CS271347B2 - Method of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-diene production - 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

Process for preparing 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione

A process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione of the general formula I, in which it is a single bond or a double bond, characterized in that the isosterol is of the formula II fermentes with a culture of a strain of microorganisms capable of degrading the sterol side chains. The compounds produced are used as intermediates for the synthesis of pharmacologically active steroids.

271 347 Italy (11) (13) B2 (51) Int. Cl. c 07 and o / oo

The invention relates to a process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione. These compounds are used for the synthesis of pharmacologically active steroids.

The present invention provides a process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione of formula I

wherein,,,,, means a single or double bond consisting in that Formula II is -sitosterol

fermented by culture of a strain of microorganisms capable of breaking down the sterile side chains.

Preferably, the process according to the invention is carried out by the use of acetosterosterol of formula (II)

It is a strain of microorganisms capable of degrading the sterol side chains of Mycobacterium or Norcardia.

A preferred embodiment of the process according to the invention is further characterized in that the tosterol of the formula II is fermented with the microorganism Mycobacterium spec. NRRL Β -3-5, Mycobacterium spec. NRRL B-3683, Mycobacterium phlei NRRL B-8154 or Mycobacterium fortuitum NRRL N-8153.

Numerous microorganisms (e.g., microorganisms of the genera Arthrobacter, Brevibacterium, Microbacterium, Protaminobacter, Bacillus, Norcardia, Streptomyces, and especially Mycobacterium) are known to have a natural ability to degrade naturally occurring

3/2-hydroxy- ^{5 5} -sterols (such as cholesterol or sitosterol) to carbon dioxide and water, the intermediate being 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione .

It is further known that the addition of inhibitors or mutants of microorganisms can control the degradation of sterol by preventing the degradation of the resulting 4-androstene-3,17-dione or 1,4-androstadiene-3,17-dione (cf. German published publications). 15 43 269 and 15 93 327 as well as U.S. Pat. No. 3,684,657).

It is therefore surprising for the skilled person that under the known conditions the side chain of -sitosterol also degrades, since it is known that the degradation of the sterol side chains causes a very complex fermentation system and it was therefore not expected that all enzymes involved in the side chain degradation Natural steroids also have the ability to cause side chain degradation of this compound. In addition, it could not be assumed that the β-double bond of α-sitosterol was hydrogenated in this degradation and that the methyl group at the 4-position of the Z @ 2 was cleaved.

Apart from the use of other starting materials, the process according to the invention is carried out under the same fermentation conditions which are also used in the known microbiological reaction of sterols to degrade side chains.

According to the present invention, the fermentation is carried out using a culture of a microorganism which is usually used to break down the sterol side chains. Suitable cultures are, for example, cultures of bacteria of the genera Arhtrobacter, Bervibacterium, Microbacterium, Protaminobacter, Streptomyces or in particular of the genera Mycobacterium, capable of degrading the sterol side chains. Suitable microorganisms include, for example: Mycrobacterium lactum IAM-1640, Protaminobacter alboflavus IAM-1040, Bacillus roseus IAM-1257, Bacillus spharicus ATTC-7055, Norcardia gardneri IAM-105, Norcardia minima IAM-374, Norcardia cirallina IFO-3338, Streptomyces rubescens IAM-74 or especially microorganisms Mycobacterium avium IF0-3082, Mycobacterium phlei IFO-3158, Mycobacterium phlei (Institut für Gesundheitswesen, Budapest No. 29), Mycobacterium phlei ATCC-354, Mycobacterium smegmatis IFO-3084, 20 Mycobacterium smegmatis (Institut fur Gesundheitswesen, Budapest No. 27), Mycobacterium smegmatis ATCC-19979 and Mycobacterium fortuitum CBS-49566.

Particularly suitable are Mycobacterium spec. NRRL B-3805, Mycobacterium spec. NRRL-3683, Mycobacterium phlei NRRL B-8154 and Mycobacterium fortuitum NRRL Β-Θ153, which can be used to fermantize sitosterol without the use of additional inhibitors of 9? -Hydroxylation.

The cultivation is carried out under the conditions usually used to cultivate these microorganisms in suitable nutrient media under aeration. Subsequently, a substrate (dissolved or preferably emulsified in a suitable solvent) is added to the cultures and fermentation is performed until the maximum conversion of the substrate is achieved.

Suitable solvents for the substrate are, for example, methanol, ethanol, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide. The emulsification of the substrate can be achieved, for example, by injecting the substrate in micronized form or (dissolved) in a water-miscible solvent (such as methanol, ethanol, acetone, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide) by injection into a water (preferably freed) calcium), which contains conventional auxiliary emulsifiers. Suitable auxiliary emulsifiers are nonionic emulsifiers, such as ethylene oxide adducts or polyglycol fatty acid esters. Suitable emulsifiers include, for example, the commercially available wetting agents Tegin Tween (R) and Span

The optimum substrate concentration, substrate addition time, and fermentation duration depend on the structure of the substrate used and the type of microorganism used. These quantities must, as is generally necessary for microbiological transformations of steroids, be determined on a case-by-case basis by preliminary experiments customary to one skilled in the art.

The 4-androstene-3,17-dione derivatives of the formula I which can be prepared by the process according to the invention are known as valuable intermediates used for the synthesis of pharmacologically active steroids.

The following examples serve to illustrate the process of the invention in more detail.

Example 1

Content of a 2 liter Elenmeyer flask with 500 ml of sterile broth containing% yeast extract

0.45% disodium hydrogen phosphate

0,34% of potassium dihydrogen phosphate and

0.20% of Tween® 80 emulsifier and adjusted to pH 6.7 were seeded with a dry culture suspension of Mycobacterium spec. NRRL B-3805 and shaken for 3 days at 30 ° C and 190 rpm.

Erlenmeyer flasks (500 ml) with 100 ml sterile broth each

2.5% corn steep liquor

0.3% dinammonium hydrogen phosphate

0.25% soya meal

0.25% of Tween® 80 emulsifier, adjusted to pH 7.0, are inoculated with 5 ml of a Mycobacterium spec culture, and the flasks are shaken for 24 hours at 30 ° C and 220 rpm. A solution of 100 mg of α-sitosterol in 3.0 ml of dimethylformamide was then added to each culture and the fermentation was continued for another 96 hours at 30 ° C.

The combined cultures were extracted with ethylene chloride, the extract was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 160 mg of 4-androstene-3,17-dione after recrystallization from diisopropyl ether.

In addition, 385 mg of unreacted β-sitosterol is recovered.

Example 2

Under the conditions described in Example 1 but using Mycobacterium spec. NRRL B-3683 was reacted with a total of 1000 mg of sitosterol. After usual work-up, in addition to 630 mg of unreacted N-sitosterol, 140 mg of 1,4-androstadiene-3,17-dione is obtained.

SUBJECT OF THE INVENTION

Claims (3)

1. A process for the preparation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione of formula w. (I) in which ... means a single bond or a double bond, characterized in that Z * -gitosterol of formula II is fermented with a culture of a strain of microorganisms capable of degrading the sterol side chains. 2. A process according to claim 1, for the preparation of 4-androatene-3,17-dione and 1,4-androstadiene-3,17-dione, characterized in that α-gitosterol is fermented with a strain of a microorganism of the genus Mycobacterium or Norcardia capable of break down the sterol side chains. 3. The method of claim 1 and 2 for the production of α-andron-3, 17-dione, lnnn-5, 17-dione, characterized in that α-gitosterol is fermented by Mycobacterlum gpec. NRRL Β -3-5, Myaobacterium spec. NRRL B-36B3, Mycohactorlum phlel NRRL B-8154, or Mycoabcterium fortuitum NRRL Β-Θ153.