CS271325B2 - Method of 1-methyl-1, 4-androstadiene-3, 17-dione production - Google Patents

Description

The present invention relates to a process for the preparation of 1-methyl-1,4-androstadiene-3,17-dione by fermentation. :

The present invention provides a process for the preparation of 1-methyl-1,4-adrostadiene, 17-dione, which comprises fermenting 1-methyl-546-androst-1-ene-3,17-dione (Metenolone) with a culture of a microorganism. of the genus Norcardia, Mycobacterium or Fusarium.

A preferred embodiment of the process for the preparation of 1-methyl-1,4-androstadiene-3,17-dione according to the invention is characterized in that the 1-methyl-5 ' -adrost-1-ene-3, 17-dione is fermented by culture of a microorganism species Norcardia corallina ATCC 14350, Mycobacterium rhodochvous ATCC 4276 or Fusarium solani ATCC 12823.

The process according to the invention is carried out under the conditions usually used in the microbiological dehydrogenation of substrates by cultures of microorganisms. Thus, generally, the most favorable fermentation conditions, such as selection of the most favorable nutrient medium, suitable solvent for the substrate or substrate suspending agent, substrate concentration, technical conditions such as temperature, aeration, pH and optimal time conditions for germination , adding the substrate and contacting the substrate with the enzyme of the microorganism, analytically, in particular by thin layer chromatography.

It has proven expedient to use concentrations of from about 100 to 2000 mg of substrate per liter of culture medium. The pH is preferably adjusted to a value in the range of from 5 to 7. The culture temperature is in the range of from 20 to 40 ° C, preferably from 25 to 35 ° C. 0.5 to 5 liters of air per minute per liter of culture broth are preferably supplied for aeration. Conversion of the substrate is conveniently monitored by analyzing extract samples. *

The microorganisms required for this fermentation are available to those skilled in the art in known collections of microorganisms. Suitable strains are Norcardia such as Norcardia corallina ATCC 14350, Mycobacterium such as Mycobacterium rodochvous ATCC 4276 or Fusarium such as Fusarium solani ATCC 12823.

After fermentation, the fermentation products are isolated in a manner known per se. Isolation can be carried out, for example, by extracting the fermentation broth with an organic solvent which is not miscible with water, such as ethyl acetate, butyl acetate or methyl isobutyl ketone, concentrating the extracts, and optionally obtaining the crude products thus obtained by chromatography and / or crystallization.

The following examples serve to illustrate the process according to the invention. However, these examples do not limit the scope of the invention in any way.

Example 1

2 liter Erlenmeyer flask containing 500 ml of nutrient solution consisting of 0,5 4 dextrose monohydrate, 0,5 4 yeast extract, 0,2% corn steep liquor and 0,1 4 peptone, pH 7,5 sterilized for 30 minutes at 120 ° C in an autoclave, inoculated with Norcardia corallina ATTC 14350 slant agar culture and shaken for 48 hours on a rotary shaker at 30 ° C.

Using a 250 ml culture culture, a 20-liter primary fermenter containing 15 liters of broth of the same composition as above was inoculated and the broth was sterilized for 30 minutes at 121 ° C and 0.11 MPa overpressure in an autoclave. . Germination is carried out with the addition of silicone SH as anti-foaming agent at 29 ° C and at an overpressure of 0.07 MPa with aeration (15 liters / min) and stirring (220 rpm) for 24 hours.

Then 0.9 liters of this culture are removed from the primary fermenter under sterile conditions and inoculated with a 20 liter main fermenter containing 14 liters of sterilized broth of the same composition as the primary fermenter. After a growth phase of 6 hours under primary fermentation conditions, a solution of 4.5 g of 1-methyl-5

CS 271 325 B2

androst-1-ene-3,17-dione in 60 ml of dimethylformamide filtered under sterile conditions and the mixture is further stirred and fermented under aeration.

After 56 hours of contact, the reaction is complete. The culture broth is extracted once with half, then twice with one third of the volume of the culture broth * using methyl isobutyl ketone, the extracts are combined and concentrated to dryness in vacuo. The residue is taken up in methanol to remove the antifoam, the insoluble fractions of the antifoam are filtered through a double pleated filter, activated carbon is added to the filtrate, the mixture is filtered and the filtrate is again concentrated to dryness. The residue was recrystallized from ethyl acetate and dried in a vacuum oven. 2.42 g (54.6% of theory) of 1-methylandrostadiene dione of melting point 164 DEG-166 DEG C. are obtained. An additional 0.45 g (10% of theory) of 1-methylan-drostadiene dione was obtained by purification of the mother liquors after crystallization by column chromatography.

Example 2

Under the conditions described in Example 1, 1-methyl-5od-androst-1-ene-3,17-dione (Metenolone) was reacted using a strain of Mycobacterium rhodochvous ATCC 4276 to give 1-methylandrostadiene dione.

Example 3 '

Under the conditions of Example 1, but using a culture medium consisting of 3 ¾ glucose, 1% corn steep liquor, 0.2% NaNOp 0.1 \ ΚΗ _{2 4 4} , 0.2 4 K ₂ HP0 ₄ , 0.05 4 MgSO ₄ . 7H ₂ O, 0.002 FeSO 4 -. 7 H ₂ O and 0.05 Ч KCl were reacted with Metenolone using the Fusarium solani strain ATCC 12823 to give 1-methylandrostadiene dione.

Claims (2)

A process for the preparation of 1-methyl-1,4-androstadiene-3,17-dione, characterized in that 1-methyl-5α-androst-1-ene-3,17-dione is fermented by culture of a Norcardia microorganism, Mycobacterium or Fusarium. 2. A process according to claim 1 for the preparation of 1-methyl-1,4-androstadiene-3,17-dione, characterized in that 1-methyl-5 ' -androst-1-ene-3,17-dione is fermented by culture. microorganisms of Norcardia corallina ATCC 14350, Mycobacterium rhodochvous ATCC 4276 or Fusarium solani ATCC 12823.