RU2425052C1 - Method of producing 6-methyleneandrost-4-ene-3,17-dione from androst-4-ene-3,17-dione, method of producing 6-methyleneandrost-1,4-diene-3,17-dione (exemestane) using obtained 6-methyleneandrost-4-ene-3,17-dione - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention relates to improved methods of producing 6-methyleneandrost-4-ene-3,17-dione and 6-methyleneandrost-1,4-diene-3,17-dione (MNN exemestane) using the obtained 6-methyleneandrost-4-ene-3,17-dione. 6-methyleneandrost-4-ene-3,17-dione is obtained using a method involving preliminary enolisation of ö4-3-ketofunction of androst-4-ene-3,17-dione with formation of 3-alkoxyandrost-3,5-dien-17-one, subsequent three-component condensation with a secondary amine and formaldehyde in the medium of a polar protonic solvent, deamination of the N,N-disubstituted amino group to form a 6-methylene group in the medium of an aprotic solvent. 6-methyleneandrost-1,4-diene-3,17-dione is obtained through 1,2-dehydrogenation of 6-methyleneandrost-4-ene-3,17-dione via microbiological transformation in a medium containing up to 40% water-miscible aprotic solvent using Nocardioides simplex cells VKM As-2033D. ^ EFFECT: high output and selectivity under mild conditions for carrying out the process. ^ 11 cl, 4 ex, 1 tbl

Description

The present invention relates to the field of organic synthesis and biotechnology, specifically relates to the production of the steroid compound 6-methyleneandrosta-1,4-diene-3,17-dione and can be used in the chemical, microbiological and pharmaceutical industries.

6-Methyleneandrosta-1,4-diene-3,17-dione (6-methylene-ADD, INN exemestane) is a highly effective modern aromatase inactivator, known under the trade name "Aromasin" (Manufacturer "Pharmacia Italia", Italy), which is widely used in the treatment of malignant neoplasms of the mammary gland [US 4808616, 1989; D. Giudici, G. Ornati, G. Briatico, F. Buzzetti, P. Lombardi and E. di Salle Journal of Steroid Biochemistry, Volume 30, Issues 1-6, 1988, Pages 391-394]. Clinical trials have shown the high efficacy and safety of exemestane in the treatment of postmenopausal women (natural or induced), suffering from metastatic breast cancer, progressing with anti-estrogen therapy, non-steroidal aromatase inhibitors or progestins [MB Stenina “Aromatase inhibitors in the treatment of disseminated breast cancer glands in menopausal patients. " Materials of the IV annual Russian oncological conference. Moscow, November 21-23, 2000].

Known methods for producing exemestane by 1,2-dehydrogenation of 6-methyleneandrost-4-en-3,17-dione (6-methylene-AD).

Known methods for 1,2-dehydrogenation of 6-methylene-AD using chemical dehydrogenating agents. Thus, methods are known for producing 6-methyleneandrost-1,4-diene-3,17-dione by dehydrogenation of 6-methylene-AD using 2,3-dichloro-5,6-dicyan-1,4-benzoquinone (DDQ) under reflux reaction mass in dioxane for 15 hours (yield 50.3%) [GB 2177700, 1987, Example 1] or in an inert solvent for 8 hours in the presence of aromatic carboxylic acid (yield 50.7%) [CN 1453288, 2003] .

Known methods for producing 6-methyleneandrost-1,4-diene-3,17-dione by dehydrogenation of 6-methylene-AD using selenium dioxide (boiling in tert-butyl alcohol for 30 hours, yield 40.2%) [GB 2177700 , 1987, Example 2] or using 2-iodoxybenzoic acid [CN 1491957, 2004].

The process of producing exemestane by the dehydrogenation of 6-methylene-AD to the 1,2-position in the presence of a quinone, silylating agent and acid having pKa in an aqueous solution of ≤1 is also known [WO 2009/077454, 2009, example 1]. In this case, 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) is used as quinone, bis (trimethylsilyl) trifluoroacetamide is used as the silylating agent, and trifluoromethanesulfonic acid is used as the strong acid; technical product Obtained in 81.8% yield, the product is recrystallized, but the yield of the purified product is not indicated. Thus, a method is known for producing 6-methyleneandrost-1,4-diene-3,17-dione by reacting 6-methylene-AD with chloranil in a convenient solvent, which is used dimethyl sulfoxide (DMSO) or N-methylpyrrolidine (NMP) [WO 2009093262 , 2009]. Moreover, from 100 g of 6-methylene-AD (Example 3), only 55 g of exemestane is obtained, 97.3% of the content.

A known method of producing 6-methyleneandrost-1,4-diene-3,17-dione, in which the dehydrogenation of 6-methylene-AD is carried out by bromination of 6-methylene-AD with bromine in an organic solvent at a temperature from 0 to 10 ° C with the formation of tribromo- compounds by debromination of the tribromo compound by reaction with an alkali metal iodide in an organic ketone solvent to form the 2-bromo derivative 6-methylene-AD and dehydrobromination of the 2-bromo derivative 6-methylene-AD by reaction with the main agent in a polar solvent (at a temperature of 120 ° C for 2.5 h, total yield minutes 40,13%) [US 4990635, 1991 Examples 2-4] (Figure 1).

Figure 1. Synthesis of exemestane from 6-methylene-AD by chemical methods

A common drawback of chemical methods of 1,2-dehydrogenation is the use of expensive and toxic reagents, harsh reaction conditions, and in most cases the need for column chromatography for purification, and, as a consequence, the low yield of exemestane.

The introduction of a 1,2-double bond into a steroid molecule using methods of microbiological transformation is known.

The implementation of complex polyenzyme processes in one technological stage under mild conditions, the relatively high yields of the desired products obtained, the possibility of inhibiting side reactions determine the prospects of using microorganisms as biocatalysts of the 1,2-dehydrogenation reaction, in particular, for the production of exemestane.

As biocatalysts of the 1,2-dehydrogenation process, cultures with 3-ketosteroid-Δ ¹ -dehydrogenase (EC 1.3.99.4) of the genera: Arthrobacter, Alternaria, Alcaligenes, Calonectria, Ophiobolus, Corynebacterium, Bacillus, Nocardia, Streptomyces, Bacterium, Bacterium Fusarium, Cylindrocarpon, Pseudomonas, Protaminobacter, Septomyxa, Didymella et al. [Charney W., Herzog H. Microbial transformation of Steroids. Academic Press. Inc. New York 1967, pp. 236-261]. Most often, strains of the species Arthrobacter simplex are used to carry out the 1,2-dehydrogenation process of steroid compounds of a number of pregnan and androstane.

Bioconversion is carried out using culture fluid, native cells (60-85% humidity) [US 4749649, 1988], dried cells obtained by treatment with acetone, dried in vacuum or air when heated [US 4704358, 1987], under lyophilization conditions, and also use cell-free extracts obtained by ultrasonic disruption or destroyed by passing frozen cells through dies under pressure [EP 0350488, 1993]. In order to exclude additional technological steps, it is preferable to use native cells.

The main problem of obtaining 1,2-dehydrogenated derivatives of a number of androstane, in particular exemestane, by the microbiological method is the low efficiency of the process, due to the low solubility of the substrate in the aquatic environment, the need to use low concentrations under these conditions to create conditions for its availability to microorganism cells and to achieve maximum complete conversion to product.

To achieve a higher concentration of steroid substrates in the medium and increase the efficiency of the 1,2-dehydrogenation process, organic solvents immiscible with water are used in an amount necessary to dissolve the starting substrate.

Thus, a method is known for converting a 1,2-saturated steroid to a 1,2-dehydrosteroid, which involves reacting a 1,2-saturated steroid with Arthrobacter simplex or Bacterium cyclooxydans in the presence of an exogenous electron carrier immiscible with water of an aromatic hydrocarbon solvent, where the oxygen concentration is maintained at the minimum oxygen concentration necessary for oxidation, or where the reaction is carried out below the flash point of the mixture, provided that the 1.2-saturated steroid has solubility in immiscible with ode aromatic hydrocarbon solvent is more than 5 g / L [US 4684610, 1987]. The process is carried out in a two-phase system using water-immiscible organic solvents: benzene, toluene, xylene.

A method for producing 1,2-dehydro-3-ketosteroid is also known, which method comprises reacting a 1,2-saturated 3-ketosteroid with Arthrobacter simplex or Bacterium cyclooxydans in the presence of an exogenous electron carrier and a water-immiscible solvent, including aromatic hydrocarbons [GB 2131811, 1984]. According to this method (examples 1 and 2), the transformation process is carried out by A. simplex cells in the presence of menadione in a medium of K ₂ HPO ₄ buffer solution (pH 7.5) containing up to 10% toluene for 4 days.

The disadvantage of these methods is the need to create and strictly maintain the minimum permissible concentration of dissolved oxygen in the medium in order to avoid an outburst of vapor of the gas-air mixture and at the same time provide optimal conditions for the oxidation of the substrate. The implementation of these methods requires special technological equipment, which virtually eliminates their practical implementation in industrial production. However, by the known methods receive androsta-1,4-diene-3,17-dione (ADD) from androst-4-ene-3,17-dione (AD), androsta-1,4,9 (11) -trien-3 , 17-dione (Δ ^{9 (11)} -ADD) from androst-4,9 (11) -diene-3,17-dione (Δ ^{9 (11)} -AD), as well as other 1,2-dehydro derivatives of the androstane series , and they do not describe the preparation of exemestane (6-methylene-ADD) from 6-methylene-AD.

It is known that exogenous electron acceptors - phenazine methosulfate (PMS), menadione (2-methyl-1,4-naphthoquinone), 1,4-naphthoquinone, menadione bisulfite - are used not only to stimulate the 1,2-dehydrogenation of steroid compounds, but also and to inhibit adverse reactions of a reducing nature and destructive activity of microorganism cells. The formation of by-products, the destruction of the target product reduce the yield of the main product.

In the processes of microbiological transformation of steroid compounds along with the use of water-immiscible organic solvents, water-miscible organic solvents such as dimethyl sulfoxide, dimethylformamide, methanol, ethanol, acetone and the like are used.

The use of dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) as water-miscible organic solvents used in microbiological 1,2-dehydrogenation processes is described.

For example, a method for producing 1,2-dehydro-Δ ⁴ -3-ketosteroid is known, which method comprises reacting a corresponding 1,2-saturated Δ ⁴ -3-ketosteroid with air-dried or heat-dried Arthrobacter simplex cells containing from 1 to 10% moisture, where the cells are dried in the absence of an organic solvent [US 4524134, 1985]. According to the method (example 2), a suspension of micronized substrate in DMF is added to a phosphate buffer solution (pH 7.5) containing a suspension of dried A. simplex ATCC 6946 cells (10 g / l) and menadione (86 mg / l). Thus DMF concentration in the medium is 2% v / v, and the 1,2-dehydrogenation is carried out with a load of substrate of 2.5 g / l for 24 hours at a temperature of ^about 31 C. However, the above method is obtained from AD, ADD, Δ ^{9 (11)} -ADD from Δ ^{9 (11)} -AD, as well as other 1,2-dehydro derivatives of the androstane and pregnane series, and it does not describe the preparation of exemestane (6-methylene-ADD) from 6-methylene-AD.

A relatively low concentration of the substrate characterizes this method of producing 1,2-dehydro derivatives as ineffective.

Also known is a method of converting 1,2-saturated 3-ketosteroids to 1,2-dehydro-3-ketosteroids, which consists in the fact that 1,2-saturated 3-ketosteroids are reacted with a preparation having a steroid-1,2-dehydrogenase activity of Arthrobacter simplex or Bacterium cyclooxydans in the presence of an exogenous electron carrier and one or more additional toxic oxygen scavengers selected from the group consisting of catalase, superoxide dismutase or platinum [US 4749649, 1988]. According to this method, the steroid substrate can be applied in the form of a powder, aqueous paste, or as a solution (or suspension) in a water-miscible organic solvent, such as DMF, DMSO, ethanol, methanol, acetone, in an amount of not more than 5% from the final volume of the medium. The method is illustrated by example 1, the description of which, however, does not specify which of the above types of application of the substrate was used (substrate load 3.5 g / l). Moreover, in a bioconversion medium, even in the presence of menadione, the accumulation of Δ ¹ -dehydrogenation product is only 50%.

Thus, the use of low concentration organic solvents miscible with water, up to 5% v / v, does not significantly increase the solubility of the substrate in the aqueous medium and its accessibility to the cells of the microorganism and does not allow to increase the efficiency of the process.

The process in question [US 4749649, 1988] also proposes the use of an exogenous catalase enzyme. The addition of catalase in an amount of 10 mg / l (equivalent to 16,000 units / l) promotes the active transformation of the substrate and allows you to increase its load to 10 g / l (example 1). For 22 hours, the degree of conversion of the substrate is 83%, for 46 hours - 95%. In addition, in the presence of catalase and menadione, a complete suppression of destructive activity occurs.

However, the cost of technology, due to the need to add an absorber (catalase enzyme, or superoxide dismutase, or platinum) to the medium, which eliminates the inhibitory effect of singlet oxygen on dehydrogenase activity, determines the main disadvantage of this method. In addition, in a known manner, ADD is obtained from HELL, Δ ^{9 (11)} -ADD from Δ ^{9 (11)} -AD, as well as other 1,2-dehydro derivatives of the androstane series, and it does not describe the preparation of exemestane (6-methylene-ADD ) from 6-methylene-AD.

It is known that to increase the solubility of steroid substrates in an aqueous medium, α, β or γ-cyclodextrins, as well as derivatives of β-cyclodextrin (βCD) methyl βCD and hydroxypropyl βCD in a concentration of 1-50 g / l are also used [Szejtli J. The use of cyclodextrins in biotechnological operations. Ed.D. Duchene, Published in France by Editions de Sante. 1991, 625 p]. In particular, a method is known for producing 1,2-dehydro derivatives of delta 4-3-ketosteroids by transformation of delta 4-3-ketosteroids using the microorganism Arthrobacter globiformis 193 in the presence of cyclodextrin, characterized in that water-soluble chemically modified derivatives of β-cyclodextrin are used as cyclodextrin [RU 2156302, 2000]. According to this method, the use of methyl β-CD allows 1,2-dehydrogenation of the substrate at a load of 20 g / L. With a weight ratio of methyl β-CD / substrate of 7/1, complete conversion of the substrate is completed in 6 hours. The content of 1,2-dehydrogenated product in the culture fluid is 95%. However, the practical relevance of this 1,2-dehydrogenation process is largely limited by the use of large quantities of expensive methyl β-CD. In addition, in a known manner get ADD from HELL (example 12) and it does not describe the production of exemestane (6-methylene-ADD) from 6-methylene-AD.

Along with the use of cyclodextrins, to increase the concentration and availability of steroid substrates for microorganisms, synthetic polymers of the N-vinylamide class are used - polyvinylpyrrolidone (PVP), polyvinylcaprolactam (PVC) and polyvinyl alcohol (PVA) [Druzhinina A.V., Andryushina V.A., Arinbasarova A.Yu., Pashkin I.I., Savinova T.S., Stytsenko T.S., Voishvillo N.E. Biotechnology, 2008, No. 1, pp. 46-50].

A known method of producing dehydroanalogue of steroids, including microbiological transformation of the corresponding Δ ⁴ -3-ketosteroids in the presence of a polymer material, followed by isolation of the target product from the reaction medium, characterized in that a homo- or copolymer of N-vinylcaprolactam with a mol. 10 ⁴ -10 ⁶ or copolymers of N-vinylcaprolactam with vinyl alcohol, vinyl acetate or vinyl methylacetamide containing up to 46%, 45% and 15%, respectively [RU 2042687, 08.27.1995]. However, the known method is claimed in general terms and the invention is not illustrated by examples; it does not describe the preparation of exemestane from 6-methylene-AD.

The intermediate in the synthesis of exemestane from androst-4-en-3,17-dione (AD) is 6-methylene-AD.

Known methods for producing 6-methylene-HELL from HELL.

In particular, a method is known for producing 6-methylene-3-oxo-Δ ⁴ -steroids, which comprises treating the 6-hydroxymethylsteroid 3-enol ether with an acidic reagent capable of regenerating Δ ⁴ -3-ketosteroids from their 3-enol ethers and simultaneously reducing 6-hydroxymethyl a 6-methylene group [US 3112305, 1963]. According to this method, 6-methylene-AD is obtained from 3-methyl enol ether 6-hydroxymethyl-AD by dehydration of 90% formic acid, which can be obtained from AD by the following reaction sequence: preparation of 3-methyl enolester AD, its interaction with Vilsmeier reagent [US 3114750 , 1963], hydrogenation of the obtained 6-formyl derivative [US 3095411, 1963] (yields not indicated) (Figure 2).

Figure 2. Synthesis of 6-methylene-HELL from HELL using the Vilsmeier reaction

There is also known a method for producing 6-methylene-Δ ⁴ -3-ketosteroid of the androstane or pregnane series, comprising the reaction of the corresponding Δ ⁴ -3-ketosteroid with a formaldehyde derivative in an inert solvent in the presence of a strong acidic condensing agent [US 4322349, 1982; Annen, Klaus; Hofmeister, Helmut; Laurent, Henry; Wiechert, Rudolf: Synthesis, 1982, No. 1 34-40] (direct γ-methyleneation method). In this case, trioxane or formaldehyde dialkyl acetal (methyl, ethyl or diisopropyl) can be used as a formaldehyde derivative, and phosphorus pentoxide or phosphorus oxychloride as a condensing agent. For example, according to this method, the interaction of blood pressure with formaldehyde diethylacetal is carried out in the presence of phosphoryl chloride and sodium acetate in an inert solvent while boiling for 5.5 hours, the product is purified by chromatography on silica gel (Figure 3).

Figure 3. Synthesis of 6-methylene-AD from AD by direct γ-methyleneation [US 4322349, 1982]

However, despite the significantly shorter path to the target product, this method does not allow to obtain a yield of 6-methylene-AD from AD higher than 68%.

A known method for producing 6-N, N-disubstituted aminomethyl derivatives of steroids [GB 1280569, 1972] and a method for producing 6-methylenesteroids from 6-N, N-disubstituted aminomethyl derivatives of steroids [GB 1280570, 1972]. The method for producing 6-N, N-disubstituted aminomethyl derivatives of steroids [GB 1280569, 1972] consists in mixing a secondary amine, formaldehyde and a steroid compound containing a 3-alkoxy-3,5-diene group in the nucleus, the reaction being carried out in tetrahydrofuran medium, and N-methylaniline, among others, is used as a secondary amine. A method for producing 6-methylene steroids [GB 1280570, 1972] is that 6- (N-methyl-N-phenylaminomethyl) -steroids obtained by a method similar to [GB 1280569, 1972] are mixed with a strong acid. According to this method, the 6- (N-methyl-N-phenylaminomethyl) -steroid derivative is dissolved in 6 N hydrochloric acid, the solution is kept for 3 hours at room temperature, and the precipitate is filtered off. However, by known methods, 6- (N-methyl-N-phenylaminomethyl) derivatives of Δ ⁴ -3-keto compounds of the pregnane series and 6- (N-methyl-N-phenylaminomethyl) derivative of 17β-hydroxy-17α-methylandrost-4.9 are obtained (11) -diene-3-one, as well as their 6-methylene analogues, and they do not describe the preparation of a 6- (N-methyl-N-phenylaminomethyl) derivative from AD and 6-methylene-AD.

The closest in essence to the proposed method for producing 6-methylene-AD from AD is a method for producing exemestane and its derivatives by reacting 3-enol ether of AD or its derivatives with 30-40% aqueous formaldehyde solution and 1-2 equivalent of an amine halide salt in an organic solvent at temperature from 30 to 50 ° C to obtain 6-methylene-AD or its derivatives, described by A. Longo and P. Lombardi [US 4990635, 1991]. The method is illustrated by example (example 1), which consists in the fact that in the synthesis of 6-methylene-AD from AD, the latter is treated with triethyl orthoformate in tetrahydrofuran medium containing ethanol (13.3%) in the presence of p-toluenesulfonic acid at a temperature of 40 ° C for 2 hours, then N-methylaniline and 40% aqueous formaldehyde solution are added, and after 2 hours at 40 ° C, the reaction mixture is treated with concentrated hydrochloric acid at room temperature. The product is crystallized by diluting the reaction mass with water. From 20 g of the starting compound, 14.8 g of 6-methylene-AD are obtained in a molar yield of 71%. (Figure 4).

Figure 4. Synthesis of 6-methylene-AD from AD using the Mannich reaction [US 4990635, 1991].

A similar method for producing 6-methylene-AD from AD was used in patents [CN 1415624, 2003; CN 1491957, 2004], while the product was obtained with a yield of 68.6%.

The disadvantages of the method described in US 4990635 are:

- long duration of the enolization phase (2 hours at 40 ° C);

- fractional feed of the p-toluenesulfonic acid catalyst in the enolization process;

- the use of a large amount of hydrochloric acid at the stage of deamination;

- low yield of 6-methylene-AD, despite the fact that the process is carried out without isolation of intermediates.

It is known that the mechanism of the Mannich reaction (when catalyzed by acid) consists in the initial formation of a highly reactive intermediate particle, the iminium cation, formed from the amine and carbonyl components, which is stabilized by acid. The high reactivity of iminium salts imposes some restrictions on the choice of solvent for the aminomethylation reaction. Usually, in the synthesis of nonsteroidal bases, Mannich prefer to use highly polar solvents (so that the salt is in solution), incapable of chemical interaction with the iminium salt: acetonitrile, dimethyl sulfoxide, dimethylformamide, etc.

In addition, the choice of a solvent for the Mannich reaction should also take into account the features of the steroid substrate. To ensure the regioselectivity of aminomethylation at the C ₆ atom and to exclude adverse reactions at the C ₂ atom located in the α-position to the 3-keto group, preliminary enolization of the Δ ⁴ -3-ketosystem is carried out. The effectiveness of the reaction depends on the solubility and stability of the latter during aminomethylation. Therefore, the choice of solvent medium is essential.

In the known methods for producing 6-methylene-substituted steroids of the pregnan and androstane series through the formation of 6- (N-methyl-N-phenylaminomethyl) -intermediate using the Mannich reaction, tetrahydrofuran is used as a solvent [GB 1280569, 1972; US 4990635, 1991; CN 1415624, 2003; CN 1491957, 2004]. So, in the patent [GB 1280569, 1972] it is noted that alcohols (methanol, ethanol, propanol, isopropanol, t-butanol), although they can be used as solvents in the aminomethylation process, polar aprotic solvents, such as tetrahydrofuran, dioxane, 1 2-dimethoxyethane is more effective. In addition, the use of aliphatic alcohols as solvents in the Mannich reaction with compounds of a non-steroidal structure is considered undesirable, since alcohols are able to react with formaldehyde and a secondary amine to form an O-aminomethylation product.

R-OH + CH ₂ O + HNR ₂ "= ROCH ₂ NR ₂ "

However, the patent [GB 1280569, 1972] does not describe the preparation of a 6- (N-methyl-N-phenylaminomethyl) derivative of HELL. In addition, there is no information on the study of the influence of the solvent medium on the parameters of the process of 6-aminomethylation of steroids in available literature.

It is also known that the splitting of Mannich bases (including steroid ones) can occur ambiguously. Along with the target product of deamination with exomethylene-compound (A), the product of the reverse process of deaminomethylation (B) can be formed, which is the starting compound in the synthesis (Figure 5).

Figure 5. Directions for the splitting of steroid Mannich bases.

Moreover, the selectivity of the deamination process substantially depends on the conditions of its implementation. Despite the fact that the treatment with concentrated mineral acid [Mannich bases, Maurilio Tramontini, Luigi Angiolini // CRC Press is considered the best option for cleaving the CN bond of phenyl substituted aminomethyl steroids (R ₁ = Ph) to form 6-methylene derivatives. Inc. 1994. P 78-80], the nature of the solvent medium and the temperature regime are also of great importance. Ensuring the regioselectivity of the cleavage of N, N-disubstituted 6-aminomethylene derivatives of AD is of great importance in deciding on the effectiveness and industrial applicability of this method. However, information on the influence of the last two factors on the selectivity of the deamination of 6-aminomethyl derivatives of steroids is practically absent in available literature. In the patent [GB 1280570, 1972] there is no information on the quality of the obtained deamination products of 6- (N-methyl-N-phenylaminomethyl) steroids. In addition, the cleavage of the 6- (N-methyl-N-phenylaminomethyl) derivative of AD with the formation of 6-methylene-AD is not described.

Also, in literature there is no information on the study of the feasibility of combining the sequential processes of enolization, aminomethylation and deamination and their carrying out without isolation of intermediates.

The closest in essence to the proposed method for producing exemestane from 6-methylene-AD is a method for producing 6-methyleneandrost-1,4-diene-3,17-dione, which consists in contacting 6-methyleneandrost-4-en-3,17- a dione with Δ ¹ dehydrogenating enzymes A. Simplex in the presence of a water-immiscible organic solvent and an exogenous electron acceptor [WO 0104342, 2001]. According to this method, whole cells or a cell-free Arthrobacter simplex ATCC 6946 extract are used to carry out the 1,2-dehydrogenation of 6-methylene-AD, with the use of whole cells being preferred. The process is carried out in a two-phase system using a water-immiscible organic solvent selected from the group of toluene, xylene, benzene, heptane, methylene chloride, n-octanol, or mixtures of these solvents in the presence of a chemical exogenous electron acceptor (4% menadione in relation to weight substrate) and catalase enzyme. Despite the fact that the authors stated that the substrate 6-methyleneandrost-4-en-3,17-dione is present in the medium at a concentration of from 10 to 125 g / l (claims 18 and 19 of the formula), in example A, illustrating the method the concentration of the starting substrate is 55.6 g / l. So, for the process 6-methyleneandrost-4-en-3,17-dione (50 g) and menadione (2.0 g) are mixed with toluene (800 ml). The aqueous phase (pH 8.7-8.9), prepared as a mixture of catalase, dipotassium phosphate, A. Simplex cell concentrate (50 ml) and water (50 ml), is added to the intensively stirred mixture in toluene. The process is carried out at ³⁰ ° C, the reaction mixture was purged with controlled mixture of air and nitrogen - air (0.1% SCHF) and nitrogen (0.3% SCHF). If necessary, A. Simplex cells are added during the reaction to complete the process. Moreover, the amount of additional cell concentrate added is not indicated. After completion of the process, the organic phase is separated, the aqueous phase is extracted with toluene. After filtration and clarification, the toluene solution is concentrated, 2.5 times the volume of octane is added to the residue, and the crystalline product is filtered off.

The disadvantages of the process described in WO 0104342 are:

- the use of the catalase enzyme in an amount of 0.03% by weight of the substrate, which increases the cost of the process and limits its industrial use;

- the use of large volumes of flammable organic solvents at the stage of transformation, which requires special fermentation equipment and compliance with special fire safety rules;

- the use of toxic organic compounds poses a threat to the health of specialists in production. The aromatic hydrocarbons used (benzene, toluene, xylene) are potent poisons that affect the blood formation function of the body. For example, toluene causes cyanosis, hypoxia, damage to the central nervous system, as well as toluene toxicomania, which also has a carcinogenic effect.

- the need to solve environmental problems;

- the complexity of the process under the necessary regulation of the composition of the gas mixture supplied to the reaction medium;

- lack of information on the selectivity of the transformation process and the efficiency of the process of isolation of the crystalline product, i.e. the degree of conversion of the substrate into the product and the yield of crystalline product (it is only indicated that the amount of untransformed initial substrate is 0.1%);

- lack of information about the quality of exemestane received;

- the absence of an example confirming the applicability of the method with a substrate load of up to 125 g / l.

The technical task of one of the inventions of the claimed group is, therefore, increasing the efficiency, manufacturability and safety of microbiological 1,2-dehydrogenation and obtaining 6-methyleneandrost-1,4-diene-3,17-dione from 6-methyleneandrost-4-ene -3,17-dione in a manner devoid of these disadvantages.

The technical task in the claimed group of inventions is to increase the yield of target products by reducing the likelihood of adverse reactions at the stage of formation of 3-enol ether AD, at the stage of introduction of N, N-disubstituted aminomethyl substituent at position C _{6 of the} androstenedione molecule, almost completely eliminating the formation of side products at the stage deamination and the formation of a 6-methylene group and the 1,2-dehydrogenation step and the preparation of 6-methyleneandrost-4-ene-3,17-dione and 6-methyleneandrost-1,4-diene-3,17-dione without The above disadvantages.

The technical problem is solved by the method of obtaining 6-methyleneandrost-4-en-3,17-dione of the formula (I)

from androstenedione of the formula (II),

including preliminary enolization of Δ ⁴ -3-ketosystem with the formation of 3-enolester of androstenedione of formula (III),

three-component condensation with formaldehyde and a secondary amine under conditions of acid catalysis with the formation of an N, N-disubstituted 6-aminomethyl derivative of androstenedione of the formula (IV),

deamination in a medium containing a strong mineral acid, characterized in that polar protic solvents are used as the medium for the aminomethylation reaction, and aprotic solvents or mixtures of aprotic solvents are used as the medium for the deamination reaction. Protonic solvents as a medium for carrying out the 6-aminomethylation reaction of the steroid substrate of a number of androstane have not previously been used in such processes. Aprotic solvents of the claimed group of solvents or mixtures thereof as a medium for the deamination of N, N-disubstituted 6-aminomethyl derivatives of androstane steroids have not been previously used in such processes.

Another invention of the claimed group is a method for producing 6-methyleneandrost-1,4-diene-3,17-dione by 1,2-dehydrogenation using 6-methyleneandrost-4-en-3,17-dione obtained as described above as the starting material the method according to the invention, the method of microbiological transformation using bacterial cells Nocardioides simplex VKM Ac-2033D and subsequent isolation of the target product from the culture fluid, characterized in that the transformation is carried out in an aqueous medium containing up to 40% by volume of an organic solvent miscible with water . The use of large concentrations of a water-miscible organic solvent in a medium for carrying out the process of 1,2-dehydrogenation of a steroid substrate is not obvious, and previously large concentrations of a water-miscible organic solvent have not been used in such processes.

The technical problem in terms of obtaining 6-methyleneandrost-1,4-diene-3,17-dione using the obtained 6-methyleneandrost-4-en-3,17-dione as the starting material (intermediate) is achieved by obtaining 6-methyleneandrost-1 , 4-diene-3,17-dione of the formula (V)

from 6-methyleneandrost-4-en-3,17-dione of the formula (I),

by the method of microbiological 1,2-dehydrogenation in a medium containing an exogenous electron acceptor and aprotic polar solvent miscible with water, using initial substrate concentrations from 5 to 100 g / l and isolating the target product from the culture fluid.

Thus, the essence of the claimed group of inventions, which includes obtaining 6-methyleneandrost-4-en-3,17-dione and then 6-methyleneandrost-1,4-diene-3,17-dione using it, is that androstenedione is first subjected to enolization of the Δ ⁴ -3-ketosystem, then three-component condensation with formaldehyde and secondary amine under acid catalysis with the formation of an N, N-disubstituted 6-aminomethylene derivative of androstenedione in a polar proton solvent, and then deamination with the formation of 6-methylene AD in an aprotic solvent (or a mixture of aprotic solvents) followed by a microbiological 1,2-dehydrogenation reaction in an aqueous medium containing up to 40% by volume of a polar aprotic solvent miscible with water.

To improve the yield and simplification of the process is subjected to aminomethylation enolefir BP position C ₆ Mannich reaction in a polar protic solvent and further regioselective cleavage Mannich base to form a 6-methylene group in an aprotic solvent (or mixture of aprotic solvents).

At the same time, three-component condensation according to the Mannich type of reaction is carried out with the release of the product from the reaction medium, and the deamination reaction is carried out under optimal conditions.

In addition, aliphatic alcohols from the group methanol, ethanol, propanol, isopropanol and the like are used as a polar proton solvent.

In addition, dialkyl ketones (e.g. acetone, methyl ethyl ketone), aromatic hydrocarbons (e.g. benzene, toluene), chlorinated hydrocarbons (e.g. dichloromethane, chloroform, dichloroethane), as well as mixtures thereof in an effective ratio, are used as an aprotic organic solvent.

In addition, N-methylaniline is used as the secondary amine, and formaldehyde is used as a 37-40% aqueous solution.

In addition, the subsequent 1,2-dehydrogenation in order to obtain 6-methyleneandrost-1,4-diene-3,17-dione is carried out using bacterial cells Nocardioides simplex VKM Ac-2033D.

In addition, dimethylformamide or dimethyl sulfoxide, preferably dimethyl sulfoxide, is used as the polar aprotic organic solvent in the 1,2-dehydrogenation. Moreover, the concentration of DMF in the aqueous medium is at least 8% vol., And the concentration of DMSO in the aqueous medium is 8-40%.

In addition, the concentration of the starting substrate during 1,2-dehydrogenation is from 5 to 100 g / l, preferably 100 g / l.

In addition, methadione in an effective amount is used as an exogenous electron acceptor.

The advantages of the proposed method are as follows:

- No need to use multiple crystallization of the main product or chromatography to purify it.

- High efficiency of the enolization process. The output at the stage of obtaining enol ether HELL is 94.4%.

- High selectivity and simplification of the aminomethylation reaction. The output at the stage of obtaining the N, N-disubstituted 6-aminomethylene derivative HELL quantitative;

- High selectivity of the deamination process. The output at the stage is 85-88%;

- The total yield of 6-methyleneandrost-4-en-3,17-dione from androstenedione is 83%.

The method of the present invention allows the process of 1,2-dehydrogenation:

- with a high load of the substrate up to 100 g / l in mild conditions;

- without the use of flammable organic solvents;

- without additional steroid compounds, ensuring the stabilization of 1,2-dehydrogenase activity;

- the method is environmentally safe, since DMSO is proposed as a water-miscible solvent - a high-boiling liquid with a low toxicity level and a flash point of 89 ° C (in a closed vessel), used in medicine in the form of aqueous solutions as a local anti-inflammatory and analgesic drug (trade name Dimexide);

- the method excludes the use of the enzyme catalase and any other absorbers of singlet oxygen;

- the method can be implemented on standard processing equipment.

Obtaining 6-methyleneandrost-4-en-3,17-dione of the formula (I) and 6-methyleneandrost-1,4-diene-3,17-dione of the formula (V) are carried out according to the scheme shown in Figure 6.

Figure 6. Synthesis of exemestane from AD through 6-methylene-AD of the claimed method

The following is an example of a detailed description of the invention.

Androstenedione of the formula (II),

they undergo enolization of the Δ ⁴ -3-ketosystem (with the release of the product of enolization (III), for example, by the action of a catalytic amount of sulfosalicylic acid. The methyl 3-enol ether of AD (III) obtained in up to 94.4% is reacted with the Mannich reagent formed in situ in conditions of acid catalysis (p-TSC) from formaldehyde and a secondary amine (N-methylaniline), in a proton polar organic solvent with the formation of an N, N-disubstituted 6-aminomethylene derivative (IV), which, after isolation from the reaction mass, is subjected to regio selective deamination under conditions of catalysis with strong mineral acid (sulfuric or hydrochloric) in an aprotic solvent (or a mixture of aprotic solvents) to form a 6-methylene derivative (I) 6-methyleneandrost-4-en-3,17 obtained in up to 88% yield -dione (I) is then converted into 6-methyleneandrosta-1,4-diene-3,17-dione (V) by the introduction of a 1,2-double bond by the method of microbiological dehydrogenation with the participation of cells of microorganisms having 3-ketosteroid-Δ ¹ -dehydrogenase activity (preferably bacterial cells Nocardioides simplex VKM Ac -2033D), while the load of the initial substrate is from 5 to 100 g / l, and the fermentation time is from 24 to 52 hours and the process is carried out in an aqueous medium containing an exogenous electron acceptor and aprotic polar solvent miscible with water - DMF or DMSO ( preferably DMSO).

The use of water-miscible organic solvents at the stage of 1,2-dehydrogenation ensures the transfer of the steroid substrate - 6-methylene-AD, or most of it into a soluble form and the process at high (50-100 g / l) substrate loads significantly reduces the effect of co-crystallization of the substrate and product and the formation of mixed crystals. There are no data in the literature on the use of water-miscible organic solvents used in high concentrations (20-40% v / v).

Analysis of the products of microbiological transformation is carried out by thin layer chromatography (TLC) and HPLC.

HPLC analysis of products was carried out on an Agilent 1100/1200 chromatograph (Agilent, Germany) with a Symmetry C18 pre-column, 5 μm, 3.9 × 20 mm and a Symmetry C18 column, 5 μm, 4.6 × 250 mm (Waters, Ireland) ; in a system with a mobile phase (v / v): 60% acetonitrile, 10% isopropanol, 0.01% acetic acid and water up to 100%, at a flow rate of 1 ml / min, a temperature of 50 ° C, and detection by absorption at 250 nm; typical retention times: for 6-methylene-AD 4.9 min and for 6-methylene-ADD (exemestane) 3.2 min.

Isolation of 6-methyleneandrost-1,4-diene-3,17-dione (V) from the culture fluid can be carried out by extraction with a water-immiscible solvent or extraction of the steroid from biomass paste with any suitable solvent after it has previously been separated from the aqueous phase. For steroid extraction, for example, a sorption extraction method, as well as any other effective method, can also be applied. The selectivity of the formation of 6-methyleneandrost-1,4-diene-3,17-dione (V) at the stage of microbiological 1,2-dehydrogenation is 95-98%. The yield of crystalline 6-methyleneandrost-1,4-diene-3,17-dione (V) at the stage of microbiological 1,2-dehydrogenation after isolation and purification is 85-87%.

The total yield of 6-methyleneandrost-1,4-diene-3,17-dione (V) from androstenedione is from 70 to 72%.

The claimed group of inventions is illustrated by the following examples, not limiting it.

The formation of 3-enol ether HELL is carried out using the minimum required amount of trialkyl orthofomyate in the medium of the corresponding absolute alcohol in the presence of an acidic catalyst. Strong organic acids, for example p-toluenesulfonic acid or sulfosalicylic acid, can be used as the acid catalyst.

The N, N-disubstituted 6-aminomethylene derivative of androstenedione of formula (IV) is obtained in an aqueous aliphatic alcohol medium with a water content of 5 to 10%.

N-methylaniline is used as the secondary amine, and formaldehyde is used as a 37% aqueous solution.

For example, aliphatic alcohols (methanol, ethanol, propanol, isopropanol, etc.) can be used as a proton solvent in the aminomethylation stage in the implementation of the invention, a method for producing 6-methyleneandrost-4-en-3,17-dione.

As an aprotic solvent in the implementation of the invention, a method for producing 6-methyleneandrost-4-en-3,17-dione at the deamination stage, for example, dialkyl ketones (e.g. acetone, methyl ethyl ketone), aromatic hydrocarbons (e.g. benzene, toluene) can be used chlorinated hydrocarbons (e.g. dichloromethane, chloroform, dichloroethane), as well as mixtures thereof in an effective ratio.

Mineral acids (preferably sulfuric or hydrochloric acid) can be used as the acid catalyst in the deamination step.

Microbiological 1,2-dehydrogenation in the implementation of the invention, a method for producing 6-methyleneandrost-1,4-diene-3,17-dione can be carried out by resting (washed) cells of bacteria of the genus Nocardioides, specifically Nocardioides simplex VKM Ac-2033D.

Miscible with water, organic polar aprotic solvents, for example DMF or DMSO, preferably DMSO, can be used as a component of the medium in carrying out the invention, a method for producing 6-methyleneandrost-1,4-en-3,17-dione.

The method is illustrated by examples.

Examples

Example 1. Obtaining 3-methoxyandrost-3,5-dien-17-one (3-enol ether AD, III).

A suspension of 100 g of AD (I) in a mixture of 85.6 ml of trimethylorthoformate and 130 ml of anhydrous methanol is stirred for 15 minutes, then 120 ml of a solution of 0.2 g of anhydrous sulfosalicylic acid in methanol are slowly added. The reaction mass is maintained at 20-25 ° C for 1 hour 15 minutes. At the end of the exposure, the reaction mass is cooled to 10-15 ° C, slowly add 75 ml of dry acetone and incubated for 1 hour at the same temperature. Then add 0.8 ml of triethylamine to a pH of 7-8, the reaction mass is cooled to 0-2 ° C and 225 ml of water are added. At the end of the exposure, the precipitate is filtered off, washed on the filter with water and a 0.25% solution of triethylamine in methanol. Obtain 99 g of 3-enol ether HELL (III) with a yield of 94.4%. Mp = 171-172 ° C (lit. 171-173 ° C [RU 2163606, 2001]). NMR ¹ H, δ, ppm: 0.90 s (3 N, 18-CH ₃ ), 0.98 s (3 H, 19-CH ₃ ), 1.02-2.52 m (17 N), 3.56 s (3 N, OCH ₃ ), 5.13 d (J = 1.2 Hz, 1 Н, Н ⁴ ), 5.22-5.25 m (1 Н, Н ⁶ ).

Example 2. Obtaining 6ξ- (N-methyl-N-phenylaminomethyl) -androst-4-en-3,17-dione (A mixture of 6α and 6β isomers, IV).

To a suspension of 30 g of the 3-enol ether of AD (III) in 150 ml of methanol, 12.9 ml of N-methylaniline and 7.45 ml of a 37% aqueous formaldehyde solution are added, then a solution of 0.75 g of p-TSC in 10 ml of methanol is slowly added. The suspension is incubated for 15-30 minutes at room temperature, then 1 hour at a temperature of 30-40 ° C until the suspension is dissolved. At the end of the reaction, the solution was poured into a 10-fold volumetric amount of water containing 0.1 g of NaOH. The precipitate is filtered off, washed with water to pH ~ 7. 40.42 g of compound IV are obtained (mixture of 6α and 6β isomers).

Found: C 80.16%; H 8.92%; N 3.71%, C ₂₇ H ₃₅ NO ₂ . Calculated: C 79.96%; H 8.70%, N 3.45%

6α-isomer: ¹ H NMR, δ, ppm: 0.92 s (3H, 18-CH ₃ ), 1.31 s (3H, 19-CH ₃ ), 2.98 s (3H, 21-CH ₃ -N), 3.25 m (1H, CH ₂ -N), 3.77 m (1H, CH ₂ -N), 5.84 s (1H, H ⁴ ), 6.50-7.25 m (5H, Ph); ¹³ C, δ, ppm: 199.02, 170.44, 148.58, 129.17, 126.53, 116.49, 112.14, 56.97, 55.33, 54.10, 52.88, 50.50, 47.24, 42.52, 39.99, 39.07, 38.15, 37.24, 35.51, 34.65, 33.85, 31.04, 30.34, 21.24, 20.13, 18.21, 13.55. 6β-isomer: ¹ H NMR, δ, ppm: 0.87 s (3H, 18-CH ₃ ), 1.21 s (3H, 19-CH ₃ ), 2.97 s (3H, CH ₃ -N ), 5.74 s (1H, H ⁴ ).

Example 3. Obtaining 6-methyleneandrost-4-en-3,17-dione (6-methylene-AD, I).

Option 1.

To a suspension of 40 g of a mixture of 6α and 6β isomers of compound IV in 200 ml of acetone was added 28.5 ml of 32% hydrochloric acid. The mass is kept at room temperature for 1 h and poured into a 10-fold volumetric amount of water. The precipitate is filtered off, washed with water to pH ~ 7. Obtain 26.7 g of 6-methylene-AD (I) with a yield of 90.7%.

After recrystallization from aqueous ethyl alcohol, 25.9 g of compound I are obtained in 88% yield.

_Mp. = 160-163 ° C (lit. 159-162 ° C [CN 1415624, 2003]).

¹ H NMR, δ, ppm: 0.86 s (3H, 18-CH ₃ ), 1.07 s (3H, 19-CH ₃ ), 5.00 d (J = 44.3 Hz, 2H, 6-C = CH ₂ ), 5.88 s (1H, H ⁴ ).

Option 2

To a suspension of 13.5 g of a mixture of 6α and 6β isomers of compound IV in a mixture of 100 ml of toluene and 50 ml of methyl ethyl ketone, 15 ml of 60% aqueous sulfuric acid are added. The mass was kept at room temperature for 1 h, the acid layer was separated and extracted twice with 20 ml of toluene. A solution of the product in toluene is washed with 60% sulfuric acid solution and water, treated with clarifying charcoal. The solvent was evaporated to dryness, the residue was crystallized from aqueous ethanol. 8.5 g of 6-methylene-AD (I) are obtained in a yield of 85.6%.

Example 4. Obtaining 6-methyleneandrost-1,4-diene-3,17-dione (V)

The strain Nocardioides simplex VKM Ac-2033D is used in the work, which is supported on an agar medium of the following composition (g / l): glucose - 10, soy peptone - 4, yeast extract - 4, MgSO ₄ - 0.5, KH ₂ PO ₄ - 2, K ₂ HPO ₄ - 4, agar - 20, distilled water, pH 7.0-7.2. Cultivation is carried out for 3-4 days at 30 ^about C.

Growing 1st inoculum

The culture was washed off from the surface of the agar medium in 5 ml of physiological solution in 750 ml of an Erlenmeyer flask with 50 ml of medium (g / l): glucose - 15, soy peptone - 6, yeast extract - 6, MgSO ₄ - 0.5, KH ₂ PO ₄ - 2, K ₂ HPO ₄ - 4, and cultured at 28-30 ^about C for 24-26 hours.

Growing a transforming culture.

5 ml of culture fluid for 24-26 hours of growth of the 1st inoculum is added to 100 ml of medium of the same composition and grown under the same conditions for 15 hours, then the synthesis inducer of steroid-Δ ^1- dehydrogenase — cortisone acetate — is introduced in the form of ethanol solution (20 mg in 1 ml of ethanol). The induction of the synthesis of Δ ¹ -dehydrogenase is carried out over the next 24-26 hours. Microbial cells were pelleted by centrifugation at 5000 g for 30 min and used for the 1-dehydrogenation process or stored at -18 ^{° C} for months until use.

Transformation of 6-methylene-AD

Option 1.

The transformation of 6-methylene-AD (I) is carried out in 250 ml flasks containing 25 ml of 0.02 M Na-phosphate buffer solution, pH 8.0. Consistently add 125 mg of the substrate in powder form and add 0.2 g of biomass (weight of dry biomass).

The bioconversion was carried out in aerated conditions at ³⁰ ° C on a rotary shaker at 200 rev / min. The process is stopped for 72 hours of cultivation when reaching the content of 6-methylene-ADD - 91-93 (± 3%).

Analysis of the conversion products is carried out by TLC and HPLC.

Option 2

The method is carried out according to option 1, but the reaction medium is represented by 0.02 M Na-phosphate buffer solution, pH 8.0 (23 ml) and DMSO (2 ml) with menadione.

Option 3

The method is carried out according to option 2, but the reaction medium is represented by 0.02 M Na-phosphate buffer solution, pH 8.0 (23 ml) and DMF (2 ml).

Options 4-9.

The methods are carried out according to option 2, but with a range of substrate concentrations from 10.0 to 100.0 g / l (Table).

The culture fluid was extracted with ethyl acetate, the extract was washed with water, dried over anhydrous MgSO ₄ , clarified with activated carbon and evaporated to dryness in vacuo. The residue is triturated with petroleum ether, the precipitate is filtered off. Get 6-methyleneandrosta-1,4-diene-3,17-dione with a yield of 85-87%.

_Mp. = 183-185 ° C (lit. 188-191 ° C [US 4904650, 1990]). NMR ¹ H, δ, ppm: 0.93 s (3H, 18-CH ₃ ), 1.16 s (3H, 19-CH ₃ ), 5.00 d (J = 24.7 Hz, 2H, 6-C = CH ₂ ) 6.16 d (J = 1.9 Hz, 1H, H ⁴ ), 6.24 dd (J = 1.9 Hz, J = 10.1 Hz, 1H, H ² ), 7.07 d (J = 10.2 Hz, 1H, H ¹ ).

Obtaining 6-methylene-ADD Option No. Volume of medium, ml Biomass, g / l (dry biomass weight) The load of the substrate, g / l Application Method The concentration of solvent,% The duration of the process, hours The content of the product in the environment,% one 25 1.0 5 powder - 72 92 ± 3 2 25 1.0 5 DMSO 8.0 48 93 ± 3 3 25 1.0 5 DMF 8.0 24 92 ± 3 four 25 2.0 10 DMSO 8.0 24 92 ± 3 5 25 4.0 twenty DMSO 14.0 24 92 ± 3 6 25 10.0 fifty DMSO twenty 27 95 ± 3 7 25 10.0 fifty DMSO 40 29th 91 ± 3 8 25 15.0 75 DMSO 40 52 91 ± 3 9 25 20.0 one hundred DMSO 40 52 92 ± 3

Claims (11)

1. The method of obtaining 6-methyleneandrost-4-en-3,17-dione of the formula (I)

from androstenedione of the formula (II)

including preliminary enolization of Δ ⁴ -3-ketosystem with the formation of 3-enolester of androstenedione of formula (III)

three-component condensation with formaldehyde and a secondary amine under conditions of acid catalysis with the formation of an N, N-disubstituted 6-aminomethyl derivative of androstenedione of the formula (IV)

deamination in a medium containing strong mineral acid, characterized in that polar protic solvents are used as the medium for the aminomethylation reaction, and aprotic solvents or mixtures of aprotic solvents are used as the medium for the deamination reaction. 2. The method according to claim 1, characterized in that aliphatic alcohols from the group of methanol, ethanol, propanol, isopropanol and the like are used as the polar proton solvent. 3. The method according to claim 1, characterized in that dialkyl ketones (for example acetone, methyl ethyl ketone), aromatic hydrocarbons (for example benzene, toluene), chlorinated hydrocarbons (for example dichloromethane, chloroform, dichloroethane) as well as mixtures thereof are used as an aprotic organic solvent in an effective ratio. 4. The method according to claim 1, characterized in that N-methylaniline is used as the secondary amine, and formaldehyde is used in the form of a 37-40% aqueous solution. 5. The method of obtaining 6-methyleneandrost-1,4-diene-3,17-dione of the formula (V)

from 6-methyleneandrost-4-en-3,17-dione of the formula (I)

by microbiological 1,2-dehydrogenation in an aqueous medium containing an exogenous electron acceptor and a water miscible solvent, using microorganism cells with 3-ketosteroid-Δ ¹ -dehydrogenase activity, characterized in that an aprotic polar solvent in concentration is used as a component of the medium up to 40 vol.%. 6. The method according to claim 5, characterized in that Nocardioides simplex BKM Ac-2033D is used as a microorganism with 3-ketosteroid-Δ ^1- dehydrogenase activity. 7. The method according to claim 5, characterized in that dimethylformamide or dimethyl sulfoxide, preferably dimethyl sulfoxide, is used as the polar aprotic organic solvent. 8. The method according to claim 5, characterized in that as an exogenous electron acceptor use menadione in an effective amount. 9. The method according to claim 5, characterized in that the concentration of the starting substrate is from 5 to 100 g / l, preferably 100 g / l. 10. The method according to claim 7, characterized in that the concentration of DMF in the aqueous medium is at least 8 vol.%. 11. The method according to claim 7, characterized in that the concentration of DMSO in the aquatic environment is 8-40%.