FI86203B - Method for production of 1-methyl-1,4-androstadiene-3,17- dione - Google Patents

Description

1 86203

Process for the preparation of 1-methyl-1,4-androstadiene-3,17-dione

The invention relates to a process for the preparation of 1-methyl-1,4-androsta-5-diene-3,17-dione, which process is characterized by the fermentation of 17β-hydroxy-1-methyl-5α-androst-1-en-3-one ( methenolone) with a culture of microorganisms belonging to the genus Nocardia, Mycobacterium or Fusarium. The process according to the invention is carried out under conditions commonly used in the microbiological dehydration of substrates by cultures of microorganisms. Thus, in general, the most preferred fermentation conditions, such as the most preferred medium, a suitable substrate solvent or suspending agent, the substrate concentration, technical conditions such as temperature, aeration and pH, and the growth of the substrate, are generally determined analytically, in particular by thin layer chromatography, in conventional preliminary experiments. 20 and selection of the best time for contacting the substrate and the microorganism enzyme.

In this case, it has proved expedient to use substrate concentrations of about 100 to 2000 mg / l of culture medium. The pH is preferably adjusted to a value in the range of 5 to 25 7. The growth temperature is in the range of 20 to 40 ° C, preferably 25 to 35 ° C. The aeration is preferably carried out by passing 0.5 to 5 l of air per minute per liter of medium. It is appropriate to monitor substrate change by analyzing sample extracts.

The microorganisms required for this fermentation are freely available to those skilled in the art from known collections of microorganisms. Suitable strains include Nocardia such as Nocardia coralline ATCC 14350, mycobacteria such as Mycobacterium rhodochrous ATCC 4276, or Fusariums 35 such as Fusarium solani ATCC 12823.

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After fermentation, the fermentation products are isolated in a manner known per se. The isolation can be carried out, for example, by extracting the fermentation broths with a water-immiscible organic solvent such as ethyl acetate, butyl acetate or methyl isobutyl ketone, concentrating the extracts and purifying the crude products thus obtained, if necessary by chromatography and / or crystallization.

It is known from FI application 842 457 that 1-methyl-1,4-androstadiene-3,17-dione is a pharmacologically active compound which inhibits estrane biosynthesis. According to the state of the art, the compound is chemically prepared from 17β-hydroxy-1-methyl-1,4-androstadien-3-one, which in turn is prepared by oxidation of the master-15 ion with SeO 2 (DE Patent 1,174,778, Chem. Abstr. 61 (1964)). ) 12059a). The disadvantage of the process is the poor yields of about 15% including both process steps.

By the process of the invention, on the other hand, 1-methyl-1,4-androstadiene-3,17-dione can be prepared by a single-step reaction in a yield of more than 60%.

It is known from the publications cited in Microbial Transformations of Steroids, Academic Press 1967, page 89, that the analogous compound 1,4-and-25 rostadiene-3,17-dione can be prepared only in very poor yield from steroids saturated at the 4,5-position, and even in the dehydration of 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione is formed only in very unsatisfactory yields.

The following embodiments illustrate the method according to the invention.

. . Example 1 In a 2 l Erlenmeyer flask containing 500 ml of sterilized cereal in an autoclave at 120 ° C for 30 minutes. 35 vintages containing 0.5% dextrose monohydrate-3 8c203, 0.5% yeast extract, 0.2% corn broth and 0.1% peptone, pH 7.5 are inoculated into an agar surface culture of Nocardia Corallina strain ATCC 14350 and shaken 48 hours on a rotary shaker at 30 ° C.

5,250 ml of the above preculture are inoculated into a 20 l pre-fermentor in which 15 l of a medium having a composition similar to that used for the preculture has been sterilized for 30 minutes at an overpressure of 1.1 bar at 121 ° C. Silicon SH is added as antifoam and the mixture is fermented for 24 hours at 29 ° C and 0.7 bar overpressure with aeration (15 l / min) and stirring (220 rpm).

The 0.9 L pre-fermenter culture is then removed under sterile conditions and inoculated into a 20 L 15 main fermentor containing 14 L of sterilized nutrient solution having the same composition as that used for the pre-fermenter culture. The culture is grown for 6 hours under pre-fermentation conditions, a sterilized solution containing 4.5 g of methenolone in 60 ml of diform-20 amide is added by filtration, and stirring and aeration are continued.

After a contact time of 56 hours, the reaction is complete. The culture broth is extracted once with half and then twice with one third of the culture volume with methyl isobutyl ketone, the extracts are concentrated and evaporated to dryness under reduced pressure. The residue is stirred to remove the antifoam in methanol, the insoluble antifoam is separated by filtration through a double corrugated filter, the solution is treated with activated carbon and re-evaporated to dryness. The residue is then recrystallized from ethyl acetate and dried in a vacuum oven. 2.42 g (54.6% of theory) of 1-methylandrostadiene dione with a melting point of 164-166 ° C are obtained. An additional 0.45 g (10% of theory) of 1-methylandrostadiene dione is obtained by purification of 35 column chromatography.

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Example 2

Under the conditions of Example 1, methenolone is reacted to 1-methylandrostadiene dione using Mycobacterium rhodochrous strain ATCC 4276.

5 Example 3

Under the conditions of Example 1, but using a medium containing 3% glucose, 1% corn steep liquor 0.2% NaNO 3, 0.1% KH 2 PO 4, 0.2% K 2 HPO 4, 0.05% MgSO 4 · 7H 2 O, 0.002% FeSO 4 · 7H 2 O and 0.05% 10 KCl, reacting methenolone to 1-methylandrostadiene dione using Fusarium solani strain ATCC 12823.

Claims (2)

A process for preparing 1-methyl-1,4-androstadien-3,17-dione, characterized in that the 17e-hydroxy-1-methyl-5a-androst-1-en-3-one (methenolone) is fermented. with a microorganism culture of the genus No-cardia, Mycobacterium or Fusarium. 2. A process for preparing 1-methyl-1,4-and-rostadiene-3,17-dione according to claim 1, characterized in that the fermentation is carried out with a microorganism culture of the strain Nocardia corallina ATCC 14350, Mycobacterium rhodochrous ATCC 4276 or Fusarium solani ATCC 12823. Il