GB2123833A - Steroid 1,2-dehydrogenation using dried microbial cells - Google Patents

Abstract

1,2-Dehydrogenation of 1,2- saturated steroids is effected with air- dried or heat-dried microbial cells (e.g. of Arthrobacter simplex or Bacterium cyclooxydans). The conversion may be effected in a weakly buffered aqueous solution in the presence of an electron carrier (e.g. menadione) and optionally an aromatic hydrocarbon.

Description

SPECIFICATION Steroid conversion The first therapeutic use of corticosteroids was demonstrated in the 1950's with the introduction of cortisone acetate treatment for rheumatoid arthritis. Further studies demonstrated that the insertion of unsaturation into the 1,2-position of hydrocortisone and cortisone caused the resultant steroids, prednisolone and prednisone, to have enhanced potency and to cause less drug-induced salt retention.

Subsequently, most other steroids used for the treatment of corticoid-responsive diseases have been synthesized so that they contain a double bond in the 1,2-position of the steroid molecule. In 1 977, two U.S. patents were issued which represent new approaches to the synthesis of corticosteroids from sterol precursors. U.S. Patent No. 4,035,236 covers a process for preparing 9- hydroxyandrostenedione via fermentation of sitosterol, stigmasterol, or cholesterol. U.S. patent No.

4,041 055 discloses a general process for the synthesis of medically useful corticosteroids from this androstene. Intermediates covered in this chemistry can possess a 3-keto-A4,911 configuration.

Following are prior art methods which disclose the bioconversion of 1,2-saturated steroids to their corresponding 1,2-dehydro steroids: U.S. Patent No. 2,837,464 "Process for Production of Dienes by Corynebacterium" Description of 1 -dehydrogenation of steroids in fermentation beers by Arthrobacter (C or ynebacterium) simplex.

U.S. Patent No. 3,360,439 entitled "Process for Preparing 1 -dehydro Steroids".

Description of 1 -dehydrogenation of steroids by use of A. simplex cells pretreated with a lower alkanol or lower alkanone such as acetone before mixing with the substrate and a hydrogen carrier.

Charney, W. and Herzog, H. 1 967. Microbial Transformation of Steroids. Academic Press, Inc., New York, pp 4-9, 236-261.

Historical background on steroid bioconversions and taxonomic listing of microorganisms known to carry out 1-dehydrogenation.

T. Yamane, H. Nakatani, E. Sada, T. Omata, A. Tanaka and S. Fukui, Biotechnology and Bioengineering, 21, 1979.

The authors performed a study of the 1-dehydrogenation of 4-androstene-3,1 7-dione by Nocardia rhodocrous in a mixture comprised of 50% benzene-heptane. The reported advantage of adding this solvent to bioconversion mixtures was to increase the reaction rate by a factor of 1 80.

However, benzene is a known carcinogen. The preferred electron acceptor for the Nocardia rhodocrous activity was phenazine methosulfate. This electron acceptor is too expensive and too unstable to render this microbial activity practical or useful in a production bioconversion process. The Nocardia rhodocrous enzyme was not active in the presence of menadione, the electron acceptor of choice for Arthrobacter simplex. The enzyme in N. rhodocrous must be different than that produced byA. simplex.

The reaction was run at an organic solvent level only 20% saturated with steroid. It was demonstrated that increasing concentration of the steroid resulted in a decrease in the reaction rate. Oxygen is an essential reagent of the 1 -dehydrogenation reaction. The solvent (50% benzene-heptane) is flammable in the presence of oxygen. The method of introduction of oxygen to the reactor without producing an explosive environment was not discussed.

K. Sonomoto, I. Jin, A. Tanka and S. Fukui; Agric. Biol. Chem., 44, 111 9-1126, 1980.

The same laboratory studied the 1-dehydrogenation of hydrocortisone byA. simplex. Two immiscible solvents, n-butanol and n-amyl alcohol, were tested but the bioconversion reaction did not proceed in the presence of these solvents. Water miscible solvents such as 10% methanol-water and 10% propylene glycol-water were chosen as preferred solvents.

The prior art does not disclose or suggest the subject improved processes.

Brief summary of the invention By using air-dried or heat-dried microbial cells that are capable of catalyzing 1 -dehydrogenation of steroids, there is obtained a more efficient conversion of 1,2-saturated steroids to their corresponding 1,2-dehydro derivatives than is obtainable by the best known prior art process in an aqueous environment, particularly in the presence of an exogenous electron acceptor.

Also disclosed and claimed is a process which involves the addition of an aromatic hydrocarbon as a water immiscible solvent to an aqueous slurry of A. simplex cells, an exogenous electron acceptor and the steroid to be dehydrogenated. The slurry is agitated and oxygen is made available such that the oxygen content in the gas space is less than the minimum oxygen for combustion; or the reaction is performed below the flash point of the organic solvent.

These procedures are superior to the prior art technology for steroid-dehydrogenation by microorganisms because of the following: a. the bioconversion results in lower unconverted steroid substrate levels; b. this process can be run at higher steroid concentrations; c. less microbial cells, for example,A. simplex, are required, to convert a given quantity of steroid; d. this process is more tolerant of organic soluble impurities present in the steroid, impurities present in the microbial cells or impurities produced from the steroid by the cells; e. reduction or total elimination of steroid degradative enzymes present in the microbial cells result in higher yields of the desired product.

Further, the solvent procedure is superior to prior technology for steroid-dehydrogenations with other microbial activities such as Nocardia rhodocrous because the A. simplex catalyzed reaction can be performed with stable and less expensive electron acceptors such as menadione.

Detailed description of the invention Microorganism The microbes which can be used in the subject process are any of the numerous well-known microbes which are known as 1-dehydrogenators. Such microbes are listed in Charney, W. and Herzog, H. 1 967. Microbial Transformation of Steroids. Academic Press, Inc., New York.

The bacteria that 1 -dehydrogenate steroids fall within two general groups establishes in Bergey's Manual of Determinative Biology, 8th edition. The procedure described herein has been successfully demonstrated for species from Arthrobacter and Corynebacterium, genera that are included in "Part 1 7. Actinomycetes s Related Organisms". The 1 -dehydrogenating species of several other genera in Part 17, such as Nocardia, Mycobacaterium, Streptomyces and Bacterium are probably useful.

Two microorganisms available to the public which are known to 1-dehydrogenate steroids have been demonstrated to lend themselves useful for this type of process. Bacterium cyclooxydans, included in U.S. Patent No.3,065,146 as a 1-dehydrogenator-ATCC number 12673, has been examined as a useful microorganism. A bacterium that has been used much more extensively for the 1 dehydrogenation of steroids is Arthrobacter simplex, ATCC 6946, which is disclosed in U.S. Patent No.

2,837,464. Thus, much of the following will use this microorganism to exemplify the invention process. It should be understood, however, that the subject process also covers the use of any 1 dehydrogenating microbe.

Growth of the microorganism The microorganisms are grown in an aqueous nutrient medium containing: a) inorganic compounds such as nitrate or ammonium salts or organic nitrogenous compounds (yeast extract, peptone, cornsteep liquor, etc). to provide nitrogen for growth.

b) a carbon and energy source such as carbohydrates and sugar derivatives, oil, fatty acids and their methyl esters, alcohols, amino acids or organic acids.

c) ions and trace elements such as sodium, potassium, magnesium, phosphate, sulfate, manganese, copper, cobalt, molybdenum, etc. in levels supplied by tap water or by the less refined medium ingredients (such as cornsteep liquor).

The organisms require oxygen present in the atmosphere for growth. The temperature range for growth is 1 0--450 C with an optimum of 2837 for A. simplex. The optimum pH for growth is around neutrality.

Production of the steroid-l -dehydrogenase The cells are induced for steroid-1 -dehydrogenase activity by the addition of a 1,2 saturated 3keto-steroid compound such as androsta-4-ene-3,1 7-dione or cortisone acetate at a level of 0.005% w/v of the medium or greater. In the presence of the inducer, incubation is continued for a period of at least 6 hours before the cells are harvested for drying.

The inducer can be added at any point during the growth cycle. Cultures grown on nutrients such as lard oil will start synthesizing the steroid-1-dehydrogenase rapidly while cultures grown on glucose require substrate depletion before enzyme synthesis will occur. Incubation is continued for a period of 6 or more hours after the inducer is added, then the cells are harvested for use in the solvent procedure or for drying.

Procedure for recovering cells for drying Cells are separated from the nutrient medium and concentrated by conventional means such as centrifugation, or flocculation, and filtration and ultrafiltration. The separated cells are then dried by placing under reduced pressures at 1 0--850C (5575 preferred), by air drying with heat, or by spray drying, or by tumble drying until a moisture content in the range of about 1 to about 10%. A moisture content of about 5% is preferred. Cells are stored at 50C until used for bioconversions. Active dried cells can also be prepared by immobilizing dried cells by standard techniques, such as entrapment within polyacrylamide gel and collagen or covalent coupling of the cells to a polyelectrolyte carrier as described in Methods in Enzymology Vol. XLIV, 1976, Academic Press, Inc., New York, pp 11-317.

Bioconversion process The bioconversion is accomplished by exposure of the prepared cells to the steroid substrate.

Typically, the cells and steroid are suspended in a weakly buffered aqueous solution with a pH in the range of pH 6-pH 10 with an optimum of pH 7.25-8.5. The amount of cells can range from 0.1-50 g/liter, the steroid is added at a weight ratio of 0.05 to 5.0 (steroid:cells). Cells levels of 8-1 0 g/liter with 5-10 g/liter steroid are preferred when the bioconversion is run in an aqueous environment. An exogenous electron carrier is added in catalytic amounts, (e.g. 5x10-4 M menadione) to stimulate the reaction. Useful compounds include menadione (2-methyl-1,4-naphthoquinone), phanazine methosulfate, dichlorophenol-indophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinones (Coenzyme Q), and vitamin K-type compounds.The mixtures incubated 0-14 days at a temperature range of 5 -45 C, During incubation, the mixture has access to molecular oxygen and is preferably stirred. The rate of 1 -dehydrogenation typically decreases with time. The bioconversion can proceed to 98-1 00% of completion in less than 24 hours using 10 g/liter substrate.

Examples of the various procedures that can be used include: a) Suspension of the steroid and menadione in a weakly buffer aqueous solution, followed by addition of the dried cells. Surfactants such as Tween 80 can be added in low concentration, e.g., 0- 5%, to aid in steroid suspension.

b) Suspension of the cells in an aqueous buffer system, followed by addition of the electron carrier dissolved in ethanol, methanol, acetone (not greater than 5% of final volume). The steroid substrate can be added as a dry powder or dissolved (suspended) in a miscible organic solvent such as dimethyl formamide, ethanol, methanol, acetone, dimethyl sulfoxide, or an immiscible organic solvent such as toluene.

c) Rehydration of the dried cells in a small volume of buffer followed by the addition of more buffer or of organic solvents. Ethanol, acetone, xylene, butyl acetate, methylene chloride, or toluene, etc. is added to give a final organic solvent content of 0-95% (vol/vol). The quantity of aromatic hydrocarbon used can range from about one-half of that necessary to dissolve the most soluble of the starting steroid or the product steroid to about 4 times that necesssary to dissolve the least soluble of the starting steroid or the product steroid. The steroid and electron carrier are added to initiate the reaction. The aromatic hydrocarbon is preferably added with the steroid. However, it may be added at later or earlier times.

d) Suspension of a wet cell cake or dilution of a fermentation beer with a weakly buffered aqueous solution, followed by addition of an exogenous elctron carrier, steroid, and an aromatic hydrocarbon.

Substrate range Compounds that are useful in the practice of this invention belong to the 3-keto-A4-androstene and 3-keto-A4-pregnene series of steroids. It is recognized that substrates for the steroid-1 dehydrogenase with have saturation between carbons C1 and C2 of the A ring, and will have a hydroxyl or keto group at position 3 on the A ring. Members of the androstene series include: 1) androsta-4-ene-3, 1 7-dione and 2) androsta-4,9(11)-diene-3,17-dione and its 6a-fluoro, 6ar-methyl, or 16-methyl derivatives; 3) 11,B-hydroxy androsta-4-ene-3,1 7-dione and its derivatives.

Among the steroids of the 3-keto-A4-pregnene series which can be used are: 1.17α-hydroxypregn-4-ene-20-yn-3-one and its 16-methyl derivatives; 2.1 1ss,21-dihydro-pregn-4,17(20)-diene-3-one and its 6a-methyl derivative; 3.20-chloro-pregn-4,9(11),17(20)-triene-21-al-3-one 4. several groups of 3,20-diketo-A4-pregnenes, including a) 11,17,21-trihidroxy compounds, such as hydrocortisone and its 6a-methyl derivative; b) 9,l 1 p-epoxy-17,21 -dihydroxy compounds, such as 9ss, -epoxy-17,21 -dihydroxy 16P-methyl-pregn-4-ene-3,20-dione;; .c) 3,20-diketo-4,9(11 )-pregnedienes such as 17,21 -dihydroxy-pregn-4,9( 11 )-diene-3,20-dione and its I 6a-methyl, 1 6-methyI or 16a:-hydroxy derivatives or 17a-acetate ester; d) 3,20-diketo-4,9(11), 4,9(11), 16-pregnetrienes, such as 21 -hydroxy-pregn-4,9( 11), 16-triene 3,20-dione and its 6-fluoro derivative.

The 21 -ester derivatives of those steroids containing 21 -hydroxyl group (#2 and #4) serve as substrates also. The preferred 21-esters consist of lower alkyl or aryl groups such as lower fatty acids, e.g. acetic acid, and monocyclic carboxylic acids, e.g., benzoic acid.

The bioconversion products and unconverted substrate can be recovered from the mixtures by conventional means. Steroids are typically recovered by filtration, followed by extraction of the filter cake with an organic solvent, such as acetone or methylene chloride. Alternatively, the whole bioconversion mixture can be extracted by mixing with a water immiscible solvent such as butyl acetate or methylene chloride. The product is then isolated from the organic solvent.

Following are the results of different bioconversions which demonstrate the superiority of the invention process over prior art processes. The bioconversions were conducted using the conditions detailed above.

Example 1 Preparation of dried cells Bacterium cyclooxydans (ATCC 12673) was inoculated into shake flasks containing a medium of cerelose, peptone, and corn steep liquor (6 g/l of each) pH 7.0. The cultures were incubated on a rotary shaker at 280C until glucose exhaustion occurred. Cortisone actate (0.5 g/l) was added at that time and the flasks incubated an additional 1 6 hrs. The cells were harvested by centrifugation, washed twice with water then placed in a low vacuum oven at 450C until dry.

Bioconversion of 6a-methyl hydrocortisone One-half gram of dried cells, prepared as described above, were rehydrated in 50 ml of 50 mM phosphate buffer pH 7.5 with stirring. Menadione was added to the cell suspension as an ethanolic solution (8.6 mg/ml ethanol) at a level of 0.025 ml/50 ml. The substrate was added as a dimethylformamide (DMF) solution (100 mg 6cz-methyl hydrocortisone/ml DMF) to a final bioconversion concentration of 0.5 g/liter. The mixture was incubated at 280C with agitation. After 4 hrs incubation, the steroid was 91% converted. The 6cr-methyl prednisolone was recovered by conventional means.

Example 2 Androsta-1 4,9(11 )-triene-3,1 7-dione production a) Preparation of biocatalyst: Arthrobacter simplex (ATCC 6946) was grown in shake flasks in a medium containing 6 g/l gluocose, 6 g/l corn steep liquor, and 6 g/l of spray dried lard water. The cultures were incubated at 280C on a rotary shaker until glucose depletion occurred. At that time, cortisone acetate (0.15 g/l) was added to induce steroid-1 -dehydrogenase synthesis. After overnight incubation, the cells were harvested by low speed centrifugation. The cell pellets were placed in a 550C low vacuum oven for 24 hr to dry. Twenty-four hours later, the dried material was transferred to an air-tight container and stored at 50C until it was needed for a bioconversion.

b) Bioconversion of androsta-4,9(11) )-diene-3,1 7-dione Dried cells (10 g/l) were dehydrated in 50 mM phosphate buffer pH 7.5 by stirring for 30 minutes.

Menadione was then added to cell suspension as a dry powder to a final concentration of 86 mg/liter.

Micronized androsta-4,9( 11)-diene-3,1 7-dione was slurried in dimethylformamide and added to the bioconversion mixture at a level of 2.5 g steroid/liter and 2% (v/v) DMF. The mixture was incubated at 31 C with agitation in the presence of air for 24 hr. After completion of the incubation, androsta 1,4,9(1 1 )-triene-3,1 7 dione was recovered by conventional means.

Example 3 The following steroidal compounds were exposed to dried cells of A. simplex in accordance with the conditions described in the preceding example in order to obtain the corresponding 1 ,2-dehydro derivatives: No. Name 1. androsta-4-ene-3,1 7-dione 2. 6α-fluoro-androsta-4,9(11)-diene-3,17-dione 3. 6a-m ethyl-a nd rosta-4,9( 1 1 )-diene-3, 1 7-dione 4. 1 6,B-methyl-androsta-4,9(1 1 )-diene-3,1 7-dione 5. 17α-hydroxypregn-4-ene-20-yn-3-one 6. 1 7α-hydroxypregn-4,9(1 1 )-diene-20-yn-3-one 7. 17α;-hydroxy-16ss-methyl-pregn-4,9(11)-diene-20-yn-3-one 8. 11ss,21 -dihydroxy-pregn-4, 1 7(20)-diene-3-one 9. 21-acetoxy-1 1 P-hydroxy-pregn-4,1 7(20)-diene-3-one 10. 6-methyl- 1 1 p,2 1 -dihydroxy-pregn-4, 1 7(20)-diene-3-one 11. 20-chloro-pregn-4,9( 11),17(20)-triene-2 l-al-3-one 12. hydrocortisone 13. 6a-methyl hydrocortisone 14. 21-acetoxy-11ss, 17-dihydroxy-16ss-methyl-pregn-4-ene-3,20-dione 15. 21-acetoxy-9α ;-fluoro-11ss, 17-dihydroxy-16ss-methyl-pregn-4-ene-3,20-dione 16. 21 -acetoxy-9 1 -epoxy- 1 7-hydroxy-l 6ss-methyl-pregn-4-ene-3,20-dione 17. 21 -acetoxy-1 7-hydroxy-pregn-4,9( 11 )-diene-3,20-dione 18. 21 -acetoxy- 1 6a, 1 7-dihydroxy-pregn-4,9( 11 )-diene-3,20-dione 19. 21 -acetoxy- 1 7-hydroxy- 1 6a-methyl-pregn-4,9( 11 )-diene-3,20-dione No. Name 20. 21-benzoyloxy-17-hydroxy-16ss-methyl-pregn-4,9(11)-diene-3,20-dione 21. 21-acetoxy-17-hydroxy-16ss-methyl-pregn-4,9(11)-diene-3,20-dione 22. 21-acetoxy-pregn-4,9(11),16(17)-triene-3,20-dione 23. 21 -acetoxy-6a-fluoro-pregn-4,9(11),16-triene-3,20-dione.

The corresponding products obtained from the conversions are as follows: No. Products 1 a androsta-1,4-diene-3,17-dione 2a 6α-fluoro-androsta-1,4,9(11)-triene-3,17-dione 3a Ga-methyl-androsta-l ,4,9(11)-triene3,1 7-dione 4a 16ss-methyl-androsta-1,4,9(11)-triene-3,17-dione 5a 17a-hydroxypregn-l ,4-diene-20-yn-3-one 6a 17a-hydroxypregn-l ,4,9(11 )-triene-20-yn-3-one 7a 1 7a-hydroxy- 16-methyl-pregn- 1,4,9(11 )-triene-20-yn-3-one 8a 11P,21 -dihydroxy-pregn-l ,4,1 7(20)-triene-3-one 9a 21-acetoxy-ll P-hydroxy-pregn-l ,4,1 7(20)-triene-3-one and 11ss,21-dihydroxy-pregn-1,4,17(20)-triene-3-one 10a 6α;-methyl-11ss,21-dihydroxy-pregn-1,4,17(20)-triene-3-one 11a 20-chloro-pregn-1,4,17(20)-tetraene-21-al-3-one 12a prednisolone 13a 6α-methyl-prednisolone 14a 21 -acetoxy-1 1 ,17-dihydroxy-16 -methyl-pregn- 1 ,4-diene-3,20-dione and 11ss,17,21-trihydroxy-16ss-methyl-pregn-1,4-diene-3,20-dione 15a 21-acetoxy-9α-fluoro-11ss-17-dihydroxy-16ss-methyl-pregn-1,4-diene-3,20-dione and 9α;-fluoro-11ss,17,21-trihydroxy-16ss-methyl-pregn-1,4-diene-3,20-dione 16a 21-acetoxy-9ss-11ss-epoxy-17-hydroxy-16ss-methyl-pregn-1,4-diene-3,20-dione and 9ss,11ss-epoxy-17,21-dihydroxy-16ss-methyl-pregn-1,4-diene-3,20-dione 17a 21-acetoxy-17-hydroxy-pregn-1,4,9(11)-triene-3,20-dione and 17,21-dihydroxy pregn-1,4, 9(11 )-triene-3,20-dione 18a 21-acetoxy-16α,17-dihydroxy-pregn-1,4,9(11)-triene-3,20-dione and 16α,17,21-trihydroxy-pregn-1,4,9(11)-triene-3,20-dione 19a 21-acetoxy-17-hydroxy-16α-methyl-pregn-1,4,9(11)-triene-3,20-dione and 17,21-dihydroxy-16α-methyl-pregn-1,4,9(11)-triene-3,20-dione 20a 21-benzoyloxy-17-hydroxy-16α;-methyl-pregn-1,4,9(11)-triene-3,20-dione 21 a 21 -acetoxy-17-hydroxy-16α-methyl-pregn-1,4,9(11)-triene-3,20-dione and 17,21-dihydroxy-16α-methyl-pregn-1,4,9(11)-triene-3,20-dione 22a 21-acetoxy-pregn-1,4,9(11),16-tetraene-3,20-dione and 21-hydroxy-pregn-1,4,9(11),16-tetraene-3,20-dione 23a 21-acetoxy-8a-fl uoro-pregn-1,4,9(11 ),1 6-tetraene-3,20-dione and 6α-fluoro-21-pregn-1,4,9(11),16-tetraene-3,20-dione.

Example 4 Two different steroids were bioconverted with cells of A. simplex in fermentation beer and with dried A. simplex cells. After each bioconversion was judged to be finished (as indicated by no further decrease in residual substrate levels), the steroid in the mixture was extracted and isolated to provide yield data for each condition. The steroids were androsta-1,4,9(11)-diene-3,17-dione (S1) and 21 acetoxypregna-4,9(11),16(17)-triene-3,20-dione (S2). The following Table gives yields obtained by different bioconversions.

Type of Chemical yield of % #' % Substrate bioconversion useful steroid(a) compound Residual Comments S1 (10 g/l) Fermetation 77.2% 1 st crop 2.6 87.4 Poor 1 dehydrogenation 2.8% 2nd crop 10.4 89.6 S1 (10 g/l) Dried cell 82.6% 1 st crop 100.0 9.9% 2nd crop 95.0 5.0 S2 (8 g/l) Fermentation 18.1 1 st crop 59.1 40.9 (b) No 2nd crop obtained S2 (8 g/l) Dried cells 79.6% 1st crop 97.8 2.1 No 2nd crop obtained (a) Useful steroid is defined as either the #' compound which can be used in further synthesis or the 1,2-dihydro substrate which can be recycled into another bioconversion to produce product.

(b) Considerable amounts of other undesirable steroid molecules were also recovered.

Example 5 Arthrobacter simplex was grown in a 5-liter "Microferm" fermentor, induced for steroid-1 dehydrogenase synthesis, and harvested by centrifugation. Portions of the recovered cell paste were subjected to two different preparation methods. The first was the procedure disclosed herein which consisted of drying the cells under reduced pressure at 550 C. The second method was the procedure recommended in U.S. Patent No. 3,360,439 for preparation of acetone-dried cells. Cells were mixed with acetone, harvested and dried at SOC under reduced pressure. The dried cell preparations were then used to bioconvert A9,11-andrestenedione in shake flasks at 10 g/liter cells and 10 g/liter steroid.

Comparision of bioconversion capacity of A. simplex cells dried by different methods Time of Bioconversion activity+ sampling % Residual (g product formed) Cell type (hours) substrate hr/g cells Acetone-dried 1 98.7 .013 4 95.5 .011 24 84.1* .007 Heat-dried 1 85.1 .149 4 23.2 .192 24 7.2* .039 +) Bioconversion activity is calculated as follows: % product formedxg substrate added =g product formed 100 g product formed/hours of incubation/g cells added=activity (*) Residual levels did not decrease with further incubation.

Example 6 By substituting the following listed substrates for 6α-methyl hydrocortisone in Example 1, or androsta-4,9(11)-diene-3,17-dione in Example 2, there are obtained the corresponding listed products: Substrates: 1.21-acetoxy-9α-fluoro-11ss,16α,17-trihydroxy-pregn-4-ene-3,20-dione; 2.21-acetoxy-6α,9α-difluoro-11ss,16α,17-trihydroxy-pregn-4-ene-3,20-dione-16,17- acetonide; 3.21-acetoxy-6α-fluoro-11ss,-hydroxy-16α-methyl-pregn-4-ene-3,20-dione; 4.21-acetoxy-6α-fluoro-11ss,17-hydroxy-pregn-4-ene-3,20-dione; 5.21-acetoxy-6α,9α-fluoro-11ss,17-dihydroxy-16α-methyl-pregn-4-ene-3,20-dione; 6.21-acetoxy-9α-fluoro-11ss,16α,17-trihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide; 7.21-acetoxy-9ss,11ss-epoxy-6α-fluoro-16α;,17-trihydroxy-pregn-4-ene-3,20-dione-16,17- acetonide; 8.21-acetoxy-9ss,11ss-epoxy-16α-hydroxy-pregn-4-ene-3,20-dione; 9. 21 -acetoxy-9p,1 1p-epoxy-1 6c',1 7-dihydroxy-pregn-4-ene-3,20-dione-1 6,1 7-acetonide; Products: 1.21-acetoxy-9α-fluoro-11ss,16α,17-trihydroxy-pregn-1,4-diene-3,20-dione and 9α-fluoro- 11ss,16α,17,21-tetrahydroxy-pregn-1,4-diene-3,20-dione; 2.21-acetoxy-6α,9α-fluoro-11ss,16α,17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17- acetonide and 6α,9α-fluoro-11ss,16α,17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17- acetonide; 3.21-acetoxy-6α-fluoro-11ss,-hydroxy-16α-methyl-pregn-1,4-diene-3,20-dione and 6α ;-fluoro- 11ss,17,21-trihydroxy-1,4-diene-3,20-dione; 4.21-acetoxy-6α-fluoro-11ss,17-hydroxy-pregn-1,4-diene-3,20-dione and 6α-fluoro- 11ss,17,21-trihydroxy-1,4-diene-3,20-dione; 5.21-acetoxy-6α,9α-difluoro-11ss,17-dihydroxy-16α-methyl-pregn-1,4-diene-3,20-dione and 6α,9α-difluoro-11ss,17,21-trihydroxy-1,4-diene-3,20-dione; 6.21-acetoxy-6α,9α-fluoro-11ss,16α,17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide; 7.21-acetoxy-9ss,11ss-epoxy-6αfluoro-16α,17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17acetonide; 8.21-acetoxy-9ss,11ss-epoxy-16-hydroxy-pregn-1,4-diene-3,20-dione and 9ss,119-epoxy16α,21-dihydroxy-pregn-1,4-diene-3,20-dione; 9.21-acetoxy-9ss,11ss-epoxy-16α;,17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide.

Example 7 Bioconversion of 11 ss-hydroxy-androsta-4-ene-3,17-dione One gram of dried A. simplex cells prepared as described in Example 2, was resuspended in 100 ml 50 mM phosphate buffer pH 7.5. Menadione, dissolved in 3A ethanol, was added to a final concentration of 86 mg/liter. One-half gram of 11 p-hydroxy-androstenedione was added to the flask.

The mixture was incubated on a rotary shaker at 31 0C. When sampled after 1 -day's incubation, two products were observed by thin layer chromatography of a methylene chloride extract. Approximately 95% of the steroid was present as 11ss-hydroxy-androsta-1,4-diene-3,17-dione. The remainder was unconverted substrate.

Example 8 Upon following the conditions of Example 7, the following androstenedione derivatives that are modified at the C-i 1 position can be 1 -dehydrogenated to the products shown: Substrate # Product (1) 11ss-hydroxy-16ss-methyl- # 11ss-hydroxy-16ss-methyl-androsta androsta-4-ene-3,17-dione 1,4-diene-3,17-dione (2) 11ss-hydroxy-16α-methyl-androsta- # 11ss-hydroxy-16α-methyl-androsta 4-ene-3,17-dione 1,4-diene-3,17-dione (3) 6α-fluoro-11ss-hydroxy-androsta- # 6α-fluoro-11ss-hydroxy-androsta 4-ene-3,17-dione 1,4-diene-3,17-dione (4) 6α-methyl-11ss-hydroxy-androsta- # 6α-methyl-11ss-hydroxy-androsta- 4-ene-3,17-dione 1,4-diene-3,17-dione (5) 11α-hydroxy-androsta-4-ene- # 11α;-hydroxy-androsta-1,4-ene 3,17-dione diene-3,17-dione (6) androsta-4-ene-3,11,17-trione # androsta-1,4-diene-3,11,17-trione.

Example 9 1) Combine in a reaction vessel 1 I basis) a) 0,9 1 of 50 mM KPO4 buffer, pH=7.5 b) 3.66 gm of dry A. simplex cells c) 0.08 gm of menadione d) 8 gm of 21 -acetoxy-pregn-4,9( 1 1),16-triene-3,20 dione e) 100 ml of toluene.

2) Agitate at 15 cal/I min.

3) Add air as necessary to maintain oxygen in the gas space of the reaction vessel at about 3 to 6%.

4) Maintain temperature at 28 C#1 OC.

5) After 25 hrs. collect the toluene phase.

For this example the steroid in the toluene phase was 94.3% 21-acetoxy-pregn-1,4,9(11),16-tetraene-3,20-dione 4.2% 21 -hydroxy-pregn- ,4,9( 11), 1 6-tetraene-3,20-dione 1.5% 21-acetoxy-pregn-1,4,9(11),16-tetraene-3,20-dione The following range of variables can be used for the above steps: Step 1) a) Buffer pH can be in the range 6 to 10.

b) Quality of A. simplex is reduced to a level just sufficient to push the reaction to completion.

Typical levels are 0.05 to 1.0 gm per gm of steroid. The microbial source of the enzyme: Arthrobacter {Corynebacterium) simplex (ATCC 6946) or Bacterium cyclooxydans (ATCC 12673).

Preparation of the microorganism:TheA simplex cells may be used in the fermentation broth or modified by collecting the cells, drying acetone treatment or immobilization, by methods well known in the art.

c) The electron acceptor can be selected from the following: Menadione (2-methyl-1,4 naphthoquinone), Menadione bisulfite, 1,4 naphthoquinone and other vitamin K-type compounds.

The level of the electron acceptor is determined by cost and reaction rate considerations. The reaction rate is linearly proportional to the menadione concentration. Typical levels are about 5 to about 40 gm/kg of steroid.

d) Steroid concentration should be as high as possible, so long as there is efficient conversion.

The steroid would belong to the 3-keto-A4-androstene or the 3-keto-54-pregnene series and preferably should have a solubility in the organic solvent of greater than 5 g/liter.

e) The aromatic hydrocarbon can be chosen from toluene, benzene or xylene. These solvents may be used together or diluted with another water immiscible solvent such as heptane, or methylene chloride.

The quantity of aromatic hydrocarbon can range from about one-half of that necessary to dissolve the most soluble of the starting steroid or the product steroid to about 4 times that necessary to dissolve the least soluble of the starting steroid or the product steroid.

For this example toluene was added at a level just sufficient to dissolve the steroid at the completion of the reaction.

The aromatic hydrocarbon is preferably added with the steroid. However, the aromatic hydrocarbon may be added at later times or earlier times.

Step 2) Agitate at as high agitation power as possible. The reaction rate is a strong function of the agitation power.

Step 3) The reaction requires oxygen as the final hydrogen (electron and proton) acceptor. However, due to the presence of the aromatic hydrocarbon, an explosive environment will exist in the gas space of the reactor. To overcome this hazard the oxygen concentration in the gas space is, advantageously, maintained well before the minimum oxygen for combustion. For benzene the minimum oxygen is 11.2%. For the xylene's it is also possible to perform the reaction below the flash point. The flash point for m-xylene, o-xylene and p-xylene are 29, 32 and 390C, respectively.

Step 4) The temperature of the reaction can be from about 0 to about 450C.

Step 5) The reaction is typically run for 2 days. It could be run for a few hours to a few weeks.

Example 10 Comparision of toluene and aqueous procedures (A) Procedure: Combine 0.4 gm ofA. simplex cells 50 ml of 50 mM KPO4 buffer, pH=7.5 Agitate for 2 hours or until uniform slurry is obtained.

Take 2-25 ml fractions and add to 125 ml flasks.

Add to each flask 0.25 ml of 5 mM menadione in 3A-alcohol 0.2 gm of androsta-4,9(1 1) diene-3,17-dione.

Add 2 ml of toluene to one of the stoppered flasks and Agitate on wrist action shaker for 4 days.

After 4 days extract with 25 ml of toluene.

Analyze extracts.

(B) Results: Procedure Aqueous Toluene (Substrate) Androstra-4,9( 11 )diene-3, 1 7-dione 17.7% 0.00% (Product) Androstra-1,4,9(11 )triene-3,1 7-dione 82.3% 100.00% Example 11 The following steroidal compounds were exposed to dried cells of A. simplex in accordance with the conditions described in Example 9 in order to obtain the corresponding 1,2-dehydro derivatives: No.Name 1. androsta-4-ene-3,17-dione 2. 6α-fluoro-androsta-4,9(11)-diene-3,17-dione 3. 6α-methyl-androsta-4,9(11)-diene-3,17-dione 4. 16ss-methyl-androsta-4,9(11)-diene-3,17-dione 5. 17α-hydroxypregn-4-ene-20-yn-3-one 6. 17α-hydroxypregn-4,9(11)-diene-20-yn-3-one 7. 17α-hydroxy-16ss-methyl-pregn-4,9(11)-diene-20-yn-3-one 8. 21 -acetoxy- 11 P-hydroxy-pregn-4, 1 7 (20)-diene-3-one 9. 20-chloro-pregn-4,9(11),17(20)-triene-21-al-3-one 10. 21-acetoxy-17-hydroxy-pregn-4,9(11)-diene-3,20-dione 11. 21-acetoxy-17-hydroxy-16α;-methyl-pregn-4,9(11)-diene-3,20-dione 12. 21-benzyloxy-17-hydroxy-16ss-methyl-pregn-4,9(11)-diene-3,20-dione 13. 21-acetoxy-17-hydroxy-16ss-methyl-pregn-4,9(11)-diene-3,20-dione 14. 21-acetoxy-pregn-4,9(11),16-triene-3,20-dione 15. 21-acetoxy-6α-fluoro-pregn-4,9(11),16(17)-triene-3,20-dione.

The corresponding products obtained from the conversions are as follows: No. Products 1a androsta-1,4-ene-3,17-dione 2a 6α-fluoro-androsta-1,4,9(11)-diene-3,17-dione 3a 6α-methyl-androsta-1,4,9(11)-diene-3,17-dione 4a 16ss-methyl-androsta-1,4,9(11)-diene-3,17-dione 5a 17α-hydroxypregn-1,4-diene-20-yn-3-one 6a 17α-hydroxypregn-1,4,9(11)-triene-20-yn-3-one 7a 17α-hydroxy-16ss-methyl-pregn-1,4,9(11)-triene-20-yn-3-one 8a 21-acetoxy-11ss-hydroxy-pregn-1,4,17(20)-triene-3-one and 11ss,21-dihydroxy-pregn-1,4,17(20)-triene-3-one 9a 20-ch loro-preg n-l ,4,9( 1 1 ), 1 7(20)-tetraene-2 1 -al-3-one 1 0a 21 -acetoxy- 1 7-hydroxy-pregn- 1,4,9(11 )-triene-3,20-dione and 17,21-dihydroxy-pregn-1,4,9(11)-triene-3,20-dione 11a 21-acetoxy-17-hydroxy-16α;-methyl-pregn-1,4,9(11)-triene-3,20-dione and 17,21-dihydroxy-16α-methyl-pregn-1,4,9(11)-triene-3,20-dione 12a 21-benzyloxy-17-hydroxy-16ss-methyl-pregn-1,4,9(11)-triene-3,20-dione 13a 21-acetoxy-17-hydroxy-16ss-methyl-pregn-1,4,9(11)-triene-3,20-dione 17,21-dihydroxy-16ss-methyl-pregn-1,4,9(11)-triene-3,20-dione 14a 21-acetoxy-pregn-1,4,9(11),16-tetraene-3,20-dione and 21-hydroxy-pregn-1,4,9(11)-tetraene-3,20-dione 15a 21 -acetoxy-6a-fluoro-pregn-l ,4,9(11),1 6-tetraene-3,20-dione and 6a-fluoro-21 -hydroxy-pregn-l ,4,9(11),1 6-tetraene-3,20-dione.

The utility of 1,2-dehydro steroids is well known. For example, see U.S. Patent 3,284,447, which discloses the utility of AlA.9(ll)prngnetrienes in the synthesis of diurectic corticosteroids substituted at carbon 1 6. U.S. Patent 4,041,055 discloses a process for the synthesis of corticosteroids from A1,4- androstenedione derivatives, demonstrating the utility of 1 ,2-dehydroandrnstenes as important intermediates in the production of medically useful steroids.

Claims (14)

Claims

1. A process for preparing a 1,2-dehydro steroid, which comprises exposing a 1,2-saturated 3keto steroid to air-dried to heat-dried cells of a 1-dehydrogenating microbe.

2. A process for preparing a 1,2-dehydro steroid, which comprises exposing a 1 2-saturated steroid to air-dried or heat-dried bacterial cells of a 1-dehydrogenating microbe, in the presence of an exogenous electron carrier.

3. A process according to claim 2, wherein the electron carrier is menadione, phenazine methosulfate, dichlorophenolindophenol, 1 ,4-naphthoquinone, menadione bisulfite, an ubiquinone (Coenzyme Q) or a vitamin K-type compound.

4. A process according to any preceding claim, wherein the air-dried or heat-dried microbial cells have a moisture content of 1 to about 10%.

5. A process according to any preceding claim, wherein the 1-dehydrogenating microbe is Arthrobacter simplex or Bacterium cyclooxydans.

6. A process for preparing a 1,2-dehydro steroid, which comprises exposing a 1,2-saturated steroid to Anthrobacter simplex or Bacterium cyclooxydans, in the presence of an exogenous electron carrier and a water-immiscible solvent comprising an aromatic hydrocarbon.

7. A process according to claim 6, wherein the electron carrier is menadione, 1,4- naphthoquinone, menadione bisulfite or a vitamin K-type compound and the aromatic hydrocarbon is toluene or xylene.

8. A process according to claim 6 or claim 7, wherein the amount of the water-immiscible solvent is from one-half of that necessary to dissolve the more soluble of the steroid substrate and the product steroid, to four times that which is necessary to dissolve the less soluble of the steroid substrate and the product steroid.

9. A process according to any of claims 6 to 8, wherein oxygen is added and the oxygen concentration is maintained below the combustion level.

10. A process according to any of claims 6 to 9, which is conducted at a temperature below the flash point of the organic solvent.

11. A process according to any of claims 6 to 10, wherein the electron carrier is menadione, 1,4- naphthoquinone, menadione bisulfite or a vitamin K-type compound, and the aromatic hydrocarbon is benzene, toluene or xylene.

12. A process according to claim ii, wherein the hydrocarbon is toluene or xylene.

13. A process according to claim 10, wherein the electron carrier is menadione, phenazine methosulfate, 1,4-naphthoquinone, menadione bisulfite or a vitamin K-type compound, and the aromatic hydrocarbon is xylene.

14. A process according to any of claims 6 to 13, wherein a mixture of water-immiscible solvents is used.

1 5. A process according to claim 1, substantially as exemplified herein.