WO1993005170A1

WO1993005170A1 - METHOD OF PREPARING 14α-HYDROXY-4-ANDROSTENE-3,17-DIONE

Info

Publication number: WO1993005170A1
Application number: PCT/EP1992/001797
Authority: WO
Inventors: Alfred Weber; Mario Kennecke
Original assignee: Schering Aktiengesellschaft Berlin Und Bergkamen
Priority date: 1991-08-28
Filing date: 1992-08-07
Publication date: 1993-03-18
Also published as: JPH07501689A; DE4129005A1; EP0600946A1

Abstract

Described is a method of preparing 14α-hydroxy-4-androstene-3,17-dione with a culture of Curvularia lunata. The method is characterized in that 4-androstene-3,17-dione is suspended in water and the suspension thus obtained fermented.

Description

Process for the preparation of 14α-hydroxy-4-androstene-3,17-dione

The invention relates to a process for the preparation of 14α-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione with a culture of Curvularia lunata, which is characterized in that the 4- Androsten-3,17-dione suspended in water and the suspension obtained fermented.

SU patent 801 517 (ref. CA. 110, 133697u) describes a process for the preparation of 14α-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione a culture of Curvularia lunata. However, this patent does not contain any reference from which public stock collection and under which number this microorganism can be obtained, so that it is not possible to rework the described method. According to the teaching of this patent, the 4-androstene-3,17-dione is dissolved in ethanol or dimethylformamide, 0.5 to 1.5 g / l of substrate is fermented for 40 to 66 hours and 35 to 70% is obviously obtained very strongly contaminated crude 14α-hydroxy-4-androsten-3,17-dione product. (You can tell that from the way it is worked up: the fermentation batch is extracted with chloroform, the chloroform phase is concentrated and the residue is boiled with hexane. The product obtained must therefore contain all extractable substances insoluble in hexane, not all of them Steroids contained in the fermentation batch but also the corresponding Curvularia ingredients and media components.)

In contrast, more than 5 g of 4-androstene-3,17-dione per liter can be reproducibly converted into 14α-hydroxy-4-androstene-3,17-dione within about 12 hours using the process according to the invention. The yield of process product is about 60% pure product.

The process according to the invention is carried out under the fermentation conditions which are also used in the known microbiological conversions of substrates with Curvularia lunata cultures. Under the culture conditions usually used for this microorganism, submerged cultures are grown in a suitable nutrient medium with aeration. Then an aqueous suspension of 4-androstene-3,17-dione is added to the culture and cemented until a maximum substrate conversion is reached. The aqueous suspension of 4-androsten-3,17-dione can be prepared in a simple manner by, for example, grinding this substance with twice at five times the amount of water with the addition of a non-ionic surfactant in a ball mill until it has a particle size of approximately 1 to 5 μ. Suitable surfactants are, for example, ethylene oxide adducts or fatty acid esters of polyglycols. Suitable surfactants include, for example, commercially available wetting agents such as Tegin ®, Tween ®, or Span ®.

After the fermentation has taken place, the culture is prepared in the customary manner, for example by extracting it with an alcohol, ketone or ester which is poorly soluble in water, concentrating the extract and purifying the residue obtained by chromatography and / or crystallization.

The further processing of the 14α-hydroxy-4-androsten-3,17-dione obtained is known to pharmacologically active steroids or is carried out by processes known per se (J. Amer. Chem. Soc. 91, 1969, 1228 ff; EP-A 0300062 and more).

The following exemplary embodiment serves to explain the method according to the invention in more detail.

Example:

a) Containing a 21 Erlenmeyer with 11 sterile nutrient medium

1 9/6 Cornsteep liqour

1.25% soy powder

- adjusted to pH 6.2 - inoculated with 5 ml of a suspension of a Curvularia lunata NRRL 2380 - culture and shaken for 60 hours at 30 ° C at 180 revolutions per minute.

b) A 501 fermenter with 301 sterile nutrient solution as indicated under a) is inoculated with 1 l of the Curvularia lunata culture and incubated for 18 hours at 30 ° C. with aeration of 2 m 2 / hour and stirring at 220 revolutions per minute.

c) Containing a 501 fermenter with 401 sterile nutrient solution

1% Cornsteep liqour

1.25% soy powder

0.3% β-cyclodextrin

- adjusted to pH 6.2 - inoculated with 41 of the Curvularia lunata preculture and incubated with aeration of 2.4 m3 per hour and stirring at 150 revolutions per minute at 30 ° C for 7 hours.

Then 250 g of 4-androstaen-3,17-dione are added to the culture as a grinding in 1000 ml of water with 25 g of Tween®80 and fermented for a further 12 hours with stirring at 220 revolutions per minute and aeration of 2.4 m ^ per hour.

d) After fermentation, the culture broth is extracted three times with 201 methyl isobutyl ketone, the extract in a rotary evaporator at max. Concentrated 50 ° C under vacuum. This is followed by purification by chromatography on a silica gel column using a hexane-acetone gradient.

150 g of 14α-hydroxy-androsta-4-en-3,17-dione with a melting point of 257-259 ° C. are obtained.

Claims

Claims:

1. Process for the preparation of 14α-hydroxy-4-androstene-3,17-dione by fermentation of 4-androstene-3,17-dione with a culture of Curvularia lunata, characterized in that the 4-androstene-3, 17-dione suspended in water and the suspensions obtained fermented.

2. Process for the preparation of 14α-hydroxy-4-androsten-3,17-dione according to claim 1, characterized in that a culture of Curvularia lunata NRRL 2380 is used for the fermentation.