RU2447154C1 - Microbiological method of producing 1,2-dehydrogenated derivatives of δ4-3-keto-steroids of androstane family in aqueus organic media - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing 1,2-dehydrogenated derivatives of Δ⁴-3-ketosteroids of the androstane family. A 1,2-dehydrogenation reaction is carried out using cells of a microorganism having high 3-ketosteroid-1-dehydrogenase activity, in a medium containing 8-40 vol. % water miscible aprotic solvent.

EFFECT: invention enables transformation of Δ⁴-3-ketosteroids and ensures high selectivity of formation of 1,2-dehydrogenated Δ⁴-3-ketosteroids at high rates of the process.

4 cl, 1 tbl, 5 ex

Description

The present invention relates to the field of biotechnology, specifically relates to the production of 1,2-dehydrogenated Δ ⁴ -3-ketosteroids of the androstane series by microbiological transformation of Δ ⁴ -3-ketosteroids with the participation of cells of microorganisms having 3-ketosteroid-1-dehydrogenase activity (3-KSD, EU 1.3.99.4), and can be used in the microbiological and pharmaceutical industries.

The 1,2-dehydrogenated Δ ⁴ -3-ketosteroids of the androstane series are not only intermediates in the synthesis of physiologically active compounds of the steroid structure, but also, having biological activity, are used as medications. So, androsta-1,4-diene-3,17-dione (ADD), being a prohormone, is able to metabolize the body into a highly active anabolic boldenone [Milewich L., Bradfield DJ, Soy LD, Masters BS, MacDonald PCJ Steroid . Biochem., 1981, 14 (11), 1115-1125]. At the same time, ADD is the initial substrate in the synthesis of almost the entire group of estrogen and progestogen drugs of the 19th nor row [Ahrem A.A., Titov Yu.A. Steroids and microorganisms. M .: Nauka, 1970]. 17β-Hydroxyandrosta-1,4-dien-3-one and 17β-hydroxy-17-methylandrosta-1,4-dien-3-one are highly active anabolic drugs (INN boldenone and methanedienone, respectively). 6-Methyleneandrosta-1,4-diene-3,17-dione (6-methylene-ADD) and 17β-hydroxy-6-methyleneandrosta-1,4-dien-3-one (6-methylene-dehydro-TC) - modern aromatase inactivators, effective agents for the treatment of imbalances between bone resorption and bone formation in postmenopausal and oophorectomized women [CA 2463142, 2004]. 6-Methylene-ADD (INN exemestane) is widely used in the treatment of malignant neoplasms of the mammary gland [US 4808616, 1989; Giudici D., Ornati G., Briatico G., Buzzetti F., Lombardi P. and di Salle EJ Steroid Biochem., 1988, 30 (1-6), 391-394], and can also be used as a preparation for postcoital contraception [WO 2007000056, 2007]. ADD, androsta-1,4,9 (11) -trien-3,17-dione and its derivatives (6α-fluoro -rosta-1,4,9 (11) -trien-3,17-dione, 6α-methylandrosta-1 4,9 (11) -diene-3,17-dione, 16β-methylandrosta-1,4,9 (11) -trien-3,17-dione, 11β-hydroxyandrosta-1,4-diene-3.17 -dione and others) can be used as intermediates in the synthesis of physiologically active compounds of the pregnan series (prednisone, dexamethasone, triamcinolone, 6α-methylprednisolone, etc.).

Androst-4-en-3,17-dione (AD) are the direct precursors of various in structure, physicochemical and pharmacological properties of 1,2-dehydrogenated steroids [RU 2079258, 1997; US 7259005, 2007; Rodina N.V., Molchanova M.A., Voishvillo N.E., Andryushina V.A., Stytsenko T.S. Adj Biochem. Microbiol., 2008, 44 (1), 56-62] and 9α-hydroxyandrost-4-en-3,17-dione (9-OH-AD) [US 4035236, 1977; RU 2077590, 1997; Rodina N.V., Andryushina V.A., Stytsenko T.S., Turova T.P., Baslerov R.V., Panteleeva A.N., Voishvillo N.E. Adj Biochem. Mikrobiol., 2009, 45 (4), 439-445], obtained from natural sterols by the method of microbiological transformation.

The introduction of functionally significant substituents into the steroid nucleus is carried out, as a rule, by chemical synthesis methods. The 1,2-dehydrogenation reaction enhancing the physiological activity of the target steroid compounds is carried out chemically or microbiologically.

The set of known disadvantages of the chemical method for producing 1,2-dehydrogenated products is well known. This is the use of expensive reagents, the multi-stage chemical reactions with the introduction of a double bond in the ring A of the steroid nucleus and the subsequent isolation of the product, severe reaction conditions (high temperature, the use of toxic compounds, some of which exhibit pronounced carcinogenic properties and pose a real threat to the health of working personnel), the need for column chromatography to purify the product and, as a consequence, its low yield. Chemical production requires solving many environmental problems.

Microbiological methods for carrying out the process of 1,2-dehydrogenation of steroids, including steroids of the androstane series, are an alternative. The use of biocatalysts makes it possible under mild conditions to carry out complex polyenzyme processes with a high yield of a product in one technological stage. High selectivity of enzymatic reactions, relative cheapness, ecological purity characterize the processes of conversion of steroid compounds with the participation of microorganisms.

Androsta-1,4-diene-3,17-dione (ADD) can also be obtained by microbiological transformation of sterols [Ahmad S., Garg SK, Johri BN Biotechnol. Adv., 1992, 10 (1), 1-67; RU 2039824, 1997; RU 2297455, 2007; US 6071714, 2000; US 7259005 B2, 2007]. However, the microbiological method for producing ADD from phytosterols with an initial load higher than 1 g / l requires certain conditions to be effective for the conversion of the substrate into a product (high reaction rate, prevention of product degradation, reaction selectivity), for example, the use of cyclodextrins [RU 2039824, 1997], liquid polymers and oils [Carvalho F., Marques M.P., de Carvalho CC, Cabral JM, Fernandes P. Bioresour. Technol., 2009, 100 (17), 4050-4053; Marques M.P.C., Carvalho F., de Carvalho S.C.C. R., Cabral JMS, Fernandes P. Food Bioprod. Process., 2010, 88 (1), 12-20; Stefanov S., Yankov D., Beschkov V. Chem. Biochem. Eng. Q., 2006, 20, 421-427] synthetic polymers of the class N-vinylamide [RU 2042687, 1995], adsorption resins [DD 291341, 1991] and the like. An alternative method for the synthesis of ADD is 1,2-dehydrogenation of AD with natural, mutant and recombinant strains of microorganisms with 3-KSD [Yamane T., Nakatani H., Sada E., Omata T., Tanaka A., Fukui S. Biotechnol. Bioeng., 1979, 21, 2133-2145; US 4,684,610, 1987; GB 2131811 A, 1984; US 4,524,134, 1985; US 4,749,649, 1988; RU 2156302, 2000; US7416866 B2, 2008]. Unlike AD, 9-OH-AD cannot be used in the microbiological process of 1,2-dehydrogenation directly due to the spontaneous aromatization of ring A of the steroid molecule and cleavage of ring B to form the 9,10-secophenol derivative - 3-hydroxy-9 , 10-secoandrosta-1,3,5 (10) -triene-3,17-dione and its subsequent degradation to CO ₂ and water [Dodson RM, Muir RDJ Am. Chem. Soc., 1961, 83, 4627-4631; Sih CJ, Wang KC, Gibson DTJ Am. Chem. Soc., 1967, 87, 1386-1388]. Therefore, pre-dehydration of the 9α-hydroxyl group is carried out with the formation of the Δ ^{9 (11)} derivative of blood pressure by chemical methods [US 3065146, 1962; US RE33364, 1990].

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, Rhodococcus et al. [Charney W., Herzog H. Microbial transformation of Steroids. Academic Press. Inc. New York 1967, p. 236-261; Kaufmann G., Schumann G., Wollweber L., Huller E., Atrat. PJ Basic. Microbiol., 1990, 30 (6), 415-423; Fujui Ch., Morii S., Kadode M., Sawamoto S., Iwami M., Itagaki EJ Biochem., 1999, 126, 662-667]. 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, natural and mutant native cells (60-85% humidity) [US 4749649, 1988; Falero A., Llanes N., Perez C., Hung B.R., Aguila B., Forseca M., Herve E. Revista CENIC Ciencias Biologicas., 2004, 35 (1), 41-43; Choi K.P., Molnar I., Murooka Y. Appl. Microbiol. Biotechnol, 1995, 43, 1044; US7259005, 2007], dried cells obtained by treatment with acetone, dried in vacuum or in air with heating [US 4749649, 1988; 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 structure-modified 1,2-dehydrogenated derivatives of steroids of the androstane series by a microbiological method in an aqueous medium is the low efficiency of the process due to the low solubility of the substrates, the limited availability of the substrate to the enzyme systems of microorganisms, the low reaction rate, and the possible inhibitory effect of the substrate or product on cell activity microorganism degradation of the product [Klibanov AM Nature, 2001, 409 (11), 241-246]. When the substrate content in the medium exceeds the solubility value, the bulk of the substrate and product are in the solid phase. In this case, the rate of the 1,2-dehydrogenation process is limited by the rate of dissolution of the substrate particles. In addition, co-crystallization of the product with the substrate occurs. In the resulting crystalline structures, the substrate becomes inaccessible to the enzyme systems of the microorganism (Kondo F., Masuo M. J. Gen. Appl. Microbiol., 1961, 7, 113-117). In this case, the content of the residual substrate in the technical target product can reach 15-20%, which determines significant losses of the product during its cleaning from the residual substrate.

It is known that to increase the solubility of steroid substrates in an aqueous medium, α-, β- or γ-cyclodextrins are used, as well as β-cyclodextrin derivatives (β-CD) - methyl-βCD and hydroxypropyl-β-CD - in a concentration of 1-50 g / l [Szejtli J. The use of cyclodextrins in biotechnological operations. Ed. D. Duchene, Published in France by Editions de Sante. 1991, p. 1-625; Wang M., Zhang L, Shen Y., Ma Y., Zheng Y., Luo JJ Mol. Cat. B: Enzymatic, 2009, 59 (1-3), 58-63; Zhang L., Wang M., Shen Y., Ma Y., Luo J. Appl. Biochem. Biotechnol., 2009, 159 (3), 642-654]. The solubility of steroids in various aqueous cyclodextrin solutions can be increased 2-1200 times compared to water [Habon I., Stadler-Szoke A., Szejtli J. Acta Biochim. Biophys. Hung., 1984, 19 (1/2), 86]. In particular, a method is known for producing 1,2-dehydro derivatives of Δ ⁴ -3-ketosteroids by transforming Δ ⁴ -3-ketosteroids using the microorganism Arthrobacter globiformis 193 in the presence of cyclodextrin, characterized in that water-soluble chemically modified β-cyclodextrin derivatives 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, ADD is obtained from HELL (Example 12) and it does not describe the preparation of 1,2-dehydrogenated derivatives of 17α-methyltestosterone, testosterone, 6-methylene testosterone, 6-methyleneandrost-4-en-3,17-dione and androsta -4.9 (11) -diene-3.17-dione.

1,2-Dehydrogenation of 17α-methyltestosterone with Pimelobacter simplex VKPM Ac-1632 cells using modified cyclodextrin derivatives is described by Druzhinina et al. [Druzhinina A.V., Andryushina V.A., Stytsenko T.S., Voishvillo N.E. Adj Biochem. Microbiol., 2008, 44 (6), 642-646]. In an environment containing methyl β-CD (pH 7.2) and 7% methanol, under aerated conditions at 28 ° C, the complete conversion of the substrate (20 g / l) with a weight ratio of substrate to methyl β-CD equal to 1: 5, proceeded in 15 hours. When replacing methyl-β-CD with hydroxypropyl-β-CD at the same concentration of thawed P. simplex cells (4.0 g / l calculated on the weight of dry biomass), the time of complete conversion was reduced to 4.5 hours. Considering the inhibitory effect of both the substrate and the 1,2-dehydrogenated product - methandrostenolone - on the enzymatic activity of bacterial microorganisms, the use of β-CD derivatives capable of forming inclusion complexes with steroid substrates and products allowed the authors to efficiently carry out 1,2-dehydrogenation of 17α- methyltestosterone in high concentration. However, the high cost of modified β-CD derivatives in the absence of their production in Russia is the main disadvantage of this method, limiting its industrial use.

Along with the use of cyclodextrins, synthetic polymers of the class of N-vinylamides - polyvinylpyrrolidone (PVP), polyvinylcaprolactam (PVC), polyvinyl alcohol (PVA) and various copolymers of N-vinylcaprolactam can be used to increase the concentration and availability of steroid substrates for microorganisms. Thus, there is a known method for 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 m is used as a polymer material. m 10 ⁴ -10 ⁶ Yes or copolymers of N-vinylcaprolactam with vinyl alcohol, vinyl acetate or vinyl methylacetamide with the content thereof up to 46%, 45% and 15%, respectively [RU 2042687, 1995]. However, the known method is claimed in general terms and the invention is not illustrated by examples.

Druzhinina et al. [Druzhinina A.V., Andryushina V.A., Arinbasarova A.Yu., Pashkin II, Savinova TS, Stytsenko TS, Voishvillo N.E. Biotechnology, 2008, 1, 46-50] describes the results of a comparative study of the effect of PVP, PVA and PVA on the 1,2-dehydrogenation of 17α-methyltestosterone with P. simplex VKPM Ac-1632 cells, according to which the selectivity of methandrostenolone formation from 17α-methyltestosterone at a concentration substrate 4 g / l in the best case (solubilization of PVP with m.m. 500 kDa or PVC with m.m. 900-2000 kDa) for 8 hours of transformation was only 70%.

There is also known a method for producing methandrostenolone, including microbiological transformation of 17α-methyltestosterone (MTS) with strains of microorganisms having 3-KSD activity in the presence of a polymer material and subsequent isolation of the target product from the culture fluid, characterized in that polyvinylpyrrolidone is used as a polymer material. As microorganisms with 3-KSD activity, Arthrobacter globiformis, Pimelobacter simplex, Rhodococcus erythropolis are used [RU 2236464, 2002]. Application of PVP with m.m. 500 kDa allowed for 1,2-dehydrogenation of MTS at a concentration of 5-6 g / l. The degree of conversion of MTS under aeration and at 28 ° C, according to HPLC, was 95-96% after 48 hours of conversion for P. simplex VKPM Ac-1632 culture, 95% after 50-52 hours of incubation for A. globiformis 193 culture and 94-95% after 50-54 hours for Rh. erythropolis VKPM Ac-1014.

The low efficiency of the process, due to the use of the substrate in a concentration not exceeding 6.0 g / l, is the main disadvantage of the considered method of producing methandrostenolone. In addition, the method is limited to the use of only one substrate and other steroid compounds of the androstane series are not considered.

Organic solvents and exogenous electron acceptors are used to intensify the 1,2-dehydrogenation process [Antonini E., Carrea G. and Cremonesi P. Enzyme Microb. Tech., 1981, 3 (4), 291-296; US 4,749,649, 1988]. 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 process of 1,2-dehydrogenation of steroid compounds, but also and to inhibit the destructive activity of microorganism cells and adverse reactions of a reducing nature occurring in the presence of reduced cofactors. The formation of by-products, the destruction of the target product reduce the yield of the main product.

When using water immiscible solvents [Straathof A.J. J. Biotechnol. Prog., 2003, 19, 755-762] the transformation is carried out in a two-phase medium. Microorganisms or enzymes are usually localized in the aqueous phase, and the organic phase concentrates the hydrophobic substrate and the product. Thus, dissolution of the substrate is achieved, inhibition by the substrate or product is eliminated, product degradation is prevented, and direct extraction of the product from the aqueous phase is also achieved [Wang Z. Appl. Microbiol. Biotechnol., 2007, 75, 1-10].

Thus, a method for producing 1,2-dehydro-3-ketosteroid is known, which consists in reacting a 1,2-saturated 3-ketosteroid with A. simplex or Bacterium cyclooxydans in the presence of an exogenous electron carrier and a water-immiscible solvent, including aromatic hydrocarbons [GB 2131811, 1984]. As substrates, androst-4-en-3,17-dione (AD), 11β-hydroxy-AD, androsta-4,9 (11) -diene-3,17-dione and its 6α-fluoro-, 6α- are mentioned methyl and 16β-methyl derivatives. However, only an example of the transformation of androst-4,9 (11) -diene-3,17-dione by A. simplex culture cells is described. According to this method (example 2), the substrate transformation process with a load of up to 8 g / l is carried out by A. simplex cells in the presence of menadione in a phosphate buffered saline (pH 7.5) containing up to 10% toluene for 4 days. The product of androsta-1,4,9 (11) -trien-3,17-dione was obtained in 100% yield. Without toluene, under the same conditions, the product yield was 82.3%.

A similar method of the same author is also known - the method of converting a 1,2-saturated steroid to a 1,2-dehydrosteroid, which involves the interaction of a 1,2-saturated steroid with A. simplex or B. cyclooxydans in the presence of an exogenous electron carrier immiscible with aromatic water hydrocarbon solvent, where the oxygen concentration is maintained at the minimum oxygen concentration required for oxidation, or where the reaction is carried out below the flash point of the mixture, provided that the 1.2-saturated steroid has a solution imost in a water-immiscible aromatic hydrocarbon solvent, 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 in the presence of exogenous electron acceptors selected from the group consisting of menadione, menadione bisulfite, 1,4-naphthoquinone or other compounds similar to vitamin K. As substrates mentioned are androst-4-ene-3,17-dione (AD), androsta-4,9 (11) -diene-3,17-dione and its 6α-fluoro, 6α-methyl and 16β-methyl derivatives. However, as in the patent [GB 2131811, 1984], only an example of the transformation of androst-4,9 (11) -diene-3,17-dione by A. simplex culture cells (example 2) in the presence of menadione is described. In a medium containing from 7.4 to 50 vol.% Toluene for 4 days of androsta-4.9 (11) -diene-3.17-dione transformation with a substrate / cell weight ratio of 1: 1 in a phosphate buffer solution (pH 7.5) up to 100% of the androsta-1,4,9 (11) -trien-3,17-dione product is formed according to the analysis of extracts.

The main disadvantage of the known methods for producing a 1,2-dehydrogenated derivative - androsta-1,4,9 (11) -trien-3,17-dione - is the low concentration of the substrate (up to 8 g / l) and the lack of information on the isolation method, and also about the output and quality of the selected product.

A known method for producing 6-methyleneandrost-1,4-diene-3,17-dione, which consists in contacting 6-methyleneandrost-4-ene-3,17-dione with Δ ¹ -dehydrogenating A. simplex enzymes in the presence of immiscible with water an organic solvent and an exogenous electron acceptor [WO 0104342, 2001]. According to this method, whole cells or cell-free A. 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 is purged with a 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 main disadvantage of the process described in WO 0104342 is 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 method of 1,2-dehydrogenation of 17α-methyltestosterone (10 g / l) by A. simplex AS 1.94 cells in a biphasic system (70% v / v) consisting of carbon tetrachloride containing tween-80 at a concentration of 0.5 g / l is described. (30% v / v) and potassium phosphate buffer solution (pH 7.0) [Antonini E., Carrea G., Cremonesi P. Enzyme Microb. Technol., 1981, 3 (4), 291-296; Bie S., Du L., Zhang L., Lu F. Proc. Biochem., 2005, 40, 3309-3313; Bie S., Lu F., Du L., Qiu Q., Zhang Y. J. Mol. Cat. B: Enzymatic, 2008, 55, 1-5; US 7259005, 2007]. The authors used menadion at a concentration of 0.2 g / L as an electron acceptor. Under aeration conditions, 95% conversion of the substrate to a 1,2-dehydrogenated derivative occurred in 48 hours at 33 ° C.

Common disadvantages of the 1,2-dehydrogenation methods in a two-phase medium using water-immiscible organic solvents are:

- 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 requirement for the use of special technological equipment virtually eliminates the practical implementation of these methods in industrial production;

- the complexity of the process under the necessary control of the composition of the gas mixture supplied to the reaction medium: 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 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. Carbon tetrachloride (CCL ₄ ), although non-combustible and fire and explosion proof, is poisonous and is a substance that is potentially carcinogenic to humans. When inhaled vapors, ingested through the gastrointestinal tract or absorption through the skin and mucous membranes, it causes damage to the liver and kidneys and disruption of the central nervous system. MPC of its vapor in the air of the working zone is 20 mg / m ³ ,

- the need to solve environmental problems: CCL ₄ , like a number of other chladones, destroys the ozone layer and in 1991 was included in the list of banned products in accordance with the amendments to the Montreal Protocol (1987), which established severe economic restrictions on its production and application.

In the processes of microbiological transformation of steroid compounds, along with the use of organic solvents which are not miscible with water, water-miscible organic solvents such as dimethyl sulfoxide, dimethylformamide, methanol, ethanol, acetone and the like are used. However, they were used solely as an auxiliary means providing the introduction of the initial substrate and / or electron acceptor in dissolved form into the transformation medium, and the concentration of such a solvent was limited to 5 vol% of the final volume of the reaction medium.

Thus, a method is known for converting 1,2-saturated 3-ketosteroids to 1,2-dehydro-3-ketosteroids, which consists in reacting 1,2-saturated 3-ketosteroids with an agent having a steroid-1,2- the dehydrogenase activity of A. simplex or B. 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]. Moreover, the 1,2-saturated 3-ketosteroid is a 1,2-saturated 3-ketoandrostane selected from the group consisting of blood pressure, androsta-4,9 (11) -diene-3,17-dione and its 6α-fluoro-, 6α-methyl and 16β-methyl derivatives; 11β-hydroxyandrost-4-en-3,17-dione and its 6α-fluoro, 6α-methyl-, 16β-methyl derivatives, as well as 11α-hydroxyandrost-4-en-3,17-dione and androst-4 en-3,11,17-trion. According to this method, the steroid substrate can be applied in the form of a powder, aqueous paste or in the form of 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% of the final volume of 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 (the load of the substrate androsta-4.9 (11) -diene-3.17-dione is 10 g / l). The considered method [US 4749649, 1988] also proposes the use of an exogenous enzyme - catalase. The addition of catalase in an amount of 10 mg / l promotes the active conversion of the substrate and allows you to increase its load up to 10 g / l (example 1). For 22 hours in the presence of menadione (5 · 10 ^-4 M) and catalase (10 mg / l), 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.

In similar conditions, the conversion of androst-4-en-3,17-dione (Example 4) is carried out with a load of 10 g / l and with a degree of conversion of the substrate of 93% in 24 hours. The method of applying the substrate is also not indicated.

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, which therefore cannot be recommended for industrial use. In addition, the production of 1,2-dehydro derivatives of testosterone, 6-methylene testosterone, 17α-methyltestosterone and 6-methyleneandrost-4-en-3,17-dione is not described in a known manner.

The closest in essence to the proposed method are the methods described in the patents [US 3091575, 1963] and [US 4524134, 1985].

The patent [US 3091575, 1963] describes a method for improving a fermentation process containing a 1,2-dehydrogenating microbial system for producing 1,2-dehydrosteroids, in which the destruction of Δ ^1,4 -3-ketosteroids is inhibited, including the addition of a substance as an inhibitor, selected from the group consisting of chromium, molybdenum and tungsten, as well as a compound containing an ortho and para-quinoid structure in an oxidized or reduced form, which compound may be an oxidized or reduced form of quinone, phenazine, thiazine, oxazine and etc. According to this method, the transformation is carried out in a medium containing methanol, in the presence of a compound of the quinoid structure, which is phenazine metasulfate, and Δ ^1,4 -3-ketosteroid - Δ ¹ -androstenedione (18 claims). However, an example of the transformation of AD into ADD by growing N. corallina cells is shown in a medium containing 10 ⁻³ M potassium dichromate (Example 8). The methanol concentration is 2% of the volume, and although after 24 hours of transformation the degree of conversion was 98%, the load of the substrate was only 0.02 g / l. In addition, substrates such as testosterone, 17α-methyltestosterone, 6-methyleneandrost-4-en-3,17-dione, 6-methylene testosterone and androsta-4,9 (11) -diene-3,17- are not considered dion.

The patent [US 4,524,134, 1985] describes a method for producing 1,2-dehydro-Δ ⁴ -3-ketosteroid, which consists in reacting the corresponding 1,2-saturated Δ ⁴ -3-ketosteroid with air-dried or heat with A. simplex cells containing 1 to 10% moisture, where the cells are dried in the absence of an organic solvent. According to the method (example 2b), a suspension of micronized androsta-4,9 (11) -diene-3,17-dione 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). The concentration of DMF in the medium is 2 vol.%, And the load of the substrate is from 2.5 g / l (example 2b) to 10 g / l (example 4). The transformation is carried out for 24 hours at a temperature of 31 ° C. As substrates for 1,2-dehydrogenation under the conditions of Example 2b, AD and 6α-fluoro, 6α-methyl and 16β-methyl derivatives of androsta-4,9 (11) -diene-3,17-dione are mentioned. However, there are no descriptions of examples of the use of these substrates in the patent [US 4,524,134, 1985]. In addition, such patents as testosterone, 17α-methyltestosterone, 6-methyleneandrost-4-en-3,17-dione, 6-methylene testosterone are not considered in this patent.

The relatively low concentration of the substrate, not exceeding 10 g / l in the case of androsta-4,9 (11) -diene-3,17-dione (example 2b) and its derivatives, characterizes this method of producing 1,2-dehydro derivatives as ineffective.

Thus, the use of organic solvents miscible with water in a low concentration of 2 vol.% Does not significantly increase the solubility of the substrate in the aquatic environment and its accessibility to the cells of the microorganism and does not allow to increase the efficiency of the process. The intensification of the process using water-miscible organic solvents in a concentration of more than 5 vol.% Can be achieved by using additionally modified polysaccharides. The possibility of using methanol at a concentration of up to 7 vol.% Was previously mentioned in the process of 1,2-dehydrogenation of 17α-methyltestosterone with a load of up to 20 g / l in a medium containing methyl-β-CD or hydroxypropyl-β-CD in a concentration of up to 100 g / l [Druzhinina A.V., Andryushina V.A., Stytsenko T.S., Voishvillo N.E. Adj Biochem. Microbiol., 2008, 44 (6), 642-646].

The technical task of the invention is to increase the efficiency, manufacturability and safety of 1,2-dehydrogenation and the creation of an effective microbiological method for producing 1,2-dehydrogenated Δ ⁴ -3-ketosteroids of a number of androstane, devoid of these disadvantages.

The technical problem is solved by the method of obtaining 1,2-dehydrogenated derivatives of Δ ⁴ -3-ketosteroids of the androstane series of general formula (I)

where R ₁ = R ₂ = H or together CH ₂ group; R ₃ = R ₄ = H or together a double bond; R ₅ = OH and R ₆ = H or CH ₃ group; R ₅ and R ₆ together are a keto group,

microbiological transformation of Δ ⁴ -3-ketosteroids of General formula (II)

where R ₁ = R ₂ = H or together CH ₂ group; R ₃ = R ₄ = H or together a double bond; R ₅ = OH and R ₆ = H or CH ₃ group; R ₅ and R ₆ together are a keto group,

in an aqueous medium containing an exogenous electron acceptor, using cells of a microorganism having 3-ketosteroid-1-dehydrogenase activity, using concentrations of the initial substrate from 5 to 100 g / l and isolating the target product from the culture fluid, characterized in that as a component of the medium using an aprotic organic solvent miscible with water in a concentration of more than 5 vol.%, specifically from 8 to 40 vol.%. The use of large concentrations of a water-miscible organic solvent in a medium for the process of 1,2-dehydrogenation of a steroid substrate is not obvious and previously large concentrations of a water-miscible organic solvent were not used in such processes.

Thus, the essence of the claimed invention lies in the fact that the introduction of a 1,2-double bond into ring A of the steroid nucleus of Δ ⁴ -3-ketosteroids of the Androstane series is carried out by the microbiological reaction of 1,2-dehydrogenation in an aqueous medium containing from 8 to 40 vol. % aprotic solvent, miscible with water, and an exogenous electron acceptor, with the participation of cells of microorganisms with 3-ketosteroid-1-dehydrogenase activity.

In addition, carrying out 1,2-dehydrogenation in order to obtain 1,2-dehydro derivatives of Δ ⁴ -3-ketosteroids of the Androstan series is carried out using bacterial cells Nocardioides simplex VKM Ac-2033D.

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

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

The technical result of the proposed method of microbiological 1,2-dehydrogenation of Δ ⁴ -3-ketosteroids of a number of androstane is as follows:

- the process is carried out at a high load of the substrate (up to 100 g / l) in mild conditions;

- the process does not use flammable and toxic organic solvents;

- the method is environmentally safe, since DMSO is proposed as a preferred solvent miscible with water - 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 with a concentration of from 10 to 90% in as an anti-inflammatory and analgesic drug (trade name "Dimexide") for external use;

- the method eliminates the use of any cyclic glucose oligomers, for example β-cyclodextrin and its modified derivatives, as well as any synthetic polymeric materials, including polymers of the class N-vinylamide;

- 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;

- the selectivity of the formation of 1,2-dehydrogenated Δ ⁴ -3-ketosteroids at the stage of microbiological 1,2-dehydrogenation is 85-98%.

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

Δ ⁴ -3-Ketosteroid of the androstane series of general formula (II)

where R ₁ = R ₂ = H or together CH ₂ group; R ₃ = R ₄ = H or together a double bond; R ₅ = OH and R ₆ = H or CH ₃ group; R ₅ and R ₆ together are a keto group,

convert to Δ ^1,4 -3-ketosteroid of the androstane series of general formula (I)

where R ₁ = R ₂ = H or together CH ₂ group; R ₃ = R ₄ = H or together a double bond; R ₅ = OH and R ₆ = H or CH ₃ group; R ₅ and R ₆ together are a keto group,

the introduction of a 1,2-double bond by the method of microbiological dehydrogenation with the participation of cells of microorganisms with 3-ketosteroid-Δ ¹ -dehydrogenase activity (preferably bacterial cells N. simplex VKM Ac-2033D), while the load of the initial substrate is from 5 to 100 g / l, and the duration of the fermentation is from 24 to 52 hours and the process is carried out in an aqueous medium containing an exogenous electron acceptor and aprotic solvent miscible with water - DMF or DMSO (preferably DMSO).

The use of DMF or DMSO in the process of 1,2-dehydrogenation ensures the conversion of the steroid substrate or most of it into the soluble form available for the enzyme systems of microorganism cells, and the process at high (up to 100 g / l) substrate loads significantly reduces the effect of co-crystallization substrate and product; and the formation of mixed crystals. The literature does not contain information on the use of high concentrations (more than 5 vol.%, Specifically from 8 to 40 vol.%) Of organic solvents miscible with water in the microbiological processes of 1,2-dehydrogenation of steroid substrates.

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

Steroid compound Retention time, min 6-Methyleneandrost-4-en-3,17-dione (6-methylene-AD) 4.9 6-Methyleneandrosta-1,4-diene-3,17-dione (exemestane, 6-methylene-ADD) 4.3 Androst-4-en-3,17-dione (HELL) 8.63 Androsta-1,4-diene-3,17-dione (ADD) 6.59 17β-hydroxyandrost-4-en-3-one (testosterone) 7.06 17β-hydroxyandrosta-1,4-dien-3-one (boldenone) 5.54 Androsta-4.9 (11) -diene-3.17-dione 8.3 Androsta-1,4,9 (11) -trien-3,17-dion 6.4 6-Methylene-17β-hydroxyandrost-4-en-3-one (6-methylene testosterone) 4.54 17β-Hydroxy-17-methylandrost-4-en-3-one (17β-methyltestosterone) 4.42 17β-Hydroxy-17-methylandrosta-1,4-dien-3-one (methandrostenolone) 3.81

Isolation of 1,2-dehydrogenated Δ ⁴ -3-ketosteroids from the culture fluid can be carried out by extraction with a water-immiscible solvent or by extracting 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 claimed group of inventions is illustrated by the following examples, not limiting it.

Microbiological 1,2-dehydrogenation in the implementation of the invention - a method for producing 1,2-dehydrogenated derivatives of Δ ⁴ -3-ketosteroids of the androstane series in aqueous-organic media - can be carried out by resting (washed) bacteria cells of the genus Nocardioides, specifically Nocardioides simplex VKM Ac- 2033D.

As a medium component in carrying out the invention, a method for producing 1,2-dehydrogenated derivatives of Δ ⁴ -3-ketosteroids of the androstane series in aqueous-organic media, water-miscible organic aprotic solvents, for example DMF or DMSO, preferably DMSO, can be used.

Steroid substrates and electron acceptors in the implementation of the invention - a method for producing 1,2-dehydrogenated derivatives of Δ ⁴ -3-ketosteroids of the androstane series - can be introduced in the form of a powder, aqueous paste, or in the form of a solution (or suspension) in a water-miscible organic solvent such as DMF, DMSO, ethanol, methanol, acetone, etc.

A sample of the culture fluid in the implementation of the invention, a method for producing 1,2-dehydrogenated derivatives of Δ ⁴ -3-ketosteroids of the androstane series, is extracted with ethyl acetate during and after the transformation process.

The method is illustrated by the following examples. The main process parameters are given in table 1.

Examples

We use the strain Nocardioides simplex VKM Ac-2033D, which is supported on an agar medium of the following composition (g / l): glucose-10, soy peptone-4, yeast extract-4, MgSO4-0.5, 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 ° C.

Growing 1st inoculum

The culture was washed off from the surface of the agar medium in 5 ml of physiological saline into 750 ml of an Erlenmeyer flask with 100 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 ° 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 cultivation is carried out under similar conditions for 10-12 hours, then a steroid-1-dehydrogenase synthesis inducer, cortisone acetate, is introduced in the form ethanol solution (20 mg in 1 ml of ethanol). Induction of the synthesis of 1,2-dehydrogenase is carried out over the next 20-24 hours. Microorganism cells are pelleted by centrifugation at 5000 g for 30 minutes and used to carry out the 1,2-dehydrogenation process or stored at -18 ° C for a month before use.

Example 1. Obtaining androsta-1,4,9 (11) -trien-3,17-dione

2.5 g of Androsta-4,9 (11) -diene-3,17-dione (in terms of 100% content of the basic substance) are transformed in 750 ml flasks in a medium containing 15 ml of 0.02 M Na-phosphate buffer solution (pH 8 , 0), 10 ml of DMSO, 0.1 g of menadione and 4.0 g of biomass (weight of dry biomass). Bioconversion is carried out under aerated conditions at 30 ° C on a rotary shaker at 200 rpm. The process is stopped for 24-30 hours of cultivation when the content of androsta-1,4,9 (11) -trien-3,17-dione is 94-95%.

Example 2. Obtaining androsta-1,4-diene-3,17-dione (ADD)

1.25 g of Androst-4-en-3,17-dione (AD) was transformed in 750 ml flasks containing 15 ml of 0.02 M Na-phosphate buffer solution (pH 8.0), 10 ml DMSO, 0.05 g of menadione and 2.0 g biomass (weight of dry biomass). Bioconversion is carried out under aerated conditions at 25 ° C on a rotary shaker at 200 rpm. The process is stopped at 22 hours of cultivation when the content of ADD is 96%.

Example 3. Obtaining 17β-hydroxyandrost-1,4-dien-3-one (boldenone)

0.5 g of Testosterone is transformed in 750 ml flasks containing 20 ml of 0.02M Na-phosphate buffer solution (pH 8.0), 5 ml of DMSO, 0.02 g of menadione and 0.8 g of biomass (weight of dry biomass). Bioconversion is carried out under aerated conditions at 25 ° C on a rotary shaker at 200 rpm. The process is stopped for 48 hours of cultivation when the content of 17β-hydroxyandrost-1,4-dien-3-one reaches 85-87%.

Example 4. Obtaining 17β-hydroxy-17-methylandrosta-1,4-dien-3-one (methandrostenolone)

0.25 g of 17β-Hydroxy-17-methylandrost-4-en-3-one (17β-methyltestosterone) is transformed in 750 ml flasks containing 22 ml of 0.02 M Na-phosphate buffer solution (pH 8.0), 3 ml DMSO, 0.02 g of phenazine methosulfate (FMS) and 0.4 g of biomass (weight of dry biomass). Bioconversion is carried out under aerated conditions at 30 ° C on a rotary shaker at 200 rpm. The process is stopped for 42-48 hours of cultivation when the methandrostenolone content reaches 92-95%.

Example 5. Obtaining 6-methyleneandrost-1,4-diene-3,17-dione (exemestane, 6-methylene-ADD)

Option 1

0.125 g of 6-Methyleneandrost-4-ene-3,17-dione (6-methylene-AD) is transformed in 250 ml flasks containing 23 ml of 0.02 M Na-phosphate buffer solution (pH 8.0), 2 ml DMSO. 0.005 g of menadione and 0.2 g of biomass (weight of dry biomass). Bioconversion is carried out under aerated conditions at 30 ° C on a rotary shaker at 200 rpm. The process is stopped for 48 hours of cultivation when the content of 6-methylene-ADD of 94-96% is reached.

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 DMF (2 ml).

Option 3

The method is carried out according to option 1, but the reaction medium contains 2.5 g of 6-methylene-AD, 15 ml of 0.02 M Na-phosphate buffer solution (pH 8.0), 10 ml of DMSO, 0.1 g of menadione and 0.5 g of biomass (dry weight biomass). The process is stopped for 52 hours of cultivation when the content of 6-methylene-ADD of 95-97% is reached.

Option 4

0.2 g of 6-Methylene testosterone is transformed in 750 ml flasks containing 18 ml of 0.02 M Na-phosphate buffer solution (pH 8.0), 2 ml of DMSO, 0.008 g of menadione and 40 g of biomass (dry biomass weight). Bioconversion is carried out under aerated conditions at 30 ° C on a rotary shaker at 200 rpm. The process is stopped for 120 hours of cultivation when the content of 6-methylene-ADD is 97-98%.

Claims (4)

1. The method of producing 1,2-dehydrogenated derivatives of Δ ⁴ -3-ketosteroids of the androstane series of general formula (I)

where R ₁ = R ₂ = H or together CH ₂ group; R ₃ = R ₄ = H or together a double bond; R ₅ = OH and R ₆ = H or CH ₃ group; R ₅ and R ₆ together are a keto group,
microbiological transformation of Δ ⁴ -3-ketosteroids of General formula (II)

where R ₁ = R ₂ = H or together CH ₂ group; R ₃ = R ₄ = H or together a double bond; R ₅ = OH and R ₆ = H or CH ₃ group; R ₅ and R ₆ together are a keto group in an aqueous medium containing an exogenous electron acceptor, using cells of a microorganism having 3-ketosteroid-Δ ¹ -dehydrogenase activity, using initial substrate concentrations of 5 to 100 g / l and isolating the target product from culture fluid, characterized in that as a component of the medium using miscible with aprotic organic solvent in a concentration of more than 5 vol.%, specifically from 8 to 40 vol.%. 2. The method according to claim 1, characterized in that Nocardioides simplex VKM Ac-2033D is used as a microorganism with 3-ketosteroid-Δ ¹ dehydrogenase activity. 3. The method according to claim 1, characterized in that as an exogenous electron acceptor use menadione or PMS in an effective amount. 4. The method according to claim 1, characterized in that dimethylformamide or dimethyl sulfoxide, preferably dimethyl sulfoxide, is used as an aprotic water-miscible organic solvent.