HU200203B - Microbiological process for producing 1,2-dehydro-steroides - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to 4.9 (1L) -androstadiene-3,17-dione; 3-oxo-23,24-dinor-4,9 (11) -choladiene-22 acid and C 1-4 alkyl ester; 3-oxo-2324-dinor-4,9 (11), 17 (20) -cholinium-22-acid and C1-C4-alkyl ester, 22-hydroxy-23,24-dinor-4,9 (1 l) - cola-3-one; 27-nor-4,9 (11) -cholastadiene-3,24-dione and 26,27-dinor-4,9 (II) cholestadiene-3,24-dione 12-position dehydrogenation by microbiology, comprising: dehydrogenation with a bacterium of the genus Arthrobacter or Mycobacterium of the genus 3ο -ο-ΔΙ dehydrogenase or fusarium genus under aerobic conditions in the presence of a macroporous polystyrene absorption resin and the resulting 1,4,9 (1 L) -androstadiene3,17- diotít; 3-oxo-23,24-dinor-1,4,4 (II) -choladiene-22-acid and C 1-4 -alkyl; 3-oxo-2324-dinor-1,4, (11), 17 (20) -cholinium-22-acid and C1-C4-alkyl ester, 22-hydroxy-23,24-dinor-1, 4.9 (II) - cola-3-one; 27-nor-1,4,4,9 (11) -cholastadiene-3,24-dione or 26,27-dinor-1,4,9 (1 L) cholestadiene-3,24-dione is optionally isolated from the fermentation broth. Scope of the description: 9 pages without figure HU 200 203 B -1-

Description

FIELD OF THE INVENTION This invention relates to a process for the dehydrogenation of steroidal synthesis intermediates of steroidal synthesis intermediates having a sterane or androstane backbone of 4,9 (1L) -diene-3-one derived from microbiological degradation products of natural sterols.

Steroids of animal and plant origin (cholesterol, β-sitosterol, campesterol) are important starting materials for the steroid industry and can be used as inexpensive intermediates in the synthesis of steroid hormones and drugs by microbiological processes.

Bacteria that utilize sterols as a carbon source break down both the sterane skeleton and the side chain. The mechanism of sterol side chain degradation has been discovered by C. J. Sih et al. Chem. Soc. 1962, S2.1957, 1982, 10474720 (1984). Acolesterol bacteria are hydroxylated at 26 carbon atoms, then oxidized to a carboxylic acid and subsequently cleaved by a β-oxidation mechanism at three sites, first C24 and C25-S, then C22-CS and C23. Between C17 and C20. The branched chain degradation of β-sitosterol and campesterol at C24 differs from cholesterol in that the side chain degrading bacterium forms a carboxyl group at C28, followed by a chain linkage between C24 and C25-C5 atoms. it also breaks the bond between C24 and C28-SS.

As a result of enzymatic conversions on the sterane backbone, 33-hydroxy-5-ene-linked sterols form 9-hydroxy-1,4-diene-3-one steroids, and then spontaneously rearranged to a commercially valuable 9,10-steroid. derivatives whose skeleton in the B-ring is broken (RM Dobson and RD Muir, J. Am. Chem. Soc. 83, 4627 (1961), K. Schubert et al., Z. Naturforsch. 15h, 584 (1960)].

The industrially desirable selective degradation of the sterol side chains was first achieved by selectively inhibiting 9a-hydroxylation leading to skeletal degradation by enzyme inhibitors to yield 1,4-androstadiene-3,17-dione after cleavage of the sterol side chain. U.S. Patent Nos. 153,173 and Hungarian Patent Nos. 153,831).

Following this work, W.J. Marscheck et al. Genetically engineered 9α-hydroxylase-deficient mutants from sterol-degrading Mycobacteria by ultraviolet irradiation, which degrade natural sterols to 1,4-androstadiene-3,17-dione. Mutants lacking either 9ahydroxylase or 3-oxo-steroid-Al-dehydrogenase have also been produced, the latter from sterols.

Form 4-androstene-3,17-dione [Appl. Microbiol. 22,72 (1972)].

MG Wovcha et al., By treatment with N-methyl-N'-nitroN-nitrosoguanidine mutagens, produced mutants from sterol-degrading Mycobacteria that lack the 3-oxo-steroid-Al-dehydrogenase enzyme and thus from the natural sterols 9a-hydroxy-4-androst. 3,17-dione, 9a-hydroxy-3-OXO-23,24-dinor-4-cholene-22-acid, 9a-hydroxy-3-oxo-23,24-dinor-4-cholene-22-acid methyl ester, 9a2-hydroxy -3-oxo-2324-dinor-4,17 (20) -choladiate-22-acid, 9a-hydroxy-3-oxo-23,24-dinor-4,17 (20) -choladiene-22-acid- methyl ester, 9a-hydroxy-3-oxo-23,24-dinor-4,17 (20) -choladiene-22-aldehyde and 9a, 22-dihydroxy-23β-dinor-4-cholen-3-one are prepared [Biochim . Biophys. Acta531.308 (1978)]. The same research group also produced a mutant that produces 9a-hydroxy-27-nor-4-cholestene-3,24-dione from β-sitosterol and 9a-hydroxy-26,26-dinor-4-cholestene-324-dione from campesterol. .

Starting from 9a-hydroxy-4-androstene-3,17-dione, V. Van Rheenen and K. P. Shephard have developed an economical industrial synthesis for the synthesis of antiinflammatory hydrocortisone [J. OTG. Chem., 44, 1582 (1979). From 9-hydroxy-4-androstene-3,17-dione, 4,9 (11) -androstadiene-3,17-dione was prepared by elimination of the hydroxyl group. Starting from the key intermediate, not only the structure of the corticoid side chain but also the presence of the 9 (1 L) double bond also provides an advantageous opportunity for the formation of the β-hydroxyl group characteristic of the anti-inflammatory corticosteroids. Products derived from the partial microbiological degradation of sterol side chains, especially 9a-hydroxy-3-oxo-23,24-dinorcollane derivatives, are also valuable starting materials for the synthesis of corticosteroids (U.S. Patent No. 4,029,549). description).

Medicinal anti-inflammatory corticosteroids usually contain two bonds at the 1-position and very often a 9a-position of a halogen atom, preferably a fluorine atom. The synthesis of such corticosteroid drugs could be based on 1,4,9 (1L) -triene-3 from natural sterols. steroid derivatives of structure. Therefore, in our research, 4,9 (11) -dien-3-one derivatives were obtained from cleavage products containing ia-hydroxyl group obtained from the natural sterols by the cleavage of the 9a-hydroxy group and microbiologically experimentally tested these compounds with 1, Dehydrogenate in 2-position.

Surprisingly, it has now been found that 9 (1 L) double bonded steroid derivatives of 9 (1 L) double bonds and naturally occurring steroid derivatives of 4,9 (11) -androstadiene have been prepared from microbiological degradation products of natural sterols. 3,17-dione is difficult to dehydrogenate with bacteria and fungi with the enzyme 3-oxo-steroidΔΙ-dehydrogenase because the steroid products of 1,4,9 (1L) -trien-3-one formed by them inhibit the enzyme function

Ai. Kominek et al. (German Patent Publication No. 33.22.120) have also found that fermentation with 4,9 (11) -adrostadiene-3,17-dione in Arthrobacter simplex is virtually impossible to produce 1,2, 9 (II) -androstatriene-3,17-dione, the dehydrogenation at the 13-position is only very slight at 2.6-10.4%. Arthlobacter simplex cell preparation prepared by air or heat drying in the presence of an electron acceptor in an aqueous medium containing dimethylformamide or preferably a water-immiscible organic solvent such as toluene succeeded in dehydrogenation of the 4,9 (11) -androstadiene-3, 17-dione at position 1,2,3. good yield-2HU 200203 Β today to carry out. Their process is more complicated and expensive than the conventional fermentation process. Conversion in the presence of toluene, since oxygen must also be introduced into the reaction mixture, is also explosive. (Exemplary embodiments of the description only illustrate the conversion of steroids with androstane and pregnane skeletons.)

A127394. The authors of European Patent Application Publication No. 4,102,123 disclose microbiologically dehydrogenation of various androstane and prengane derivatives. The conversion is carried out by electron acceptors (such as menadione, menadione bisulfite, 1,4-naphthoquinone, phenazine methosulfate, or quitamin compounds) as well as the resulting oxygen species (such as hydrogen peroxide, superoxide anion hydroxyl radical, oxygen radical). removal or inactivation scavangers (catalase, peroxidase or superoxide dismutase enzymes in the presence of mannitol, α-tocopherol or platinum catalyst) are cultured in Arthrobacter simplex bacteria. The above additions used during the conversion significantly increase the cost of the process.

No. 190,784. Hungarian Patent No. 5,198,125 discloses a process for intensifying microbiological conversion of various steroids of the androstane, pregnane, cholestane, or stigmastane type or cardardenolide type. The authors achieve the intensification of the transformations by carrying out a microbiological conversion in the presence of α-, β- and γ-cyclodextrin. The exemplary embodiments also disclose the 13-position dehydrogenation of hydrocortisone and 17α-methyl testosterone among various types of conversion. No data are provided in the specification for the dehydrogenation of 13-positioned compounds of a collan backbone.

Accordingly, it is an object of the present invention to provide a safe and economical fermentation process which allows for the dehydrogenation of androstane and cyano-skeletal compounds at the 13-position without product inhibition that inhibits microbiological conversion.

In our research to this end, it has been observed that macroporous polystyrene-type adsorption resins added to the fermene promote the dehydrogenation of 43 (II) -androstadiene-3,17-dione at the 13-position.

In the course of our further research, the products of the partial microbiological degradation of the side chain of natural sterols (cholesterol, β-sitosterol, campesterol): 9a-hydroxy-3-oxo-2334-dinor-4-cholene-22-acid, 9a-hydroxy-27- 9 (II) double from nor-4-cholestene-334-dione and their methyl esters, 9a-hydroxy-27-nor-4-cholestene-334-dione and 9a-2637nor-4-cholestene-3,24-dione linkages were prepared. The 9 (11L) double bond can be formed by acid dehydration, preferably using, for example, polyphosphoric acid or chlorosulfonic acid as the elimination agent. The 9α-hydroxy group is cleaved with the above reagents directly in good yields for most sterol degradation products. However, for 9a32-dihydroxy-2334dinor-4-cholen-3-one, the hydroxy group 22 should be protected before the 9a-hydroxy group is eliminated. This may be accomplished by acetylating 9a, 22-dihydrol-23,24-dinor-4-cholene-3-one with acetic anhydride, for example in a pyridine medium, to give 9a-hydroxy-22-acetoxy-2334-dinor-4-cholene 4.

3 is subjected to dehydration. The 22acetoxy-2334-dinor-4,9 (11) -choladiene-3-one thus prepared can be deacetylated, for example, with potassium hydroxide to give 22-hydroxy-2334-dinor-4,9 (11) -choladiene-3-one.

Of the 9 (11) dehydro derivatives derived from sterol degradation products, 3-oxo-23,24-dinor-4,9 (II) -choladiene-22-acid and its alkyl esters of 1 to 4 carbon atoms, 3-oxo-2334-dinor-, 4.9 (II), 17 (21) -cholecollen-22-acid and C1-C4 alkyl esters, 22-hydroxy-2334-dinor-4,9 (II) -choladiene-3-one, 27-nor4 No data have been found in the literature for the microbiological dehydrogenation of 9 (II) -cholestadiene-3,24-dione and 2637-dinor4,9 (11) -cholestadiene at 334-dione.

It has now been found that these steroid derivatives are preferably dehydrogenated with microorganisms having 3-oxo-steroid alpha-dehydrogenase activity in the presence of majcroporous polystyrene adsorption resins.

Accordingly, the present invention relates to a process for the preparation of 4,9 (11) -androstadiene-3,17-dione; 3-oxo-23,24-dinor-4,9 (II) -choladiene-22-acid and C 1 -C 4 alkyl ester; 3-oxo-2334-dinor-4,9 (11), 17 (20) -cholecroleic-22-acid and C1-C4 alkyl ester; 22-hydroxy-23,24-dinor-4,9 (ll) -choladien-3-one; Microbiological 13-dehydrogenation of 27-nor4.9 (II) -cholestadiene-334-dione and 26,37-dinor4.9 (II) -cholestadiene-3,24-dione, comprising the step of aerobically in the presence of a macroporous polystyrene-type adsorption resin with a bacterium of the genus Arthrobacter or Mycobacterium or a fungus of the genus Fusarium, and the resulting 1,4,9 (11) -androstatriene-3,17- dione;

3-oxo-2334-dinor-1,4,9 (II) -cholecroleic-22-acid and C 1 -C 4 alkyl ester; 3-oxo-23,24-dinoro-1,4,9 (1 L), 17 (20) -colcetetraenoic 22-acid and C 1 -C 4 alkyl ester; 22-hydroxy-23,24-dinor-4,9 (ll) -kolatrién-3-one; 27-nor-1,4,9 (II) -cholestatriene-3,24-dione or 26,27-dinor-1,4,9 (II) -cholestatriene-334-dione or 2637-dinor-1,4, If desired, 9 (II) cholestatriene-334-dione is isolated from the fermentation broth.

Any microorganism having 3-oxo-steroid Al-dehydrogenase activity can be used in the practice of the invention, preferably bacteria, such as those belonging to the genus Arthrobacter, Corynebacterium and Mycobacterium; or mushrooms, e.g. Fusarium species present.

For example, the following microorganisms deposited in the National Collection of Agricultural and Industrial Microorganisms of the University of Horticulture and Food Industry are preferred in the process of the present invention: Arthrobacter simplex [NCAI (P) 000066], Mycobacterium lacticola [NACAIM B (P) 000037], Mycobacterium P (P) 000038], Fusarium caucasicum [NCAIMF (P) 000090].

Growth of microorganisms and 13-position dehydrogenation were performed in shake culture (300 rpm, 8 cm flat-plate shaker) and mentors.

The culture media used vary depending on the microorganism carrying out the transformation: el3

-320U 200203 Β corn jelly, yeast extract and asparagine are useful sources of nitrogen; and carbon sources include glycerol and glucose. Typical nutrient media and alkali metal and alkaline earth metal phosphate, nitrate and sulfate salts include cobalt, zinc, copper, nickel and manganese salts as trace elements.

The bacterial strains carrying out the 1,2-position dehydrogenation are preferably cultured at 37-37 ° C and the fungi are cultured at 25-30 ° C.

In some 15-position dehydrogenating microorganisms, such as Arthrobacter simplex, the amount of the 3-oxo-steroid Al-dehydrogenase can be significantly increased by induction. Formation of the 3-oxo-steroid Al-dehydrogenase enzyme is conveniently induced by incubation in the presence of 4-androstene-3,17-dione, but any 3-oxo-4-ene or 3-oxo-1,4-diene steroid may also be used as an inducer. Adehydrogenation may also be carried out with a concentrated culture of the induced strains, but it is preferable to use a 10-20 fold dilution of the strains in water for conversion.

In a preferred embodiment of the process of the invention, the lipophilic adsorption resins of the macroporous polystyrene type (Amberliter polymeric adsorbents, Rohm and Haas, GmbH, Frankfurt; Duolite adsorbent resins, Doulite International SA, Vitry-sur-Seine; Diaion adsorbent resins, Mitsubishi Co., Tokyo) are used to facilitate the 1,2-position dehydrogenation process. The adsorption resins should preferably be added in an amount of 1-31% to the fermentation broth prior to the start of steroid conversion.

The dehydrogenated steroid products in the 15-position are preferably isolated from the above by extraction with chlorinated hydrocarbons, organic esters, water-immiscible organic ketones and organic alcohols. Prior to extraction of the 23,24-dinorocholanoic acid derivatives, the fermentation broth should be acidified to increase the extraction efficiency. The crude product obtained by evaporation of the extracts can be purified by recrystallization, if necessary, by chromatography.

The structure of the compounds of the present invention was confirmed by infrared, 1 H-NMR and mass spectroscopy.

TLC separations were carried out using silica gel adsorbent (Kieselgel G (Reanal, Budapest) and Kieselgel 60 HF254 + 366 (Reanal, Budapest) 2: 1).

Of the steroids having the structure of 4,9 (11) -dien-3-one, which is the starting material for the process of the invention, only 4,9 (11) -androstadiene-3,17-dione and 27-nor-4,9 (II) cholestadiene-3,24-dione and 26,27-dinor-4,9 (II) -cholestadiene-3,24-dione are known in the art. Preferably, the first compound is disclosed in U.S. Patent No. 189,351. The latter can be prepared according to J.C. Knight and M.G. Wovcha (Steroids, 36,723 (1980)). The preparation of the novel starting compounds is described in connection with each embodiment.

The following examples illustrate the invention.

Example 1

1 ml of a suspension of Arthrobacter simplex [NCAIM B (000066]) on potato dextrose slant agar for 1-2 weeks was inoculated with 100 ml CS medium sterilized in 500 ml Erlenmeyer flasks in 10 ml sterile water, as follows:

potassium dihydrogen phosphate 4.4 g disodium hydrogen phosphate 8.8 g yeast extract (dry matter) 10.0 g

1000 ml tap water.

The medium was adjusted to pH 7.0 before sterilization. The medium was sterilized at 121 ° C for 25 minutes.

The culture was grown for 2 days on a flat shaker at 37 ° C and 5 ml of the culture were inoculated with 5 500 ml Erlenmeyer flasks containing 100-100 ml of KA12 sterile medium.

The composition of the KAS 12 medium is as follows:

corn jam (dry matter) 2.0 g asparagine 1.0 g yeast extract (dry matter) 0.2 g glucose 5.0 g potassium dihydrogen phosphate 1.0 g trace element solution * 10.0 ml

100 ml tap water.

. The medium was adjusted to pH 6.5-7.0 before sterilization. The medium was sterilized at 121 ° C for 25 minutes.

* Composition of the trace element solution: calcium chloride water (1: 6) 1.0 g cobalt (IT) nitrate water (1: 6) 10.0 mg zinc sulfate water (1: 7) 88.0 mg of copper (II) sulfate water (1: 5) 3.9 mg of nickel sulfate water (1: 7) 1.0 mg of sodium tetraborate water (1:10) 0.8 mg of manganese (II) chloride water (1: 4) 0.72 mg ammonium phosphorus molybdenate water (1: 6) 0.37 mg iron (II) sulfate water (1: 7) 1.0 mg

1000 ml of distilled water.

The resulting cultures were shaken on a flat shaker at 37 ° C for one day, then 10 mg of 4-androstene-3,17-dione in 1 ml of acetone was added to each flask and shaking was continued for 6 hours. The inoculum cultures were then pooled and sterile added in 45 L of sterile water to a 10 L laboratory fermenter containing 90 g of Amberlite XAD-2 adsorption resin. Thereafter, 6 g of 4.9 (1 L) -andiOdadiene-3,17-dione in 75 ml of tetrahydrofuran was added. Thereafter, the steroid conversion was carried out for 24 hours at 37 ° C with stirring at 500 rpm with a flow of sterile air of 180 liters / hour. During this time, 4.9 (1 L) -androstadiene-3,17-dione was analyzed by TLC. is completely converted to 1,4,9 (II) -andiostadiene-3,17-dione. TLC (6: 4) n-hexane: ethyl acetate was used to develop the starting material and the product.

After completion of the fermentation, the fermentation broth is extracted first with 15 L of dichloromethane and then 5 L of dichloromethane. The combined dichloromethane extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting crude product (5.75 g) was recrystallized from acetone to give 3.65 g of pure 1,4,9 (11) -androstatriene-3,17-dione.

-4HU 200203 Β

Mp: 164-167 ° C.

[cx] d = + 102 * (c = 1, chloroform).

Ultraviolet spectrum (ethanol): λ = 238 nm (ε =

15900).

Infrared (KBr):? C = O 1730.1655 (ketones 17-5 and 3),? C = O 1620.1602 cm -1 ¹ H NMR Spectrum: (CDCl 3): 73 (HI),? 2 (H 2), 6.0 (H-4), AMXm (Ji ^ = 10, J _2, 4 = 2, J _{2, 4} - O Hz), 5.6 m (H-ll), 1, 4 s (2H-19), 0.95 s (3H-18) ppm. 10

Mass spectrum: Molecular ion: 282.

Typical ions (m / z): 282, 267, 249, 239, 225,

224.209.91.

The recrystallization mother liquor was evaporated in vacuo and the evaporation residue was purified by preparative thin layer chromatography using ethyl acetate-hexane (1: 1) as developing solvent. An additional 1.18 g of chromatographically pure 1,4,9 (1 L) -androstatriene-3,17-dione is obtained.

Example 2

Following the procedure described in Example 1, a culture of 4-androstene-3,17-dione-induced arthrobacter simplex [NCAIM B (P) 000066] was prepared. 1010 ml of this culture was sterile transferred into 10 500 ml Erienmeyer flasks and diluted with 90-90 ml of 1.8-1.8 g Amberlite XAD-2 adsorption resin in sterile water.

To the resulting cultures were added 100 mg of 3-oxo-23,24-dinor-4,9 (11) -choladiene-22-acid in 2 ml of ethanol per flask. The cultures were shaken for two days at 37 ° C on a 30 shaker and then the cultures were pooled and the pH of the fermentation broth was adjusted to 2N sulfuric acid.

The extract was extracted with ethyl acetate (3 x 200 mL). The ethyl acetate extracts were combined and concentrated in vacuo. The crude product (35 g, 1.05 g) was purified by preparative thin layer chromatography (developing solvent: benzobaceton (8: 2)). The resulting 720 mg chromatographically uniform product is dissolved in 20 ml of methanol and then added with 1 ml of 10% methanolic hydrochloric acid at 0 ° C. Then the acidified solution

It is diluted with 100 ml of ethyl acetate, washed twice with 25 ml of water, and the liver bar is concentrated in vacuo. That's how it got

3-oxo-23,24-dynr-1,4,9 (II) -cholecroleic-22-acid is crystallized from acetone.

Mp: 220-225 ° C.

Ultraviolet spectrum (ethanol): λ max 238 nm.

IR (KBr):? OH 3500-2500,? C = O 1705 (COOH), 1660 (ketone 3),? C = C 1620,

1600 cm ^-1 . 50 H-NMR Spectrum: (CDCl3 δ: 7.4 (HI), 6.2 (H2), 6.0 (H-4), AMXm (Ju = 10Hz, J2.4 = 2Hz, J2.4OH2) , 5.5 m (H-11), 1.4 s (3H-19), 1.15 d (3H-21),

0.7 s (3H-18) ppm.

Mass spectra: Molecular ion: 340. 55

Typical ions (m / z): 340, 325, 323, 312, 174,

105.93.91.

The starting 3-oxo23,24-dinor-4,9 (II) -choladiene-22-acid used as the starting material for the fermentation can be prepared from 9a-hydroxy-3-oxo-23,24-dinor-4-cholene-22-acid, for example as follows. live:

9 g of 9a-hydroxy-3-oxo-23,24-dinor-4-cholene-22-acid is dissolved in 20 ml of dichloromethane, at -5 DEG C., while stirring, 1/2 ml of chlorosulfonic acid is added dropwise. the solution is stirred at this temperature for 30 minutes. Water (25 mL) was added to the reaction mixture with stirring and the phases were separated. The aqueous phase was extracted with dichloromethane (2 x 10 mL). The dichloromethane extracts were then combined and concentrated in vacuo. The evaporation residue (2.05 g) was purified by preparative thin layer chromatography (developing solvent: benzene: acetone (8: 2)). The chromatographically uniform crystals obtained are dissolved in 30 ml of methanol and 2 ml of 10% methanolic hydrochloric acid are added. The acidified methanolic solution was then diluted with ethyl acetate (120 mL), washed with water (2 x 25 mL) and evaporated in vacuo. The resulting 13 g of 3oxo-23,24-dinor-4,9 (II) -choladiene-22-acid is crystallized from methanol.

Mp 225-230'C.

Ultraviolet spectrum (ethanol): λ ≤ 238 nm.

Infrared (KBr): vOH 3500-2400, vC = O 1720 (COOH), 1650 (ketone 3), vC = C 1610 cm ^-1 .

1 H NMR Spectrum: (CDCl 3 δ): 5.7 s (H-4), 5.5 m (H-11), 1/2 d (3H-21), 135 s (3H-19), δ , 7 s (3H-18) ppm.

Mass spectrum: Molecular ion: 342.

Typical ions (m / z): 342, 327, 300, 269, 176,

105,93,91.

Example 3

Using the procedure described in Example 1, a culture of Arthrobacter simplex [NCAIM B (P) 000066] induced by 4-androstene-3,17-dione was prepared. 1010 ml of this culture were sterile transferred into 10 500 ml Erienmeyer flasks and diluted with 90-90 ml of sterile water containing 1Λ-13 g Amberlite XAD-2 resin. To the resulting cultures, 100 mg of 3-oxo2334-dinor-4 was dissolved in 2 ml of ethanol. 9 (II), 17 (20) -cholecrolene-22-acid is added per flask. The cultures are shaken on a shaker at 37 ° C for two days. The cultures are pooled and the pH of the fermentation broth is adjusted to 2 with 2N sulfuric acid. The fermentation broth was extracted with ethyl acetate (3 x 200 mL). The ethyl acetate extracts were combined and concentrated in vacuo. The crude product (960 mg) was purified by preparative thin layer chromatography (developing solvent: benzohacetone (8: 2)). The chromatographically uniform product (710 mg) was dissolved in methanol (20 ml) and 10% hydrochloric acid in methanol (1 ml) was added at 0 ° C. The acidified solution was then diluted with ethyl acetate (100 mL), washed with water (2 x 25 mL) and evaporated in vacuo. The resulting 3-oxo-2334-dinor-1,4,9 (II), 17 (20) -cholethetraeno-22-acid was crystallized from methanol.

240-244 'C.

Ultraviolet spectrum (ethanol): Xnux 235 nm.

Infrared (KBr): vOH 3600-2500, v C = O 1690 (COOH), 1665 (ketone 3), v C = C 1630, 1610 cm ^-1 .

1 H-NMR Spectrum: (CDCl 3 δ): 1/2 (H 1), 6.3 (H 2), 6.1 (H-4), AMXm (Ju = 10Hz, J ₂ , 4 = 2Hz, J ~ 0Hz), 5.5 m (H-11), 1.95 s (3H-21), 1.4 s (3H-19), 035 s (3H-18) ppm.

Mass spectrum: Molecular ion: 338.

Typical ions (m / z): 338, 323, 310, 211, 165,

145,121,91.

The starting material for the fermentation is 3-oxo-5HU 200203 Β

23.24-dinor-4,9 (II), 17 (20) -cholecrene-22-acid from 9α-hydroxy-3-oxo-2334-dinor-4,17 (20) -cholenen-22-acid can be prepared, for example, as follows. :

9 g of 9a-hydroxy-3-oxo-2334-dinor-4,17 (20) -choladiene-22-acid is dissolved in 20 ml of dichloromethane and 1 (2 ml of chlorosulphonic acid) is added dropwise with stirring at -5 ° C. After stirring for 30 minutes at the same temperature, water (25 mL) was added with stirring, the layers were separated, the aqueous layer was extracted with dichloromethane (2 x 10 mL), and the dichloromethane extracts were combined and concentrated in vacuo. The residue (05 g) was purified by preparative thin layer chromatography (developing solvent: benzoacetone (8: 2)) to give a chromatographically uniform product, dissolved in 30 ml of methanol and 2 ml of 10% methanolic hydrochloric acid, followed by 120 ml of ethyl acetate. The mixture was diluted and the resulting mixture was washed with water (2 x 25 mL) and concentrated in vacuo.

1.6 g of 3-oxo-2334-dinor-4,9 (II), 17 (20) -cholecroleic acid are crystallized from methanol.

Mp: 238-239 'C.

Ultraviolet spectrum (ethanol): λmax 236 nm.

IR (KBr): vOH 3400 to 2400,? C = O 1675 (COOH and 3-ketone),? C = C 1610 cm ^1st

1 H NMR (CDCl 3 δ): 5.7 s (H-4), 55 m (H-11), 2.0 s (3H-21), 13 s (3H-19), 0.9 s (3H-18) ppm.

Mass spectrum: Molecular ion: 340.

Typical ions (m / z): 340, 325, 307, 295, 227,

213,105,91.

Example 4

Using the procedure described in Example 1, a culture of Arthrobacter simplex [NCAIM B (P) 000066] induced by 4-androstene-3,17-dione was prepared. 1010 ml of this culture was sterile transferred into 5 Erlenmeyer flasks and diluted with 90-90 ml of sterile water containing 1.8-1.8 g Amberlite XAD-2 resin. To the resulting cultures was added 100 mg of 3-oxo-2334-dinor-4,9 (II) -choladiene-22-acid methyl ester in 2 ml of ethanol. The cultures were shaken for two days at 37 ° C on a flat shaker. The cultures were then pooled and the fermentation broth extracted with ethyl acetate (2 x 100 mL). The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate and evaporated in vacuo. The crude product obtained was purified by preparative thin layer chromatography (developing solvent: benzene: acetone (8: 2)). There was thus obtained 370 mg of chromatographically uniform methyl ester of 3-oxo-23,24-dinorl, 4,9 (II) -cholecrene-22-acid.

Mp: 174-176 ° C.

Ultraviolet spectrum (ethanol): λη »« 238 nm.

Infrared (KBr): v C = O 1738 (ester), v C = O 1661 (ketone 3), v C = C 1626.1603 cm ^-1 .

1 H-NMR Spectrum: (CDCl 3): 7.15 (H 1), 63 (H-2), 6.0 (H-4), AMXm (J = 10Hz, J ₂ , 4 = 2Hz, J -OH2), 5.4 m (H-11), 135 s (3H-19), 1.15 d (3H21), 0.7s (3H-18) ppm.

TO spectra: Molecular ion: 354.

Typical ions (m / z): 345,339,121,93,91,59.

The 3-oxo23.24-dinor-4,9 (II) -choladiene-22-acid methyl ester used as the starting material for the fermentation was

Example 2 Prepared from 3-oxo-2334-dinor-4,9 (11) -choladiene-22 acid by diazomethane esterification.

M.p .: 194-200'C.

Ultraviolet spectrum (ethanol): λmax 238 nm. infrared (KBr): v C = O 1735 (ester), v C = O 1670 (ketone 3), v C = C 1618 cm ^-1 .

1 H NMR (CDCl 3 δ): 5.7 s (H-4), 5.4 m (H-11), 135 s (3 H-19), 1.18 d (3 H-21), δ, 7 s (3H18) ppm.

Example 5

Following the procedure described in Example 1, a culture of Arthrobacter simplex (MNG 66) induced by 4-androstene-3,17-dione was prepared. 10 to 10 mL of this culture is sterile weighed into 5 Erlenmeyer flasks and diluted with 90 to 90 mL of sterile water containing 13 to 15 g Amberlite XAD-2 resin. To the resulting cultures was added 100 mg of 3-oxo-23,24-dinor4,9 (II), 17 (20) -cholecrene-22-acid methyl ester (100 mg) in ethanol (2 ml). shaken. The cultures were then pooled and the fermentation broth extracted with ethyl acetate (2 x 100 mL). The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product obtained was purified by preparative thin layer chromatography (developing solvent: benzene: acetone (8: 2)) to give 405 mg of chromatographically homogeneous 3-oxo-2334-dinor-1,4,9 (11), 17 (20) -choletetraene-22. acid methyl ester.

M.p .: 130'C.

Ultraviolet spectrum (ethanol): λ <η * χ 235 nm.

IR (KBr): vC = O 1710 (ester), 1670 (3-ketone),? C = C 1625.1605 cm ^1st

1 H-NMR Spectrum: (CDCl 3): 73 (H 1), 63 (H 2), 6.05 (H 2 t), AMXm (J 1 = 10Hz, J2.4 = 2 / 2z, J1, 4 ~ 0Hz), 55 m (H-11), 3.7 s (OCH 3), 1.9 s (3H-21),

1.4 s (3H-19), 0.95 s (3H-18) ppm.

Mass Spectrum: Molecular Ion: 352.

Typical ions (m / z): 352, 337, 324, 321, 305, 293, 211, 147.

The 3-oxo-2334-dinor-4,9 (II), 17 (20) -cholecrene-22-acid methyl ester used as the starting material for the fermentation was prepared as described in Example 3, using 3-oxo-2334-dinor-4.9 (II). ; It was prepared from 17 (20) -collrene-22-acid by diazomethane esterification.

Mp 143 ° C.

Ultraviolet spectrum (ethanol): λmax 237 nm.

Infrared (KBr): v C = O 1705 (ester), v C = O 1670 (ketone 3), v C = C 1630.1610 cm ^-1 .

1 H NMR (CDCl 3 δ): 5.7 s (H-4), 5.45 m (H-11), 3.65 s (OCH 3), 1.9 s (3H-21), 135 s (3H19), 0.9 s (3H-18) ppm.

Example 6

Following the procedure described in Example 1, a culture of Arthrobacter simplex induced by 4-androstene-3,17-dione was prepared. 10 ml of this culture is sterile weighed into 10 500 ml Erlenmeyer flasks and 90-90 ml

It is supplemented with sterile water containing 1.8 to 1.8 g of Amberlite XAD-2 adsorption resin. To the resulting cultures was added 100 mg of 22-hydroxy-2334-dinor-4,9 (II) -choladien-3-one in 2 ml of ethyl alcohol and the cultures were placed on a flat shaker at 37 ° C.

Shake for 6 days 200203 Β. The cultures were then combined and the broth was extracted with ethyl acetate (3 x 200 mL). The ethyl acetate extracts were combined in vacuo and the crude product was purified by thin layer chromatography (developing solvent: n-heptane: ethyl acetate (1: 1)). The thus obtained 750 mg of 22-hydroxy-2324-dinor-1,4,9 (11) cholatriene-3-one is recrystallized from methanol.

M.p .: 192-196 'C.

Ultraviolet spectrum (ethanol): 239 nm.

Infrared (KBr): vOH 3439, vOO1666, vC = C 1622.1605 cm.

1 H-NMR Spectrum: (CDCl 3 δ): 7.15 (H 1), 62 (H-2), 6.05 (H-4), AMXm (J 4 = 10Hz, J ₂ , 4 = 2Hz, J 4 ~ 0Hz), 5.45m (H-11), 3.45 ABXm (2H-22), 1.35s (3H-19), 1.05d (3H-21), 0.7S ( 3H-18) ppm.

Mass spectrum: Molecular ion: 326.

Typical ions (m / z): 326,121,107,105,93,91, 55.41.

The 22hydroxy-23,24-dinor-4,9 (1 L> colladien-3-one) used as the starting material for the fermentation can be prepared as follows:

9a, 22-Dihydroxy-2324-dinor-4-cholen-3-one g is dissolved in 10 ml of abs. Acetic anhydride (3 ml) was added in pyridine. The mixture was allowed to stand at room temperature for 3 hours, then poured into water (100 ml) and extracted with ethyl acetate (3 x 50 ml). The ethyl acetate extract was combined and concentrated in vacuo. The product thus obtained was recrystallized from ketone to give 2.6 g of 9a-hydroxy-22-acetoxy-2324dinor-4-cholen-3-one.

Mp: 150-153 ° C.

Ultraviolet spectrum (ethanol): λ max 242 nm.

IR (KBr): vOH 3481,? C = O 1734 (acetate),? C = O 1659 (ketone 3),? C-C 1620 cm?

1 H NMR (CDCl 3): δs (H-4), 3.9 ABXm (2H-22), 2.1s (3H-acetate), 155s (3H-19), 1.05 d (3H-21), 0.78 s (3H-18) ppm.

g 9a-hydroxy-22-acetoxy-2324-dinor-4-cholesterol ·

3-one is dissolved in 30 ml of dichloromethane and the solution is cooled to -10 ° C. 15 ml of sterile sulfonic acid are added dropwise with stirring and stirring is continued for 30 minutes at -10 ° C. Water (20 mL) was added and the phases were separated. The aqueous phase was extracted with dichloromethane (2 x 10 mL), and the dichloromethane extracts were combined and concentrated in vacuo. The bipolar residue was purified by thin layer chromatography (developing solvent: n-heptane: ethyl acetate (6: 4)). The chromatographically pure product was recrystallized from methanol to give 1.9 g of 22-acetoxy-2324-dinor-4,9 (11) -choladien-3-one.

M.p. 89-91 'C.

Ultraviolet spectrum (ethanol): λ ™ ™ 238 nm.

Infrared (KBr): v C = O 1742 (acetate), v C = O 1659 (ketone 3), v C = C 1610 cm ^-1 .

1 H NMR Spectrum: (CDCl 3): 5.75 s (H-4), 5.45 m (H-11), 3.9 ABXm (2H-22), 2.05 s (3H-acetate), 155 s (3H-19), 1.0 d (3H-21), 0.7 s (3H-18) ppm.

22-Acetoxy-2324-dinoro-4 ((II) -choladiene-3-g) was dissolved in 10 ml of ethyl alcohol and 5 ml of 2N ethyl alcohol potassium hydroxide was added. The reaction mixture was stirred at room temperature for 30 minutes and then evaporated in vacuo. The crude product was purified by thin layer chromatography (developer 12 n-heptane: ethyl acetate (6: 4)) to crystallize the chromatographically pure product from methanol to give 1.3 g of 22-hydroxy-23,24-dinor4.9 (1 L). -choladiene-3-one is obtained.

Mp: 160-165 ° C.

Ultraviolet spectrum (ethanol): λ «η» χ 236 nm. Infrared (KBr): vOH 3466, v C = O 1664 (ketone 3), v C = C 1614 cm ^-1 .

1 H NMR (CDCl 3): 5.7 s (H-4), 5.45 m (H-11), 152 s (3 H-19), 1.0 d (3 H-21), 0.65 s ( 3H18) ppm.

Example 7

Following the procedure described in Example 1, a culture of Arthrobacter simplex induced by 4-androstene-3,17-dione was prepared. 10 ml of this culture is sterile transferred into 10 500 ml Erlenmeyer flasks and made up to 90 ml with 90 ml sterile water containing 15 to 1.8 g Amberlite XAD-2 adsorption resin. To the resulting cultures were added 100 mg of 27-nor4.9 (II) -cholestadiene-3,24-dione in 2 ml of ethyl alcohol per flask. The cultures were shaken on a shaker for two days at 37 ° C. The cultures were then combined and the broth was extracted with ethyl acetate (3 x 200 mL). The ethyl acetate extracts were combined and concentrated in vacuo. The crude product thus obtained (12 g) was obtained by thin layer chromatography [developer. N-heptane: ethyl acetate (1: 1)]. The chromatographically pure 27-nor-1,4,9 (II) -cholestatriene-3,24-dione was recrystallized from methanol to give 580 mg of product.

Mp 118-120 ° C.

Ultraviolet Spectrum (Ethanol): λ max 239 nm. Infrared (KBr): v C = O 1718 (ketone 24), v C = O 1657 (ketone 3), v C = C 1620.1600 cm -1 1 H-NMR spectrum (CDCl 3): 72 (HI), 625 (H-2), 6.0 (H-4), AMXm (J1 = 10Hz, J2.4 = 2Hz, J1, 4 ~ 0Hz), 5.45m (H-11), 155 s (3H-19), 1.05 t (3H-26), 058d (3H-21), 0.65 s (3H-18) ppm.

Mass spectrum: Molecular ion: 380.

Typical ions (m / z): 380, 365, 265, 253, 225,

212,121,57.

Example 8

Following the procedure described in Example 1, a culture of Arthrobacter simplex induced by 4-androstene-3,17-dione was prepared. 10 to 10 ml of this culture is sterile transferred into 10 500 ml Erlenmeyer flasks and made up to 90-90 ml with sterile water containing 15 to 1.8 g Amberlite XAD-2 adsorption resin. The resulting cultures were treated with 100 mg of 2627-dinor42 (II) -cholestadiene-3,24-dione adagolun in 2 ml of ethyl alcohol per flask. The cultures were shaken at 37 'C on a slipper for two days. The cultures were then pooled and the broth was extracted with ethyl acetate (3 x 200 mL). The ethyl acetate extracts were combined and concentrated in vacuo. This gave 950 mg of crude product which was purified by TLC (n-heptane: ethyl acetate (1: 1)). Recrystallization from methanol of 2627dinor-1,4,9 (II) -cholestatriene-324-dione by chromatography gives 620 mg of product.

130-132'C.

Ultraviolet spectrum (ethanol): λm x 239 nm.

-7HU 200203 Β

Infrared (KBr): νC = O 1718 (ketone 24), νC = O 1661 (ketone 3), νC = C 1624.1603 cm-1 H-NMR spectrum: (CDCl 3 δ): δ 15 (Hl), 62 (H-2), 6.0 (H-4), AMXm (Ju = 10HZ; J2.4 = 2Hz, J4.4-O), 5.4 m (H-11), 2.4 s (3H-25), 1.4 s (2H19), 0.88 d (3H-21), 0.7 s (3H-18) ppm.

Mass Spectrum: Molecular ion: 366.

Typical ions (m / z): 366. 351. 349, 225, 212,

173,121,43.

Example 9

From 1 to 2 ml of cell suspensions of Mycobacterium lacticola [NCAIM B (P) 000037] and Mycobacterium nugmatis [NCAIM B (P) 000038] on potato dextrose inclined agar with 1 ml of 10 ml of sterile water, respectively, in 500 ml. 100-100 ml of MFG medium sterilized in Erlenmeyer flasks containing 15.0 g of glycerol 10.0 g of corn jam (dry matter) was inoculated.

100 ml tap water.

The pH of the medium before sterilization is 6.5. The medium was sterilized at 121 ° C for 25 minutes.

The inoculum cultures were grown on a shaker at 37 ° C for two days, and the cultures were inoculated with 5 to 5 ml of 100 to 100 ml of MKA2 medium sterilized in 500 ml Erlenmeyer flasks with the following composition:

asparagine 1.0 g potassium dihydrogen phosphate 1.0 g glycerol 5.0 g corn jam (dry matter) 10.0 g as given in Example 1 trace element solution 1.0 ml 1000 ml tap water.

The pH of the medium before sterilization was 6.3. The medium was sterilized at 121 ° C for 25 minutes.

The cultures were shaken at 37 ° C on a flat shaker for two days, then 1.8 g of Amberlite XAD-2 adsorption resin and 100 mg of 4.9 (II) -androstadiene-3,17-dione in 12 ml of tetrahydrofuran were added to each flask. The cultures were then shaken for three days and the amount of 1,4,9 (11) -androstatriene-3,17-dione produced in the culture was determined by quantitative thin layer chromatography. Developed with n-heptane: ethyl acetate (4: 6). After separation by chromatography, the amount of product dissolved from the layer is determined by ultraviolet spectrophotometry. (Mycobacterium lacticola cultures) produced 65 mg, and Mycobacterium nugmatis cultures 48 mg, 1,4,9 (11) -androsiatriene-3,17-dione.

HLpélda

One ml of a cell suspension of Fusarium caucasicum [NCAIM F (P) 00009] on potato slant agar was inoculated with 100 ml Richard's medium sterilized in 500 ml Erlenmeyer flasks with 1 ml of 10 ml sterile water in the following composition:

glucose 50.0 g potassium dihydrogen phosphate 0.5 g magnesium sulfate water (1: 7) 2.5 g potassium nitrate 1.0 g ammonium nitrate 10.0 g

1000 ml tap water.

The pH of the medium before sterilization is 5.0. The medium is sterilized at 121 ° C for 25 minutes.

The inoculum culture was incubated at 28 ° C for one day with shaking on a shaker and the culture was inoculated with 5 ml of 100 ml F medium in a 500 ml Erlenmeyer flask with the following composition:

glucose 0.5 g peptone to 15.0 g corn (dry matter) 6.0 g

1000 ml tap water.

The medium was adjusted to pH 6.9 before sterilization. The medium was sterilized at 121 ° C for 25 minutes.

The culture was shaken for one day at 28 ° C with the help of a steamer. Thereafter, 10 ml of the culture was sterile transferred into a 500 ml Erlenmeyer flask and 90 ml of sterile water containing 1.8 g of Amberlite XAD-2 adsorption resin followed by 100 mg of 4.9 (II) -androstadiene-3.17 in 12 ml of tetrahydrofuran. -dione is added. After shaking for two days, the culture produces 72 mg of 1.42 (11) androstatriene-3,17-dione as determined by thin layer chromatography as described in Example 9.

Claims (5)

PATENT CLAIMS Method 1: 4.9 (1 L) -androstadiene-3,17-dione; 3oxo-23,24-dinor-4,9 (II) -choladiene-22-acid and C 1 -C 4 alkyl ester; 3-oxo-23,24-dinor-4,9 (11), 17 (20) -cholecroleic-22-acid and C 1 -C 4 alkyl ester, 22-hydroxy-23,24-dinor-4,9 (11) -choladiene -3-one; Microbiological dehydrogenation of 27-nor-4,9 (II) -cholastadiene-3,24-dione and 2627-dinor-4,9 (II) -cholestadiene-324-dione, characterized in that -ΔΙ dehydrogenase activity with Arthrobacter or Mycobacterium or Fusarium fungus under aerobic conditions in the presence of a macroporous polysaccharide absorption resin to yield 1,42 (11) -androstadiene-3,17-dione; 3-oxo-23,24-dinor-1,4,9 (II) -choladenic acid 22 and C 1 -C 4 alkyl ester; 3-oxo-2332-dinor-1,4,9 (11), 17 (20) -cholecroleic-22-acid and its C 1 -C 4 alkyl ester, 22-hydroxy-2324-dinor-1,42 (II) -choladiene-3 -you; 27-nor-1,42 (II) -cholastadiene-324-dione or 2627-dinor-1,4,9 (II) -cholestadiene-324-dione is optionally isolated from the fermentation broth. 2. The method of claim 1, wherein the milobiobiological transformation is carried out by culturing Arthrobacter simplex (NCAIM B (P) 000066). 3. The method of claim 1, wherein the microbiological conversion is carried out by culturing Mycobacterium lacticola [NCAIM B (P) 000037]. 4. The process of claim 1, wherein said microbiological conversion is carried out by culturing Microbacterium nugmatis [NCAIM B (P) 000038]. 5. The method of claim 1, wherein the microbiological transformation is performed by culturing Fusarium caucasicum [NCAIM B (F) 000090]. 15 16 Zuk. before culturing the microorganism cultures 4- 6. A method according to any one of claims 1 to 4 androstene-3,17-dione. characterized by the fact that microbiological transformation 5 7