DD213242A5 - PROCESS FOR CONVERTING 1,2-STAINED STEROIDS IN 1,2-DEHYDROSTEROIDS - Google Patents

Abstract

Described is an improved microbiological conversion for the preparation of 1,2-dehydro-steroids from their corresponding 1,2-saturated derivatives. An advantage of the method described is that air or heat-dried microbial cells or microorganism cells can be used as supplier or source for the steroid-1-dehydrogenase activity. Advantageous in the context of the described method is further that during the course of the reaction, a water-immiscible organic solvent in the form of an aromatic hydrocarbon can be used.

Description

Process for converting 1,2-saturated steroids to 1,2-dehydrosteroids

Field of application of the invention;

The invention relates to an improved process for the conversion of 1,2-saturated steroids into 1,2-dehydro-steroids *

Characteristic of the known technical solutions:

The first therapeutic use of corticosteroids was reported in the fifties. This was to perform a cortisone acetate treatment of rheumatoid arthritis. Further studies showed that the incorporation of an unsaturated bond in the 1,2-position of hydrocortisone and cortisone resulted in the prednisolone and prednisone steroids obtained being more potent and producing less drug-reduced salt retention. Thereafter, most other steroids used in the treatment of corticosteroid-responsive diseases were also provided with a double bond in the 1,2-position of the steroid molecule. Since 1977, two attempts have been made to synthesize corticosteroids from sterol precursors. US Pat. No. 4,035,236, for example, discloses a process for preparing 9dL-hydroxyandrostenedione via a fermentation of sitosterol, stigmasterol or cholesterol. From US Patent 4,041,055 a process for the synthesis of medically valuable corticosteroids from androstene is known * These intermediates occurring

A Q f * 4

can have 3-keto-A configuration.

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

U.S. Patent 2,837,464 "Method of Making Dienes by Corynebacterium".

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

US Pat. No. 3,360,439 "Process for the preparation of 2-dehydro-steroids".

Described is a 1-dehydration of steroids with

Help of A. simplex cells, which are mixed before mixing the 15

Substrate with a hydrogen carrier with a short chain alkanol or short chain alkanone, e.g. Acetone, pretreated.

W.Charney and H. Hezog "Microbial Transformation of Steroids", Academic Press, Inc., New York, pp. 4-9, 236-261 (1967).

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

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

30

The authors investigated the 1-dehydrogenation of 4-androstene-3,17-dione using Nocardia rhodocrous in a mixture of 50% 3-enol 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 was phenazine methosulfate in the experiments performed. This one. The electron acceptor is too expensive and too unstable to be able to exploit this microorganism activity in large scale biological conversion processes. The Nocardia rhodocrous enzyme was not active in the presence of menadione, the electron acceptor of choice for Arthrobacter simplex. Thus, the enzyme formed by N. rhodocrous must differ from the enzyme formed by A.simplex. The reaction was carried out in a steroid saturated solvent system with only 20% organic solvent. It was found that with increasing concentration of the steroid, the reaction rate decreased. Oxygen forms 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 the occurrence of an explosive environment is not explained in the cited reference.

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

The authors mentioned investigated the 1-dehydrogenation of hydrocortisone by means of A.simplex. Two water immiscible solvents, namely n-butanol and η-amyl alcohol were tested. However, no bioconversion occurs in the presence of these solvents. As preferred solvents, water-miscible solvents, e.g. Mixtures of 10% methanol and 90% water and 10% propylene glycol and 90% water used.

The cited references neither describe nor suggest the process according to the invention.

Object of the invention:

The invention is intended to provide a method of the aforementioned type which is improved over the prior art,

Explanation of the essence of the invention;

The novel process is characterized in that on the 1,2-saturated steroids in the presence of an exogenous electron carrier and a water-immiscible solvent in the form of an aromatic hydrocarbon optionally with the addition of a below the minimum oxygen concentration required for combustion Arthrobacter simplex and / or Bacterium cyclooxydans.

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

a) The bioconversion results in lower levels of unconverted steroid substrate;

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

c) to convert a given amount of steroid. one needs fewer microorganism cells, e.g. simple cells;

d) the process according to the invention is less sensitive to organic soluble contaminants contained in the steroid, in the microorganism cells or in the steroid formed by the cells.

5 cleanings 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

 10

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

microorganisms:

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

Microorganisms, the numerous microorganisms known for a 1-dehydrogenation are used. Such microorganisms are described by W.Charney and H. Hezog in "Microbial Transformation of Steroids", publ

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

The bacteria dehydrating steroids in the 1-position generally fall into two groups, cf. "Sergey's Manual of Determinative Biology", 8th ed. The method described above was successfully demonstrated on species of Arthrobacter and Corynebacterium genera of "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

35 usable.

Two publicly available microorganisms known to dehydrate steroids in the 1-position have been found useful in this type of process. For example, Bacterium cyclooxydans (ATCC No. 12673), which is known as a 1-dehydrogenating bacterium from US Pat. No. 3,065,146, has been tested and found to be useful, a bacterium which has been studied much more intensively for the 1-dehydrogenation of steroids The bacterium Arthrobacter simplex (ATCC No. 6946), which is known from US Pat. No. 2,837,464, is therefore used to illustrate the process according to the invention to the greatest extent possible, but the process according to the invention can of course also be used with other 1-dehydrogenated

15 perform microorganisms.

Growth of the microorganism:

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 and the like. (Nitrogen source for growth); 25

b) a source of carbon and energy z.3. 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 with tap water or less purified nutrient media, e.g. corn steep liquor,

35, let grow.

For growth, the organisms need in the atmosphere. Sphere contained oxygen. The temperature range for growth is 10 - 45 ° C with an optimum range of 28 to 37 C for A.simplex. The optimum pH for growth is approximately at the neutral point.

Recovery of steroid 1-dehydrogenase:

The cells are supplemented by addition of a 1,2-saturated 3-ketosteroid compound, e.g. of androsta-4-ene-3,17-dione

¹ ^ or cortisone acetate in an amount of 0.005% (w / v) of the medium or above to stimulate steroid-1 dehydrogenase activity. In the presence of the "stimulating agent" incubation is continued for at least 6 hours before the cells are harvested for drying.

The "stimulant" may be added at any time during the growth cycle. Cultures grown on nutrient media such as bacon oil begin rapidly with the synthesis of steroid 1-dehydrogenase, while cultures grown on glucose require substrate depletion before enzyme synthesis. After addition of the "excitation agent", incubation is continued for 6 hours or more, after which the cells are incubated for use in the solution

25 medium or harvested for drying.

Measures for collecting cells for drying:

The cells are separated from the nutrient medium and incubated in the usual manner, e.g. by centrifuging or

3® flocculation and filtration and ultrafiltration concentrated. Thereafter, the separated cells are dried by applying 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

^ 5 has a moisture content of about 1 to about 10%.

dried. Preferably, the moisture content is'. about 5%. The cells are then stored at 5 ° C until used for bioconversion. Active dried cells are also obtained by immobilizing 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, cf. "Methods in Enzymology", 3and XLIV, 1976, Academic Press, Inc., New York, pp. 11-317.

Biological conversion:

The bioconversion occurs by exposing the cell preparations to the steroid substrate. Typically, the cells and steroid are suspended in a weakly buffered aqueous solution having a pH in the range of 6-10, preferably 7.25-8.5. The amount of cells is 0.1 - 50 g / l. The steroid may be added in a weight ratio (steroid: cells) of 0.05 to 5.0. Cell levels of 8-10 g / L with 5-10 g / L steroid are preferred when biological conversion occurs in an aqueous environment. To stimulate the reaction, an exogenous eletro-

carrier in a catalytic amount (for example

-4 5x10 M menadione). Suitable compounds are menadione (2-methyl-1,4-naphthoquinone), phenazine methosulfate, dichlorophenol-indophenol, 1,4-naphthoquinone, menadione bisulfite, ubiquinone (coenzyme Q) and vitamin K-type 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. The rate of 1-dehydrogenation typically decreases over time. Using

From 10 g / L substrate, the bioconversion may be complete in less than 98-100%.

The following are examples of different practicable procedures:

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 low concentration, for example, a concentration of 0-5%, are added.

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 may be added as a dry powder or in a miscible organic solvent such as dimethylformamide, ethanol, methanol, acetone or dimethylsulfoxide, or in a non-miscible organic solvent such as toluene, dissolved or suspended.

c) rehydration of the dried cells in a small buffer volume and subsequent addition of more buffer or an organic solvent. In this case, 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 of the amount required to dissolve the most soluble initial steroid or steroid formed to about four times the amount of dissolution

amount of the least soluble starting steroid or steroid formed. The steroid and the electron carrier are added to initiate the reaction. The addition of the aromatic carbon-hydrogen is preferably carried out together with the steroid, but it can also be done sooner 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.

15 substrate area:

Usable in the context of the method according to the invention

Compounds belongs to the 3-keto-A -androsten- and 3-keto-Δ-pregnenreihe of steroids. Substrates for the steroid 1-dehydrogenase are known to be saturated between the carbon atoms C ₁ and C ~ of the ring A and have in the 3-position of the ring A a hydroxy or keto group. Examples of compounds of the Androstenreihe are:

1) androsta-4-ene-3,17-dione and

2) androsta-4,9 (11) -diene-3,17-dione and its -6a ^ fluoro, 6a-methyl or 16-methyl derivatives;

3) 11S-hydroxyandrosta-4-ene-3,1,7-dione and its derivatives.

4 Usable steroids of the 3-keto-A-pregnene 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 its βα-methyl derivatives;

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

4 4. different groups of 3,20-diketo-A rainstorms,

as

a) 11,17,21-trihydroxy 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

ene-3,20-dione;

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

d) 3,20-diketo-4,9 (11), 4,9 (11), 16-pregnetrienes, for example 21-hydroxy-pregn-4,9 (11), 16-triene-3,20-dione and

its 6a fluoro derivative.

Also suitable as substrates are the 21-ester derivatives of those steroids which have a hydroxyl group in the 21-position (Nos. 2 and 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 the usual manner. The steroids are typically filtered off, after which the filter cake is extracted with an organic solvent such as acetone or methylene chloride. On the other hand, the entire biological mixture may be mixed by mixing with a water-immiscible solvent, e.g. Butyl acetate or methylene chloride. The reaction precursor is then isolated from the organic solvent.

- 12 embodiments:

The following Seispiele to illustrate the invention in more detail.

example 1

Preparation of the dried cells:

Inoculation with Bacterium cyclooxydans (ATCC No. 12673) is carried out in shake flasks with a medium of cerelose, peptone and corn steep liquor (in each case 6 g / l) of a pH of 7.0. The cultures are incubated on a rotary shaker at 28 ⁰ C until the appearance of a Glueöseerschöpfung. When glucose depletion occurs, 0.5 g / l cortisone acetate is added and the shake flasks are incubated again for 16 h. 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 dried.

Bioconversion of ooC methyl hydrocortisone.

Half a gram of the dried cells prepared as described above are rehydrated with stirring in 50 ml. Of a 50 mM phosphate buffer of pH 7.5. To the cell suspension menadione is added 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 6 ^ -methylhydrocortisone / ml dimethylformamide) to a final bio-conversion concentration of 0.5 g / l. Now the mixture is incubated with stirring at 28 ⁰ C. After 4 hours of incubation, the steroid is converted to 91 % . The methylprednisolone is recovered in the usual manner.

1 example. .2

Preparation of androsta-1,4,9 (11} -triene-3,17-dione

a) Preparation of the biocatalyst: 5

Arthrobacter simplex (ATCC No. 6946) is grown in shake flasks in a medium containing 6 g / l glucose, 6 g / l corn steep liquor, and 6 g / l spray-dried bacon. The

·. λ cultures are incubated on a rotary shaker at 28 C until a glucose depletion occurs. At this point, 0/15 g / l cortisone acetate is added to initiate steroid-1 dehydrogenase synthesis. After an incubation over

2g night the cells are harvested by low speed centrifugation. The cell pellets are then placed in a low vacuum oven at 55 ° C for 24 hours to dry. 24 hours later, the dried

Transfer the Q te material to an airtight container and store at 5 C until needed for biological conversion.

b) Biological conversion of androsta-4,9 (11) -diene-3,1 7-dione:

25.

10 g / L of dried cells are rehydrated in 50 mM phosphate buffer of pH 7.5 with stirring for 30 minutes. Thereafter, the cell suspension is mixed with so much dry powdered menadione,

₃ q that a final concentration of 86 mg / 1 is reached. Further, micronized androsta-4,9 (11) -diene-3,17-dione is slurried in dimethylformamide and, in the form of the slurry, is added to the biological conversion mixture in an amount of 2.5 g steroid / 1 and 2%.

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

with agitation, incubated in the presence of air for 24 h at 31 ° C. After completion of the incubation, the androsta-1,4,9 (11) -triene-3,17-dione is purified in the usual manner.

example

In a similar manner as in Example 2 are on

folate steroids: 10

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-Kydroxy-pregn-4,9 (11) -dien-2 O -yyn-3-one

7. 17a-hydroxy-16β-methyl-pregn-4,9 (11) -dien-20-yn

³ - ° ⁿ

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

9. 21-Acetoxy-11β-hydroxy-pregn-4,1 7 (20) -dien-3-one

10. 6a-methyl-11β, 21-dihydroxy-pregn-4,17 (20) -dien-3-one 25 11. 20-chloro-pregn-4,9 (11), 17 (20) -triene-21 -al-3-one

12. Hydrocortisone

13. 6a-methyl hydrocortisone

14. 21-Acetoxy-11β, 17-dihydroxy-16β-methyl-pregn-4-ene-30 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- ₃₅ 4-ene-3,20-dione

15 17. 21-Acetoxy-17-hydroxy-pregn-4,9 (11) -diene-3,20-dione

18. 21-Acetoxy-16α, 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. 21-Acetoxy-17-hydroxy-16β-methyl-pregn-4,9 (11) -diene-3,20-dione

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

23. 21-Acetoxy-6α-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 17a-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-16S-methyl-pregn-1,4,9 (11) -trien-20-yn

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

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

11ß, 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) -tetraene-21-al-3-one

1 12a prednisolone

13a βα-methyl prednisolone

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

dien-3,20-dion and

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

15a 21-acetoxy-9a-fluoro-11β, u7-dihydroxy-16β-methyl

pregn-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

9SS, 11.beta.-epoxy-17,21-dihydroxy-16ss-methyl-pregn-1,4-diene-3,20-dione

17a 21-acetoxy-17-hydroxy-pregn-1,4,9 (11) -triene-3,2-dione and 17,21-dihydroxy-pregn-1,4,9 (11) -triene-3,20- dion

18a 21-acetoxy-iea, 17-dihydroxy-pregn-1,4,9 (11) -triene

3,20-dion and

16a, 1-7,21-Trihydroxy-pregn-1, 4,9 (11) -triene-3,20-dione

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

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

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

trien-3,20-dion 30

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

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

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

2 3a 21-acetoxy-5ct-fluoro-pregn-1,4,9 (11), 16-tetraene-3,20-

dion and

6a-fluoro-21-hydroxy-pregn-1,4,9 (11), 16-tetraene

3,20-dione

dry ropes of A. simplex exposed ·.

Example 4

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

Comparison of isolated yields from different types of biological transformations

substratum

g / l

Type of biological chemical yield% Δ ¹ % residue Remark Conversion to valuable compound

Steroid (a)

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

bad 1-dehydration

g / l

OAc fermentation

dried cells

18.1% first charge 59.1

no second batch

7 9.6% first batch 97.8

no second batch

40.9

2.1

(B)

(a) A "useful steroid" is a Δ ¹ compound useful in a further synthesis or the 1,2-dihydrosubstrate, which are recycled to a further biological conversion for product recovery

5 can, understand.

(b) Substantial amounts of other unwanted steroid molecules are also found.

Example 5

Arthrobacter simplex is grown in a 5 liter "Microferm" fermentation flask, stimulated for steroid-1 dehydrogenase synthesis, and harvested by centrifugation. Parts of the cell paste obtained are processed by two different methods. In the first method, the cells are dried under reduced pressure at 55 ° C. In the second method, 3,360,439 acetone-dried cells are prepared according to US Pat. The cells are mixed with acetone, harvested and dried at 5 C under reduced pressure.

Thereafter, the dry cell preparations become biological

9 11

see conversion of A'-endrostenedione into shake flasks used at concentrations of 10 g / l cells and 10 g / l steroid.

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

5 Cell type Trial duration (h) % Residual substrate Biological conversion activity ⁺ (g product produced) h / g cells 10 dried acetone 1 4 98.7 95.5 0.013 0.011 24 84.1 * 0,007 heat dried 1 4 85.1 23.2 0.149 0.192 15 24 7.2 * 0,039

2Q (+) The biological conversion activity is calculated as follows:

% product formed χ g added substrate

100

= g product formed

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

(*) The residual substrate amounts no longer decrease on further incubation.

21 I Example 6

When replacing 6a.-Methylhydrocort.ison 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, 16α, 17-trihydroxy-pregn-4-ene

3,20-dione;

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

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

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

^ c 5. 21-acetoxy-6α, 9α-difluoro-11β, 17-dihydroxy-16α-methyl-pregn-4-ene-3,20-dione;

6. 21-acetoxy-9a-fluoro-11β, 16α, 17-trihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide;

7. 21-Acetoxy-9β, -IIβ-epoxy-6a-fluoro-1 6a, 1,7-dihydroxy-

Pegn-4-ene-3,20-dione-16,17-acetonide;

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

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

you get the following products:

1. 21-acetoxy-9a-fluoro-11β, 16a-17-trihydroxy-pregn-1,4-diene-3,20-dione and

9a-fluoro-11beta, 16a, 17,21-tetrahydroxy-pregn ~ 1,4-diene-3,20-dione;

2. 21-acetoxy-6a, 9a-difluoro-11ß, 16a, 17-trinydroxy-pregn 1,4-diene-3,20-dione-16,17-acetonide and

6a, 9a-fluoro-11β, 16β-tetrahydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide;

3. 21-acetoxy-6a-fluoro-11β-hydroxy-16a-methyl-pregn-1,4-diene-3,20-dione and

6a-fluoro-11beta, 21-dihydroxy-16a-methyl-pregn-1,4-diene-3,20-dione;

4. 21-Acetoxy-6α-fluoro-11β, 17-hydroxy-pregn-1,4-diene-3,20-dione and 6α-fluoro-11β, 17,21-trihydroxy-1,4-diene 3/20-dione;

5. 21-acetoxy-6a, 9a-difluoro-11β, 17-dihydroxy-16a-methyl

pregn-1,4-diene-3,20-dione and 10

6a, 9a-difluoro-11β, 1, 7,21-trihydroxy-1,4-diene-3,20-dione;

6. 21-acetoxy-9a, fluoro-11β, 16a> 17-trihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide;

7. 21-acetoxy-9β, 11β-epoxy-6α-fluoro-16α, 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

9SS, 11.beta.-epoxy-16a, 21-dihydroxy-pregn-1, -4-diene-3,20-

dione;

9. 21-Acetoxy-98,11β-epoxy-16a, 17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide.

Example 7

Biological conversion of 11β-hydroxyandrosta-4-ene-3,17-dione.

One gram of Example 2 dried A.simplex cells is resuspended in 100 ml of 50 mM phosphate buffer of pH 7.5. This is followed by the addition of menadione dissolved in 3A ethanol in a final concentration of 86 mg / l. After addition of 1/2 g of 11β-hydroxyandrostene

dion in the bottle, the mixture is on a rotary-35

ler incubated at a temperature of 31 ⁰ C. When samples are taken after 1 day of incubation, two products appear on a thin layer chromatograinin of a methylene chloride extract. About 95% of the steroid consists of 11β-hydroxyandrosta-1,4-diene-3,17-dione, the remainder being unreacted substrate.

example

According to Example 7, the following can be dehydrogenated at C ₁₁ modified androstenediones 1:

Substrate -> Product

(1) 11β-hydroxy-16β-methylandrosta-4-ene-3,17-dione *

11.beta.-hydroxy-16ss-methylandrosta-1,4-diene-3,17-dione

(2) 11β-Hydroxy-16α-methylandrosta-4-ene-3,17-dione> 11β-hydroxy-16α-methylandrosta-1,4-diene-3,17-dione

(3) 6α-fluoro-11β-hydroxyandrosta-4-ene-3,1,7-dione>

6a-Flüor-11.beta.-hydroxyandrosta-1,4-diene-3,17-dione

(4) 6a-Methyl-11β-hydroxyandrosta-4-ene-3,17-dione 6a-methyl-11β-hydroxyandrosta-1,4-diene-3,17-dione

(5) 11a-Hydroxyandrosta-4-ene-3,17-dione * 11a-hydroxyandrosta-1,4-diene-3,17-dione. and

(6) androsta-4-en-3 _f 11,17-trione

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

example

1. Into a reaction vessel (on a 1 liter basis)

1 basic ingredients combined:

a) 0.9 1 50 nm KPO ₄ ~ pH 7.5 buffer

b) 3.66 g dry A.simplex cells

c) 0.08 g of 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. 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. 15 5. After 25 h, the toluene phase is separated off.

In this example, the steroid exists in the toluene phase

94.3% 21-acetoxy-pregn-1,4,9 (11), 16-tetraene-3,20-dione, 4.2% 21-hydroxy-pregn-1,4,9 (11), 16- tetraene-3,20-dione and 1.5% 21-acetoxy-pregn-4,9 (11), 16-triene-3,20-dione.

The various steps described can be as follows

be varied: 25

step 1

a) buffer pH range: 6-10.

b) The amount of A.simplex is reduced so much that 30

it is just enough to complete the reaction. Typical A.simplex levels are 0.05-1.0 g / g steroid. The microbiological enzyme supplier is: Arthrobacter (Corynebacterium) simplex (ATCC No. 6946)

or Bacterium cyclooxydans (ATCC No. 12S73).

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

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

The content of the electron acceptor is determined by its cost and consideration of the reaction rate. 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 there is only one effective conversion.

4 Steroids are those of the 3-keto-A -androsten-

4 or 3-keto-A-pregnene series. Preferably, the steroid should have a solubility in the organic solvent of greater than 5 g / L.

e) Suitable aromatic hydrocarbons are toluene, benzene and / or xylene. These solvents may be used alone, mixed with each other, or diluted with other water-immiscible solvents such as heptane or methylene chloride.

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

1 steroid required amount.

In the present example, just enough toluene is added that the steroid is just dissolved upon completion of the reaction.

The addition of the aromatic hydrocarbon takes place. preferably together with the steroid. However, the aromatic hydrocarbon may be added later or earlier.

Level 2

You can also move with high kinetic energy. The reaction rate is clearly related

to the kinetic energy.

level 3

The reaction requires oxygen as the final hydrogen (electron and proton) acceptor

However, presence of the aromatic hydrocarbon exists in the gas space of the reactor an explosive environment. In order to eliminate or substantially reduce this risk of oxygen concentration in the gas space, the oxygen concentration should be lower than that used for combustion.

minimum concentration. For benzene, the minimum oxygen concentration is 11.2%. With xylene, it is also possible to carry out the reaction below the flash point. The flashpoints for

m-xylene, o-xylene and p-xylene are 29, 32 and 39 ° C. 30

Level 4

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

 35

1 level 5

The conversion typically takes 2 days, but it can be done for a few hours to a few weeks.

Example 10

Comparison of tests in toluene and aqueous phase.

in ^A * measures :

First, 0.4 g of A.simplex cells are combined with 50 ml of pH 50 pH 50 mM KPO. Buffer, and the whole is agitated for 2 hours or until a uniform slurry is obtained. Two 25 ml fractions are now taken and transferred to 125 ml bottles. 0.25 ml of 5 mM menadione are then added to each flask in -3A-AlKOhOl "and 0.2 g of androsta-4,9 (11) -diene-3,17-dione are placed in one of the stoppered bottles ml of toluene, then the bottles are agitated for 4 days by means of an articulated vibrator, followed by extraction with 25 ml of toluene after 4 days Analysis of the extracts gives the following results.

25 B. Results:

method

in aqueous in toluene medium

30 (substrate)

Androsta-4,9 (11) -diene-3,17-dione 17.7% 0.00%

(Product) Androsta-4,9 (11) -triene-3,17-dione 82.3% 100.00%

28 Example 11

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

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-2G-yn-3-one

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

7. 17a-hydroxy-16β-methyl-pregn-4,9 (11) -dien-20-yn-3-one

8. 21-Aee% axy-iiss-hydroxy-pregn-4,17 (20) -dien-3-one

9. 20-Chloro-pregn-4, 9 (11), 17 (20) -tri-ene-21-al-3-one

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

11. 21-Acetoxy-17-hydroxy-16a-methyl-pregn-4,9 (11) -diene- ¹⁵ 3,20-dione

12. 21-Benzoyloxy-17-hydroxy-16β-methyl-pregn-4,9 (11) -diene-3,20-dione

13. 21-Acetoxy-17-hydroxy-16β-methyl-pregn-4,9 (11) -diene-3,20-dione

14. 21-Acetoxy-pregn-4,9 (11), 16-triene-3,20-dione

15. 21-Acetoxy-6α-fluoro-pregn-4,9 (11), 16 (17) -triene-3,20-dione

in the following products:

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 17a-hydroxypregn-1,4-dien-20-yn-3-one

6a 17a-Hydroxy-pregn-1,4,9 (11) -trien-20-yn-3-one

7a 17a-hydroxy-16β-methyl-pregn-1,4,9 (11) -trien-20-yn-3-one

8a 21-acetoxy-11β-hydroxy-pregn-1,4,17 (20) -trien-3-one 35 and

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

29 9a 20-chloro-pregn-1,4,9 (11), 17 (20) -tetraene-21-al-3-one

10a 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 5

11a 21-acetoxy-17-hydroxy-16a-methyl-pregn-1,4, 9 (11) -

trien-3,20-dion and

17,21-dihydroxy-1βα-methy1-pregn-1,4,9 (11) -triene

3,20-dione

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

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

17,21-dihydroxy-16ss-methy1-pregn-1,4,9 (11) -triene-3,20-dione

14a 21-Acetoxy-pregn-1,4,9 (11) -tetraene-3,20-dione and 21-hydroxy-pregn-1,4,9 (11), 16-tetraene-3,20-dione

15a 21-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

transferred.

² ^ The utility of 1,2-dehydrosteroids. known. For example, it is known from US Pat. No. 3,284,447,

1 4 9 (11)

Δ ''' pregnetrienes in the synthesis of diuretic, coc substituted corticosteroids. From US-PS 4,041,055 a process for the synthesis of corticosteroids from Δ '-Androstendionderivaten is known that 1,2-Dehydroandrostene are important intermediates in the production of medically valuable steroids.

35

Claims (6)

A process for the conversion of 1,2-saturated steroids to 1,2-dehydro-steroids characterized by comprising the 1,2-saturated steroids in the presence of an exogenous electron carrier and a water-immiscible solvent in the form of an aromatic hydrocarbon optionally with the addition of a below the minimum oxygen concentration required for combustion Artrobacter simples and / or Bacterium cyclooxydans, 2. The method according to item 1, characterized in that menadione, 1,4-naphthoquinone, menadione bisulfite and another compound of the vitamin K type and as the aromatic hydrocarbon benzene, toluene and / or xylene used as slektronenträger. 3. The method according to item 1, characterized in that one carries out the reaction at a temperature below the flash point of the organic solvent, 4. Method according to item 1, characterized in that one uses as much water-immiscible solvent in the form of an aromatic hydrocarbon that its amount about half of the dissolution of the least soluble to dissolve the best soluble steroid substrate about four times the amount soluble steroid substrate or formed steroid. 5. The method according to item 1, characterized by diluting the water-immiscible solvent with another, immiscible with water solvent. 6. Method according to. Item 1, characterized in that air-dried or heat-dried cells of Ärthrobacter simplex and / or Bacterium cyclic used.