GB2131811A

GB2131811A - Microbial 1,2-dehydrogenation of steroids

Info

Publication number: GB2131811A
Application number: GB08402640A
Authority: GB
Inventors: Timothy Wendell Evans
Original assignee: Upjohn Co
Current assignee: Pharmacia and Upjohn Co
Priority date: 1982-07-30
Filing date: 1984-02-01
Publication date: 1984-06-27
Also published as: CH657871A5; DD213242A5; JPH0614874B2; JPS5939299A; FR2531101B1; JPH06225792A; JPH0824596B2; GB2123833B; DE3322120C2; IE55533B1; FR2531101A1; GB8402640D0; GB8317422D0; NL8302596A; GB2123833A; GB2131811B; IE831767L; DE3322120A1

Abstract

A process for preparing a 1,2- dehydro-3-keto-steroid, comprises exposing a 1,2-saturated-3-keto- steroid to Arthrobacter simplex or Bacterium cyclooxydans, in the presence of an exogenous electron carrier and a water-immiscible solvent comprising an aromatic hydrocarbon. The electron carrier may be menadione, menadione bisulphite, 1,4-naphthoquinone, a vitamin K-type compound, or phenazine methosulphate.

Description

1 GB 2 131 811 A 1

SPECIFICATION Steroid bioconversion

The first therapeutic use of corticosteroids was demonstrated in the 1 950'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 gave the resultant steroids, 5 prednisolone and prednisone, enhanced potency and reduced drug-induced salt retention.

Subsequently, most other steroids used for the treatment of corticoidresponsive diseases have been synthesised so that they contain a double bond in the 1,2-position of the steroid molecule.

US-A-3284447 discloses the utility of pregna-1,4,9(1 1)-trienes in the synthesis of diurectic corticosteroids substituted at carbon 16. US-A-4041055 discloses a process for the synthesis of 10 corticosteroids from androsta-1,4-dienedione derivatives, demonstrating the utility of androst-1 -enes as important intermediates in the production of medically useful steroids.

The bioconversion of 1,2-saturated steroids to the corresponding 1,2dehydro steroids is disclosed in US-A-2837464, describing the 1 -dehyd roge nation of steroids in fermentation beers by Arthrobacter (Corynebacterium) simplex,. in US-A-3360439, describing the 1 -dehydrogenation of 15 steroids by using A. simplex cells pre-treated with a lower alkanol or lower alkanone such as acetone before mixing with the substrate and a hydrogen carrier; and by Charney and Herzog in "Microbial Transformation of Steroids", Academic Press Inc., New York, pp 4-9, 23626 1.

US-A-4035236 discloses a process for preparing 9a-hydroxyandrostenedione via fermentation of sitosterol, stigmasterol or cholesterol. US-A-4041055 discloses a general process 20 for the synthesis of medically useful corticosteroids from this androstene. Intermediates involved in this chemistry can possess a 3-keto-4,.9(1 1)-diene configuration.

Steroid bioconversions, and the taxonomy of micro-organisms which can cause 1 - dehydrogenation, are described by Yamane et aL, Biotechnology and Bioengineering 21 (1979) and by Sonomoto et al, Argric. Biol. Chem. 44 (1980) 1119-1126. Sonomoto et aL 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.

Yamane et aL studied the 1 -dehydrogenation of 4-androstene-3,17-dione by Nocardia rhodorcrous in a mixture comprising 50% benzene-heptane. The reported advantage of adding this solvent to bloconversion mixtures was to increase the reaction rate by a factor of 180. The preferred electron acceptor for the Nocardia rhodocrous activity was phenazine methosulphate. The Nocardia rhodocrous enzyme was not active in the presence of menadione. The reaction was run at an organic solvent level only 20% saturated with steroid. It was demonstrated that increasing concentration of the 35 steroid resulted in a decrease in the reaction rate. Oxygen was an essential reagent for the 1 - dehydrogenation reaction. The solvent was 50% benzene-heptane. However, benzene is a known carcinogen. Phenazine methosulphate is too expensive and too unstable to render the reported microbial activity practical or useful in a production bioconversion process. The solvent is inflammable in the presence of oxygen. The method by which oxygen was introduced into the reactor, without 40 producing an explosive environment, was not discussed.

British Patent Application No. 8317422 (Serial No. 2123833) describes and claims a process for preparing a 1,2-dehydro-3-keto-steroid, which comprises exposing a 1,2- saturated-3-keto-steroid to air-dried or heat-dried cells of a 1 -dehydrogenating microbe. The reaction is preferably conducted in the presence of an exogenous electron carrier.

According to the present invention, a process for preparing a 1,2-dehydro3-keto-steroid comprises exposing a 1,2-saturated-3-keto-steroid to Arthrobacter simplex or Bacterium cyclooxydans, in the presence of an exogenous electron carrier and a water-immiscible solvent comprising an aromatic hydrocarbon.

Advantages of the invention over the prior art are that the bioconversion results in lower 50 unconverted steroid substrate levels, the process can be run at higher steroid concentrations, fewer microbial cells, e.g. A. simplex, are required to convert a given quantity of steroid, and the process is more tolerant of organic-soluble impurities present in the steroid, in the microbial cells, or produced from the steroid by the cells. Further, A. simplex-catalysed reactions can be performed with more stable and less expensive electron acceptors, such as menadione, than have been required for Nocardia 55 rhodocrous. The enzyme in N. rhodocrous must be different from that produced byA. simplex.

In the invention, the reaction mixture may be a slurry which is agitated. Oxygen is made available so that the oxygen content in the gas space is less than the minimum amount of oxygen for combustion; alternatively, the reaction is performed at a temperature below the flash-point of the organic solvent.

Two micro-organisms which are publicly available, and are known as 1 dehydrogenation agents for steroids, are suitable for use in the invention. These are Bacterium cyclooxydans, ATCC 12673, and Arthrobacter simplex, ATCC 6946. The former has been examined as a useful microorganism, but the 2 GB 2 131 811 A 2 latter has been used much more extensively, for the 1 -dehydrogenation of steroids. These microorganisms are respectively disclosed in US-A-3065146 and US-A-2837464.

The micro-organisms are grown in an aqueous nutrient medium containing:

(a) inorganic compounds such as nitrate or ammonium salts or organic nitrogenous compounds, 5 e.g. yeast extract, peptone or comsteep liquor, 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, sulphate, manganese, copper, cobalt or molybdenum, in levels supplied by tap water or by the less refined med;um ingredients (such as comsteep liquor).

The organisms require oxygen present in the atmosphere for growth. The temperature range for growth is 10-45'C forA. simplex. The optimum pH for growth is around neutrality.

The cells are induced for steroid-1 -dehydrogenase activity by the addition of a 1,2saturated-3 ketosteroid compound such as androst-4-ers-3,1 7-dione or cortisor.3 acetate at a level of 0.005% w/v of the medium, or higher. The inducer can be added at any point during the growth cycle. Cultures grown on nutrients such as lard oil will start synthesising 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 at least 6 hours after the inducer is added. The cells are then harvested.

For drying, cells are separated from the nutrient medium and concentrated by conventional means such as centrifugation or flocculation, filtration or ultrafiltration. The separated cells are then dried under reduced pressure at 1-85 C, preferably 55-75 C, by air-drying with heat, spray-drying or tumble-drying, to a moisture content of 1 to 10, preferably 5%. Cells are stored at 5 C until used for bioconversion. Active dried cells can also be prepared by immobilising dried cells by standard techniques, such as entrapment within polyacrylamide gel and collagen or covalent coupling of the cells to a poiyelectrolyte carrier as described in Methods in Enzymology, Vol. XLIV, 1976, Academic Press, Inc., New York, pp 11-317.

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

Examples of the various procedures that can be used include:

(a) Suspension of the cells in an aqueous buffer system, followed by addition of the electron carrier dissolved in ethanol, methanol or acetone (not greater than 5% of final volume). The steroid 30 substrate can be added as a dry powder or dissolved (suspended) in a water-immiscible organic solvent such as toluene.

(b) Rehydration of dried cells in a small volume of buffer followed by the addition of more buffer or of organic solvent. Xylene or toluene, for example, is added to give a final organic solvent content of 0-95% (vol/vol). 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 later or earlier.

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

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-! mmiscible solvent such as butyl acetate or methylene chloride. The product is then isolated from the organic solvent.

By way of illustration, the process of the invention may be conducted by combining, in a reaction 45 vessel, a buffer providing a pH of 6 to 10; the micro-organism, in an amount sufficient to push the reaction to completion; the electron acceptor; 3-keto-androst-4-ene or 3keto-pregn-4-ene steroid; and aromatic hydrocarbon The amount of the micro-organism is typically 0.05 to 1 g/g steroid (A. simplex cells may be used in the fermentation broth or modified by collecting the cells, drying, acetone treatment or immobilisation, by methods well known in the art). The amount of the electron acceptor is determined by cost and in consideration of the reaction rate (e.g. linearly proportional to menadione concentration), and typically from 5 to 40 g/kg steroid. Preferred electron acceptors are menadione, menadione bisulphite, 1,4-naphthoquinone and vitamin K-type compounds. Phenazine methosulphate may also be used.

The concentration of the steroid is as high as possible, compatible with efficient conversion. The steroid prepferably has a solubility in the organic solvent of more than 5 g/1.

The aromatic hydrocarbon is preferably toluene, benzene or a xylene (which may be diluted with another water-i m miscible solvent such as heptane or methylene chloride). The amount of aromatic hydrocarbon is preferably from one-half of the amount necessary to dissolve the more soluble of the 60 starting and product steroids, to four times the amount necessary to dissolve the less soluble of the starting and product steroids.

The combined materials are then agitated at as high an agitation power as is possible. The reaction rate is strongly dependent on the agitation power.

The reaction requires oxygen as the final hydrogen (electron and proton) acceptor. However, 65 4 7 c 1 11 3 GB 2 131 811 A 3 owing to the presence of the aromatic hydrocarbon, an explosive environment can exist in the gas space of the reactor. Advantageously, to overcome this hazard, the oxygen concentration in the gas space is maintained well below the minimum oxygen concentration for combustion. For benzene, the minimum oxygen is 11.2% For the xylenes, it is also possible to perform the reaction below the flash point. The flash points for m-xylene, o-xylene and p-xylene are 29, 32 and 39 C, respectively.

The reaction temperature may be from 0-45 C. The reaction may be run for from a few hours to a few weeks but, typically, for 2 days.

Substrate steroids for use in this invention belong to the 3-keto-androst4-ene and 3-keto-pregn 4-ene series of steroids. Members of the androstene series include androst-4-ene-3,17-dione, androsta-4,9(1 1)-diene-3,17-dione and its 6a-fluoro, 6a-methyl or 16- methyl derivatives, and 1 lp- 10 hydroxyandrost-4-ene-3,1 7-dione and its derivatives. Among steroids of the 3-keto-pregn-4-ene series which can be used are 17a-hydroxypregn-4-en-20-yn-3-one and its 1 6- methyl derivatives; 1 1P,21 dihydroxypregna-4,17(20)-dien-3-one and its 6a-methyl derivative; 20- chloropregna-4,9(11),17(20) trien-21-al-3-one; and various 3,20-diketo-pregn-4-enes, including 11,17, 21 -trihydroxy compounds such as hydrocortisone and its 6a-methyl derivative; 9p,l lp-epoxy-17,21dihydroxy compounds, such 15 as9A,llp-epoxy-17,21-dihydroxy-16p-methylpregn-4-ene-3,20-dione;3,20diketo-4,9(11)- pregnadienes such as 17a,21 -dihydroxypregna-4,9(1 1)-diene-3,20-dione and its 1 6a-methyl, 1 6p methyl or 1 6a-hydroxy derivatives or 17a-acetate ester; and 3,20-diketo- 4,9(11), 1 6-pregnatrienes such as 21 -hydroxypregna-4,9(11),1 6-triene-3,20-dione and its 6a-fluoro derivative.

The 21 -ester derivatives of those steroids containing a 2 1 -hydroxyl group serve as substrates also. The preferred 21 -esters are derived from lower fatty acids, e.g. acetic acid, or monocyclic carboxylic acids, e.g. benzoic acid.

The following Examples illustrate the invention.

Example 1

0.9 litre 50 mM K2HPO, buffer (pH=7.5), 3.66 g dryA. simplex cells, 80 mg menadione, 8 g 2 1 - 25 acetoxypregna-4,9(11),16-triene-3,20-dione and 100 mi toluene were combined in a reaction vessel (1 fitre basis) and agitated at 15 cal/1.min. The amount of toluene was just sufficient to dissolve the steroid, on completion of the reaction. Air was added, as necessary, to maintain the oxygen concentration in the gas space of the reaction vessel at about 3 to 6%. The temperature was maintained at 28:F 1 C.

After 25 hours, the toluene phase was collected. This phase contained 94. 3% 2 1 -acetoxypregna1,4,901), 1 6-tetraene-3,20-dione, 4.2% 21 hydroxypregna-1,4,9(11), 1 6-tetraene-3,20-dione and 1.5% 21 acetoxypregna-4,9(11),16-triene-3,20-dione.

Example 2

0.4 gA. simplex cells and 50 mi 50 mM K,HPO, buffer (pH=7.5) were combined and agitated for 35 2 hours or until a uniform slurry was obtained. A 25 mi fraction was introduced into a 125 mi flask, and 0-25 mi 5 mM menadione in 3A alcohol, 0.2 g androsta-4,9(1 1)-diene-3,17- dione and 2 mi toluene were then added. The flask was stoppered and agitated on a wrist action shaker for 4 days. The reaction mixture was then extracted with 25 mi toluene. The product, androsta-1,4,9(1 1)-triene-3,17 dione, was obtained in 100% yield. 40 By comparison, using another 25 mi sample in another 125 mi flask, and following the same procedure but without the addition of toluene, the yield of product was 82.3%. Accordingly, in this aqueous procedure, 17.7% of the substrate steroid remained.

The following is a list of steroids which can be treated in accordance with the procedure of Example 1: androst-4-ene-3,17-dione 6a-fluoroandrosta-4,90 1)-diene-3,17- dione 6a-methylandrosta-4,9(1 1)-diene-3,17-dione 16p-methylandrosta-4, 9(1 1)-diene-3,17-dione 17a-hydroxypregn-4-en-20-yn-3-one 1 7a- hydroxypregna-4,9(1 1)-dien-20-yn-3-one 1 7a-hydroxy-1 6-methylpregna-4, 9(1 1)-dien-20-yn-3-one 21 -acetoxy- 11 P-hydroxypregna-4,1 7(20)-diene-3- one 20chloropregna-4,9(11),17(20)-trien-21 -al-3-one 21 -acetoxy-1 7hydroxypregna-4,9(1 1)-diene-3,20-dione 21 -acetoxy-1 7-hydroxy-1 6 a-m ethyl preg na-4,9 (1 1)-diene-3,20-dione 21 -benzyloxy- 1 7-hydroxy-1 6pmethylpregna-4,9(1 1)-diene-3,20-dione 2 1 -acetoxy-1 7-hydroxy-1 6pmethylpregna-4,9(1 1)-cliene-3,20-dione 21 -acetoxypregna-4,9(1 1),1 6triene-3,20-dione 21 -acetoxy-6a-fluoropregna-4,9(11),1 6(1 7)-triene-3, 20-dione These substrates are convered to the following products, respectively: androsta-1,4-diene-3,1 7-dione 6a-fluoroandrosta-1,4,90 1)triene-3,1 7-dione 4 GB 2 131 811 A 4 6a-methylandrosta-1,4,9(1 1)-triene-3,117-dione 1 6p-methylandrosta-1,4, 9(1 1)-triene-3,1 7-dione 17a-hydroxypregna-1,4-dien-20-yn-3-one 17a- hydroxypregna-1,4,9(1 1)-trien-20-yn-3-one 17a-hydroxy-1 6p-methylpregna- 1,4,9(1 1)-trien-20-yn-3-one 1 lp,21-dihydroxypregna-1,4,17(20)-triene-3- one and its 21-acetate 20-chloropregna-1,4,9(11),17(20)-tetraen-21 -a]-3- one 1 7,21-dihydroxypregna-1,4,9(1 1)-triene-3,20-dione and its 21 acetate 17,21 -dihydroxy-1 6a-methylpregna-1,4,9(1 1)-triene-3,20-dione and its 2 1 -acetate 21 -benzoyloxy-1 7-hydroxy-1 6p-methylpregna-1,4,9(1 1)-triene-3,20-dione 17,2 1 -dihydroxy-1 6p-methylpregna-1,4,9(1 1)triene-3,20-dione and its 2 1 -acetate 21-hydroxypregna-1,4,9(11),16tetraene-3,20-dione and its 21-acetate 6a-fluoro-21-hydroxypregna-1,4,9(1 1),16-tetraene-3,20-dione and its 21-acetate

Claims

1. A process for preparing a 1,2-dehydro-3-keto-steroid, which comprises exposing a 1,2- 15 saturated-3-keto-steroid to Arthrobacter simplex or Bacterium cyclooxydans, in the presence of an exogenous electron carrier and a water-immiscible solvent comprising an aromatic hydrocarbon.

2. A process according to claim wherein the amount of the water-im miscible 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 20 product steroid.

3. A process according to claim 1 or claim 2, wherein oxygen is added and the oxygen concentration is maintained below the combustion level.

4. A process according to any preceding claim, which is conducted at a temperature below the flash point of the organic solvent.

5. A process according to any preceding claim, wherein the aromatic hydrocarbon is benzene, toluene or a xylene.

6. A process according to any preceding claim, wherein the electron carrier is menadione, 1,4- naphthoquinone, menadione bisulphite or a vitamin K-type compound. 30

7. A process according to any preceding claim, wherein a mixture of water- immiscible solvents is 30 used.

8. A process according to claim 1, substantially as described in either of Examples 1 and 2.

Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1984. Published by the Patent Office.

Southampton Buildings, London, WC2A TAY, from which copies may be obtained.

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