DE1200815B - Process for the preparation of 11alpha, 17alpha, 21-trihydroxy-9beta, 10alpha-pregn-4-one - Google Patents

Description

Process for the preparation of 11a, 17a, 21-trihydroxy-9,8, 10a-pregn-4-en-3,20-dione. The present invention relates to a process for the preparation of 11a, 17a, 21-trihydroxy-9β, 10a-pregn-4-en-3,20-dione, which has the formula is equivalent to. In contrast to normal IOß, 9a steroids, in which the four rings of the carbon skeleton lie approximately in one plane, the steroid skeleton of the connection that can be produced according to the invention has a kink along the connecting line of the carbon atoms 9 and 8. As a result of this, the configuration of the methyl groups and hydrogen atoms on carbon atoms 10, 9, 8, 13 and 14 is not always the same as that of normal steroids. The configuration of these groups and hydrogen atoms on the carbon atoms mentioned is 10a, 9β, 8β in the case of the compound 10a, 9β, 8β which can be prepared according to the invention. 13 (3. 14a, while with normal steroids it is 10?. 9a, 8?, 13?, 14a.

The compound which can be prepared according to the invention has surprising pharmacological properties Properties. So this substance has no anti-inflammatory effect, no influence on the electrolyte excretion of the body, but a glucocorticoid effect. These properties differ significantly from those of the corresponding compound of the normal range, which has no pharmacologically valuable properties.

The compound that can be produced according to the invention also has no uterotropic properties and no anti-uterotropic effect.

It is also an important starting product for production other 9ß.I0a steroids. It can e.g. B. by oxidation of the 11-hydroxy group 17a, 21-dihydroxy-9ii, lOa-pregn-4-en-3,11,20-trione, or, by methylation on the carbon atom 2 can e.g. B. by reaction with diethyl oxalate and sodium hydride and then Reaction with methyl iodide in the presence of dried potassium carbonate 2-methyl-1 1 a, 17a, 21-trihydroxy-9β, 10a-pregn-4-ene-3,20-dione can be obtained.

The inventive method is characterized in that one 17a, 21 - Dihydroxy-9ß, IOa-pregn-4-en-3,20-dione in a manner known per se with a Culture of a microorganism or the oxidizing enzymes obtained from it, which in connections of the normal steroid series (9a, 10ß) has a 11a or 11ß-permanent Ability to introduce hydroxyl group, incubated under aerobic conditions.

As such, microorganisms of the sex Moniliaceae can be called will. Of this gender, the following varieties can be mentioned in particular, z. B.

Aspergillus such as A. niger, A. nidulans; Cunninghamella such as C. blakesleeana; Absidia such as A. glauca; Pycnosporium sp. QM 703; Pestalotia such as P. foedans; Beauveria ; Fusarium such as F. elegans; Helminthosporium such as H. sativum; Colletotrichum, like C. pisi; Rhizopus, such as R. nigricans. In particular, were in coming about the invention achieved favorable results with Aspergillus ochraceus.

The method according to the invention is in a Wei: °. carried out, which corresponds to the known microbiological conversions, e.g. B. in that the Under the appropriate conditions with a culture of one of the above Mold type and / or its enzyme systems is brought into contact. For this one lets z. B. first a j culture of the mold species under aerobic conditions in one Grow nutrient solution, after which a substrate that contains the steroid to be oxygenated, which can be added in solution or suspension, the oxybiontic dissimilation effect of the resulting mycelium. The nutrient solution consists essentially of a carbon and a nitrogen source, e.g. B. a carbohydrate, such as glucose, Maltose or starch, and an organic nitrogen source, such as cornmeal . water or yeast extract, albumin hydrolysates, amino acids, or an inorganic one Nitrogen source, such as ammonium salts or alkali metal nitrates. The means that the steroid to be oxygenated and one or more of the aforementioned food sources contains, if desired, an antifoam agent, e.g. B. glyceryl monostearate, can be added.

The suitable fermentation temperature is usually between 20 and 28 ° C, although higher or lower temperatures between 15 and 35 ° C, im are generally also useful.

The pH of the agent can be adjusted in the normal way and is preferably brought to a value between 6 and 7.

The time required for the steroid to oxidize may vary between vary widely, but usually an oxygenation period of 10 to 48 hours optimal for a complete implementation.

The 11-hydroxysteroid compound obtained after completion of the oxygenation process can in one of the usual ways from the agent and / or the mycelium, preferably by extraction with water-immiscible organic solvents such as Diethyl ether, ethyl acetate, amyl acetate, methyl isobutyl ketone or other esters and Ketones, to be isolated. In particular, methyl isobutyl ketone is a suitable extractant.

The oxygenated steroid can also be obtained by chromatographic methods, optionally in combination with extraction from the fermentation agent, isolated and getting cleaned.

According to the invention, the 11-hydroxysteroid can also be obtained thereby be that one spores of the microorganisms in question on solutions or dispersions the specified starting steroids can act.

The compound obtained can normally be converted into tablets, pills, Capsules and liquids for oral administration and in liquid form for parenteral Administration to be processed.

EXAMPLE A nutrient solution containing 120 g of thickened corn soft water and 120 g of glucose in 61 distilled water was adjusted to pH 6.5 with the aid of an aqueous 2N sodium hydroxide solution and after 20 minutes of sterilization at a temperature of 120 ° C. with an on oat malt agar inoculated grown culture of Aspergillus ochraceus. This inoculation culture was shaken in twelve shake flasks of 21 capacity each for 36 hours at 26 ° C on a rotating shaker (250 rpm), after which the resulting mycelium mixture was transferred to a stainless steel fermentation vessel of 5001 with a stirrer and aerator , in which the following fermentation agent sterilized under the above-mentioned proportions was present: maize softened water (calculated on dry matter) ....................... 1110 g glucose ... ........................ 1110 g tap water. . _. . . . ... .......... 2201 pH 6.5 (sodium hydroxide solution) This fermentation agent was added to 66 g of Retro-comp. 24 hours after inoculation. S (17a, 21-dihydroxy-9 / 3,10a-pregn-4-ene 3,20-dione) dissolved in 22.2 l of methyl cellosolve, added, followed by another 41 hours of fermentation at 26 ° C (speed 160 / min ., Air supply: 0.9 m3 / m2 floor area / min.).

The filtrate of the culture (2601) was then five times each with 651 methyl isobutyl ketone extracted. The total extract was evaporated in vacuo to 101 and activated with Coal treated. The solution then turned to a partially crystalline in vacuo Remnants evaporated. This residue was recrystallized from 150 ml of dichloroethane, from which 39 g of crystalline product resulted. The mother liquor was evaporated to dryness, and the residue was crystallized from 35 ml of dichloroethane, giving a further 12.8 g of crystalline product were obtained.

The combined crystals (51.8 g) were crystallized twice, each from about 1 1 dichloroethane. In this way 44.16 g of end product was obtained, that as 11 a-Hydroxy-Retro-comp. S plus 1 mole of dichloroethane was identified.

The crystal solvent could be removed therefrom by drying in vacuo (0.5 mm HZ) at 80 ° C. (6 hours). The product had the following physical constants: melting point 189 to 194 ° C. (dried preparation 191.5 to 194.3 ° C.). UV absorption spectrum in methanol max.

242 m # L, E = 16 600 (both of the dried and the undried Preparation). [a] ″ = -61.7 ° C (c = 1, dioxane) (dried preparation -72.3 ° C).

Elemental analysis: C 59.890%, H 7.390%, Cl 15; 620%.

Calculated for C23H340 @ C12: C 59.870%, H 7.430%, Cl 15.36%.

IR = 33 1 0, 1693, 1642, 16 1 1, 1417, 1275, 1238, 1213, 1117, 1077, 1057, 1000, 949, 870th at .1 "« 242 = 476.

Claims (2)

Claims: 1. Process for the preparation of lla, 17a, 21 trihydroxy-9f, i0a-pregn-4-en-3,20-dione, characterized in that 17a, 21-dihydroxy 9β, 10a-pregn-4-en-3,20-dione in in a manner known per se with a culture of a Microorganism or the oxidizing enzymes obtained therefrom, which in compounds of the normal Steroid series (9a, 103) are able to introduce a 11a or II (3-position hydroxyl group, incubated under aerobic conditions. 2. The method according to claim 1, characterized in that that Aspergillus ochraceus is used as a hydroxylating microorganism. In Reference U.S. Patent No. 3,019,370.