CH637993A5 - Process for the preparation of a mixture of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione - Google Patents

Description

In the context of the present invention, new microorganism mutants, namely Mycobacterium fortuitum NRRL B-8153 and Mycobacterium phlei NRRL B-8154, are used in particular for the selective degradation of steroids with 17-alkyl side chains with 2 to 10 carbon atoms, including ADD and AD. Examples of suitable steroid substrates are sitosterols, cholesterols, stigmasterols, campesterol and similar steroids with 17-alkyl side chains with 2 to 10 carbon atoms inclusive. These ste-roids can either be used in pure form or in raw form.

As already mentioned, their ability to selectively break down steroids with 17-alkyl side chains with 2 to 10 carbon atoms, which are characterized and accumulate in the nutrient broth ADD and AD, by mutation of microorganisms of the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces.

To carry out the method according to the invention, the species Mycobacterium fortuitum ATCC 6842 and Mycobacterium phlei UC 3533 were mutated to new laboratory microorganism mutants in the manner described later. In addition to listing ATCC 6842, the 1974 ATCC catalog explains the following: «J.C. Cruz 2. Cold abscess. Acta Med. Rio de Janeiro 1: 1 (1936). Medium 90 37C ». M. fortuitum ATCC 6842 does not selectively break down sterols into low-molecular compounds, for example CO2 + H2O. This microorganism is therefore not suitable for selective steroid breakdown.

When mutating M. fortuitum ATCC 6842 and M. phlei UC 3533 (UC stands for “The Upjohn Company's culture collection”) using nitrosoguanidine, new mutants are obtained that selectively add steroids with 17-alkyl side chains with 2 to 10 carbon atoms to ADD and AD are able to reduce. These microorganism mutants have been identified by the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA (the microorganism mutants were deposited at this depository in the permanent collection on February 12, 1976) with the numbers NRRL B-8153 and NRRL B-8154. A subculture of these microorganisms is on s

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637 993

Request from this depository freely available. However, it should be pointed out that the availability or delivery of the cultures does not constitute a license to a patent for the present invention.

The microorganisms used in the process according to the invention differ from the species Mycobacterium NRRL B-3805 known from US Pat. No. 3,759,791. The Mycobacterium species NRRL B-3805 shows the general properties of Mycobacterium vaccae. The latter type is a distinctly different species from the Mycobacterium species M. fortuitum and M. phlei used according to the invention. For a comparison of these microorganisms, see "Bergey's Manual of Determinative Bacteriology", 8th edition, The Williams and Wilkins Company, 1974, pages 695 and 696.

The morphology and chemical or drug sensitivity of M. fortuitum NRRL B-8153 and M. phlei NRRL B-8154 cannot be distinguished from the corresponding properties of the starting types M. fortuitum ATCC 6842 and M. phlei UC 3533. Both M. fortuitum and M. phlei cultures represent acid-fast, immobile, non-spore-forming Bacilli from the Mycobacteriaceae family of the Actinomycetales order. According to the Runyons classification [cf. E.H. Runyon «Med. Clin. North America », 43, 273 (1959)], M. fortuitum is a non-chromogenic, group IV Mycobacterium, i.e., it grows rapidly at low temperature to form unpigmented colonies on relatively simple media. M. phlei is also a group IV Mycobacterium, which, however, forms deep yellow to orange colonies when grown on simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8153 can be clearly distinguished from one another with regard to their effect on steroid molecules. As already stated, the use of M. fortuitum ATCC 6842 leads to a non-selective degradation of steroids, whereas the use of M. fortuitum NRRL B-8153 results in selective steroid degradation. This property of M. fortuitum NRRL B-8153 represents a highly valuable property. M. phlei UC 3533 and M. phlei NRRL B-8154 also differ in their effects on steroid molecules. M. phlei UC 3533 causes non-selective steroid degradation, while M. phlei NRRL B-8154 causes selective steroid degradation.

The mutation of M. fortuitum ATCC 6842 and M. phlei UC 3533 to M. fortuitum NRRL B-8153 and M. phlei NRRL B-8154 takes place with the help of nitrosoguanidine. The details of the mutation measures will be explained in more detail below. Although mutation methods are generally known, there are no teachings or even assumptions in the relevant art regarding the types of mutants that may be available when the mutation measures followed in the present case are carried out. The mutation and conversion or transformation measures explained in more detail below are described in detail for a Mycobacterium, but it is readily possible to apply similar or equivalent measures to microorganisms of the other genera mentioned.

The selective conversion can be carried out in a growing culture of M. fortuitum NRRL B-8153 or M. phlei NRRL B-8154 either by adding the respective steroid substrate to the culture during the incubation period or by adding the respective steroid substrate to the nutrient medium before Carry out inoculation. The steroid can be added either alone or in combination with another steroid. The preferred, but not absolutely necessary, concentration range for the steroid in the culture is about 0.1 to about 100 g / l. The culture is grown or grown in a nutrient medium with a carbon source such as an assimilable carbohydrate and a nitrogen source such as an assimilable nitrogen compound or a protein-like material. Preferred carbon suppliers are glucose, brown sugar, cane sugar, glycerin, starch, corn starch, lactose, dextrin, molasses and the like. Preferred nitrogen suppliers are corn steep liquor, yeast, autolysed brewing yeast with milk solids, soybean meal, cottonseed meal, corn meal, milk solids, pancreatic digestion products of casein, fish meal, distillery residues, animal peptone liquids, meat and bone waste, ammonium salts and the like. Combinations of these carbon and nitrogen suppliers can advantageously be used. The fermentation or fermentation media do not need trace metals, e.g. Zinc, magnesium, manganese, cobalt, iron and the like can be added, since tap water and unpurified components are normally used as components of the medium before the medium is sterilized.

The conversion process generally takes about 72 hours to 15 days. The incubation temperature usually ranges from about 25 ° to about 37 ° C, preferably 30 ° C for M. fortuitum NRRL B-8153 and preferably 35 ° C for M. phlei NRRL B-8154. The contents of the conversion vessel are aerated with sterilized air and preferably moved to facilitate the growth of the respective microorganism. In this way, the efficiency of the conversion process can be increased.

After the conversion, as determined by thin layer chromatography using commercially available silica gel plates and a solvent system consisting of two volumes of ethyl acetate and three volumes of cyclohexane, the converted steroid can be isolated in a conventional manner. For example, the fermentation or fermentation or conversion mixture including the fermentation broth and the microorganism cells can be extracted with a water-immiscible organic solvent for steroids. Suitable solvents are preferably dichloromethane, methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, ether, amyl acetate, benzene and the like.

On the other hand, the fermentation or fermentation broth and the cells can first be separated from one another in the customary known manner, for example by filtration or centrifugation, and then extracted separately with suitable solvents. The cells can be extracted either with a water-miscible or with a water-immiscible solvent. The cell-free fermentation and fermentation broth can be extracted with water-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtrate is distilled to dryness in vacuo. The distillation residue containing the desired converted steroids can be dissolved in a minimum amount of a 20:80 ethyl acetate / cyclohexane mixture. The resulting solution can then be chromatographed on dry silica gel using a 20:80 ethyl acetate / benzene mixture as the developer. ADD and AD can be separated from the silica gel by elution with a 15:85 ethyl acetate / chloroform mixture. The individual compounds can be isolated from the mixture by evaporating the solvent and recrystallizing from hexane.

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The compounds ADD and AD which are obtained as desired end products when carrying out the processes according to the invention are known steroid intermediates. Thus, these compounds are suitable as intermediates in the synthesis of suitable steroid hormones. ADD can be used, for example, to produce estrone using the process known from US Pat. No. 3,274,183. Testosterone can be obtained from AD according to the teachings of US Pat. Nos. 2,143,453.2,253,798, 2,264,888 and 2,356,154.

The following examples are intended to illustrate the process according to the invention in more detail. Unless otherwise stated, all percentages mean “percentages by weight”. Furthermore, all quantities in the case of solvent mixtures, unless otherwise stated, mean volumes.

example 1

Production of the mutant M. fortuitum NRRL B-8153 from M. fortuitum ATCC 6842:

(a) Nitrosoguanidine mutagenesis:

M. fortuitum ATCC 6842 cells are grown at a temperature of 28 ° C in a sterile seed medium of the following composition:

Nutrient broth (Difco) 8 g / 1

Yeast extract 1 g / 1

Glycerin 5 g / 1

made up to 11 with distilled water

The pH of the medium is adjusted to 7.0 with 1N NaOH before sterilization at 121 ° C. for 20 minutes.

The cells are grown to a density of about 5 x 108 / ml, transferred to beads by centrifugation, and then washed with an equal volume of sterile 0.1M sodium citrate, pH 5.6. The washed cells are resuspended in the same volume of citrate buffer, after which an aliquot is separated for titer determination (i.e. for cell counting). Finally, nitrosoguanidine is added to a final concentration of 50 µg / ml. The cell suspension is incubated in a water bath for 30 minutes at a temperature of 37 ° C., after which an aliquot is separated off again to determine the titer. The rest is centrifuged off and washed with an equal volume of sterile O, lm potassium phosphate with a pH of 7.0. Finally, the cells are supplied in a sterile minimal salt medium without a carbon supplier of the following composition:

NH4NO3 1.0 g / 1

K2HPO4 0.25 g / 1

MgS04-7H20 0.25 g / 1

NaCl 0.005 g / 1

FeS04-7H: 0 0.001 g / 1

made up to 11 with distilled water

resuspended. The pH of this medium is adjusted to 7.0 with IN HCl before sterilizing at 121 ° C. for 20 minutes. Finally the cells are spread out to select the mutants.

(b) Selection and isolation of the mutant M. fortuitum NRRL B-8153:

The cells mutagenized in the manner described are diluted and spread on plates which contain a medium of the following composition (modified according to Fraser and Jerrel, 1963 "J. Biol. Chem.", 205.291 to 295):

Glycerin 10.0 g / 1

K2HPO4 0.5 g / 1

NH4C1 1.0 g / 1

MgSC> 4 • 7H2O 0.5 g / 1

5 FeCb • 6H2O 0.05 g / 1

made up to 11 with distilled water

After adding 15 g / 1 agar, the medium is heated in an autoclave to a temperature of 121 ° C. for 30 minutes and then poured into sterile Petri dishes. The growth or cultivation on this medium eliminates most of the nutritional auxotrophs formed by the mutagenesis process. For example, cultures that require vitamins, growth factors and the like to grow on chemically defined media are eliminated. After about 7 days of incubation at a temperature of 28 ° C., the colonies formed are replicated on test plates suitable for selecting mutants and then back on the control plates 20 containing glycerol-based medium. The test panels are made according to the publication by G.E. Peterson, H.L. Lewis and J.R. Davis "Preparation of uniform dispersions of cholesterol and other water-insoluble carbon sources in agar media", J. Lipid Research 3,275 to 276 (1962). The minimum salt 25 medium on these plates is under (a) of Example 1

described. After adding 15 g / 1 agar and 1.0 g / 1 of a suitable carbon supplier, steroids, such as sitosterol or androstenedione (AD), are added, and the suspension obtained is then heated in an autoclave at 121 ° C. for 30 minutes. The sterile, hot mixture is then poured into a sterile mixing vessel, mixed for several minutes and finally poured into sterile Petri dishes. These measures can cause difficulties due to the formation of foam, but these can be reduced by looking through the hot mixture and by flaming the surface of the molten agar plates. In this way, uniform dispersions of water-insoluble carbon suppliers are obtained, which facilitates the production of very homogeneous but opaque agar plates. 40 The colonies grown on the control plates, but not the colonies grown on the AD containing the only carbon suppliers, are cleaned by streaking on nutrient agar plates. After growing at a temperature of 28 ° C, 45 individual clones are picked from the nutrient agar plates with the aid of sterile toothpicks and tested again by inoculating mesh-like plates with AD as carbon suppliers. Purified isolates that still have a different phenotype from the mother culture are then evaluated in 50 shake flasks.

(c) Shake bottle evaluation:

500 ml shaking bottles with 100 ml 55 of a biotransformation agent of the following composition are used:

NH4C1

3.0 g / 1

urea

0.5 g / 1

Cerelose

10.0 g / 1

KH2PO4

0.5 g / 1

MgS04-7H20

0.5 g / 1

FeCb • 6H2O

0.5 g / 1

CaCOs

3.0 g / 1

Tween 80 *

0.5 g / 1

filled up with water from the tap

1 liter

* Atlas Refinery, Inc., Newark, New Jersey

Then 1 g / 1 soy flour and then 30 g / 1 sitosterol are mixed into the medium. After heating the bottles in an autoclave to 121 ° C. for 30 minutes, they are cooled to 28 ° C. and then inoculated with 10 ml of a seed produced as follows:

The purified isolates from step (b) are grown at a temperature of 28 ° C on sloping agar. For inoculating a 500 ml bottle with 100 ml of sterile seeds of the following composition:

Nutrient broth (Difco) 8 g / 1

Yeast extract 1 g / 1

Glycerin 5 g / 1

made up to 11 with distilled water

an eyelet full of cells removed from an inclined agar is used. The pH of the seed medium is adjusted to 7.0 in an autoclave with In NaOH before heating the bottles to 121 ° C. for 30 minutes. The seed bottles are incubated for 72 hours at a temperature of 28 ° C.

As already mentioned, 10 ml of grown seeds are used to inoculate all 500 ml bottles, each with 100 ml of sterile transformation medium. The bottles are then incubated at a temperature of 28 ° to 30 ° C on a rotary vibrator, aliquots being taken from time to time. These samples each comprise 10 ml and are extracted by shaking with three volumes of methylene chloride after discharge. Portions of the extracts are by thin layer chromatography using silica gel and the solvent system described, i.e. two volumes of ethyl acetate and three volumes of cyclohexane, as well as analyzed by gas / liquid chromatography. Detection of the presence of ADD and AD confirms the selective breakdown of sitosterol by the new mutant from the mother Mycobacterium M. fortuitum ATCC 6842.

Example 2

Conversion of sitosterol to ADD and AD:

The medium used corresponds to the medium of Example 1 (c). This medium is sterilized by heating in an autoclave at 121 ° C. for 30 minutes, then cooled to 30 ° C. and finally inoculated with 10 parts of a seed culture of the mutant Mycobacterium M. fortuitum NRRL B-8153, which had been prepared in accordance with Example 1 (c) . The inoculated mixture is incubated with agitation at a temperature of 30 ° C for 336 h to promote submerged growth. After the incubation, the mixture is extracted with dichloromethane. The extract obtained is dried over anhydrous sodium sulfate and then freed from the solvent by vacuum distillation. The distillation residue obtained is then in a minimum amount of a 20:80 ethyl acetate / cyclohexane

637993

Mixture was dissolved and the solution was chromatographed on dry silica gel using a 20:80 ethyl acetate / benzene solvent system. The presence of androst-4-en-3,17-dione and Androsta-l, 4-dien-3, l7-dione results from a thin layer chromatogram. The compounds obtained are separated from the silica gel by elution with a 15:85 ethyl acetate / chloroform solvent system, whereupon the individual compounds are isolated by evaporating the solvent and recrystallizing the evaporation residue from hexane.

Example 3

A mixture of ADD and AD is also obtained by replacing M. fortuitum NRRL B-8153 with M. phlei NRRL B-8154 in Example 2 and changing the incubation temperature from 30 ° C. to 35 ° C.

Example 4

Replacing the sitosterol with cholesterol in Examples 2 and 3 gives a mixture of ADD and AD.

Example 5

Replacing the sitosterol with stigmasterol in Examples 2 and 3 gives a mixture of ADD and AD.

Example 6

Replacing the sitosterol with campesterin in Examples 2 and 3 gives a mixture of ADD and. AD.

Example 7

By adding a combination of any steroids from Examples 2 to 6 to the sitosterol in Examples 2 and 3 or by replacing the sitosterol in Examples 2 and 3 with a combination of any steroids from Examples 2 to 6, a mixture of ADD and is also obtained AD.

Example 8

By replacing Mycobacterium fortuitum ATCC 6842 or Mycobacterium phlei UC 3533 in Example 1 with a microorganism of the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia or Strepto-myces, microorganism mutants are obtained through mutations selective degradation of steroids with 17-alkyl side chains with 2 to 10 carbon atoms inclusive and for the accumulation of ADD and AD in the fermentation broth.

Example 9

A mixture of ADD and AD is likewise obtained by substituting M. fortuitum NRRL B-8153 or M. phlei NRRL B-8154 in Examples 2 to 7 with the mutants obtained according to Example 8.

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Claims (8)

637 993 1. A process for the preparation of a mixture of Androsta-l, 4-diene-3,17-dione and Androst-4-en-3,17-dione, characterized in that a microorganism mutant of Mycobacterium fortuitum or Mycobacterium phlei, the Its ability to selectively break down steroids with 17-alkyl side chains with 2 to 10 carbon atoms inclusive and to form and accumulate androsta-l, 4-diene-3,17-dione and androst-4-en-3,17- dion in the fermentation broth, grown in an aqueous nutrient medium under aerobic conditions in the presence of a steroid with 2 to 10 carbon atoms in the 17-alkyl side chain. 2. The method according to claim 1 for the preparation of a mixture of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione, characterized in that Mycobacterium fortuitum NRRL B-8153 in one aqueous nutrient medium under aerobic conditions in the presence of a steroid with 2 to 10 carbon atoms in the 17-alkyl side chain breeds. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that said steroid is selected from the group consisting of sitosterol, cholesterol, stigmasterol and cam pesterin. 4. The method according to claim 1 for the preparation of a mixture of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione, characterized in that Mycobacterium phlei NRRL B-8154 in one aqueous nutrient medium under aerobic conditions in the presence of a steroid with 2 to 10 carbon atoms in the 17-alkyl side chain. 5. The method according to claim 4, characterized in that said steroid is selected from the group consisting of sitosterol, cholesterol, stigmasterol and cam pesterin. 6. Means for performing the method according to claim 1, characterized in that one uses a microorganism mutant of Mycobacterium fortuitum or of Mycobacterium phlei. 7. Composition according to claim 6, characterized in that Mycobacterium fortuitum NRRL B-8153 is used. 8. Composition according to claim 6, characterized in that Mycobacterium phlei NRRL B-8154 is used. The conversion of steroids by microorganisms has been extensively studied and published in the literature. The earliest relevant work apparently comes from Mamoli and Vercellone in 1937 “Reports” 70,470 and “Reports” 70,2079. AaO reports the reduction of 17-ketosteroids to 17β-hydroxy-steroids using fermentation yeast. In 1952 (see US Pat. No. 2,602,769), Peterson and Murray report the 1 la hydroxylation of progesterone using the fungus Rhizopus nigricans. In 1972 (cf. US Pat. No. 3,684,657) Kraychy and co-workers report the selective microbiological degradation of steroid 17-alkyl residues by fermenting steroids with at least 8 carbon atoms in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 to form androst-4-en-3,17-dione, androst-l, 4-dien-3, l7-dione and 20a-hydroxymethyl-pregna-l, 4-dien-3-one. 1973 (see U.S. Patent No. 3,759,791) Marsheck and coworkers report the selective microbiological preparation of androst-4-en-3,17-dione by fermenting a steroid of the cholestane or stigmastane series with at least 8 carbon atoms in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805. Mutants which are characterized by their ability to selectively break down steroids with 17-alkyl side chains with 2 to 10 carbon atoms, inclusive, and in the fermentation broth (fermentation beer) androsta-l, 4-diene-3,17-dione (hereinafter referred to as ADD ) and Androst-4-en-3,17-dione (hereinafter referred to as AD) can be obtained from the microorganisms of the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, according to the mutation measures or other mutation measures explained in more detail below. Mycobacterium, Nocardia, Protaminobacter, Serratia and Strepto-myces, preferably Mycobacterium. Examples of species of these genera are M. phlei, M. smegmatis, M. rhodo-chrous, M. mucosum, M. fortuitum and M. butyricum. The process according to the invention for producing a mixture of Androsta-l, 4-diene-3, 17-dione and Androst-4-en-3,17-dione is characterized in that a microorganism mutant from Mycobacterium fortuitum or from Micobacterium phlei, the Its ability to selectively break down steroids with 17-alkyl side chains with 2 to 10 carbon atoms inclusive and to form and accumulate androsta-1,4-diene-3,17-dione and androst-4-ene-3,17- dion in the fermentation broth, in an aqueous nutrient medium under aerobic conditions in the presence of a steroid with 2 to 10 carbon atoms in the 17-alkyl side chain.