CH638563A5 - Process for the preparation of a mixture of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione - Google Patents

Abstract

A mixture of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione is obtained by culturing a microorganism mutant from the group consisting of Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia and Streptomyces, which mutant is distinguished by its capability for the selective degradation of steroids having 17-alkyl side chains having from 2 up to and including 10 carbon atoms and for the formation or accumulation of androsta-1,4-diene-3,17-dione and androst-4-ene-3,17-dione in the fermentation broth, in an aqueous nutrient medium under aerobic conditions in the presence of a steroid having from 2 up to and including 10 carbon atoms in the 17-alkyl side chain. The compounds obtained as desired final products when carrying out the process according to the invention are known steroid intermediates. These compounds are thus suitable as intermediates in the synthesis of suitable steroid hormones.

Description

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PATENT CLAIM In the context of the present invention, in particular

Process for the preparation of a mixture of Androstaedere new microorganism mutants, namely Mycobacte-l, 4-dien-3,17-dione and Androst-4-en-3,17-dione, characterized by rium fortuitum NRRL B-8153 and Mycobacterium phlei that a microorganism mutant NRRL B-8154 for the selective degradation of steroids with 17 from the group Arthrobacter, Bacillus, Brevibacterium, Cory- 5 alkyl side chains with 2 to 10 carbonato-nebacterium, Microbacterium, Nocardia, Protaminobacter, men to ADD and AD used. Examples of suitable steroid Serratia and Streptomyces, which are characterized by their ability to substrate are sitosterols, cholesterols, stigmasterols, camselective degradation of steroids with 17-alkyl side chains with 2 pesterin and similar steroids with 17-alkyl side chains with 2 to 10 carbon atoms inclusive and for formation up to and including 10 carbon atoms. This steroid sub or accumulation of androsta-l, 4-diene-3,17-dione and io strate can be distinguished either in pure form or in raw form for androst-4-ene-3,17-dione in the fermentation broth in one operation reach.

aqueous nutrient medium under aerobic conditions. As already mentioned, their ability to maintain a steroid with 2 to 10 carbon atoms selectively degrades steroids with 17-alkyl side chains with 2 men in the 17-alkyl side chain. up to and including 10 carbon atoms labeled and

15 mutants accumulating 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.

The conversion of steroids by microorganisms 20 To carry out the method according to the invention has been extensively investigated and the species Myco-published in the manner described later. The earliest relevant work is obviously bacterium fortuitum ATCC 6842 and Mycobacterium phlei visibly mutated by Mamoli and Vercellone in 1937, UC 3533 into new laboratory microorganism mutants.

“Reports” 70,470 and “Reports” 70,2079. In addition to the list, the reduction of 17-ketosteroids to 17ß-hydroxysteroids 25 from ATCC 6842 explains the following: «J.C. Cruz 2. Cold abscess. Acta reported using fermentation yeast. In 1952 (cf. US Pat. Med. Rio de Janeiro 1: 1 (1936). Medium 90 37C ». M. fortui-2602769) Peterson and Murray report on the 1 la hydride ATCC 6842 does not build sterols selectively to low molecular weight xylation of progesterone with the help of the fungus Rhizopus nigrilar compounds, for example CO2 + H2O. So cans. In 1972 (see US Pat. No. 3,684,657) Kraychy and co-workers said that this microorganism was not suitable for selective workers on the selective microbiological degradation of steroid degradation.

roidic 17-alkyl residues by fermenting steroids with the mutation of M. fortuitum ATCC 6842 and M.

at least 8 carbon atoms in the 17-alkyl side chain phlei UC 3533 (UC stands for “The culture collection of The with Mycobacterium sp. NRRL B-3683 to form Upjohn Company”) new nitrosoguanidine is obtained

Androst-4-en-3,17-dione, androst-l, 4-diene-3,17-dione and 20a mutants, which selectively steroids with 17-alkyl side chains with 2 hydroxymethyl-pregna-l, 4-diene-3 -on. 1973 (cf. US Pat. No. 35 up to and including 10 carbon atoms for ADD and AD

3759791) report Marsheck and co-workers about the selective mining ability. These microorganism mutants have been microbiologically prepared from androst-4-en-3, 17-dione by the Northern Regional Research Laboratory, U.S. by fermenting a steroid from the Cholestan or Stigma Department of Agriculture, Peoria, Illinois, USA (the stan row with at least 8 carbon atoms in the 17-alkyl microorganism mutants were placed on this deposit side chain with Mycobacterium sp. NRRL B-3805. 40 in the Permanent collection deposited on February 12, 1976)

Mutants that are assigned the numbers NRRL B-8153 and NRRL B-8154 by their ability to selectively break down, from steroids with 17-alkyl side chains with 2 to inclusive. A subculture of these microorganisms is characterized by the request of 10 carbon atoms and in which this Depository freely available. However, it is fermentation beer Androsta-l, 4-dien-3,17-dione (note that the availability or delivery of the following ADD indicates) and Androst-4-en-3, l 7-dion (in the case of 45 crops, no license to a product that is referred to as AD after this one), one obtains a justified patent according to the invention.

Mutation measures explained in more detail below or the mutation measures from microorganisms of the microorganisms used, which are different within the scope of the method according to the invention, differ from the species Mycobacterium terium, Microbacterium, Mycobacterium known from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebac or the aforementioned US Pat. No. 3,759,791 , Nocardia, Protami-50 NRRL B-3805. The Mycobacterium species NRRL B-3805 shows nobacter, Serratia and Streptomyces, preferably Mycobac- the general properties of Mycobacterium vaccae. terium. Examples of species of these genera are M. phlei, M. The latter species is one of the smegmatis, M. rhodochrous, M. mueosum, M. fortuitum and Mycobacterium species M. fortuitum and M. phlei. M. butyricum. different kind. With regard to a comparison of these microorganisms

For the method according to the invention for producing a 55 nism, see “Bergey's Manual of Determinative Bacterio-Mixture of Androsta-l, 4-dien-3, l7-dion and Androst-l0gy”, 8th Edition, The Williams and Wilkins Company, 1974,

4-en-3, l 7-dione is characterized in that it has a pages 695 and 696.

Microorganism mutants from the Arthrobacter group, The morphology and the chemicals or drug

Bacillus, Brevibacterium, Corynebacterium, Microbacterium, sensitivity of M. fortuitum NRRL B-8153 and M. phlei Nocardia, Protaminobacter, Serratia and Streptomyces, the eo NRRL B-8154 are characterized by their ability to selectively break down steroids. indistinguishable from the starting species M. fortuitum ATCC 6842 and M. phlei UC with 17-alkyl side chains with 2 to 10 Koh- 3533 inclusive. Both M. fortuitum and M. lenstoffatom and for the formation or accumulation of phlei cultures provide acid-proof, immobile, non-spores-

Androsta-1,4-diene-3,17-dione and androst-4-en-3, l 7-dione in forming Bacilli from the family Mycobacteriaceae of the Ord-the fermentation broth, in an aqueous nutrient medium it act Actomyomycetales the Runyons classification under aerobic conditions in the presence of a steroid with 2 (cf. EH Runyon "Med. Clin. North America", 43.273 up to and including 10 carbon atoms in the 17-alkylsei- [1959]) is M. fortuitum a non-chromotene chain is growing. genes Mycobacterium belonging to group IV, i.e. it

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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 mentioned, when using M. fortuitum ATCC 6842 there is a non-selective degradation of steroids, when using M. fortuitum NRRL B-8153, however, a selective steroid degradation takes place. 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 effect 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 is carried out 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 accomplished 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 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 cultured 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 proteinaceous 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 must 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 has been completed, determined by thin layer chromatography using commercially available silica gel plates and a solvent system comprising 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 or 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.

The compounds ADD and AD which are obtained as desired end products when carrying out the process 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, 2253798, 2264888 and 2356154.

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 1 1 with distilled water

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15

20th

25th

30th

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40

45

50

55

60

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The pH of the medium is adjusted to 7.0 with IN 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 up to a final concentration of 50 p. g / ml added. 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

FeS04-7H20 0.001 g / 1

made up to 1 1 with distilled water

mixed and finally poured into sterile Petri dishes. These measures can lead to difficulties due to foam formation, but these can be reduced by mixing 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.

10 The colonies grown on the control plates, but not the colonies grown on the test plates containing AD as the sole carbon supplier, are cleaned by streaking on nutrient agar plates. After growing at a temperature of 28 ° C, 15 sterile toothpicks are used to pick individual clones from the nutrient agar plates and to test them again by inoculating mesh-like patterned plates with AD as the carbon supplier. Purified isolates, which still have a different phenotype from the mother culture, are then evaluated in shake bottles 20.

(c) Shake bottle evaluation:

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

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):

NH4CI 3.0 g / 1

Urea 0.5 g / 1

Cerelose 10.0 g / 1

30 KH2PO4 0.5 g / 1

MgS04-7H20 0.5 g / 1

FeCl3-6H20 0.5 g / 1

CaCOa 3.0 g / 1

Tween 80 * 0.5 g / 1

35 made up to 1 liter with tap water

* Atlas Refinery, Inc., Newark, New Jersey

Glycerin 10.0 g / 1

K2HPO4 0.5 g / 1 40

NH4CI 1.0 g / 1

MgS04-7H20 0.5 g / 1

FeCl3-6H20 0.05 g / 1

made up to 1 1 with distilled water

45

After adding 15 g / 1 agar, the medium is heated in an autoclave to a temperature of 121 ° C. for 20 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 55 test plates suitable for selecting mutants and then back on the control plates containing glycerol-based medium. The test panels are made according to the publication by G.E. Peter-son, H.L. Lewis and J.R. Davis "Preparation of uniform dispersions of cholesterol and other water-insoluble carbon sour-60 ces in agar media", J. Lipid Research 3,275 to 276 (1962). The minimum salt medium on these plates is described in (a) of Example 1. 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, 65 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 for several minutes

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:

Broth (Difco)

Yeast extract

Make up glycerin with distilled water

8 g / 1 lg / 1 5 g / 1 11

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 bottle 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.

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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. 5

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 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), inoculates. The inoculated mixture is incubated under agitation for 336 hours at a temperature of 30 ° C 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 dissolved in a minimum amount of a 20:80 ethyl acetate / cyclohexane mixture, and the solution is chromatographed on dry silica gel using a 20:80 ethyl acetate / benzene solvent system. The presence of androst-4-en-3, l7-dione and 25 androsta-l, 4-diene-3,17-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. 35 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 Streptomyces, microorganism mutants are obtained which are degraded by their ability to break down of steroids with 17-alkyl side chains with 2 to 10 carbon atoms and for the accumulation of ADD and AD in the fermentation broth.

Example 9

By replacing the M. fortuitum NRRL B-8153 or M. phlei NRRL B-8154 in Examples 2 to 7 with the mutants obtained according to Example 8, a mixture of ADD and AD is also obtained.

G