

a) Corn staple breaker 1% Soybean powder 1.25% Adjust to pH value 6.2 In a 2 liter factory containing 1 liter of sterile medium containing Burarialnata NRRL2380 Culture Sono! ! ! Seed 5 liters of suspension, 30℃ The mixture was shaken at 180 revolutions per minute for 60 hours.

b) 50 liter fermentor with 40 liters of sterile culture medium as described in a) was inoculated with 11 liters of Calburial Innata growth medium and incubated at 30'C for 18 hours. The greenhouse was kept under aeration of 2 m3 per hour and stirring at 220 revolutions per minute.

C) Corn staple breaker 1% Soybean powder 1.25% β-cyclodextrin 0.3% Adjust to pH value 6.2 In a 50 liter fermenter containing 40 liters of sterile culture medium containing Inoculate 4 liters of Rialnata preculture, aerate 2.4 m' per hour and aerate for 1 minute. The mixture was wetted at 30° C. for 7 hours with stirring at 150 rpm.

Next, 250 g of the cultured bacteria 4-androstene-3,17-dione was ground. and added to 10,100 O of water containing 8,025 g of Tween(R), and further added at 12 o'clock. Fermentation was carried out under stirring at 220 revolutions per minute and aeration of 2.4 m' per hour.

d) After carrying out the fermentation, the liquid of the culture is treated with methyl isobutyl ketone 2 Extract 3 times with 0 liters and concentrate the extract under vacuum in a rotary evaporator at a maximum of 50 °C. did. Followed by chromatography on silica gel using a hexane-acetone gradient. 14α-hydroxy-and, melting point 257-259°C, purified by 150 g of rost-4-ene-3,17-dione were obtained.

International search report. , 71. . . 5/n17゜7PCT/EP 92101797

Claims (2)

[Claims] 1. 4-androstene-3,17-di- 14α-hydroxy-4-androstene-3,1 by fermenting In the method for producing 7-dione, 4-androstene-3,17-dione is 14α-hydrohydride, characterized in that it is suspended in water and the resulting suspension is fermented. A method for producing roxy-4-androstene-3,17-dione. 2. For fermentation, a culture of Calbra Rialannata NRRL2380 was used. 14α-hydroxy-4-androstene-3,17-di according to claim 1, On manufacturing method.