DD249284B1 - PROCESS FOR PREPARING 9ALPHA-HYDROXY-ANDROST-4-EN-3,17-DION - Google Patents

Description

Field of application of the invention The invention relates to a process for the preparation of 9a-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) from sterols

microbiological conversion.

9-OH-AD is a key product for the production of steroid drugs. From this product can be highly effective

Steroids of the pregnane series can be obtained with a wide range of uses. The field of application of the invention lies in the pharmaceutical research and industry. Characteristic of the known technical solutions

It is known from the literature first, a microbial process for the preparation of 9-OH-AD from sterols (see, the patents DEtAS 2647895 and DD 127455), in which one under the registration Nm. NRRL B-8119 and DSM 752 registered microorganism of the species Mycobacterium fortuitum for the sterol transformation to o.g. Target product is used. ··, .. · ;. ; / * *. , ·

This method has some significant disadvantages: on the one hand, a microorganism is used which is to be expected to be the facultatively pathogenic species of the genus Mycobacterium (RE Buchanan et al., Bergey's Manual of Determinative Bacteriology, 1975, DP Nicholson and WRServier, Am. Rev. Resp. Dis. 104 [1971] 747-750). From the use of this microorganism results in a significant additional technical effort for health, labor and environmental protection. On the other hand, this technical solution still has the disadvantage that even with very long fermentation times (14 to 15 days) only an unsatisfactory product formation is disadvantageous also suggests in this solution to book that a separation of Ferrnentationsprodukr.es 9-OH-AD is required by unreacted sterol, since in an extraction of the fermentation broth in addition to the target product and substrate in the extract is included.

A first attempt to overcome one and the latter disadvantage of this method reported M. Aoki u. a. (see JP Kokai 77 57161). used for the processing of the fermentation broth nonpolar adsorber of the XAD type and in this way selectively separated the product formed could largely.

-2- 249 2 & 4

A further attempt to overcome the disadvantages of the basic process according to DE 2647895 or DD ¹ 27455 is also the technical solution of the patent DD 232167 ago, in which using the strain M. fortuitum ZIMET10 849 and the addition of a finely divided solid, hydrophobic organic Polymer to the fermentation broth in fermentation times of 2 to 6 days now sine satisfactory 9-OH-AD formation is achieved, but in which still a microorganism from a species is used, which in turn also just contains facultative pathogenic strains. Finally, the microbial process for 9-OH-AD production according to JP-KTK 80 85 397 is known, in which the selective sterol transformation is carried out with the aid of a strain of the species Mycobacterium vaccae (strain M. vaccae MCI-1104). Although in this technical solution an apathogenic strain is used, however, even after long fermentation times again only a little satisfactory Prcduktbildung takes place (from 20g cholesterol within 8 days, for example, only 0.33 g 9-OH-AD).

Object of the invention

The aim of the present invention is to produce from sterols in good yields 9a-hydroxy-androst-4-ene-3,17-dione (9-OH-AD), an intermediate for the production of steroidal drugs.

Explanation of the essence of the invention

The invention has for its object to describe an improved microbial process for the preparation of Sa-hydroxy-androst-4-ene-3,17-dione (9-OH-AD), this product with high sterol throughput and significantly reduced fermentation times in good yields.

According to the invention this object is achieved by microbial transformation with strains of the species Mycobacterium vaccae dadurcn that for the transformation of the respective sterol or Sterolgemisches strain M. vaccae ZIMET11052 or strain

M. vaccae ZlMET 11053 can be used. The strains used according to the method have been deposited in the depository for microorganisms of the GDR, Central Institute for Microbiology and Experimental Therapy of the AdW of the GDR, Beutenbergstrasse 11, DDR - Jena 6900 under the above-mentioned registration numbers.

For the purpose of the invention, strain M. vaccae ZIMET11052 or strain M. vaccae ZIMET11 053 is fermented in an aqueous nutrient medium in the various variants of the invention under aerobic and sterile culture conditions, which in addition to sterol a Kohlenstcffquelle (such as glycerol, glucose, sucrose, starch or Lactose), a nitrogen source (such as yeast extract, urea or casein hydrolyzate) and mineral salts. Sterols such as sitosterol, cholesterol, carnpesterol, stigmasterol or ergosterol or mixtures of these sterols are used for this fermentation. The sterol is introduced either as such or in the form of a sterol surfactant preparation into the aqueous fermentation medium, with the sterol feed concentration being selected in the range of 1.0 g / l to 50 g / l of fermentation medium. The optional sterol surfactant preparations are provided by mixing the respective sterol by wet milling, lyophilization or spray drying with a surfactant of the groups A or B identified below, the surfactant having a content of from 1.0 to 20% by weight the finished preparation. Among surfactants of groups A and B, polyethylene oxide (PEO) -polyprophylene oxide (PPO) block copolymers with a proportion of 30% to 70% of polypropylene glycol or the PEO-PPO adducts of N, N "are used, as in one of our earlier patent applications. Tetra - (- oxypropylpropyol) -N '- (2-oxypropyl) -diethylentriamins understood.

The fermentation is conveniently carried out in the stages pre- and main cultivation; it is carried out at temperatures of 25 ° C to 35 ° C for a period of 48 hours to 96 hours, with the pH chosen to be in the range of pH 6.0 to pH 9.0

Furthermore, an adsorbent of the type of an organic polymer in an application concentration of 10 g / l to 160 g / l is optionally added to the fermentation medium, either from the beginning or in the course of cultivation. Preferably, an organic polymer produced from ethylvinyl and divinylbenzene, which has a specific surface area of at least 200 m ² / g, is used as adsorber. Specifically, in the process variants with adsorber use, an organic polymer, formed from ethylvinyl and divinylbenzene, with polar groups, in particular with polar groups based on acrylic acid esters, is used.

The fermentation can be carried out in round-bottomed flasks and steep-breasted bottles of various contents, in stirred glass fermenters and in V2A steel tanks.

After completion of the fermentation takes place the recovery of the formed 9-OH-AD in the process variant without Adsorberzusatz in a conventional manner by extraction with an organic solvent, preferably with butyl acetate, followed by crystallization.

In the variant with adsorber, the workup with the separation of the adsorber from the culture solution, preferably by filtration, initiated, and by elution of the adsorber with organic solvent such as methanol, ethanol or acetone or aqueous solutions of such agents, by concentration of the eluate About 10% of the initial volume with re-filtration and by recrystallization is finally '9-OH-AD obtained.

In a preferred embodiment of the method, wherein the cells from the strain ZIMET11053 in a medium with the addition of 140g / l adsorber above labeling for 96 hours at 28 ⁰ C have been fermented, resulted in dependence on the sterol amount used in Rundkolbehmaßstab 9-OH AD yields in the range of 35% to 50% by weight (analytical data), with a residual sterol content of less than 5% by weight. As it turned out, under these process conditions the use of an adsorbent ο. G. Texture not only a beneficial effect on the workup of the formed 9-OH-AD; Rather, this also results in an increase in product formation itself and thus an increase in sterol utilization.

The advantages of the method are summarized in that

- The product 9-OH-AD can be produced after short fermentation times and in good yields using each one as a non-pathogen-classified microorganism strain and that

- In the process variant with Adsorberzufügung not only the product 9-OH-AD is removed from the culture solution, but in addition still further increased yields can be achieved (see Table).

embodiments Example 1: Mycobacterium vaccae ZIMET11053 is cultured on slants of the composition for 7 days at 28 ° C: 20g glycerol;

5g Bacto peptones; 5g meat extract; 15g agar-agar; Distilled water ad 1000ml; pH 7.0 (tube culture).

With the washed-off cells of each tube, 500 ml stalk flasks are inoculated, the following each 50 ml of nutrient solution Composition contains: 10g glycerol; 10g yeast extract; 0.5 g KH ₂ PO ₄ ; 1.5 g (NH ₄ ) ₂ HP0 ₄ ; 0.2 g of MgSO ₄ .7H ₂ O;

0.01 g of FeSO ₄ .7H ₂ O; 0.002 g ZnSO ₄ .7H ₂ O; Distilled water ad 1000ml; pH 7.0.

After 72 hours of shaking on a rotary table at 200 rpm and 28X, 5 ml of each preculture are added Inoculation of the fermentation culture used. Ten 500 ml round-bottomed flasks are each charged with 7 g of moist adsorbent of the type of an organic polymer (formed from Ethyl vinyl and divinylbenzene, with acrylic acid ester based polar groups, containing 10 ml of sitosterol surfactant preparation

(100 g of sitosterol, 10 g of group B surfactant, distilled water ad 1000 ml, wet-milled) and charged with 50 ml of fermentation medium. The fermentation medium is a solution of 5 g of glucose; 10g yeast extract; 1.3gK ₂ HPO ₄ · 3H ₃ O; 1.5 g (NH ₄ I ₂ HPO _4; 0.2 g MgSO ₄ · 7H ₂ O 0.01 g FeSO ₄ · 7H ₂ O; 0,002g ZnSO ₄ .7H ₂ O; Distilled water ad 1000 ml; pH 8, 0 used.

The thus prepared flasks are sterilized for 20 minutes at 120 ⁰ C and inoculated after cooling, then a

96-hour fermentation on the rotary table at 200 rev / min and 28 ° C made.

The average total yield of 9 OH-AD per round-bottomed flask, determined by thin-layer chromatography, was 330 mg. Example 2:

According to the conditions of Example 1 for tube and preculture strain Mycobacterium vaccae ZIMET11052 is grown.

Ten 500 ml stems and round bottom flasks are dissolved with 4 g wet adsorber of the above labeling, with 500 mg sitosterol surfactant preparation (500 mg sitosterol, 50 mg surfactant group B, in benzene * and lyophilized) and with 50 ml fermentation medium of the composition of Example 1 fed.

The yield of 9-OH-AD averaged 90.6 mg per round bottom flask after 96 hours fermentation on the rotary table at 28 ° C.

Example 3: Strain Mycobacterium vaccae ZIMET11053 is prepared according to the conditions of Example 1 in tube and liquid pre-culture

grown.

The conditions for the fermentation culture are selected analogously to Example 1, but the adsorber additive is omitted. After 96 hours of fermentation on the rotary table at 200 rpm and 28 ° C, an average of 120 mg of 9-OH-AD were obtained Round piston formed. Example 4: The culture in tube and liquid culture of the strain Mycobacterium vaccae ZIMET11053 is carried out according to that in Example 1

mentioned standard.

In each case, 5 g of adsorbents of the organic polymer type (formed from ethylvinyl) are prepared in ten 500 ml stellite flasks.

and divinylbenzene, without polar groups), 5 ml of sitosterol surfactant preparation of the above composition and 50 ml

Fermentation medium (composition as in Example 1). The fermentation is carried out for 72 hours at 28 ° C on the rotary table after the flasks at 120 ^e C for 20 minutes

sterilized and then inoculated.

From the culture solution and the adsorber were obtained on average per round-bottomed flask 208mg 9-OH-AD.

comparison

the performance parameter in the production of 9-OH-AD by the strains Mycobacterium vaccae ZIMHT11052 and ZIMET11053 with the published prior art

publication tribe scale adsorber sitosterol Ferment.- yield (G / l) at first- Time 9-OH-AD concentr. (H) (G / l) (G / l) Patent M. vaccae 500 ml DD249284A1 ZIMET round-bottomed flask example 1 11053 a 50 ml 140 20 96 6.6 Patent M. vaccae 500 ml DD 249 284 A1 ZIMET round-bottomed flask Example 2 11052 a 50 ml 80 10 96 1.8 Patent M. vaccae 500 ml DD 249 284 A1 ZIMET round-bottomed flask Example 3 11053 a 50 ml - 20 96 2.4 Patent M. vaccae 500 ml DD 249 284 A1 ZIMET round-bottomed flask Example 4 11053 a 50 ml 100 10 72 4.2 Patent M. vaccae shake JP 8085397 11CJ-1104 bottles (Mitsubishi) 11053 - 20 200 0.83 Patent M.fortui- 500 ml DD127455 tum shake (Upjohn) NRRL bottles SS-8119 a 100 ml - 10 336 ?

Claims (11)

claims: 1. A process for the preparation of 9a-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) by microbial transformation of sterols with strains of the species Mycobacterium vaccae under aerobic and sterile conditions in aqueous nutrient media containing a Carbon and nitrogen source and mineral salts, characterized in that for the transformation of the sterol strain M. vaccae ZIMET11052 or strain M. vaccae ZIMET11 053 are used. 2. The method according to claim 1, characterized in that an absorber of the organic polymer type is used. 3. The method according to claim 1 or 2, characterized in that the sterol is introduced in the form of a sterol surfactant preparation. 4. The method according to claim 3, characterized in that in the sterol surfactant preparation, a surfactant from the group of polyethylene oxide-polypropylene oxide block copolymers is used in a proportion of 30% to 70% polypropylene glycol. 5. The method according to claim 3, characterized in that in the sterol surfactant preparation, a surfactant from the group of polyethylene oxide-polypropylene oxide adducts of N, N "tetra - (- oxypropylpropyol) -N '- (2-oxypropyl) -diethylenetriamine is used. 6. The method according to claim 1 to 5, characterized in that the sterol is introduced into the nutrient medium in a use concentration of 1 g / l to 50 g / l. 7. The method according to claim 2, characterized in that an organic polymer having a specific surface area of at least 200m ² / g, obtained from Ethylvinyl- and Oivinylbenzen used as an adsorber. 8. The method according to claim 7, characterized in that an organic polymer having polar groups is used as adsorber. 9. The method according to claim 8, characterized in that ajs adsorber is an organic polymer having polar groups based on acrylic esters is used. 10. The method according to any one of claims 7 to 9, characterized in that the adsorber is added to the aqueous medium 'in a concentration of 10g / l to 160g / l. 11. The method according to any one of claims 7 to 10, characterized in that 9-OH-AD is isolated in such a way that the adsorber separated from the culture solution, then eluted with organic solvent and the pure product is recovered from the eluate by recrystallization ,