DD254025A5 - PROCESS FOR PREPARING 4-ANDROST-3,17-DION AND 1,4-ANDROSTADIENE-3,17-DION - Google Patents

Abstract

Process for manufacturing 4-androstene-3,17 dion and 1,4 androstadiene-3,17-dion of the general formula (I) where ..... is a simple bond or a double bond, characterized by the fact that alpha -sitosterine of formula (II) is fermented with a culture of a strain of micro-organisms suitable for breaking down the lateral sterine chain.

Description

Field of application of the invention

The invention relates to a process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione.

The compounds according to the invention are valuable intermediates for the preparation pharmacologically

effective steroids.

Characteristic of the known state of the art

It is known that numerous microorganisms (such as those of the genera Ärthrobacter, Brevibacterium, Microobaterium, Protaminobacter, Bacillus, Norcardia, Streptomyces and especially Mycobacterium) have the natural ability to naturally occurring 3/3-hydroxy-A ⁵ -sterins (such Cholesterol or sitosterol) to carbon dioxide and water and that in this degradation intermediate 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione are formed.

It is also known that it is possible with the aid of inhibitor additives or mutated microorganisms to direct the degradation of sterols such that degradation of the formed 4-androstene-3,17-dione or 1,4-androstadiene-3,17-dione is avoided (see DE-OS 1 543269 and 1 593 327 and US-PS 3,684,657).

Object of the invention

The aim of the invention is to provide a simple and economical process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione.

Explanation of the essence of the invention

The invention has for its object to produce 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione by side chain degradation of Qf-sitosterol.

According to the invention 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione of the general formula ^

    "o

... a single bond or a double bond meant the way that one made a-sitosterol

fermented with a culture of a steroid-degradable microbial strain. For the expert it is surprising that under the known conditions, the side chain of sitosterol is degraded, because it is known that the side chain degradation of sterols thereby causes a very complex fermentation system and one could not expect that all participate in the side chain degradation of natural steroids Enzyme have the ability to cause the side chain degradation of this compound. Moreover, one could not foresee that in this degradation, the A ⁷ double bond of a-sitosterol is hydrogenated and the methyl group which is in position 4a is split off. Apart from the use of other starting compounds, the process according to the invention is carried out under the same fermentation conditions which are also used in the known microbial side chain degradation reactions of sterols.

According to the invention, the fermentation is carried out using the microorganism culture which is usually used for the side chain degradation of sterols.

Suitable cultures are, for example, for the side chain degradation of sterols capable of bacterial cultures of the genera Arthrobacter, Brevibacterium, Microbacterium, Protaminobacter, Streptomyces or especially the genus Mycobacterium. Examples of suitable microorganisms include: Microbacterium lactum IAM-1640, Protaminobacter alboflavus IAM-1040, Bacillus roseus IAM-1257, Bacillus sphicricus ATTC-7055, Norcardia gardneri IAM-105, Norcardia minima IAM-374, Norcardia corallina IFO-3338, Streptomyces rubescens IAM-74 or in particular the microorganisms Mycobacterium avium IFO-3082, Mycobacterium phlei IFO-3158, Mycobacterium phlei (Institute for Healthcare, Budapest No. 29), Mycobacterium phlei ATCC-354, Mycobacterium smegmatis IFO-3084, Mycobacterium smegmatis ATCC-20 , Mycobacterium smegmatis (Institute of Public Health, Budapest No.27), Mycobacterium smegmatis

ATCC-19979 and Mycobacterium fortuitum CBS-49566.

Particularly preferred microorganisms Mycobacterium spec. NRRL B-3805, Mycobacterium spec. NRRL B-3683, Mycobacterium phlei NRRL B-8154 and Mycobacterium fortuitum NRRL B-8153 with the aid of which a-sitosterol can be carried out without the use of additional 9-hydroxylation-inhibiting inhibitors.

Culturing conditions commonly used for these microorganisms are cultivated in a suitable nutrient medium under aeration, submerged cultures. Then the substrates are added to the cultures (dissolved in a suitable solvent or preferably in emulsified form) and fermented until a maximum substrate conversion is achieved.

Suitable substrate solvents are for example methanol, ethanol, glycol monomethyl ether, dimethylformamide or Dunethylsulfoxyd). Emulsification of the substrate can be effected, for example, by dissolving it in micronized form or in a water-miscible solvent (such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide or dimethyl sulfoxide) with vigorous turbulence in (preferably decalcified) water, which is the usual Emulsifying aids contains an effect. Suitable emulsifying aids are nonionic emulsifiers, such as, for example, ethyleneoxyadducts or fatty acid esters of polyglycols. As suitable emulsifiers, the commercial wetting agents Tegin ^(R) , Tween ^(RI and Span ^{| R | may be mentioned by way of} example.

The optimum substrate concentration, substrate addition time and fermentation time will depend on the structure of the substrate used and the type of microorganism used. These variables must, as is generally required in microbiological Steriodumwandlungen, in individual cases by preliminary experiments, as they are known in the art, are determined.

The 4-androstene-3,17-dione derivatives of the general formula I which can be prepared by the process according to the invention are known to be valuable intermediates which are commercially used today for the synthesis of phamakologically active silicides.

embodiment

The following examples serve to illustrate the process according to the invention.

example 1

A 2 l Erlenmeyer flask containing 500 ml of sterile nutrient medium containing

1% yeast extract

0.45% disodium hydrogen phosphate

0.34% potassium dihydrogen phosphate and

0.2% Tween ^IR1 80 - adjusted to pH 6.7-

is treated with a suspension of a Mycobacterium spec. Inoculated NRRL B-3805 dry culture and shaken for 3 days at 30 ° C at 190 revolutions per minute

 10 Erlenmeyer flasks (500ml) each containing 100ml of sterile nutrient medium containing 2.5% Comsteep liquor

0.3% diammonium hydrogen phosphate

0.25% soy flour and

0.25% Tween ^IR) 80 - adjusted to pH 7.0 -

are each 5ml of Mycobacterium spec. Inoculated seed culture and shaken for 24 hours at 30 ⁰ C at 220 revolutions per minute. Then, 100 mg of a-sitosterol dissolved in 3.0 ml of dimethylformamide are added to each culture and fermented for a further 96 hours at 30 ° C.

The combined cultures are extracted with ethylene chloride, the extract is concentrated under reduced pressure, the residue is purified by chromatography on a silica gel column and, after recrystallization from diisopropyl ether, 160 mg of 4-androstene-3,17-dione are obtained

 In addition, 385 mg of unreacted α-sitosterol are recovered.

Example 2

Under the conditions of Example 1, but using Mycobacterium spec. NRRL B-3683, a total of 1000 mg of a-sitosterol are reacted and treated, and in addition to 630 mg of unreacted α-sitosterol, 140 mg of 1,4-androstadiene-3,17-dione are obtained.

Claims (4)

claims: 1. A process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione of the general formula _έ ... symbolizes a single bond or a double bond, characterized in that a-sitosterol fermented with a culture of a steroid-degradable microbial strain; 2. A process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione according to claim 1, characterized in that a-sitosterol with a side chain degradation of sterols capable of microorganism strain of the genus Mycobacterium or Norcardia fermented. 3. Process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione according to claims 1 and 2, characterized in that a-sitosterol with Mycobacterium spec. NRRL B-3805, Mycobacterium spec. NRRL B-3683, Mycobacterium phlei NRRL B-8154 or Mycobacterium fortuitum NRRL B-8153 fermented.