DE4129005A1 - METHOD FOR PRODUCING 14 (ALPHA) -HYDROXY-4-ANDROSTEN-3,17-DION - Google Patents

Abstract

Described is a method of preparing 14 alpha -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

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

SU patent specification 8 01 517 (ref. C.A. 110, 1 33 697u) describes a process for the production of 14α-hydroxy-4-androsten-3,17-dione by fermentation of 4-androsten-3,17-dione with a culture of Curvularia lunata. However, there is no reference in this patent contain from which public trunk collection and under which number this Microorganism can be obtained, so that a rework of the described Procedure is not possible. According to the teaching of this patent, 4-rusting is solved 3,17-dione in ethanol or dimethylformamide, fermented 0.5 to 1.5 g / l substrate 40 to 66 For hours and receives 35 to 70% obviously very contaminated 14α-hydroxy-4- androsten-3,17-dione crude product. (You can tell that from the type of processing: The fermentation batch is extracted with chloroform, the chloroform phase is concentrated and the residue boiled out with hexane. The product obtained must therefore all extractable, in Contain hexane insoluble substances, these are not only all in the fermentation batch contained steroids but also the corresponding Curvularia ingredients and Media components.)

In contrast, more than 5 g can be obtained using the method according to the invention 4-Androsten-3,17-dione per liter reproducible in about 12 hours To convert 14α-hydroxy-4-androsten-3,17-dione. The yield of process product is about 60% pure product.

The process according to the invention is carried out under the fermentation conditions, which is also used in the known microbiological conversions of substrates Curvularia lunata uses cultures.

Under the culture conditions usually used for this microorganism are grown in a suitable nutrient medium with aeration submerged cultures. Then an aqueous suspension of 4-androsten-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, for example by using this substance twice with five times the amount of water with the addition of a nonionic surfactant in a ball mill grinds until it has a grain size from about 1 to 5 µm. Suitable surfactants are, for example, ethylene oxide adducts or Fatty acid esters of polyglycols. Suitable surfactants are, for example commercially available wetting agents such as Tegin®, Tween®, or Span®.

After fermentation has taken place, the culture is prepared in the usual manner, for example by extracting them with an alcohol, ketone or ester that is difficult to dissolve in water, the extract is concentrated and the residue obtained by chromatography and / or Purifies crystallization.

The processing of the 14α-hydroxy-4-androsten-3,17-dione obtained pharmacologically active steroids is known or is carried out according to known Process (J. Amer. Chem. Soc., 91, 1969, 1228 ff; EP-A 03 00 062 and others).

The following embodiment serves to explain the invention Procedure.  

example

• a) Containing a 2 l Erlenmeyer with 1 l sterile nutrient medium
  1% Cornsteep liqour,
  1.25% soy powder,
  - adjusted to pH 6.2 -
  is 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 50 l fermenter with 30 l of 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 ³ per hour and stirring at 220 revolutions per minute .
• c) A 50 l fermenter containing 40 l of sterile nutrient solution
  1% Cornsteep liqour,
  1.25% soy powder,
  0.3% β-cyclodextrin
  - adjusted to pH 6.2 -

is inoculated with 4 l of the Curvularia lunata preculture and incubated with aeration of 2.4 m ³ 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 fermentation is continued for a further 12 hours with stirring at 220 revolutions per minute and aeration of 2.4 m ³ per hour.

• d) After fermentation, the culture broth is three times with 20 l of methyl isobutyl ketone extracted, the extract in a rotary evaporator at max. Concentrated 50 ° C under vacuum. It is then purified 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 (2)

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 used.