GB1590651A - Preparation of 17-ketosteroids by microbiological degradation of 17-hydrocarbylsteroids - Google Patents

Description

(54) PREPARATION OF 17-KETOSTEROIDS BY MICROBIOLOGICAL DEGRADATION OF 1 7-HYDROCARBYLSTEROIDS (71) We, THE UPJOHN COMPANY, a corporation organised and existing under the Laws of the State of Delaware, United States of America, of 301 Henrietta Street, Kalamazoo, State of Michigan, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a microbiological process for preparing androsta-1,4-dione-3,17dione (hereinafter referred to as ADD) and androst-4-ene-3,17-dione (hereinafter referred to as AD).

The transformation of steroids by microorganisms has been widely studied and documented. Apparently, the earliest such work was by Mamoli and Vercellone in 1937, Ber.

70, 470 and Ber. 70, 2079. They disclosed the reduction of 17-ketosteroids to 17,ss-hydroxy- steroids by fermenting yeast. U.S. Patent Specification No. 2,602,769 (1952) discloses the 11e hydroxylation of progesterone with the fungus Rhizopus nigricans. British Patent Specification No. 1,092,145 discloses a process for preparing ADD and AD from a suitable steroid by the action of microorganisms having a steroldecomposing ability in the presence of a chelating agent capable of forming a chelate with iron and/or copper. British Patent Specifications Nos. 1,113,887 and 1,208,078 disclose a process for degrading steroids to ADD and, in small amounts, AD, by using a microorganism of the Mycobacterium, Arthrobacter or Nocardia species, in the presence of heavy metal ions which are specified as nickel, cadmium, cobalt, lead or selenite ions. In the earliest of the above British Specifications, the presence of the chelating agent inhibits degradation of the steroid beyond ADD, and the same function is achieved by the presence of the heavy metal or selenite ions in the second and third of the above-mentioned British Patent Specifications.

In "Microbiological Transformations of Steroids - a Handbook" by W. Chamey and H. L. Herzog, Academic Press (1967) page 51, it is disclosed that cholesterol was degraded in poor yield by Nocardia sp. into 3-ketobisnor4-cholenic acid, 3-ketobisnor- 1,4-choladienic acid, AD and ADD. It is clear that neither AD nor ADD is accumulated in the fermentation medium.

British Patent Specification No. 1,327,888 discloses the selective mircobiological degradation of 17-alkyl steroids by fermenting a steroid containing at least 8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B3683 to prepare AD, ADD and 2O hydroxymethylpregna-l,Sdien-3-one. British Patent Specification No. 1,329,387 discloses the selective microbiological preparation of androst-4-ene-3,17-dione by fermenting a steroid of the cholestane or stigmastane series containing at least 8 carbon atoms in the 17alkyl side chain with Mycobacterium sp. NRRL B--3805.

According to the present invention, a process for preparing a fermentation beer comprising a mixture of AD and ADD comprises cultivating a mutant obtained from a microorganism in an aqueous nutrient medium under aerobic conditions in the presence of one or more 17-(C2 to hydrocarbyl) steroids, the mutant being characterised by its ability to selectively degrade 17-hydrocarbyl steroids as defined above and accumulate AD and ADD in the fermentation beer in the absence of added chelating agents and heavy metal and selenite ions. The microorganisms which can be used in this invention are mutants obtained from steroid-degrading microorganisms of the following genera by using the mutation procedures disclosed below, or other mutation procedures: Arthrobacter, Bacillus, Brevi- bacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces. Preferred species are Arthrobacter simplex, Arthrobacter globiformis, Bacillus sphaericus, Bacillus roseus, Brevibacterium lipolyticum, Coryncbactci-ium equi, Corynebacterium sepedonicum, Nocardia corallina, Nocardia erythropolis, Nocardia gardneri, Protaminobacter alboflavous, Serratia marcescens, Streptomyces rubescens, Strew to myces tanashiensis and Streptomyces venezuellae. The preferred genus is Mycobacterium.

Exemplary species of this genus are M. phlei, M. smegmatis, M. rhodochrous, M. mucosum, M. fortuitum and M. butyricum. Novel mutant microorganisms having the required property are Mycobacterium tortuitum, NRRL B8153, and Mycobacterium phlei, NRRL B8154, which are both described and claimed in British Patent Application No. 8007922 (Serial No. 1,590,652).

Suitable steroid substrates are androst-5-en3-ols having a C2 10 hydrocarbyl, e.g. C2 l0 alkyl, substituent at the 17-position. Preferred steroids are sitosterols (particularly gB-sito- sterol), cholesterol, stigmasterol and campesterol. These steroid substrates can be in either pure or crude form.

The mutation of M. fortuitum ATCC 6842 and M. phlei UC 3533 to give M. fortuitum NRRL B-8153 and M. phlei NRRL B8154, respectively, was accomplished by the use of nitrosoguanidine. The details of the procedure are described in British Application No.

8007922 (Serial No. 1,590,652) the mutation and transformation procedures disclosed therein are detailed for a Mycobacterium species, it should be understood that similar or equivalent procedures can be used with microorganisms of the other genera disclosed herein.

The fermentation beer preparation process of this invention can be effected in a growing culture of M. fortuitum NRRL B-8153 or M. phlei NRRL B-8 154 either by adding the selected steroid substrate to the culture during the incubation period or by incorporating it in the nutrient medium prior to inoculation. The steroid can be added singly or in combination with another steroid. The concentration of the steroid in the culture is preferably from 0.1 to 100 g/l. The culture is grown in a nutrient medium containing a carbon source such as an assimilable carbohydrate, and a nitrogen source such as an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin and molasses. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps and ammonium salts. Combmations of these carbon and nitrogen sources can be used advantageously. Trace metals such as zinc, magnesium, manganese, cobalt and iron need not be added to the fermentation medium since tap water and unpurified ingredients are used as components of the medium prior to sterilisation of the medium.

The transformation process may take from 72 hours to 15 days or more. The incubation temperature can range from 25"C to 37"C., with 30"C being preferred for NRRL B-8153 and 35"C for NRRL B-8154. The contents are aerated with sterilised air and agitated to facilitate growth of the microorganism, thereby' enhancing the effectiveness of the transformation process.

Upon completion of the transformation pro cess, as evidenced by thin layer chromatography using silica gel plates (E. Merck, Darmstadt) and a solvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane, the desired transformed steroids are recovered by means well known in the art. For example, the fermentation (transformation) reaction mixture, including the fermentation liquor and cells, can be extracted with a water-immiscible organic solvent for steroids. Suitable solvents are dichloromethane (preferred), methylene chloride, chloroform, carbon tetrachloride ethylene chloride, trichloroethylene, ether, amyl acetate and benzene.

Alternatively, the fermentation liquor and cells can be first separated by convention methods, e.g. filtration or centrifugation, and then separately extracted with suitable solvents.

The cells can be extracted with either watermiscible or water-immiscible solvents. The fermentation liquor, freed of cells, can be extracted with water-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtrate vacuum distilled to dryness. The resulting residue containing the desired transformed steroids then can be dissolved in a minimum of ethyl acetatecyclohexane (20:80 v/v). This solution then can be chromatographed on dry silica gel using the solvent system ethyl acetate-benzene (20: 80 v/v). ADD and AD can be separated from the silica gel by elution with the solvent system ethyl acetate-chloroform (15 : 85 v/v). The compounds then can be isolated as separate entities by evaporation of the solvent and recrystallisation from hexane.

ADD and AD are useful as intermediates in the synthesis of useful steroidal hormones. For example, ADD can be used to make estrone according to the process disclosed in U.S.

Patent Specification No. 3,274,183. Also, AD can be used to make testosterone according to the processes disclosed in U.S. Patent Specifications Nos. 2,143,453; 2,253,798; 2,264,888 and 2,356,154.

The following Examples illustrate the invention. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1.

Transformation of Sitosterol to ADD and AD.

The medium used in the same as in part (c) of the Example in British Patent Applica tion No. 8007922 (Serial No. 1,590,652). This medium is sterilised by autoclaving for 30 minutes at 121"C whereupon it is cooled to 30"C and then inoculated with 10 ml of a seed culture of the mutant mycobacterium M.

fortuitum NRRL B-8153, prepared as described in part (c) of the Example of British Patent Application No. 8007922 (Serial No.

1,590,652). The inoculated mixture is incubated at 30"C for 336 hours with agitation to promote submerged growth. Following incubation, the mixture is extracted with dichloromethane. The extract is dried over anhydrous sodium sulfate and the solvent is removed by vacuum distillation. The resulting residue is dissolved in a minimum of ethyl acetatecyclohexane (20:80). This solution is then chromatographed on dry silica gel using the solvent system ethyl acetate-benzene (20:80).

The presence of androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione is shown by thin layer chromatography. These compounds are separated from the silica gel by elution with the solvent system ethyl acetate-chloroform (15: 85). The compounds are then isolated by evaporation of the solvent and recrystallisation from hexane.

Example 2.

By substituting M. phlei NRRL B-8154 for M. fortuitum NRRL B-8 153 in Example 1, and an incubation temperature of 35"C for 30"C., also in Example 1, there is obtained a mixture of ADD and AD.

Results similar to those achieved in Example 1 can be obtained by replacing sitosterol by, or adding thereto, any of the other steroids named above, or a mixture thereof, and/or by replacing the microorganism M. fortuitum NRRL B-8153 by any mutant obtained by the procedure of the Example of British Patent Application No. 8007922 (Serial No. 1,590,652) from any of the general or specific steroidproducing microorganisms named above.

WHAT WE CLAIM IS:- 1. A process for preparing a fermentation beer comprising a mixture of androsta-1,4diene-3,17-dione and androst-4-ene-3,17-dione which comprises cultivating a mutant obtained from a microorganism selected from Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces in an aqueous nutrient medium under aerobic conditions in the presence of one or more 17-(C,-1, hydrocarbyl) steroids, the mutant being characterised by its ability to selectively degrade 17-hydrocarbyl steroids as defined above and accumulate androsta-1,4diene-3,17-dione and androst-4-ene-3, 1 7-dione in the fermentation beer in the absence of added chelating agents and heavy metal and selenite ions.

2. A process according to claim 1 in which the microorganism is a Mycobacterium for tuitum mutant.

3. A process according to claim 2 in which the microorganism is Mycobacterium fortuitum NRRL B-8153.

4. A process according to claim 1 in which the microorganism is a Mycobacterium phlei mutant.

5. A process according to claim 4 in which the microorganism is Mycobacterium phlei NRRL B-8154.

6. A process for preparing a fermentation beer comprising a mixture of androsta-1,4diene-3,17-dione and androst-4-ene-3,17-dione which comprises cultivating a microorganism selected from Mycobacterium fortuitum NRRL B-8153 and Mycobacterium phlei NRRL B-8 154 in an aqueous nutrient. medium under aerobic conditions in the presence of one or more 17-(C1.0 hydrocarbyl) steroids.

7. A process according to any preceding claim in which only one of the steroids is present.

8. A process according to claim 7 in which the steroid is 8-sitosterol.

9. A process according to claim 7 in which the steroid is cholesterol.

10. A process according to claim 7 in which the steroid is stigmasterol.

11. A process according to claim 7 in which the steroid is campesterol.

12. A process according to any of claims 1 to 6 in which a mixture of two or more of the steroids is present.

13. A process according to claim 12 in which the steroids in the mixture are selected from 8-sitosterol, cholesterol, stigmasterol and campesterol.

14. A process according to claim 1 substantially as described in Example 1 or Example 2.

15. Androsta-1,4-diene-3,17-dione when isolated from a fermentation beer prepared by a process according to any preceding claim.

16. Androst-4-ene-3,17-dione when isolated from a fermentation beer prepared by a process according to any of claims 1 to 4.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (16)

**WARNING** start of CLMS field may overlap end of DESC **. tion No. 8007922 (Serial No. 1,590,652). This medium is sterilised by autoclaving for 30 minutes at 121"C whereupon it is cooled to 30"C and then inoculated with 10 ml of a seed culture of the mutant mycobacterium M. fortuitum NRRL B-8153, prepared as described in part (c) of the Example of British Patent Application No. 8007922 (Serial No. 1,590,652). The inoculated mixture is incubated at 30"C for 336 hours with agitation to promote submerged growth. Following incubation, the mixture is extracted with dichloromethane. The extract is dried over anhydrous sodium sulfate and the solvent is removed by vacuum distillation. The resulting residue is dissolved in a minimum of ethyl acetatecyclohexane (20:80). This solution is then chromatographed on dry silica gel using the solvent system ethyl acetate-benzene (20:80). The presence of androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione is shown by thin layer chromatography. These compounds are separated from the silica gel by elution with the solvent system ethyl acetate-chloroform (15: 85). The compounds are then isolated by evaporation of the solvent and recrystallisation from hexane. Example 2. By substituting M. phlei NRRL B-8154 for M. fortuitum NRRL B-8 153 in Example 1, and an incubation temperature of 35"C for 30"C., also in Example 1, there is obtained a mixture of ADD and AD. Results similar to those achieved in Example 1 can be obtained by replacing sitosterol by, or adding thereto, any of the other steroids named above, or a mixture thereof, and/or by replacing the microorganism M. fortuitum NRRL B-8153 by any mutant obtained by the procedure of the Example of British Patent Application No. 8007922 (Serial No. 1,590,652) from any of the general or specific steroidproducing microorganisms named above. WHAT WE CLAIM IS:-

1. A process for preparing a fermentation beer comprising a mixture of androsta-1,4diene-3,17-dione and androst-4-ene-3,17-dione which comprises cultivating a mutant obtained from a microorganism selected from Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces in an aqueous nutrient medium under aerobic conditions in the presence of one or more 17-(C,-1, hydrocarbyl) steroids, the mutant being characterised by its ability to selectively degrade 17-hydrocarbyl steroids as defined above and accumulate androsta-1,4diene-3,17-dione and androst-4-ene-3, 1 7-dione in the fermentation beer in the absence of added chelating agents and heavy metal and selenite ions.

2. A process according to claim 1 in which the microorganism is a Mycobacterium for tuitum mutant.

3. A process according to claim 2 in which the microorganism is Mycobacterium fortuitum NRRL B-8153.

4. A process according to claim 1 in which the microorganism is a Mycobacterium phlei mutant.

5. A process according to claim 4 in which the microorganism is Mycobacterium phlei NRRL B-8154.

6. A process for preparing a fermentation beer comprising a mixture of androsta-1,4diene-3,17-dione and androst-4-ene-3,17-dione which comprises cultivating a microorganism selected from Mycobacterium fortuitum NRRL B-8153 and Mycobacterium phlei NRRL B-8 154 in an aqueous nutrient. medium under aerobic conditions in the presence of one or more 17-(C1.0 hydrocarbyl) steroids.

7. A process according to any preceding claim in which only one of the steroids is present.

8. A process according to claim 7 in which the steroid is 8-sitosterol.

9. A process according to claim 7 in which the steroid is cholesterol.

10. A process according to claim 7 in which the steroid is stigmasterol.

11. A process according to claim 7 in which the steroid is campesterol.

12. A process according to any of claims 1 to 6 in which a mixture of two or more of the steroids is present.

13. A process according to claim 12 in which the steroids in the mixture are selected from 8-sitosterol, cholesterol, stigmasterol and campesterol.

14. A process according to claim 1 substantially as described in Example 1 or Example 2.

15. Androsta-1,4-diene-3,17-dione when isolated from a fermentation beer prepared by a process according to any preceding claim.

16. Androst-4-ene-3,17-dione when isolated from a fermentation beer prepared by a process according to any of claims 1 to 4.