DD291341A5 - PROCESS FOR THE PREPARATION OF ANDROST-4-EN-3,17-DION, ANDROSTA-1,4-DIEN3,17-DION AND 9ALPHA-HYDROXY-ANDROST-4-EN-3,17-DION - Google Patents

Abstract

The invention relates to a microbial process for the preparation of * * (ADD) and 9a-hydroxy-androst-4-ene-3,17-dione, 9-OH-AD, which is characterized in that mixtures of complete and incomplete fermented sterols with a culture of a 3-hydroxy-D5-sterol side-chain degrading microorganism. {9a-hydroxy-androst-4-ene-3,17-dione * androst-4-ene-3,17-dione * * (ADD), mixtures of complete and incomplete sterols, microbial side chain degradation}

Description

Field of application of the invention

The present invention relates to a microbial process for the preparation of androst-4-ene-3,17-dione, androsta-1,4-dione-3,17-dione and 9a-hydroxy-androst-4-ene-3,17- dion. These products are important starting materials for the synthesis of androstane pregnane derivatives and estratrienes, which are used as active ingredients in drug manufacturing.

Characteristics of the known prior art

It is known that microorganisms utilize naturally occurring 3β-hydroxy-A ⁵ steroids (eg, cholesterol, sitosterol, campesterol, stigmasterol, ergosterol) as energy and carbon sources and degrade them to carbon dioxide and water. Intermediate AD, ADD and 9-OH-AD are formed during this degradation. In addition, it is known that enriched with the aid of modified substrates, inhibitor additives or mutated microorganisms from 3ß-hydroxy-A ^B -terols AD, ADD or 9-OH-AD (see patents: EP 274147, US-PS 4755463, GB-PS 862701, DE-OS 3521111, DD-WP 248143). A major enzymatic step is the dehydrogenation of the hydroxyl group to the keto group at C-3 and the isomerization of the double bond from C-5 to C-4 (Tallalay, P., and Wang, NS: Biochim, Biophys, Acta 18, 300-301). Furthermore, it was found that Mycobacterium Spec. NRRL B-3683 has the ability to introduce an A ⁴ double bond into 5a-cholestan-3β-ol (P.Hesselink, Sterol side ohain cleavage by Mycobacterium, Groningen, 1988, 45-46). The mechanism of this enzymatic 4-dehydrogenation has not been elucidated.

Object of the invention

The aim of the invention is to produce the title compound in an economically favorable manner by microbial side chain degradation.

Explanation of the essence of the invention

Surprisingly, it has been found that those known for the microbial side chain degradation of 3 ß-hydroxy-A ^s -terols

Process conditions and substrate mixtures of completely and not voflstänoig saturated sterols at any mixing ratio with constant product formation rate to AD, ADD and 9-OH-AD are degraded. This finding is surprising in that for both groups of sterols other enzymes or enzyme systems are necessary to produce the title compound. Likewise, it was not foreseeable that completely and not fully saturated sterols would be simultaneously reacted to the title compounds.

Apart from the use of the substrate mixture, the inventive method is constructed as it is customary for the microbial side chain degradation of 3ß-hydroxy-A ⁶ sterols.

According to the invention, the process is carried out in its basic form as follows:

A microorganism capable of microbial site killing of sterols, preferably an organism selected from the group of bacterial genera Arthrobacter, Bacillus, Brevibscterium, Coryulbacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces, in particular the strains Mycobacterium spec. NRRL B-3805, Mycobacterium spec. NRRL B-3683, Mycobacterium vaccae ZIMET11094, Mycobacterium vaccae ZIMET11053 and Mycobacterium vaccae ZIMET11052 are added to a fermentation batch.

This fermentation mixture contains the nutrients required for the growth of microorganisms, such as carbon sources such. As glucose, glycerol, etc., nitrogen sources such. As yeast extract, ammonium sulfate, etc., various mineral salts, optionally polymeric adsorbent resins and finally the mixture of saturated and not completely saturated sterols in a particular preparation form.

The preparation of the substrate preparation is carried out by known methods, e.g. Wet milling, spray drying, freeze drying, etc. Optionally, surfactants are used to prepare the substrate preparations.

The fermentation is carried out for 48 hours to 120 hours at 25 ° C to 35 ⁰ C and an acidity in the range of pH 5 to pH 9 under aerobic conditions.

The fermentation is necessary, completed by 30 minutes heating at 90 ⁰ C.

embodiments example 1 Mycobacterium spec. NRRL B-3683 is grown 5d at 31 ⁰ C on slants of the following composition:

20g glucose, 5g Bacto-peptone, 5g meat extract, 15g agar agar, distilled water. ad 1000ml, pH = 7.0.

This culture is used to inoculate a 500 ml steep breast bottle with 50 ml nutrient medium of the following composition:

10g yeast extract, 10g glucose, 1.5g of (NH ₄ J ₂ HPO _4, 0.5g KH ₂ PO ₄ 0.5 g K ₂ HPO _4, 0.2 g MgSO ₄ .7H ₂ 0,0,01 g FeSO ₄ · 7H ₂ O and 0.02g

ZnSO ₄ .7H ₂ O, Aqua ad 1000 ml pH = 7.0. After incubation for 48 hours at 8 ° C. on a rotary shaker, 5 ml of this preculture are added each time Inptung the transformation medium used. For this purpose, twenty BOC ml steep breast bottles each with 4.0g of a

polymeric adsorbent resin (Wofatit EP62), 500 mg substrate mixture (250 mg β-sito ^c (erol and 250 mg β-sitostanol) inlyophylized form and charged with 50 ml of fermentation medium.

The fermentation medium is a solution of 5.0 g glucose, 3.0 g (NH ₄ I ₂ SO ₄ , 0.3 g urea, 2.0 g KH ₂ PO ₄ , 8.0 g K ₂ HPO ₄ , 0.2 g MgSO ₄ -7 H ₂ 0,0,01 g FeSO ₄ .7H ₂ O and 0,002g ZnSO ₄ · 7H ₂ O, distilled water. ad 1000ml pH = 7.0 used. Subsequently, will

a 72-hour fermentation on a rotary table made.

Thereafter, the fermentation is terminated by heating to 9O ⁰ C. The resin is separated from the culture solution and mixed with methane!

eluted. The average yield of ADD determined by thin-layer chromatography is 4.3 g / l.

Example 2 With otherwise the same procedure as in Example 1, the strain Mycobacterium vaccae ZIMET11094 will be used. When used

of 10 g per liter Storol mixture gives 4.0 g / l ADD.

Example 3 Following the procedure in Example 1, the strain Mycobacterium spec. NRRL B-3805 used. The

The average total yield of AD determined by thin-layer chromatography is 3.5 g / l.

Example 4 According to the conditions of Example 1, the strain Mycobacterium vaccae ZIMET11053 is grown. When using 10g / l Sterol mixture is obtained 4.4 g / l 9-OH-AD. Example 5 With otherwise the same procedure as in Example 2, the substrate used is a mixture of 7 g / l β-sitostanol and 3 g / l β-sitosterol

used. The amount of ADD determined by thin-layer chromatography is 4.0 g / l ADD.

example With otherwise the same procedure as in Example 2, the substrate used is a mixture of 3 g / l β-sitostanol and 7 g / l β-sitosterol

used. This gives 4.2 g / l ADD.

Example 7 With otherwise the same procedure as in Example 2, the substrate used is a mixture of 1 g / l β-sitostanol and 9 g / l β-sitosterol

used. The amount of ADD determined by thin-layer chromatography is 4.1 g / l ADD.

Claims (3)

A process for the preparation of androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione and 9a-hydroxy-androst-4-ene-3,17-dione, characterized in that mixtures of completely and not completely saturated sterols are fermented with a microorganism which is capable of the side-chain degradation of 3β-hydroxy-A ⁶ sterols. 2. The method according to claim 1, characterized in that with Mycobacterium spec. NRRL B-3805, Mycobacterium spe'v NRRL B-3683, Mycobacterium vaccae ZIMET11094, Mycobacterium vaccae ZIMET11053 or Mycobacterium vaccae ZIMET11052 fermented. 3. The method according to claim 1 and 2, characterized in that one uses completely saturated sterols in any mixing ratio with not completely saturated sterols.