SK279666B6 - Method of preparing 17-oxosteroids - Google Patents

Abstract

Described is a method of preparing 17-oxosteroids by the fermentative oxidation of 17beta-hydroxysteroids, characterized in that a bacterial culture of the species Mycobacterium spec. NRRL B-3805, Mycobacterium spec. NRRL B-3683, Mycobacterium phlei NRRL B-8154 or Mycobacterium fortuitum NRRL B-8153 is used for the fermentation.

Description

Technical field

The invention relates to a process for the preparation of 17-oxosteroids by fermentative oxidation of 17β-hydroxysteroids.

BACKGROUND OF THE INVENTION

The method of fermentative production of 17-oxosteroids has been known for a long time. As early as 1938, A. Vercellone and L. Momoli described a process for the production of 4-androsten-3,17-dione by fermentation of 5-androsten-3, 17β-diol (Ber. 71, 1938, 152-155). This method provides high yields of the product of the process when suitable microorganisms are selected (Czech 87.068; ref. C.A. 54, 1960, 2441). There are also many publications describing that 17-oxosteroids can be produced (such as 4-androsten-3,17-dione, 1,4-androstadiene-3,17-dione or 3-hydroxy-1, 3,5,10 (-estratrien-17-one) to obtain high yields by fermentative degradation of the side chains of substrates that carry the hydrocarbon chain at the 17β position.

Methods by which the 17β-hydroxysteroids can be oxidized microbiologically in good yield to 17-oxosteroids without additional conversion of steroids are known are very little known. N.Welsh and C.Heusghem describe a method by which estradiol can be converted to estrone in 95% yield, but this method is not reproducible because the organism used by the authors is not available to the public (Compt. Rend. Soc. Biol .., 142 (1948, 1074).

SUMMARY OF THE INVENTION

There has now been a great need for such a fermentative process, since chemical oxidation processes are indeed very expensive. Otherwise, chemical processes have the drawback that they are carried out using reagents such as pyridine and sulfur trioxide, the removal of which is very problematic from an environmental point of view.

It has now been found that, surprisingly, it is possible to produce 17-oxosteroids in high yields by using bacterial cultures of Mycobacterium sp. NRRL B-3805, Mycobacterium sp NRRL B-3683, Mycobacterium phlei NRRL B-8154 or Mycobacterium fortuitum NRRL B-8153.

The process of the present invention is carried out under the same conditions as known feeder transformations, using the microorganisms of these bacterial cultures.

Under the conditions commonly used for said bacterial cultures, a submerged culture is grown in a suitable nutrient medium under aeration. Subsequently, the substrate (dissolved in a suitable solvent or in an emulsified form) is added to the culture and fermented 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. For example, the emulsification of the substrate may be carried out by spraying it in micronized form or dissolved in a water-miscible solvent (such as methanol, ethyl alcohol, acetone, glycol monomethyl ether, dimethylformamide or dimethylsulfoxide), injected into water (preferably cooled) under strong turbulence, which contains conventional emulsifying aids. Suitable emulsifying auxiliaries are non-ionic emulsifiers such as adducts of ethylene oxide or polyglycol fatty acid esters. Suitable emulsifiers include commercially available wetting agents Tegin®, Tween® and Span®.

The optimum substrate concentration, substrate addition time and fermentation time are dependent upon the type of substrate used and the microorganism used and the fermentation conditions. These quantities must be continuously detected by preliminary experiments, as is generally known to those skilled in the art of microbiological transformation of steroids, for each possible individual case.

Preferably, 17β-hydroxysteroids of formula (I) are used as substrates for carrying out the process of the present invention.

in which it means

R 1 is a hydrogen atom or a methyl group

The St residue of an estran-17β-lead derivative containing up to 5 non-accumulated double bonds substituted at the 3-position by a phenolic hydroxyl, lower alkoxy, lower 1-alkoxy-alkoxy, 2-tetrahydropyranyloxy, or lower alkylenedioxy group as well as optionally in position 5 , 10 with an epoxy group and at position 19 with a methyl group, respectively. A lower alkoxy group is preferably a group containing up to 4 carbon atoms. Suitable alkoxy groups are, for example, methoxy or tert-butyloxy.

The term lower 1-alkoxy-alkoxy means preferably lower alkoxy-methoxy having up to 4 carbon atoms and alkoxy. An example is 1-methoxymethoxy. The term lower alkylenedioxy is especially understood to mean the group having 2 to 6 carbon atoms, such as, for example, ethylenedioxy or 2,2-dimethyl-propylenedioxy.

A substrate which is suitable for carrying out the process of the present invention is, for example, a derivative of formula (Ia)

SK 279666-6

in which it means

R 2 is a hydrogen atom or an alkyl group of up to 4 carbon atoms; and

I I

-A-B-C-D- groups of partial formulas

I I II

-CH 2 -CH 2 -CH-CH-, -ch ₂ -ch = c-ch

Or -CH = CH-C = C and

R 1 is as defined above.

The estran-17-one derivatives or the 3-hydroxy compounds obtained from these substrates, which are obtained by ether cleavage, are known to be pharmacologically active and can furthermore be reduced to the corresponding 17 [alpha] -hydroxy compounds or converted to the corresponding 17 [beta] -hydroxy- etinylzlúčeniny.

Suitable substrates are furthermore estran derivatives of formula (Ib)

in which it means

R 3 is an alkyl group of up to 4 carbon atoms, a single or double bond; and

R 1 is as defined above

The 17-oxo-steroids formed from these compounds can, as is known, serve for the preparation of pharmacologically active 17α-ethynyl-17β-hydroxy-4-ester-3ones or their 18-methyl-homologues.

Compounds of formula (Ic) are suitable substrates to be considered.

production of pregnane derivatives, as is known to those skilled in the art.

Finally, estran-17β-ol derivatives of formula (Id) may also be contemplated.

in which it means

R 9 is a hydrogen atom as well as R 7 and R 8 together are lower alkylenedioxy, or

R 7 and R 9 are two hydrogen atoms or together a carbon-carbon bond a

R8 is lower alkoxy, lower 1-alkoxy-alkoxy or 2-tetrahydropyranyloxy and R1 and R5 are as defined above

The ketones obtained from these compounds can be converted to antigestagenically active steroids as described in EP-A-0129499.

DETAILED DESCRIPTION OF THE INVENTION

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

Example 1

(a) Erlenmeyer flask of 500 ml capacity, filled with 100 ml of sterile nutrient medium containing 1% yeast extract

0.45% disodium phosphate dihydrate

0,34% of potassium dihydrogen phosphate and

0.2% tween 80, is inoculated with a suspension of Mycobacterium sp. NRRL B-3683 and shaken at 180 rpm at 30 ° C for 72 hours.

(b) 50 Erlenmeyer flasks (100) ml each containing 20 ml sterile medium composed of 0,5% comsteep liquor

0.05% glucose monohydrate

0.2% yeast extract is inoculated at pH 7 with 1 ml of a pre-cultured Mycobacterium sp. and these flasks are incubated for 24 hours on a rotary shaker at 220 rpm and at 30 ° C.

Thereafter, each culture was mixed with 0.02 g of 3,3- (2,2-dimethyltrimethylenedioxy) -5,10 α-epoxy-5α-estra-9 (11) -en-17β-ol dissolved in 0.2 mL dimethylformamide and sterile filtered, and fermented for an additional 48 hours.

c) Extract the combined cultures with methyl isobutyl ketone and concentrate the extract in a rotary shaker at a maximum of 50 ° C under vacuum. Then purify by silica gel column chromatography.

in which it means

R4 is a hydrogen atom or a methyl group, I <5 and R8 together are a lower alkyldioxo group or R5 a hydrogen atom and

R 6 is lower alkoxy, lower 1-alkoxy-alkoxy or 2-tetrahydropyranyl and

R1 is as defined above

The 17-oxosteroids obtained from these compounds can be further processed in the same manner as the steroids from estran-17β-ol derivatives of formula (Ic); on the other hand, they can also serve to

0.85 g of 3,3 (2,2-dimethyltrimethylenedioxy) -5,10α-epoxy-5α-estra-9 (11) -en-170-one is obtained, which is identical to HPLC with an authentic sample.

Example 2

a) The production of the inoculum culture proceeds as described in Example 1a)

(b) 50 ml 100 ml Erlenmeyer flasks are filled with 20 ml sterile nutrient medium

2.5% comsteep liquor

0.25% soya meal

0.3% ammonium hydrogen phosphate

The pH of the 0.25% tween 80 is adjusted to 6.5 and inoculated with 1 ml of a pre-cultured Mycobacterium sp. Culture and incubated on a rotary shaker at 220 rpm for 24 hours at 30 ° C.

After incubation, 0.002 g of α-equilol dissolved in 0.2 ml of dimethylformamide is added to each culture and fermented for another 72 hours at 30 ° C.

c) Isolation of the product proceeds as described in Example 1c).

In this way, 0.095 g of equilin is obtained, which is identical to an authentic sample by HPLC.

Claims (2)

PATENT CLAIMS Process for the production of 17-oxosteroids by fermentative oxidation of 17β-hydroxysteroids, characterized in that bacterial cultures of Mycobacterium sp. NRRL B-3805, Mycobacterium sp. NRRL B-3683, Mycobacterium phlei NRRL B-8154, or Mycobacterium fortuitum NRRL B-8153. Process according to claim 1, characterized in that the 17β-hydroxysteroid of formula (I) is oxidized with - R 1 is a hydrogen atom or a methyl group The St radical of an estran-17β-ol derivative containing up to 5 non-accumulated double bonds substituted at the 3-position with a phenolic hydroxyl, lower alkoxy, lower 1-alkoxy-alkoxy, 2-tetrahydropyranyloxy or lower alkylenedioxy group as well as optionally in the 5-position, 10 is optionally substituted with epoxy and in position 19 with methyl.