CH657871A5 - METHOD FOR CONVERTING 1,2-saturated 3-keto steroids IN 1,2-DEHYDROSTEROIDE. - Google Patents

Description

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PATENT CLAIMS

1. A process for converting 1,2-saturated 3-keto-steroids into 1,2-dehydrosteroids, characterized in that air-dried or heat-dried cells of 1-dehydrating microorganisms are allowed to act on the 1,2-saturated steroids.

2. The method according to claim 1, characterized in that one uses air-dried or heat-dried microorganism cells with a moisture content of 1 to 10%.

3. The method according to claim 1, characterized in that Arthrobacter simplex and / or Bacterium cyclooxydans are used as 1-dehydrating microorganisms.

4. The method according to claim 1, characterized in that air-dried or heat-dried bacterial cells of 1-dehydrating microorganisms are allowed to act on the 1,2-saturated steroids in the presence of an exogenous electron carrier.

5. The method according to claim 4, characterized in that the exogenous electron carrier used is menadione, phena-tin methosulfate, dichlorophenolindophenol, 1,4-naphtho-quinone, menadione bisulfite, ubiquinones or compounds of the vitamin K type.

6. The method according to claim 4, characterized in that one uses air-dried or heat-dried microorganism cells with a moisture content of 1 to 10%.

7. The method according to claim 4, characterized in that Arthrobacter simplex and / or Bacterium cyclooxydans is used as the 1-dehydrating microorganisms.

8. Process for the conversion of 1,2-saturated 3-keto-steroids into 1,2-dehydrosteroids, characterized in that the 1,2-saturated steroids in the presence of an exogenous electron carrier and a water-immiscible solvent in the form an aromatic hydrocarbon Arthrobacter simplex and / or Bacterium cyclooxydans.

9. The method according to claim 8, characterized in that the electron carrier used is menadione, 1,4-naphtho-quinone, menadione bisulfite or another compound of the vitamin K type and toluene and / or xylene as the aromatic hydrocarbon.

10. The method according to claim 8, characterized in that one uses so much water-immiscible solvent in the form of an aromatic hydrocarbon that its amount about half the amount required to dissolve the most soluble steroid substrate or steroid formed up to about four times the amount is the amount required to dissolve the least soluble steroid substrate or steroid formed.

11. The method according to claim 8, characterized in that one works by adding an amount of oxygen below the minimum oxygen concentration required for combustion.

12. The method according to claim 10, characterized in that one uses as the electron carrier menadione, 1,4-naphtho-quinone, menadione bisulfite and another compound of the vitamin K type and as an aromatic hydrocarbon benzene, toluene and / or xylene.

13. The method according to claim 8, characterized in that one carries out below the flash point of the organic solvent.

14. The method according to claim 13, characterized in that the electron carrier used is menadione, phenazinmethosul-fat, 1,4-naphthoquinone, menadione bisulfite or another compound of the vitamin K type and xylene as the aromatic hydrocarbon.

15. The method according to any one of claims 8, 11 or 13,

characterized in that the water-immiscible solvent is diluted with another water-immiscible solvent.

The first therapeutic use of corticosteroids was reported in the 1950s. This was a cortisone acetate treatment for rheumatoid arthritis. Further research showed that the incorporation of an unsaturated bond in the 1,2-position of hydrocortisone and cortisone led to the steroids obtained prednisolone and prednisone having an increased effect and a lower drug-reduced salt retention. Thereafter, most of the other steroids used in the treatment of corticoid-responsive disorders were also provided with a double bond in the 1,2-position of the steroid molecule. Two attempts to synthesize corticosteroids from sterol precursors have been known since 1977. For example, US Pat. No. 4,035,236 discloses a process for the preparation of 9a-hydroxyandrostenedione by fermentation of sitosterol, stigmasterol or cholesterol. A process for the synthesis of medically valuable corticosteroids from androstene is known from US Pat. No. 4,041,055. Intermediates occurring here can

3-Keto-A4'9 (") configuration.

Known methods for the biological conversion of 1,2-saturated steroids into the corresponding 1,2-dehydrosteroids are:

US Pat. No. 2,837,464 "Process for the preparation of dienes by Corynebacterium".

A 1-dehydrogenation of steroids in various fermentation broths by Arthrobacter (Corynebacterium) simplex is described.

US Pat. No. 3,360,439 "Process for the production of 2-de-hydrosteroids".

A 1-dehydrogenation of steroids is described using A. simplex cells, which are mixed with a short-chain alkanol or short-chain alkanone, e.g. Acetone, have been pretreated.

W. Charney and H. Herzog, “Microbial Conversion of Steroids,” published by Academic Press, Inc., New York, pages 4-9, 236-261 (1967).

In the latter reference, the historical background of biological steroid conversions and a taxonomic list of microorganisms suitable for carrying out 1-dehydrogenation are given.

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

The authors examined 1-dehydration of

4-Androsten-3, I7-dione using Nocardia rhodocrous in a mixture of 50% benzene and 50% heptane. By adding this solvent mixture to the biological conversion mixture, the reaction rate should be increased by a factor of 180. However, benzene is known to be carcinogenic. The preferred electron acceptor for Nocardia rhodocrous activity in the experiments carried out was phenazine methosulfate. This electron acceptor is too expensive and too unstable to be able to make practical use of this microorganism activity in large-scale biological conversion processes. The Nocardia rhodo-crous enzyme was not active in the presence of menadione, the electron acceptor of choice for Arthrobacter simplex. Thus, the enzyme produced by N. rhodocrous must differ from the enzyme produced by A.simplex. The

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Reaction was carried out in a steroid saturated solvent system with only 20% organic solvent. It was found that the reaction rate decreased with increasing concentration of the steroid. Oxygen is an essential reagent in the 1-dehydrogenation reaction. The solvent (50% benzene / 50% heptane) is flammable in the presence of oxygen. How the oxygen was introduced into the reactor without creating an explosive environment is not explained in the cited literature reference.

K. Sonomoto, I. Jin, A. Tanaka and S. Fukui «Agric. Biol. Chem. », 44, 1119-1126 (1980).

The authors mentioned examined the 1-dehydrogenation of hydrocortisone using A.simplex. Two water-immiscible solvents, n-butanol and n-amyl alcohol, were tested. However, no bio-conversion takes place in the presence of these solvents. Water-miscible solvents, e.g. Mixtures of 10% methanol and 90% water and 10% propylene glycol and 90% water are used.

The cited references neither describe the methods according to the invention nor are they suggested.

The first method for converting 1,2-saturated 3-keto steroids into 1,2-dehydrosteroids is characterized in that air-dried or heat-dried cells of 1-dehydrating microorganisms are acted on the 1,2-saturated steroids. These cells can have a moisture content of 1 to 10%. Air and heat-dried cells of the microorganisms Arthrobacter simplex and / or Bacterium cyclooxydans are preferably used.

In a preferred embodiment of this method, air-dried or heat-dried bacterial cells from 1-dehydrating microorganisms are allowed to act in the presence of an exogenous electron carrier. Menadione, phenazine methosulfate, dichlorophenolindophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinones or compounds of the vitamin K type can be used as exogenous electron carriers.

The second method according to the invention also relates to the conversion of 1,2-saturated 3-keto steroids into 1,2-dehydrosteroids. This process is characterized in that the 1,2-saturated steroids are allowed to act in the presence of an exogenous electron carrier and a water-immiscible solvent in the form of an aromatic hydrocarbon Arthrobacter simplex and / or Bacterium cyclooxydans.

Toluene and / or xylene are preferably used as aromatic hydrocarbons.

In a preferred embodiment of this process, the addition of an amount of oxygen below the minimum oxygen concentration required for combustion is carried out, and in another preferred embodiment the reaction according to the invention is carried out below the flash point of the organic solvent.

Using air-dried or heat-dried microbial or microorganism cells which are able to catalyze 1-dehydrogenation of steroids, the invention thus achieves a more effective conversion of 1,2-saturated 3-keto steroids into their corresponding 1,2-dehydro derivatives than in the context of in an aqueous environment, especially in the presence of an exogenous electron acceptor known processes.

The second method does not require air-dried or heat-dried microbial or microorganism cells, but you work in the presence of an exogenous electron carrier and a water-immiscible solvent in the form of an aromatic hydrocarbon and use Arthrobacter simplex and / or Bacterium cyclooxydans.

An aqueous slurry of A. simplex cells is preferably used. The slurry is usually agitated. The oxygen supply can take place in such a way that the oxygen content in the gas space remains below the minimum oxygen concentration for combustion. On the other hand, the reaction can also be carried out (at a temperature) below the flash point of the organic solvent.

These measures are superior to the known technology of steroid dehydration by microorganisms in the following:

a) Bio-conversion leads to lower levels of unconverted steroid substrate;

b) the methods according to the invention can be carried out at higher steroid concentrations;

c) fewer microorganism cells are required to convert a given amount of steroid, e.g. A. simplex cells;

d) the methods according to the invention are less sensitive to the organic soluble contaminants contained in the steroid, in the microorganism cells or in the steroid formed by the cells, and e) a reduction or complete elimination of steroid-degrading enzymes contained in the microbial cells leads to higher yields of the desired Reaction product.

The solvent measures are the known technology of steroid dehydration with other microorganism activities, e.g. Nocardia rhodocrous, because the A.simplex-catalyzed reaction can be carried out with more stable and less expensive electron acceptors such as menadione.

Microorganisms:

In the process according to the invention, the numerous microorganisms known for 1-dehydrogenation can be used as microorganisms. Such microorganisms are described by W. Charney and H. Herzog in “Microbial Transformation of Steroids”, published by Academic Press, Inc., New York (1967).

The bacteria that dehydrate steroids in the 1-position generally fall into two groups, cf. “Bergey's Manual of Determinative Biology”, 8th edition. The above-mentioned The described method was successfully demonstrated on species from Arthrobacter and Corynebacterium genera from «Part 17, Actinomycetes & Related Organisms». The 1-dehydrating species of various other genera in part 17, e.g. Nocardia, Mycobacterium, Streptomyces and Bacterium are also likely to be useful.

Two microorganisms available to the public, known to dehydrate steroids in the 1-position, have been found to be useful in this type of process. For example, the bacterium cyclooxydans (ATCC No. 12673) known from US Pat. No. 3,065,146 as 1-dehydrogenating bacterium was tested and found to be useful. A bacterium which has been investigated even more intensively for the 1-dehydrogenation of steroids is the bacterium Arthrobacter simplex known from US Pat. No. 2,837,464 (ATCC No. 6946). In the following, this microorganism is largely used to illustrate the method according to the invention. Of course, the method according to the invention can also be carried out with other 1-dehydrating microorganisms.

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Microorganism growth:

The microorganisms are in an aqueous nutrient medium with:

a) inorganic compounds, e.g. Nitrates or ammonium salts, or organic nitrogen-containing compounds, e.g. Yeast extract, peptone, corn steep liquor and the like. (Nitrogen source for growth);

b) a carbon and energy source, e.g. Carbohydrates and sugar derivatives, oil, fatty acids and their methyl esters, alcohols, amino acids or organic acids, and c) ions and trace elements, e.g. Sodium, potassium, magnesium, phosphates, sulfates, manganese, copper, cobalt, molybdenum and the like. in amounts such as those with tap water or less purified components of the nutrient medium, e.g. the corn steep liquor,

let grow.

The organisms need oxygen in the atmosphere for growth. The temperature range for growth is 10-45 ° C with an optimal range of 28 to 37 ° C for A.simplex. The optimal pH for growth is around the neutral point.

Preferred recovery of steroid 1-dehydrogenase:

The cells are grown by adding a 1,2-saturated 3-ketosteroid compound, e.g. of androsta-4-en-3,17-dione or cortisone acetate in an amount of 0.005% (w / v) of the medium or above to induce steroid-1-dehydrogenase activity. Incubate for at least 6 hours in the presence of the "stimulant" before harvesting the cells to dry.

The "stimulant" can be added at any time during the growth cycle. Cultures grown on nutrient media such as bacon oil begin to synthesize steroid-1-dehydrogenase rapidly, while cultures grown on glucose require substrate depletion before enzyme synthesis takes place. After adding the "excitation agent", incubation is continued for 6 hours or longer, after which the cells are harvested for use in the solvent process or for drying.

Preferred measures for collecting cells to dry:

The cells are separated from the nutrient medium and processed in the usual known manner, e.g. concentrated by centrifugation or flocculation and filtration and ultrafiltration. The separated cells are then dried by drying them under reduced pressure at 1 to 85 ° C., preferably 55 to 75 ° C., by air drying with heating, by spray drying or by drum drying to a moisture content of 1 to 10%. The moisture content is preferably about 5%. The cells are then stored at 5 ° C until they are used for bio-conversion. Active dried cells are also obtained by immobilizing dried cells according to standard procedures, e.g. by inclusion in a polyacrylamide gel and collagen or by covalent coupling of the cells to a polyelectrolyte carrier, cf. Methods in Enzymology, volume XLIV, 1976, published by Academic Press, Inc., New York, pages 11-317.

Preferred biological conversion:

The bio-conversion takes place by allowing the cell preparations to act on the steroid substrate. Typically, the cells and the steroid are suspended in a weakly buffered aqueous solution with a pH in the range of 6-10, preferably 7.25-8.5. The number of cells is

0.1-50 g / 1. The steroid can be added in a weight ratio (steroid: cells) of 0.05 to 5.0. Cell quantities of 8-10 g / 1 with 5-10 g / 1 steroid are preferred if the biological conversion takes place in an aqueous environment. To stimulate the reaction, an exogenous electron carrier is added in a catalytic amount (for example 5 x IO-4 M menadione). Suitable compounds are menadione (2-methyl-l, 4-naphthoquinone), phenazine methosulfate, dichlorophenol indophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinones (coenzyme Q) and compounds of the vitamin K type. The mixtures are incubated at a temperature of 5-45 ° C for 0 to 14 days. Molecular oxygen may be allowed to enter the mixture during the incubation. The mixture is preferably stirred during the incubation. The rate of 1-dehydrogenation typically decreases over time. When using 10 g / 1 substrate, the bio-conversion can be 98-100% complete in less than 24 hours.

The following are examples of various process measures that can be carried out:

a) Suspension of the steroid and menadione in a weakly buffered aqueous solution and subsequent addition of the dried cells. To improve the steroid suspension, surfactants, e.g. Tween 80 in a low concentration, for example a concentration of 0-5%, can be added.

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

c) rehydrating the dried cells in a small buffer volume and then adding more buffer or an organic solvent. So much ethanol, acetone, xylene, butyl acetate, methylene chloride or toluene is added that the final content of organic solvent is 0-95% (vol / vol). The amount of aromatic hydrocarbon used ranges from about half the amount required to dissolve the most soluble starting steroid or steroid formed to about four times the amount required to dissolve the least soluble starting steroid or steroid formed. The steroid and the electron carrier are added to initiate the reaction. The aromatic hydrocarbon is preferably added together with the steroid, but it can also be added earlier or later.

d) suspension of a wet cell cake or dilution of a fermentation broth with a weakly buffered aqueous solution and subsequent addition of an exogenous electron carrier, a steroid and an aromatic hydrocarbon.

Substrate area:

Compounds which can be used in the process according to the invention belong to the 3-keto-A4-androsten- and 3-keto-A "-prain series of steroids. It is known that substrates for the steroid-1-dehydrogenase are saturated between the carbon atoms Ci and C2 of the ring A and have a hydroxy or keto group in the 3-position of ring A. Examples of compounds of the androstene series are:

1. Androsta-4-en-3,17-dione and

2. Androsta-4,9 (1 l) -diene-3,17-dione and its 6a-fluoro-6a-methyl or 16-methyl derivatives;

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3. 1 lß-Hydroxyandrosta-4-en-3,17-dione and its derivatives.

Usable steroids of the 3-keto-A4-pregnen series are:

1. 17a-hydroxy-pregn-4-en-20-yn-3-one and its 16-methyl derivatives;

2. 1 lß, 21-dihydroxy-pregn-4,17 (20) -dien-3-one and its 6a-methyl derivatives;

3. 20-chloro-pregn-4,9 (1 l), 17 (20) -trien-21-al-3-one;

4. various groups of 3.20-Diketo-A4-Pregnenen,

like a) 11,17,21-trihydroxy compounds, e.g. Hydrocortisone and its 6a-methyl derivative;

b) 9β, lß-epoxy-17,21-dihydroxy compounds, e.g. 9β, 11β-epoxy-17,21-dihydroxy-16ß-methyl-pregn-4-en-3,20-dione;

c) 3.20-diketo-4.9 (1 L) -pregnediene, e.g. 17a, 21-di-hydroxy-pregn-4,9 (1 l) -diene-3,20-dione and its 16a-methyl, 16ß-methyl or 16a-hydroxy derivative or the 17a-acetate ester;

d) 3,20-diketo-4,9 (11), 4,9 (1 l), 16-pregnetrienes, e.g. 21-Hydroxy-pregn-4,9 (l 1), 16-triene-3,20-dione and its 6a-fluoro derivative.

Also suitable as substrates are the 21-stere derivatives of those steroids which have a hydroxyl group in the 21-position (No. 2 and No. 4). The preferred 21 esters contain short chain alkyl or aryl groups of short chain fatty acids, e.g. Acetic acid and monocyclic carboxylic acids e.g. Benzoic acid.

The biological conversion products and unconverted substrate can be isolated from the reaction mixtures in a conventional manner. The steroids are typically filtered off and the filter cake is extracted with an organic solvent such as acetone or methylene chloride. On the other hand, the entire biological conversion mixture can be mixed by mixing with a water-immiscible solvent, e.g. Butyl acetate or methylene chloride can be extracted. The reaction product can then be isolated from the organic solvent.

The following examples are intended to illustrate the invention in more detail.

example 1

Production of the dried cells:

Inoculation with Bacterium cyclooxydans (ATCC No. 12673) takes place in shaking bottles with a medium of cerelose, peptone and corn steep liquor (6 g / 1 each) with a pH of 7.0. The cultures are incubated on a rotary vibrator at 28 ° C. until glucose exhaustion occurs. When glucose depletion occurs, 0.5 g / 1 cortisone acetate is added, after which the shaking flasks are incubated again for 16 hours. The cells are harvested by centrifugation. The centrifuged cells are washed twice with water and then stored in a low vacuum oven at 45 ° C until dry.

Bioconversion of 6a-methylhydrocortisone

Half a gram of the dried cells produced in the manner described is rehydrated with stirring in 50 ml of a 50 mM phosphate buffer with a pH of 7.5. Menadione is added to the cell suspension as an ethanolic solution (8.6 mg / ml ethanol) in an amount of 0.025 ml / 50 ml. The substrate is added as a solution in dimethylformamide (100 mg 6a-methylhydrocortisone / ml dimethylformamide) up to a final organic conversion concentration of 0.5 g / 1. Now the mixture is incubated with agitation at 28 ° C. After 4 hours of incubation, the steroid is 91% converted. The 6cc-methylprednisolone is obtained in the usual known manner.

Example 2

Production of Androsta-l, 4,9 (l l) -triene-3,17-dione a) Production of the biocatalyst:

Arthrobacter simplex (ATCC No. 6946) is grown in shake flasks in a medium containing 6 g / 1 glucose, 6 g / 1 corn steep liquor and 6 g / 1 spray-dried bacon water. The cultures are incubated on a rotary vibrator at 28 ° C until glucose depletion occurs. At this point, 0.15 g / 1 cortisone acetate is added to initiate steroid-1-dehydrogenase synthesis. After overnight incubation, the cells are harvested by low speed centrifugation. The cell pellets are then placed in a low vacuum oven at 55 ° C to dry for 24 hours. 24 hours later, the dried material is transferred to an airtight container and stored in it at 5 ° C until use for biological conversion.

b) Biological conversion of Androsta-4,9 (l l) -diene-3,17-dione:

10 g / l of dried cells are rehydrated in 50 mM phosphate buffer with a pH of 7.5 while stirring for 30 minutes. Then the cell suspension is mixed with so much dry powdery menadione that a final concentration of 86 mg / 1 is reached. Furthermore, micronized androsta-4,9 (ll) -diene-3,17-dione is slurried in demethylformamide and, in the form of the slurry, the biological conversion mixture in an amount of 2.5 g steroid / 1 and 2% (v / v) dimethylformamide admitted. The mixture is then incubated at 31 ° C. for 24 hours while moving in the presence of air. After the incubation has ended, the and rosta-1,4,9 (1 l) -triene-3,17-dione is presented pure in the usual known manner.

Example 3

In the same way as in Example 2, the following steroids are used:

1. Androsta-4-en-3,17-dione

2. 6a-fluoro-androsta-4,9 (1 l) -diene-3,17-dione

3. 6a-methyl-androsta-4,9 (l) -diene-3,17-dione

4.16β-methyl-androsta-4,9 (1 l) -diene-3,17-dione

5. 17a-Hydroxy-pregn-4-en-20-yn-3-one

6. 17a-Hydroxy-pregn-4,9 (11) -dien-20-yn-3-one

7. 17cc-Hydroxy-16ß-methyl-pregn-4,9 (1 l) -dien-20-yn-3-one

8.1 lets, 21-dihydroxy-pregn-4,17 (20) -dien-3-one

9. 21-acetoxy-lß-hydroxy-pregn-4,17 (20) -dien-3-one

10.6a-methyl-lß, 21-dihydroxy-pregn-4,17 (20) -dien-3-one

11. 20-chloro-pregn-4,9 (1 l), 17 (20) -trien-21-al-3-one

12. Hydrocortisone

13. 6a-methylhydrocortisone

14. 21-Acetoxy-lß, 17-dihydroxy-16ß-methyl-pregn-4-en-3,20-dione

15.21-Acetoxy-9ct-fluoro-11β, 17-dihydroxy-16ß-methyl-pregn-4-en-3.20-dione

16.21-acetoxy-9β, lß-epoxy-17-hydroxy-16ß-methyl-pregn-4-en-3,20-dione

17. 21-acetoxy-17-hydroxy-pregn-4,9 (1 l) -diene-3,20-dione

18. 21-Acetoxy-16a, 17-dihydroxy-pregn-4,9 (1 l) -diene-3,20-dione

19.21-Acetoxy-17-hydroxy-16a-methyl 1-pregn-4.9 (1 l) -diene-3.20-dione

20. 21-Benzoyloxy-17-hydroxy-16β-methyl-pregn-4.9 (1 L) -diene-3.20-dione

21.21 - acetoxy-17-hydroxy-16ß-methyl-pregn-

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4.9 (1 L) -diene-3.20-dione

22.21-acetoxy-pregn-4.9 (ll), 16 (17) -triene-3.20-dione and 23.21-acetoxy-6ci-fluoro-pregn-4.9 (ll), 16-triene-3.20 -dione to obtain the corresponding 1,2-dehydro derivatives: la androsta-l, 4-diene-3,17-dione 2a 6a-fluoro-androsta-l, 4,9 (ll) -triene-3,17-dione 3a 6a-methyl-androsta-l, 4,9 (ll) -triene-3,17-dione 4a 16ß-methyl-androsta-1,4,9 (1 l) -triene-3,17-dione 5a 17ct-hydroxy -pregn-1,4-dien-20-yn-3-one 6a 17a-hydroxy-pregn-l, 4.9 (ll) -trien-20-yn-3-one 7a 17a-hydroxy-16ß-methyl- pregn-l, 4,9 (ll) -trien-20-yn-3-one

8a 1 lß, 21-dihydroxy-pregn-l, 4.17 (20) -trien-3-one 9a 21-acetoxy-l lß-hydroxy-pregn-1,4,17 (20) -trien-3-one and

11β, 21 -dihydroxy-pregn-1,4,17 (20) -trien-3-one 10a 6cc-methyl-lß, 21-dihydroxy-pregn-l, 4,17 (20) -trien-3- on

I la 20-chloro-pregn-l, 4.9 (l l), 17 (20) -tetraen-21-al-3-one 12a prednisolone

13a 6a-methylprednisolone

14a 21-acetoxy-lß, l7-dihydroxy-16ß-methyl-pregn-l, 4-diene-3,20-dione and

IIβ, 17.21-Trihydroxy-16ß-methyl-pregn-1,4-diene-3.20-dione

15a 21 - acetoxy-9a-fluoro-11β, p.7-dihydroxy-16ß-methyl-pregn-l, 4-diene-3,20-dione and

9cc-fluoro-1 1β, 17.21-trihydroxy-16ß-methyl-pregn-1,4-diene-3,20-dione

16a 21 -acetoxy-9ß, 11β-epoxy-17-hydroxy-16ß-methyl-pregn-l, 4-diene-3,20-dione and

9ß, lIß-Epoxy-17,21-dihydroxy-16ß-methyl-pregn-l, 4-diene-3,20-dione

17a 21-acetoxy-17-hydroxy-pregn-l, 4.9 (l l) -triene-3.20 dione and

17.21-dihydroxy-pregn-1,4,9 (11) -triene-3,20-dione 18a 21-acetoxy-16cc, 17-dihydroxy-pregn-l, 4,9 (ll) -triene-s 3 , 20-dion and

16a, 17.21 -Trihydroxy-pregn-1,4,9 (1 l) -triene-3,20-dione 19a 21 - acetoxy-17-hydroxy-16 <x-methyl-pregn-1,4,9 ( 1 l) -triene-3,20-dione and

17.21 -dihydroxy-16 <x-methy 1-pregn-1,4,9 (11) -triene-io 3,20-dione

20a 21-benzoyloxy-17-hydroxy-16β-methyl-pregn-1,4,9 (1 l) -triene-3,20-dione

21a 21-acetoxy-17-hydroxy-16ß-methyl-pregn-1,4,9 (1 l) -triene-3,20-dione and 15 17,2l-dihydroxy-16ß-methyl-pregn-l, 4, 9 (ll) -triene-3.20-dione

22a 21-acetoxy-pregn-l, 4.9 (ll), 16-tetraen-3.20-dione and 21-hydroxy-pregn-1,4.9 (11), 16-tetraen-3.20-dione 23 a 21 - acetoxy-6a-fluoro-pregn-1,4,9 (11), 16-tetraen-20 3,20-dione and

6cc-Fluor-21-hydroxy-pregn-1,4,9 (U), 16-tetraen-3,20-di on let dry cells of A.simplex act.

Example 4

25 With A.simplex cells in a fermentation broth and with dried A. simplex cells, two different steroids are subjected to a biological conversion. After each biological conversion is considered complete (no further decrease in the amount of residual substrate), the steroid contained in each mixture is extracted and isolated (in order to obtain data for the individual experiments). The following table summarizes the results obtained:

Comparison of the isolated yields from different types of biological transformations

Substrate Type of biological chemical yield% A '% residue Remark

Conversion of valuable steroid (a) compound

0 $ ^

O

10 g / 1

Fermentation of dried cells Fermentation of dried cells

77.2% first batch 2.8% second batch

82.6% first batch 9.9% second batch 18.1% first batch no second batch

79.6% first batch no second batch

2.6 10.4

100.0

95.0

59.1

97.8

87.4 89.6

poor 1-dehydration

5.0 40.9

(b)

2.1

(a) A “usable steroid” is to be understood as an À'-compound that can be used in a further synthesis or the 1,2-dihydro substrate, which can be recycled into a further biological conversion for product production.

(b) Considerable amounts of other undesirable steroid molecules are also found.

Example 5

Arthrobacter simplex is grown in a 5 1 «Micro-ferm» fermentation vessel, stimulated to steroid-1-dehydrogenase synthesis and by centrifugation

65 harvested. Parts of the cell paste obtained are worked up by two different methods. In the first method, the cells are dried under reduced pressure at 55 ° C. In the second method, the

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U.S. Patent 3,360,439 acetone dried cells. The cells are mixed with acetone, harvested and dried at 5 ° C under reduced pressure. The dry cell preparations are then used for the biological conversion of A9 '"- androstenedione in shake flasks at concentrations of 10 g / 1 cells and 10 g / 1 steroid.

Comparison of the biological conversion capacity of A-simplex cells that have been dried by different methods

Cell type

Test duration (h)

% Residual substrate

Biological

Conversion activity * (g product formed) h / g cells acetone-

1

98.7

0.013

dries

4th

95.5

0.011

24th

84.1 *

0.007

warm

1

85.1

0.149

dries

4th

23.2

0.192

24th

7.2 *

0.039

(+) The biological conversion activity is calculated as follows:

% product formed x g substrate added

100

= g product formed g product formed / duration of incubation in h / g cells added = activity

(*) The remaining substrate amounts no longer decrease with further incubation.

Example 6

When 6cc-methylhydrocortisone in Example 1 or Androsta-4,9 (1 l) -diene-3,17-dione in Example 2 was replaced by the following substrates:

1. 21-acetoxy-9a-fluoro-lß, 16a, 17-trihydroxy-pregn-4-en-3,20-dione;

2,21-acetoxy-6a-9a-difluoro-lß, 16a, 17-trihydroxy-pregn-4-en-3,20-dione-16,17-acetonide;

3. 21-acetoxy-6a-fluoro-11β-hydroxy-16a-methyl-pregn-4-en-3,20-dione;

4. 21-acetoxy-6a-fluoro-lß, 17-hydroxy-pregn-4-en-3,20-dione;

5. 21-acetoxy-6cc-9a-difluoro-lß, 17-dihydroxy-16a-methyl-pregn-4-en-3,20-dione;

6. 21-acetoxy-9a-fluoro-lß, 16a, 17-trihydroxy-pregn-4-en-3,20-dione-16,17-acetonide;

7. 21-acetoxy-9β, lß-epoxy-6a-fluoro-16 <x, 17-dihydroxy-pegn-4-en-3,20-dione-16,17-acetonide;

8. 21-Acetoxy-9ß, l lß-epoxy-16a-hydroxy-pregn-4-en-3,20-dione and

9. 21-acetoxy-9β, 1 lß-epoxy-16a, 17-dihydroxy-pregn-4-en-3,20-dione-16,17-acetonide gives the following products:

1. 21-acetoxy-9a-fluoro-lß, 16a-17-trihydroxy-pregn-l, 4-diene-3,20-dione and

9a-fluoro-11β, 16a, 17.21-tetrahydroxy-pregn-1,4-diene-3.20-dione;

2. 21-acetoxy-6a, 9a-difluoro-lß, 16a, 17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide and

6a, 9a-fluoro-lß, 16a, 17,21-tetrahydroxy-pregn-l, 4-diene-3,20-dione-16,17-acetonide;

3. 21-Acetoxy-6a-fluoro-1β-hydroxy-16a-methyl-pregn-1, 4-diene-3,20-dione and

6a-fluoro-lß, 21-dihydroxy-16a-methyl-pregn-l, 4-diene-3,20-dione;

4.21-acetoxy-6a-fluoro-lß, 17-hydroxy-pregn-l, 4-diene-3,20-dione and

5 6a-fluoro-lß, 17.21-trihydroxy-l, 4-diene-3.20-dione;

5. 21-acetoxy-6a, 9a-difluoro-lß, 17-dihydroxy-16a-methyl-pregn-1,4-diene-3,20-dione and

6a, 9a-difluoro-11β, 17.21-trihydroxy-1,4-diene-3.20-dione;

6. 21-acetoxy-9a, fluoro-1ß, 16a, 17-trihydroxy-pregn-lo 1,4-diene-3,20-dione-16,17-acetonide;

7. 21-acetoxy-9β, lß-epoxy-6a-fluoro-16a, 17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide;

8. 21-Acetoxy-9ß, l lß-epoxy-16-hydroxy-pregn-l, 4-diene-3,20-dione and

15 9β, 11β-epoxy-16a, 21 -dihydroxy-pregn-1,4-diene-3,20-dione;

9. 21-Acetoxy-9ß, l lß-epoxy-16a, 17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide.

20 Example 7

Biological conversion of 1β-Hydroxyandrosta-4-en-3,17-dione

One gram of dried A.sim-plex cells according to Example 2 is resuspended in 100 ml of 50 mM phosphate buffer with a pH value of 7.5. Thereafter, menadione dissolved in 3A ethanol is added in a final concentration of 86 mg / 1. After adding Vi g 1 lß-hydroxyan-drostendione to the bottle, the mixture is incubated on a rotary shaker at a temperature of 31 ° C. If samples are taken after the 1-day incubation, two products appear on a thin-layer chromatogram of a methylene chloride extract. About 95% of the steroid consists of 11β-Hydroxyandrosta-l, 4-diene-3,17-dione, the rest of unconverted substrate.

35

Example 8

According to Example 7, the following androstenediones modified on the Cn can be 1-dehydrogenated:

40 substrate product

1 lß-Hydroxy-16ß-methylandrosta-4-en-3,17-dione 11 ß-Hydroxy-16ß-methylandrosta-1,4-diene-3,17-dione

1 lß-Hydroxy-16a-methylandrosta-4-en-3,17-dione - * 45 1 lß-Hydroxy-16a-methylandrosta-l, 4-diene-3,17-dione

6a-fluoro-1ß-hydroxyandrosta-4-en-3,17-dione -> ■ 6a-fluoro-11ß-hydroxyandrosta-1,4-diene-3,17-dione

6a-methyl-lß-hydroxyandrosta-4-en-3, l 7-dione 6a-methyl-11 ß-hydroxyandrosta-1,4-diene-3,17-dione 50 1 la-hydroxyandrosta-4-en-3 , l 7-dione 1 la-hydroxyandrosta-l, 4-diene-3, l7-dione and

Androsta-4-en-3,11,17-trione Andorsta-1,4-diene-3,11,17-trione.

55 Example 9

1. The following components are combined (on a 1 liter basis) in a reaction vessel:

a) 0.9 1 50 mM KPCh buffer with a pH of 7.5

b) 3.66 g dry A.simplex cells b0 c) 0.08 g menadione d) 8 g 21-acetoxa-pregn-4.9 (I l), 16-triene-3.20-dione e) 100 ml toluene.

2. It is moved at 15 cal / 1 min.

3. Access of so much air that the oxygen content in the gas space of the reaction vessel is about 3-6%.

4. The temperature is kept at 28 ° ± 1 ° C.

5. After 25 h the toluene phase is separated off.

In this example, the steroid is in the toluene phase

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out

94.3% 21-acetoxy-pregn-l, 4.9 (l l), 16-tetraen-3,20-dione

4.2% 21-hydroxy-pregn-1,4,9 (1 l), 16-tetraen-3,20-dione and

1.5% 21-acetoxy-pregn-l, 9 (l l), 16-triene-3,20-dione.

The different stages described can be varied as follows:

step 1

a) Buffer pH range: 6-10.

b) The amount of A.simplex is reduced to such an extent that it is just sufficient to allow the reaction to proceed to completion. Typical amounts of A. simplex are 0.05-1.0 g / g steroid. The microbiological enzyme supplier is: Arthrobacter (Corynebacterium) simplex (ATCC No. 6946) or Bacterium cyclooxydans (ATCC No. 12 673).

Production of the microorganisms: The A.simplex cells can be used in the fermentation broth or modified by collecting the cells, drying, acetone treatment or immobilization in a conventional manner.

c) Menadione- (2-methyl-l, 4-naphthoquinone), menadione bisulfite, 1,4-naphthoquinone or other vitamin K-like compounds are suitable as electron acceptors.

The content of the electron acceptor is determined by its cost and reaction rate considerations. The reaction rate is linearly proportional to the menadione concentration. Typical amounts are about 5 to about 40 g / kg steroid.

d) The steroid concentration should be as high as possible as long as an effective conversion is taking place.

Suitable steroids are those of the 3-Keto-A4-androsten- or 3-Keto-A4-pregnen series. Preferably, the steroid should have a solubility in the organic solvent of more than 5 g / l.

e) Toluene, benzene and / or xylene are suitable as aromatic hydrocarbons. These solvents can be used alone, as a mixture with one another or in dilution with other water-immiscible solvents, such as heptane or methylene chloride.

The amount of aromatic hydrocarbon ranges from about half the amount required to dissolve the most soluble starting steroid or steroid formed to about four times the amount required to dissolve the least soluble starting steroid or steroid formed.

In the present example, just enough toluene is added that the steroid is dissolved when the reaction is complete.

The aromatic hydrocarbon is preferably added together with the steroid. However, the aromatic hydrocarbon can also be added later or earlier.

Level 2

You can also move with high kinetic energy. The reaction speed is clearly related to the kinetic energy.

level 3

The implementation requires oxygen as the final hydrogen (electron and proton) acceptor. However, due to the presence of the aromatic hydrocarbon, an explosive environment exists in the gas space of the reactor. In order to eliminate or substantially reduce this risk of oxygen concentration in the gas space, the oxygen concentration should be kept below the minimum concentration required for combustion. The minimum oxygen concentration for benzene is 11.2%. In the case of xylene, it is also possible to carry out the reaction below the flash point. The flash points for m-xylene, o-xylene and p-xylene are 29.32 and 39 ° C.

Level 4

The temperature of the reaction can range from 0 ° to 45 ° C.

Level 5

The implementation typically takes 2 days, but it can also take a few hours to a few weeks.

Example 10

Comparison of experiments in toluene and aqueous phase.

A. Measures:

First, 0.4 g of A. simplex cells are combined with 50 ml of 50 mM KP04 buffer with a pH of 7.5, followed by agitation for 2 hours or until a uniform slurry is reached. Two 25 ml fractions are now removed and transferred to 125 ml bottles. 0.25 ml of 5 mM menadione in 3A alcohol and 0.2 g of Androsta-4.9 (1 l) -diene-3.17-dione are then introduced into each bottle. 2 ml of toluene are poured into one of the stoppered bottles, and the bottles are moved for 4 days using an articulated shaker. After 4 days, extraction with 25 ml of toluene is carried out. An analysis of the extracts gives the following results.

B. Results:

Process in aqueous in toluene

medium

(Substrate)

Androsta-4.9 (l l) -diene-3, l 7-dione 17.7% 0.00% (product)

Andorsta-4.9 (l l) -triene-3, l 7-dione 82.3% 100.00%

Example 11

According to Example 9, the following steroids are made using dried A.simplex cells:

1. Androsta-4-en-3,17-dione

2. 6a-fluoro-androsta-4,9 (1 l) -diene-3,17-dione

3. 6a-methyl-androsta-4,9 (l) -diene-3,17-dione

4. 16β-methyl-androsta-4,9 (1 l) -diene-3,17-dione

5. 17a-Hydroxy-pregn-4-en-20-yn-3-one

6. 17 <x-hydroxy-pregn-4,9 (l) -dien-20-yn-3-one

7. 17a-Hydroxy-16ß-methyl-pregn-4,9 (1 l) -dien-20-yn-3-one

8.21-acetoxy-lß-hydroxy-pregn-4,17 (10) -dien-3-one

9. 20-chloro-pregn-4,9 (1 l), 17 (20) -trien-21-al-3-one

10. 21-Acetoxy-17-hydroxy-pregn-4.9 (l) -diene-3.20-dione

11. 21-acetoxy-l 7-hydroxy-16a-methyl-pregn-4,9 (1 l) -diene-3,20-dione

12.21 -Benzoyloxy- 17-hydroxy-16ß-methyl-pregn-4,9 (1 l) -diene-3,20-dione

13.21-Acetoxy-17-hydroxy-16β-methyl-pregn-4.9 (1 l) -diene-3.20-dione

14.21-acetoxy-pregn-4.9 (ll), 16-triene-3.20-dione 15.21-acetoxy-6cc-fluoro-pregn-4.9 (ll), 16 (17) -triene-3.20- dion in the following products:

la Androsta-l, 4-diene-3, l 7-dione 2a 6a-fluoro-androsta-l, 4.9 (ll) -triene-3.17-dione 3a 6a-methyl-androsta-l, 4.9 (ll) -triene-3,17-dione

5

10th

15

20th

25th

30th

35

40

45

50

55

60

b5

9

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4a 16β-methyl-androsta-l, 4.9 (ll) -triene-3,17-dione 5 a 17a-hydroxypregn-1,4-dien-20-yn-3-one 6a 17a-hydroxy-pregn-l , 4,9 (ll) -trien-20-yn-3-one 7a 17a-hydroxy-16ß-methyl-pregn-l, 4,9 (ll) -trien-20-yn-3-one

8a 21 - acetoxy-11β-hydroxy-pregn-1,4,17 (20) -trien-3-one and

11β, 21 -dihydroxy-pregn-1,4,17 (20) -trien-3-one 9a 20-chloro-pregn-1,4,9 (1 l), 17 (20) -tetraen-21-al -3-one 10a 21-acetoxy-17-hydroxy-pregn-1,4,9 (ll) -triene-3,20-dione and

17.21 -Dihydroxy-pregn-1,4,9 (1 l) -triene-3,20-dione 1 la 21 -acetoxy-17-hydroxy-16a-methyl-pregn-1,4,9 (1 l) -triene-3.20-dione and

17,21 -dihydroxy-16a-methyl-pregn-1,4,9 (11) -triene-3,20-dione

12a 21 -Benzoyloxy-17-hydroxy-16ß-methyl-pregn-1,4,9 (1 l) -triene-3,20-dione

13a 21 -acetoxy-17-hydroxy-16ß-methyl-pregn-1,4,9 (1 l) -triene-3,20-dione and

17,21 -dihydroxy-16ß-methyl-pregn-1,4,9 (1 l) -triene-3,20-dione

5 14a 21 - acetoxy-pregn-1,4,9 (11) tetraen-3,20-dione and 21-hydroxy-pregn-1,4,9 (11), 16-tetraen-3,20-dione 15a 21 -Acetoxy-6a-fluoro-pregn-l, 4.9 (ll), 16-tetraen-3.20-dione and

6cc-Fluoro-21-hydroxy-pregn-1,4,9 (11), 16-tetraen-3,20-dione io.

The utility of 1,2-dehydrosteroids is known. For example, it is known from US Pat. No. 3,284,447 to use A1 -4'9 (11 Prego et ri en e in the synthesis of diuretically active cis-substituted corticosteroids. I5 A process is known from US Pat. No. 4,041,055 known for the synthesis of corticosteroids from A '^ - androstenedione derivatives, ie 1,2-dehydroandrostenes are important intermediates in the production of medically valuable steroids.

G