DE1205092B - Process for the production of 3-keto-steroids saturated in ring A by microbiological means - Google Patents

Description

Process for the production of 3-keto-steroids saturated in ring A by microbiological means. It is already known to convert A4m3-keto-steroids into corresponding A1, 4-3-keto-steroids by treatment with microorganisms. There are compilations of the microorganisms used here, for example. B. in Angewandte Chemie, 1957, p. 461, and Experientia, 1955, p. 469 and 478. It is also known that Bacillus sphaericus and Corynebacterium simplex A6-3-hydroxy-steroids in the corresponding, dI, 4-3 -Keto-steroids. In this reaction, the l (2) -dehydrogenation runs parallel to the oxidation of the 3-hydroxyl group, so that no 3-keto steroids saturated in the l (2) position can be produced in this way.

It has now been found that the group of microorganisms which is otherwise used for 1-dehydrogenation of steroids surprisingly does not carry out 1-dehydrogenation if 3-hydroxy steroids are used as starting material, from the carbon atom 5 of which there is no double bond, which are therefore unsaturated neither in ring A nor in the 5 (6) position. According to the invention, only the 3-hydroxyl group is oxidized to the 3-keto group in such a case. The reaction then stops and dehydration in the 1-position does not take place.

, Object of the invention is thus a process for preparing saturated in ring A 3-ketosteroids, which consists in reacting a 3-hydroxysteroid, which neither in 5 (6) position, a double bond in the ring A, by per se known treatment with such microorganisms or enzymes isolated therefrom, which are known to convert A4-3-keto-steroids or Asm3-hydroxy-steroids into AI, 4-3-keto-steroids, to the corresponding 3-keto- saturated in ring A steroid oxidized.

For the process according to the invention, all microorganisms can be used which are capable of converting steroids unsaturated in the 4- or 5-position with an oxygen function in the 3-position into the corresponding, I 1, 4-3-keto steroids. So the implementation z. B. can be carried out with microorganisms from the following genera: Alternaria, Didymella, Caloneetria, Colletotrichum, Cylindrocarpon, Fusarium, Ophiobolus, Septomyxa, Vermicularia, Acetobacter, Aerobacter, Alcaligenes, Baeillus, Corynebacterium, Erysipelothrix, Protaminiapora, Nromonosacterium, Listeria, Micocacteria Pseudomonas or Streptomyces. Particularly good results can be achieved when using Bacillus sphaericus, Corynebacterium simplex and Pseudomonas testosteroni.

The process according to the invention can be carried out according to the usual methods of fermentation technology. In general, the microorganism is cultured in a suitable nutrient solution under aerobic conditions submerged in shake flasks or fermenters. The temperature is kept between 20 and 35'C depending on the needs of the organism used.

The steroid to be converted is conveniently in a water miscible and solvents that are less toxic to the microorganism, such as methanol, ethanol, Acetone, dimethylformamide or propylene glycol are added. The addition can be either to the uninoculated nutrient solution or to the growing culture. Thereon the culture is kept under the same conditions for some time for incubation. It is also possible to filter the fungal cultures before adding the steroids and then incubating the steroids with the aqueous suspension of the mycelium.

After the fermentation, which can be followed by paper or thin-layer chromatography, the steroids are separated from the culture filtrate using customary methods. The end products are preferably extracted with organic solvents such as chloroform, methylene chloride or ethyl acetate. Instead of extraction, the steroids can also be adsorbed, e.g. B. can be isolated by adding activated carbon. The end products are adsorbed almost quantitatively and, after filtration, eluted from the filter cake with a solvent. Occasionally it is necessary to separate the resulting mixture of steroids, e.g. B. by chromatography over aluminum oxide, silica gel or kieselguhr. As a starting material, steroids of the various steroid series can be used, e.g. B. those of the testan, androstan-, pregnan, 18- or 19-nor-androstan-, cardenolide, bufadienolide, bile acid and sapogenin series. The steroids can, except in ring A and in the 5 (6) position, contain double bonds, e.g. B. in one or more of the positions 6, 7, 9 (11), 11, 14, 15, 16, 17 (20), 20, 20 (22) or 22. Furthermore, the starting materials can have substituents in one or more of the positions 1, 2, 4, 5, 6, 7, 9, 11, 12, 14, 16, 17, 18, 19, 20, 21 wear. Particularly suitable substituents are: alkyl, alkylidene, hydroxy, oxo, oxido, amino, halogen, cyano, merepto or thio substituents. These substituents can optionally be present in a functionally modified form. Hydroxy, amino and mercapto groups can be alkylated or acylated. Hydroxyl groups on two adjacent carbon atoms can be present as a cyclic ketal with a carbonyl compound. Oxo and thio groups can be ketalized or in the form of enol acylate, enol ether or enamine.

Particularly preferred starting materials for the process according to the invention are: androsterone, 3-epi-androsterone, 17x-methylandrostane-3ß, 17fl-diol, 17 # x-methyl-19-nor-androstane-3ß, 17ß-diol, 5ß-androsterone , Androstan-3, x, 17ß-diol, Androstan-3fl, 17ß-diol, Androstan-3fl-ol-16-one, Androstan-3fl, 16ß-diol, 5ß-! Androstan-3oc, 16ix, 17ß-triol, 16-androsten-3ß-ol, 16-androsten-3x-ol and the corresponding 16- and 17-esters, 5oc-pregnan-3ß-ol-20-one, pregnan-3oc, 20, x-diol, pregnan-11 , 20-dione-3x-ol, 5x-pregnan-3x, 20x-diol, 5cc-pregnan-3β, 20cc-diol, pregnan-3oc-ol-20-one, 5oc-pregnan-3β-ol-20-one , 5x-Pregnan-3 (x-ol-20-on, 5x-Pregnan-3fl, 20ß-diol, 5ß-Pregnan-3oc, 6x-diol-20-on, 5oc-Pregnan-3ß, 16x, 20 # - triol, pregnan-3, x, 17oc-diol-11,20-dione, 5o; -pregnan-3fl, 11fl, 17, x, 20β, 21-pentol, 5oc-pregnan-20-one-3x, llβ, 17oc , 21-tetrol, 5P-pregnan-11,20-dione-3fl, 17, x, 21-triol, 5x-pregnan-3β, 11ß, 20oc, 21-tetrol, 5x-pregnan-3fl, 17, y, 20β , 21-tetrol, 5x-pregnan-3β, 17 # x, 20cc-triol, 5x-pregnan-3β, 21-diol-11,20-dione, 5x-pregna n-20-one-3fl, 11fl, 21-triol and its 21-esters, methyl deoxycholic acid, methyl 3x-hydroxy-11-cholenic acid, 5oc-pregnane-11,20-dione-3β, 5,17oc, 21-tetrol , 16cc-methyl-pregnan-3fl-ol-20-one, digitoxigenin, periplogenin, bufalin, epi-sarmentogenin, digoxigenin.

The compounds produced by the process of the invention can either be used as medicaments because of their physiological activity or serve as intermediates for the production of pharmaceuticals. Example 1 A culture of Corynebaeterium simplex is grown as a shaking culture in a 1-1 Erlenmeyer flask with 200 μl of a nutrient solution of 0.501 yeast extract ( buffered to pH 6.8 with Sörensen phosphate). After 16 hours of growth at 28 ° C., 70 mg of epi-androsterone, dissolved in 4 nil of methanol, are added and the mixture is shaken at the same temperature. After 24 hours, the culture solution is extracted three times with the same volume of chloroform and the combined extracts are concentrated. According to the thin-layer chromatogram, no more starting material can be detected. The concentrated extract is filtered through a small column of silica gel, freed from the solvent and recrystallized from ether-pentane. Melting point of the androstane-3,17-dione obtained 129 to 130'C.

Example 2 A small fermenter with 121 sterile nutrient solution of 10 % yeast extract ( buffered to pH 6.8 with Sörensen phosphate) is inoculated with 800 ml of Bacillus sphaericus shaking culture. The culture, which grows with vigorous aeration and stirring at 28 ° C., is given after 17 hours an addition of 4 g of digitoxigenin in 200 ml of methanol. The conversion is followed by thin-layer chromatography; 14 hours after the addition, no more starting material can be detected, only the less polar reaction product. The batch is terminated after 19 hours and stirred three times with the same volume of chloroform. The combined extracts are dried over sodium sulfate and, after filtration, freed from the solvent. The residue is treated with petroleum ether; the undissolved part is decanted and recrystallized from ethyl acetate: melting point of the digitoxigenone obtained 200'C; 216 m # t, EI .. 430.5 (ethanol); [x] +30 ' D (chloroform); UV absorption in 96 % sulfuric acid at 339 and 410 m # t.

Example 3 A culture of Bacillus sphaericus is grown as a shaking culture in a 1-1 Erlenmeyer flask with 200 ml of a nutrient solution of 10 % yeast extract, pH 6.5. After 8 hours of growth at 28 ° C., 70 mg of 17oc-methyl-19-nor-androstane-3ß, 17fldiol in 4 ml of methanol are added. 40 hours after the addition, the culture solution is extracted three times with 200 ml of chloroform; the concentrated extract is filtered through a small column of silica gel, freed from the solvent and recrystallized from ethyl acetate. The resulting 171x-methyl-19-nor-androstan-17fl-ol-3-one melts at 145 to 147 ° C.

Example 4 Analogously to Example 1 , 70 mg of pregnan-3oc-ol-20-one are added to a shaking culture of Corynebaeterium simplex. After a running time of 20 hours, it is extracted with chloroform analogously to Example 1 and pure pregnane-3,20-dione is obtained after recrystallization from ethyl acetate. Melting point 120 to 122'C. Example 5 Analogously to Example 3 , 70 mg of pregnan-20-one-3β, 11β, 21-triol-21-acetate are added to a shaking culture of Bacillus sphaericus. After 40 hours, the mixture is extracted with chloroform and, after recrystallization from ethyl acetate, pure pregnan-11ß, 21-diol-3,20-dione-21-acetate is obtained. Melting point 158'C.

Example 6 Analogously to Example 1 , 70 mg of 5β-androstane-3p, 17β-diol are added to a shaking culture of Corynebaeterium simplex. The reaction has ended after 15 hours; the culture solution is extracted analogously to Example 1 with chloroform. After recrystallization from acetone-water, 37 mg of pure 5fl-androstan-17fl-ol-3-one with a melting point of 139 to 140 ° C are obtained; [ix] T + 33 ' (ethanol).

Claims (2)

Patent claims: 1. Process for the preparation of 3-keto-steroids saturated in ring A by a microbiological route, characterized in that a 3-hydroxy-steroid which has a double bond neither in ring A nor in the 5 (6) position, incubated with such microorganisms or enzymes obtained therefrom, which are known to convert A44-keto- or A5-3-hydroxy-steroids into Al, 4 # 3-keto-steroids. 2. The method according to claim 1, characterized in that one uses Bacillus sphaericus or Corynebacterium simplex. Publications considered: Experientia, Vol. 7 (1951), pp. 81ff .; Journ. At the. Chem. Soc. 79: 2658 (1957).