DE3322120A1 - METHOD FOR CONVERTING 1,2-SATURED 3-KETOSTEROIDS IN 1,2-DEHYDROSTEROIDS - Google Patents

Description

Process for converting 1,2-saturated 3-keto steroids into 1,2-dehydrosteroids

the first therapeutic use of corticosteroids was reported in the 1950s. This was a cortisone acetate treatment for rheumatoid arthritis. Further investigations showed that the incorporation of an unsaturated bond in the 1,2-position of hydrocortisone and cortisone led to the resulting steroids prednisolone and prednisone receiving an increased effect and causing less drug-reduced salt retention. Thereafter, most of the other steroids that have been used in the treatment of corticoid-responsive diseases were also given a double bond in the 1,2-position of the steroid molecule. Since 1977, two attempts to synthesize corticosteroids from sterol precursors have become known. From the U.S. PS. 4,035,236, for example, a process for the production of 9a-hydroxyandrostenedione by fermentation of sitosterol, stigmasterol or cholesterol is known. US Pat. No. 4,041,055 discloses a process for the synthesis of medically valuable corticosteroids from androstene. Intermediate products that occur here can have the 3-keto-A ⁴ ' ^{9 (11} * configuration.

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Known processes for the biological conversion of 1,2-saturated Steroids in the corresponding 1,2-dehydrosteroids are:

U.S. Patent 2,837,464 "Process for the Production of Dienes by Corynebacterium".

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

U.S. Patent 3,360,439 "Process for the Preparation of 2-Dehydrosteroids".

Describes a 1-dehydration of steroids with Aid of A. simplex cells, which were prepared before mixing the

·: -

Substrate with a hydrogen carrier with a short-chain Alkanol or short-chain alkanone, e.g. acetone, have been pretreated.

W.Charney and H.Herzog "Microbial Conversion of Steroids", Academic Press. Inc., New York, Pp. 4-9, 236-261 (1967).

The latter reference sets out the historical background of biological steroid conversions and a Taxonomic listing of microorganisms suitable for carrying out a 1-dehydration is given.

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

The authors examined the 1-dehydrogenation of 4-androstene-3,17-dione using Nocardia rhodocrous in a mixture of 50% benzene and 50% heptane. By adding this mixture of solvents to the biological Conversion mixture should speed the reaction

can be increased by a factor of 180 »however, benzene is known to be carcinogenic. The preferred electron acceptor for Nocardia rhodocrous activity was at the experiments carried out with phenazine methosulfate. This Electron acceptor is too expensive and too unstable to use in large-scale biological conversion processes to be able to use this microorganism activity in practice. The Nocardia rhodocrous enzyme was present of menadione, the electron acceptor of choice for Ärthrobacter simplex, inactive. So it has to be distinguish the enzyme produced by N. rhodocrous from the enzyme produced by A. simplex. The implementation was in a steroid-saturated solvent system with only 20% organic solvent. It was found that the rate of reaction decreased as the concentration of the steroid increased. Oxygen is an essential reagent in the 1-dehydrogenation reaction. The solvent (50% Benzene / 50% heptane) is flammable in the presence of oxygen. Like the oxygen in the reactor without being created was introduced in an explosive environment, is not explained in the cited literature.

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

The authors mentioned examined 1-dehydration of hydrocortisone using A. simplex. There were two with Water immiscible solvents, namely n-butanol and η-amyl alcohol, tested. In the presence of these solvents however, there is no bioconversion. As. preferred solvents were water-miscible solvents, ZoB. Mixtures of 10% methanol and 90% water and 10% propylene glycol and 90% water are used.

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The cited references describe neither the invention Procedure nor do they suggest this.

Using air-dried or heat-dried Microbial or microorganism cells / capable of catalyzing 1-dehydrogenation of steroids a more effective conversion of 1,2-saturated steroids into their corresponding 1,2-dehydro derivatives than in the context of those working in an aqueous environment, especially in the presence of an exogenous electron acceptor known procedures.

The invention also relates to a method in which an aromatic hydrocarbon as water-immiscible solvent of an aqueous slurry of A. simplex cells, an exogenous Electron acceptor and the steroid to be dehydrated is added. The slurry is agitated. the Oxygen is supplied in such a way that the oxygen content in the Gasraura below the minimum oxygen concentration remains for a burn. On the other hand, the reaction can also (at a temperature) below the Flash point of the organic solvent can be carried out.

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

a) The bioconversion leads to lower levels of ° unconverted steroid substrate;

b) the inventive method can at higher Steroid concentrations are carried out;

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

d) the method according to the invention is less sensitive opposite in the steroid, in the microorganism cells or in the one formed by the cells Steroid contained organic soluble impurities and

e) a reduction or complete elimination of steroid-degrading substances contained in the microbial cells Enzymes lead to higher yields of the desired reaction product.

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The solvent measures are the well-known technology of steroid dehydration with other microorganism activities, e.g. Nocardia rhodocrous, because the reaction catalyzed by A.simpelx with more stable and cheaper electron acceptors such as menadione can be performed.

Microorganisms:

In the context of the method according to the invention, as

The numerous microorganisms required for 1-dehydration known microorganisms are used. Such microorganisms are described by W. Charney and H. Herzog in "Microbial Transformation of Steroids", publisher

Academic Press, Inc., New York, (1967). 25th

The bacteria that dehydrate steroids in position 1, generally fall into two groups, see "Bergey's Manual of Determinative Biology ", 8th edition. The method described loc. cit. could be successfully used on species Arthrobacter and Corynebacterium genera from "Part 17, Actinomycetes & Related Organisms" will be demonstrated. The 1-dehydrogenendai species of various other genera in Part 17, e.g., Nocardia, Mycobacterium, Streptomyces and Bacterium are also likely useful.

4ο.

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

Microorganism growth:

The microorganisms are in an aqueous nutrient medium with:

a) inorganic compounds, e.g. nitrates or

Ammonium salts, or organic nitrogenous compounds, e.g. yeast extract, peptone, corn steep liquor, etc. (source of nitrogen for growth) j
25th

b) a carbon and energy source, for example 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 quantities such as those found in tap water or less purified components of the nutrient medium, e.g. corn steep liquor, are supplied,

let grow.

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

Obtaining steroid 1 dehydrogenase:

The cells are saturated by the addition of a 1,2 3-ketosteroid compound, e.g. from Androsta-4-en-3, 1 7-diori or cortisone acetate in an amount of 0.005% (w / v) of the medium or above to steroid-1-dehydrogenase activity stimulated. In the presence of the "stimulant", incubation is continued for at least 6 hours before the cells harvested to dry.

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The "stimulant" can be added at any point during the growth cycle. Cultures, grown on culture media such as lard oil, begin with the synthesis of the steroid 1-dehydro-

20 'recover quickly while cultures growing on glucose require substrate depletion before enzyme synthesis occurs. After adding the "stimulant" incubation is continued for δ h or longer, after which the cells are used in the solvent procedure or harvested to dry.

Measures to collect cells for drying:

The cells are separated from the nutrient medium and in the customary known manner, e.g. by centrifugation or

³ ^ Flocculation and Filtration and Ultrafiltration Concentrated. The separated cells are then dried by drying them under reduced pressure at 1 ° to 85 ° C, preferably 55 to 7 5 ° Ci by air drying with heating, by spray drying or by drum drying to a moisture content of about 1 to about 10%.

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dried. Preferably the moisture content is about 5%. The cells are then stored at 5 ° C until they are used for bioconversion. Active dried cells can also be obtained by immobilization dried cells by standard methods, e.g., by inclusion in a polyacrylamide gel and collagen, or by covalent coupling of the cells to a polyelectrolyte carrier, see "Methods in Enzymology", Volume XLIV, 1976, Academic Press, Inc., New York, Pages 11-317.

Biological conversion:

The bioconversion takes place by letting the cell preparations act on the steroid substrate. In typical 5 way, the cells and the steroid are in a weakly buffered aqueous solution of a pH in the range from 6-10, preferably 7.25-8.5, suspended. The cell volume is 0.1 - 50 g / l. The steroid can be in a weight ratio (steroid: cells) from 0.05 to 5.0 can be added. Cell quantities of 8-10 g / l 5-10 g / L steroid is preferred when the biological conversion occurs in an aqueous environment. To stimulate the reaction, an exogenous electron carrier is used in a catalytic amount (for example

-4
5 χ 10 "M menadione) added. Suitable compounds are menadione (2-methyl-1,4-naphthoquinone), phenazine methosulphate, dichlorophenol indophenol, 1,4-naphthoquinone, menadione bisulphite, ubiquinone (coenzyme Q) and vitamin K compounds The mixtures are incubated at a temperature of 5-45 ° C. for 0 to 14 days.. During the incubation, access of molecular oxygen to the mixture is possible. Preferably, the mixture is stirred during the incubation. Dehydration typically decreases over time, and with use

from 10 g / l substrate, the bioconversion can be complete in less than 98 - 100 % .

Examples of various feasible procedural measures are given below:

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

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

c) rehydration of the dried cells in a small volume of buffer and subsequent addition of more buffer or an organic solvent. As much ethanol, acetone, xylene, butyl acetate, Methylene chloride or toluene added so that the final organic solvent content is 0 - 95% (Vol / Vol) is. The amount of aromatic used Hydrocarbon ranges from about half that required to dissolve the most soluble parent steroid or formed steroid up to about four times the amount required to dissolve

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the least soluble parent steroid or bil deten steroid required amount. The steroid and the electron carrier are used to initiate the implementation admitted. The aromatic carbon-hydrogen is preferably added together with the Steroid, but it can also be done sooner or later.

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

Substrate area:

Usable in the process according to the invention

4th
Compounds belongs to the 3-keto-Δ-android and 3-keto-Δ -pregnen series of steroids. Substrates for steroid 1-dehydrogenase are known to be _{saturated between the carbon atoms C 1} and CU of ring A and have a hydroxyl 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

"5- 2) Androsta-4, 9 (11). -diene-3, 1 7-dione and its 6a-fluorine-, 6a-methyl or 16-methyl derivatives;

3) 11ß-Hydroxyandrosta-4-en-3,17-dione and its derivatives .

4th
Usable steroids of the 3-Keto-A -pregnen series are:

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

2. 11β, 21-dihydroxy-pregn-4,17 (20) -dien-3-one and his 6a-methyl derivatives;

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

4 4. different groups of 3,20-diketo-A -Pregnenes, how

a) 11,17,21-tri-hydroxy compounds, e.g. hydro

cortisone and its 6a-methyl derivative;

b) 9β, 11β-epoxy-17, 21-dihydroxy compounds, e.g.

9β, 11β-epoxy-17,21-dihydroxy-16β-methyl-pregn-4-en-3,20-dione;

c) 3,20-diketo-4,9 (11) -pregnedienes, e.g. 17a, 21-dihydroxy-pregn-4,9 (11) -diene-3,20-dione and its 16α-methyl, 16β-methyl or 16α-hydroxy derivative or the 17a-acetate ester ;.

d) 3,20-diketo-4,9 (11), 4,9 (11)., 16-pregnetrienes, e.g. 2.1-Hydroxy-pregh-4,9 (11), 1 6-triene-3, 20-dione and his 6a-fluorderivato

The 21-ester derivatives are also suitable as substrates 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 acids Carboxylic acids, e.g., benzoic acid.

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

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

example

Preparation of the dried cells:

Inoculation with Bacterium cyclooxydans (ATCC No. 12673)

success in shake flasks with a medium made of cerelose, 10

Peptone and corn steep liquor (6 g / L each), pH 7.0. The cultures are up to occurrence a glucose depletion incubated on a rotary shaker at 28 ° C. When the glucose depletion occurs

an addition of 0.5 g / l cortisone acetate, whereupon the 15th

Shake flasks are incubated again for 16 hours. the

The cells are harvested by centrifugation. The centrifuged Cells are washed twice with water and then in a low vacuum Oven stored at 4 5 ° C until dry. 20th

Bio-conversion of βα-methylhydrocortisone.

Half a gram of that prepared in the manner described dried cells is rehydrated with stirring in 50 ml of a 50 mM phosphate buffer of pH 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 added. The substrate is added as a solution in dimethylformamide (100 mg 6a-methylhydro-

gg cortisone / ml dimethylformamide) up to a final bio-conversion concentration of 0.5 g / l. The mixture is then incubated at 28 ° C. with agitation. After 4 hours of incubation the steroid is 91% converted. The 6a-methylprednisolone is used in the usual known manner

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example

Preparation of Androsta-1,4,9 (11) -triene-3,17-dione

a) Production of the biocatalyst:

Arthrobacter simplex (ATCC No. 6946) is placed in shake flasks in a medium with 6 g / l glucose, Grown 6 g / L corn steep liquor and 6 g / L spray dried bacon water. the Cultures are incubated on a rotary shaker at 28 ° C. until glucose depletion occurs. At this point, 0.15 g / l is added Cortisön acetate 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 of one temperature for 24 hours to dry of 55 ° C. 24 hours later, the dried material is transferred to an airtight container and stored therein at 5 ° C until used for biological conversion.

b) Biological conversion of Androsta-4,9 (11) -diene-3,17-dione:
.

10 g / l dried cells are stirred for 30 minutes in 50 mM phosphate buffer with a pH of 7.5 rehydrated. Then the cell suspension is mixed with so much dry powdered menadione that that a final concentration of 86 mg / 1 is reached. Micronized androsta-4,9 (11) -diene-3,17-dione is also used slurried in dimethylformamide and in the form of the slurry to the biological conversion mixture in an amount of 2.5 g steroid / 1 and 2%

qc (v / v) dimethylformamide added. Now the mixture is

incubated with agitation in the presence of air for 24 hours at 31 ° C. When the incubation is complete, the Androsta-1, 4, 9 (11) -triene-3,17-dione in the usual known Wise portrayed purely.

example

In a manner similar to Example 2, the following steroids are used:

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

2. 6a-fluoro-androsta-4,9 (11) -diene-3,17-dione

3. 6a-methyl-androsta-4,9 (11) -diene-3,17-dione

- 4. 16β-methyl-androsta-4,9 (11) -diene-3,17-dione

5. T7a-hydroxy-pregn-4-en-20-yn-3-one.

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

7th 17oc-Hydroxy-16β-methyl-pregn-4,9 (11) -dien-20-yn- _2D 3-one

8. 11β, 21-dihydroxy-pregn-4,17 (20) -dien-3-one

9. 21-acetoxy-11β-hydroxy-pregn-4,17 (20) -dien-3-one

10. 6α-methyl-11β, 21-dihydroxy-pregn-4,17 (20) -dien-3-one 11. 20-chloro-pregn-4,9 (11), 17 (20) -trien-21-al-3-one

12. Hydrocortisone

13. 6a-methylhydrocortisone

14. 21-Acetoxy-11β, 17-dihydroxy-16β-methyl-pregn-4-en-. 3,20-dione

15. 21-Acetoxy-9a-fluoro-11β, 17-dihydroxy-16β-methylpregn-4-ene-3,20-dione

16. 21-Acetoxy-9β, 11β-epoxy-17-hydroxy-16β-methyl-pregn-4-en-3,20-dione

17o 21-acetoxy = 17-hydroxy-pregn-4,9 (11) -diene-3 _f 20-dione

18. 21-acetoxy-16a, 17-dihydroxy-pregn-4,9 (11) -diene-3,20-dione

19. 21-Acetoxy-17-hydroxy-16a-methyl-pregn-4,9 (11) -diene-3,20-dione

20. 21-Benzoyloxy-17-hydroxy-16β-methyl-pregn-4,9 (11) -diene-3,20-dione

21. 2i-acetoxy-17-hydroxy-16β-methyl-pregn-4,9 (11) - · Dien-3, 20-dione

22. 21-acetoxy-pregn-4,9 (11), 16 (17) -triene-3,20-dione and

23. 21-acetoxy-6a-fluoro-pregn-4,9 (11), 16-triene-3,20-dione ·

to obtain the corresponding 1,2-dehydro derivatives: 1a androsta-1,4-diene-3,17-dione 2a 6a-fluoro-androsta-1,4,9 (11) -triene-3,17-dione 3a 6a-Methyl-androsta-1,4,9 (11) -triene-3,17-dione 4a 16β-methyl-androsta-1,4,9 (11) -triene-3,17-dione 5a 17cL-hydroxy-pregn-1,4-dien-20-yn-3-one 6a 17a-hydroxy-pregn-1,4,9 (11) -trien-20-yn-3-one

.

7a 17a-hydroxy-16ß-methyl ~ pregn-1,4,9 (11) -trien-20-yn-

3-on
8a 11β, 21-dihydroxy-pregn-1,4,17 (20) -trien-3-one

9a 21-acetoxy-11β-hydroxy-pregn-1,4,17 (20) -trien-3-ones and

11β, 21-dihydroxy-pregn-1,4,17 (20) -trien-3-one

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

11a 20-chloro-pregn-1,4,9 (11), 17 (20) -tetraen-21-al-3-one

12a prednisolone

13a 6a-methylprednisolone

14a 21-acetoxy-11ß, 17-dihydroxy-16ß-methyl-pregn-1, 4-

diene-3,20-dione and
5

11β, 17,21-trihydroxy-16β-methyl-pregn-1,4-diene-3,20-dione

15a 21-acetoxy-9a-fluoro-11β, u7-dihydroxy-16β-methylpregn-1 , 4-diene-3,20-dione and

9a-fluoro-11ß, 17,21-trihydroxy-16ß-methyl-pregn-1,4-diene-3,20-dione

16a 21-acetoxy-9β, 11β-epoxy-17-hydroxy-16β-methyl-pregn-1,4-diene-3,20-dione and

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

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

19a 21-acetoxy-17-hydroxy-16α-methyl-pregn-1,4,9 (11) - ² ^ triene-3,20-dione and

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

20a 21-Benzoyloxy-17-hydroxy-16ß-methyl-pregn-1,4,9 (11) -

triene-3,20-dione
30th

21a 21-acetoxy-17-hydroxy-16β-methyl-pregn-1,4,9 (11) -triene-3,20-dione and

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

22a 21-acetoxy-pregn-1,4,9 (11), 16-tetraene-3,20-dione and 2i-hydroxy-pregn-1 , 4,9 (11) , 16-tetraene-3,20-dione

: 3 3 2 2 1 2

23a 2T-acetoxy-6a-fluoro-pregn-1,4,9 (11), 16-tetraene-3.20 dione and 6a-fluoro ~ 21-hydroxy-pregn-1,4,9 (11), 16-tetraene-3 ", 20-dione

allowed to act on dry cells of A "simplex.

example

With cells from A. simplex in a fermentation broth and with ge. Dried A. siraplex cells become two different Steroids undergo biological conversion. Considered complete after any biological transformation became (no further decrease in the amount of residual substrate) j becomes the steroid contained in each mixture

extracted and isolated (to obtain data for each experiment). The following table summarizes the obtained results together:

Comparison of the isolated yields from different types of biological conversions

Substrate

g / l

Type of biological chemical yield % Δ ¹ % remainder Comment Conversion of valuable compound

Steroid (a) dung

fermentation

dried cells

77.2% first batch 2.6

2.8% second batch 10.4

82.6% first batch 100.0 9.9% second batch 95.0

87.4

89.6

5.0

poor 1-dehydration

g / l

OAc fermentation

dried cells 18.1% first batch 59.1 no second batch

79.6% first batch 97.8

no second batch

40.9

2.1

(b)

(a) A "useful steroid" is a Δ "compound that can be used in a further synthesis or the 1,2-dihydric substrate, which can be fed back into a further biological conversion for product recovery can understand.

(b) There will also be significant amounts of other undesirable Steroid molecules found.

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Example 5

Arthrobacter simplex is placed in a 5 liter "Microferm" fermentation vessel grown, stimulated to a steroid-1 dehydrogenase synthesis and centrifuged harvested. Parts of the cell paste obtained are worked up by two different methods. The first In the process, the cells are dried at 55 ° C. under reduced pressure. The second procedure is similar U.S. Patent 3,360,439 produced acetone-dried cells. The cells are mixed with acetone, harvested and dried at 5 C under reduced pressure.

Then the dry cell preparations are used for biological

9 11
see conversion of Δ '-androstendione in shake flasks at concentrations of 10 g / l cells and 10 g / l steroid used.

Comparison of the biological conversion capacity of A. simplex cells that were dried by different methods

Cell type

Test duration (h)

% Residual biological substrate substrate activity +

(g formed product)

h / g cells

acetone
dries 1
4th 98.7
95.5 0.013
0.011. 24 84.1 * 0.007 warming
dries 1
4th 85.1
23.2 0.149
0.192 24 7.2 * 0.039

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

% product formed χ g substrate added

100

= g product formed

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

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

Example 6

When replacing 6a-methylhydrocortisone in Example 1 or Androsta-4,9 (11) -diene-3,17-dione in Example 2 by the following Substrates:

1 "21-acetoxy-9a-fluoro-11β, 16a, 17-trihydroxy-pregn-4-en-3,20-dione;

2. 21-acetoxy-6a, 9a-difluoro-11ß, 16a, 17-trihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide;

3 _O 21-acetoxy-öa-fluoro-11ß-hydroxy-16a-methyl-pregn-4-en-3,20-dione;

4. 21-acetoxy-6a-fluoro-11β, 17-hydroxy-pregn-4-en-3,20-dione;

5. 21-acetoxy-6a, 9a-difluoro-11ß, 17-dihydroxy-16a-methylpregn-4-en-3 , 20-dione;

6 ο 21-acetoxy-9a-fluoro-11ß, 16a, 17-trihydroxy-pregn-4-en-3,20-dione-16,17-acetonide;

7. 21-acetoxy-9β, 11β-epoxy-6a-fluoro-16a, 17-dihydroxypegn-4-en-3,20-dione-16,17-acetonide;

8. 21-Acetoxy-9β, 11β-epoxy-16a-hydroxy-pregn-4-ene-3,20-dione and

9. 21-Acetoxy-9β, 11β-epoxy-16a, 17-dihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide

you get the following products:

1, 21-acetoxy-9a-fluoro-11ß, 16a-17-trihydroxy-pregn-1 , a- diene-3,20-dione and

9a-fluoro-11beta, 16a, 1 1, 21-tetrahydroxy-pregn-1,4-diene-3,20-dione;

2 ". 21 -acetoxy-6a, 9a-difluoro = 11β, 16a, 17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide and 6a, 9a-fluoro -11 ß "16öS, 17 _f 21-tetrahydroxy-pregn-1,4-diene-3,20-UiOn = Io ^ 17-acetonide;

H.

3. 21-Acetoxy-6a-fluoro-11ß-hydroxy-16a-methyl-pregn-1 _f 4-diene-3,20-dione and

6a-fluoro-118,21-dihydroxy-16a-methy1-pregn-1,4-diene-3,20-dione;

4. 21-acetoxy-6a-fluoro-11ß, 17-hydroxy-pregn-1,4-diene-3 _f 20-dione and
6a-fluoro-11ß, 17,21-trihydroxy-1,4-diene-3 _f 20-dione;

5. 21-acetoxy-βα, 9a-difluoro-11β, 17-dihydroxy-16a-methyl pregn-1,4-diene-3,20-dione and

6a, 9a-difluoro-11β, 1 Ί, 21-trihydroxy-1,4-diene-3,20-dione;

. 21-acetoxy-90., Fluor-11β, 16a, 17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide;

IS 7, 21-acetoxy-96,11S-epoxy-6a-fluoro-16a, 17-dihydroxypregn-1,4-diene-3,20-dione-16,17-acetonide;

8. 21-Acetoxy-9β, 11β-epoxy-16-hydroxy-pregn-1,4-diene-3,20-dione and

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

9. 21-Acetoxy-9β, 11β-epoxy-16a, 17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide.

Example 7

Biological conversion of 11ß-Hydroxyandrosta-4-en-3,17-dione.

One gram of A. simplex cells dried according to Example 2 is resuspended in 100 ml of 50 mM phosphate buffer, pH 7.5. Then in is added 3A Menadione dissolved in ethanol at a final concentration of 86 mg / 1. After adding 1/2 g of 11ß-Hydroxyandrosten-

The mixture is poured into the bottle on a rotary shaker 35

2?

lex incubated at a temperature of 31 ° C. “If samples are taken after 1 day of incubation, ^{two products appear on a thin-layer chromatogram of a methylene chloride extract Λ} · '. About 95% of the steroid consists of 11ß-Hydroxyandrosta »1 _f 4-diene-3,17-dione, the rest of unconverted substrate,

Example 8

According to Example 7 can be the following at the C ₁ --modifizierte ..Ändrostendione T. dehy drier en:

Substrate ■ »Product

(1) 11β-Hydroxy-T6β-methylandrosta-4-en-3 ₍ , 17-dione ■> 11β-hydroxy-1ββ-methylandrosta-1,4-diene-3,17-dione

(2) 11 ~ ß-Hydro2sy J.6a-methylandrosta-4-ene-3,17-dione - * ■ 11 dione 7-ß-hydroxy-1 6a-methylandrosta-1, 4-diene "3.1

(3) 6a-fluoro-nß "= - hydroxyatidros" ta ™ -4-en-3,17-dione - »

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

(4) 6a-methyl-11 ß-hydroxyandrosta-4-en-3,1 7-dione * 6a-methyl-11β-hydroxyandrosta ~ -1,4-diene-3, 17-dione

^: T1 a-Hydrosyandrosi © - ^ ^: ^ -ea-3/17-dione 1 1 & = Hydroxyandrosta-1 _ff 4-? 4ien-3,17-dione and

(6) Androsta-4-ene-3,11 "17-trione

Aedot1,4-diene-r3 ^ 1 X ₄ 17-trione.

Example 9
1 .. In a reaction vessel, /! On a .1 liter basis) follow

The following components are combined:

a) 0.9 1 50 mM KPO. buffer, pH 7.5

b) 3.66 g dry A. Simplex cells

c) 0.08 g menadione

d) 8 g of 21-acetoxy-pregn-4,9 (11), 16-triene-3,20-dione e) 100 ml of toluene.

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

3. Admission of so much air that the oxygen content about 3-6% in the gas space of the reaction vessel. amounts to. ·

4. The temperature is kept at 28 ° -1 ° C. 5. After 25 hours, the toluene phase is separated off.

In this example>: the steroid in the toluene phase the end

94.3% 21-AcetO3cy-pxegn-1V4 _T 9 <T1 | , 16-tetraen-r3 "20-dione, 4.2% 21-hydroxy-pxegnr-V, i |, 9 (1t) - _f 16-te ± xaen-3 _r 20-dione and" 1 _f 5% 21 -Acetoxy-pregn-4,9 (11), 16-triene-3,20-dione.

"" The various stages described can be as follows

"can be varied:
■ 25

Level 1 ■

a) Pxiffer pH range € - '10. - ■

b) The length of A-s ± niple * is reduced so much that

. .

■ 'it is just enough * the. Reaction complete. · ■ -

permit. Typical ^ Ä.simplex amounts are O, O5 --1, 0 g / g-steroid. The microbiological enzyme supplier is: Arthrobacter (Corynebacterium) simplex (ATCC No. 6946) or Bacterium cyclooxydans (ATCC No. 12573).

Production of the microorganism Di « A ^ siaplex cells

can be used in the fermentation broth or by collecting the. Cells, drying, acetone treatment or immobilization be modified in a conventional manner. 5

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

The content of the electron acceptor is determined by its Cost and speed of response considerations certainly. The rate of reaction is linearly proportional to the menadione concentration.

Typical amounts are from about 5 to about 40 g / kg steroid.

d | The steroid concentration should be as high as possible se ± n “as long as an effective conversion takes place.

As: Steroids are suitable such as 3-Ket.o - & * - androsten-

4th
or 3 ^ keto & pregnen series. Preferably that should ..

Steroid has a solubility in the organic solvent of more than 5 g / l.

2b e) Suitable aromatic hydrocarbons are toluene, benzene and / or xylene. These solvents can be used alone, mixed with one another or diluted with other water-immiscible solvents such as heptane

· - · - ■ or methylene chloride "· are used.

'The' amount of aroseti.0chem hydrocarbon ranges from about half animal to dissolve the most soluble Home or educated steroid, required Amount up to about four times the amount of the .zum. Dissolving the The least soluble or formed initial steroid

ί. L. I C \ J

Steroid required amount.

In the present example just enough toluene is added that the steroid is just at the end of the reaction is resolved.

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

Level 2

. "You can also move with high kinetic energy

The speed of reaction is clearly related to kinetic energy.

level 3

The conversion requires oxygen as the final hydrogen- (Electron and proton ^ acceptor. As a result "· -

Presence of the aromatic hydrocarbon exists however, an explosive environment in the gas space of the reactor. To this danger of the oxygen concentration in the gas space should be switched off or reduced as far as possible, the

Oxygen concentration below that for combustion er-25

required minimum casicentration are maintained. For Benzene, the minimum oxygen concentration is 11.2%. ■ In the case of xylene, it is also possible to carry out the implementation below of the flash point. The flash points for

Sa-xylene, o-xylene, and p-xylene are 29. _r 32 and 39 ° C. 30th

Level 4

The temperature of the reaction can range from -0 to 45.degree.
35

322120

Level 5

The implementation typically takes 2 days, but it can can also be carried out for a few hours to a few weeks.

Example 10

Comparison of tests in toluene and aqueous phase.

A. Measures :

First, 0.4 g of A complex cells are mixed with 50 ml of 50 mM KPO. Buffer of pH 7.5 combined, whereupon agitated for 2 hours or until a uniform slurry is obtained. It will now be two 25 ml fractions removed and transferred to 125 ml bottles. Then 0.25 ml 5mM menadione in 3A alcohol and 0.2g Androsta-4,9 (11) -diene-3,1 7 — dion registered "Irt one of the plugs Bottles are filled with 2 ml of toluene, whereupon the Bottles 4 ^ ag <s ^ i.ang by means of a joint vibrator away "after 4 days there is an extraction with 25 ml of toluene. An analysis of the extracts gives the following results.

B "Results:

procedure

in aqueous in toluene medium

.30.! Substrate) ^;

(Product).
Ändrosta-4,9 (11) -trien-3,17-dione 82.3% 100.00%

Example 11

According to Example 9, the following steroids are obtained with the aid of dried A. simplex cells:

_ 1. Androsta-4-ene-3,17-dione

2. 6a-fluoro-androsta-4,9 (11) -diene-3,17-dione

3. 6a-methyl-androsta-4,9 (11) -diene-3,17-dione

4. 16β-methyl-androsta-4,9 (11) -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. 17α-Hydroxy-16β-roethyl-pregn-4,9 (11) -dien-2O-yn-3-one

8. 21 -Aeetoxy-i1β-thiydroxy-pregn-4,17 (20) -dien-3-one

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

10. 21 -Acetoxy-17-hydroxy-pregn-4,9 (11) -diene-3, 20-dione

11. 21-Acetoxy-17-hydroxy-16a-methyl-pr.egn-4, 9 (11) -diene-3,20-dione

12. 21-Benzoyloxy-17-hydroxy-16β-methyl-pregn-4,9 (11) -diene-3,2O-dipn _r .

13.- 21-Acetoxy-i l ^ * hydroxy-1 68-methyl-pregn-4, 9 (11) -diene-3,20-dione:

14. 21-acetoxy-pregn-4 1S 111)> 36-txien-3,20-dione

15. 21-acetoxy-6a-fluoro-pregn-9V9 (11), 16 (1-7) -triene-3, 20-dione

in the following products:
1a androsta-1/4-diene-3,17-dione

- ² S 2a. 6a-_Fluor-androsta-1,4 _f 9 {11) -txien-3,17-dione 3a 6ct-methyl-ändrosta- 1,4,9 (11) -triene-3,17-dione 4a 16ß-methyl- androsta-1 _f 4 _f 9 (11) -triene-3,17-dione

5a 17a-hydroxypregn-1, 4-dien-2O-yn-3-one

6a 17a-Hydroxy-pregn-1 ♦ 4,9 <11) -trien-2O-yn-3-one · ,

7a 17a-Hydroxy-16 £ -methy1-pregn-1,4,9 (11) -trien-2O-yn-3-one

. 8a 21-acetoxy-1iss-hydroxy-pregn-1,4,17 (20) -trien-3-one 35

11β, 21-dihydroxy-pregn-1,4,17 (20) -trien-3-one

9a 2Q-Chlpr.-pregn-1, 4, 9 (11), 17 (20) -tetraen-21-al-3-one

10a 2 i-acetoxy - ^ - hydroxy-pregn-1,4,9 {11) -triene-3,20-dione and

17,21-dihydroxy-pregn-1,4,9 (11) -triene-3,20-dione 5

11a 21-acetoxy-17-hydroxy-16α-methyl-pregn-1,4,9 (11) -triene-3,20-dione and

17,21-dihydroxy-10a-methyl-pregn-1,4,9 (11) -triene-3,20-dione

12a 21-Benzoyloxy-17-hydroxy-16B-methyl-pregn-1,4,9 (11) -triene-3,20-dione

13a 21-acetoxy-17-hydroxy-16β-ynethyl-pregn-1,4,9 (11) -triene-3,20-dione and

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

14a 21-acetoxy-pregn-i ,, · 4 # 9i 11) -tetraen-3, 20-dione and 21-hydroxy-ira: egn-1i4 _/ 9 <1'1,), T6-tetraene-3,. 20-dione

15a 21- Acetoxy ^. Sa * f lnor-s: g-1 _r 4 _# 9 <11). _# 1 £ -tetraene-3.20-.dion .. and ·. · "·.. ^ 'I. - ■' -.

6a »Flupr-21-hydroxy-pregn-1,4,9'Mi), 16-tetra-3,20-dione

convicted.

² ^ The usefulness of 1,2-dehydrosteroids is known.

It is known, for example, from US Pat. No. 3,284,447, to use Δ ' ^r · -Pregnetrienes in the synthesis of diuretically more diuretic, C ^ g-substituted corticosteroids. From US Pat. No. 4,041,055 there is a method of synthesis

30. from Cortl cos te roxcfen from \ £ **. ·. —Androsteadlonder derivatives. Known, ie 1,2-dehydroandrostenes are important intermediates in the manufacture of medically valuable steroids.

Claims (15)

PATENT CLAIMS 1. Process for the conversion of 1,2-saturated 3-ketc steroids in 1,2-dehydrosteroids, thereby marked. draws that one is on the 1,2-saturated steroids air-dried or heat-dried cells of 1-dehydrating microorganisms can act. 2. The method according to claim 1-, characterized in that that one air-dried or heat-dried microorganism cells a moisture content of about 1 to about 10% is used. 3 .. The method according to claim 1, characterized in that that as 1-dehydrating microorganisms Arthrobacter simplex and / or. Bacterium cyclooxydans used. ■ 4. Process for the conversion of 1,2-saturated Steroids in 1,2-dehydrosteroids, thereby marked. draws that one is on the 1,2-saturated steroids air-dried or heat-dried bacterial cells of 1-dehydrating microorganisms in the presence an exogenous electron carrier can act. ■ 5. The method according to claim 4, characterized in that the exogenous electron carrier is menadione, Phenazine methosulfate, dichlorophenol indophenol, 1,4-naphthoquinone, Menadione bisulfite, ubiquinone (co- i- i. I C \ J enzyme Q) or compounds of the vitamin K type are used. 6. The method according to claim 4, characterized in that that one air-dried or heat-dried microorganism cells a moisture content of about 1 to about 10% is used. 7. The method according to claim 4, characterized in that there is used as 1-dehydrogenating microorganisms Arthrobacter simplex and / or Bacterium cyclooxydans are used. 8. Improved process for converting 1,2-saturated Steroids in 1,2-dehydrosteroids, characterized in that one on the 1,2-saturated Steroids in the presence of an exogenous electron carrier and a solvent immiscible with water in the form of an aromatic hydrocarbon Arthrobacter simplex and / or Bacterium allows cyclooxydans to act. 9. The method according to claim 8, characterized in that that menadione, 1,4-naphthoquinone, Menadione bisulfite or another compound of the vitamin K type and as an aromatic hydrocarbon Toluene and / or xylene are used. 10. The method according to claim 8, characterized in that there is so much water-immiscible solvent used in the form of an aromatic hydrocarbon, that its amount is about half that of the Dissolve the most soluble steroid substrate or formed steroid to the required amount about four times the amount needed to dissolve the least most soluble steroid substrate or steroid formed is the amount required. 11. Improved Process for Converting 1,2-Saturates Steroids in 1,2-dehydrosteroids, characterized in that one on the 1,2-saturated Steroids in the presence of an exogenous electron carrier and a solvent immiscible with water in the form of an aromatic hydrocarbon substance with the addition of one below for combustion required minimum oxygen concentration lying oxygen quantity Arthrobacter simplex and / or allows Bacterium cyclooxydans to act. 12. The method according to claim 10, characterized in that that the electron carrier is menadione - 1,4-naphthoquinone, Menadione bisulfite and another compound of the vitamin K type and as an aromatic hydrocarbon Benzene, toluene and / or xylene are used. 13. Improved Process for Converting 1,2-Saturated Steroids in 1,2-dehydrosteroids, characterized in that one on the 1,2-saturated Steroids in the presence of an exogenous electron carrier and a solvent immiscible with water in the form of an aromatic hydrocarbon Arthrobacter simplex and / or Bacterium cyclooxydans lets act and the reaction (at a temperature) below the flash point of the organic Solvent » 14. The method according to claim 13, characterized in that that the electron carrier is menadione, phenazine methosulphate, 1,4 »naphthol highinone, menadione bisulphite or any other compound of the vitamin K type and U L L IZU 1 used as aromatic hydrocarbon xylene, 15. The method according to claims 8, 11 or 13, characterized characterized in that the water-immiscible solvent with another, with water Immiscible solvent diluted.