Microbiological method of producing 1,2-dehydrogenated derivatives of Δ4-3-keto-steroids of androstane family in aqueus organic media

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

 

The present invention relates to the field of biotechnology, specifically for producing 1,2-dehydrogenated Δ4-3-ketosteroids number of androstane microbiological transformation Δ4-3-ketosteroids with the participation of the microorganism cells with 3-ketosteroid-1-dehydrogenase activity (3-CFC, EU 1.3.99.4), and can be used in microbiological and pharmaceutical industries.

1,2-Dehydrogenated Δ4-3-ketosteroids number of androstane not only are intermediates in the synthesis of physiologically active compounds of steroid structure, but, having biological activity, are used as medicines. So, androsta-1,4-diene-3,17-dione (ADD), being a prohormone that can result from metabolism to turn the body into a highly active anabolic boldenone [L. Milewich, Bradfield D.J., Soy L.D., Masters B.S., MacDonald P.C. J. Steroid. Biochem., 1981, 14(11), 1115-1125]. At the same time ADD is the initial substrate in the synthesis of almost all groups of estrogen and gestagenic preparations 19-nor series [Akhrem A.A., Titov Y.A. Steroids and microorganisms. M.: Nauka, 1970]. 17β-Hydroxyandrost-1,4-Dien-3-one and 17β-hydroxy-17-methylandrosta-1,4-Dien-3-one - a highly potent anabolic drugs (INN boldenone and methandienone respectively). 6-Medienanstalt-1,4-diene-3,17-dione (6-methylene-ADD) and 17β-hydroxy-6-methylene is the Drost-1,4-Dien-3-one (6-methylene-degidro-TC) - modern inactivator aromatase, an effective means for treatment of violations of the balance between bone resorption and bone formation in postmenopausal and oophor-acetominoven women [SA 2463142, 2004]. 6-Methylene-ADD (INN exemestane) is widely used in the treatment of malignant tumors of the breast [US 4808616, 1989; D. Giudici, Ornati g, Briatico G., F. Buzzetti, Lombardi P. and di Salle E. J. Steroid Biochem., 1988, 30(1-6), 391-394], and can also be used as a drug for postcoital contraception [WO 2007000056, 2007]. ADD, androsta-1,4,9(11)-triene-3,17-dione and its derivatives (6α-forandret-1,4,9(11)-triene-3,17-dione, 6α-methylandrosta-1,4,9(11)-diene-3,17-dione, 16β-methylandrosta-1,4,9(11)-triene-3,17-dione, 11β-hydroxyandrost-1,4-diene-3,17-dione and others) can be used as intermediates in the synthesis of physiologically active compounds of the series pregnane (prednisolone, dexamethasone, triamcinolone, 6α-methylprednisolone, and etc).

The direct predecessors of different structure, physico-chemical and pharmacological properties of 1,2-dehydrogenated steroids are androst-4-ene-3,17-dione (AD) [EN 2079258, 1997; US 7259005, 2007; birthplace NV, Molchanov M.A., Voishvillo N.E., Andryushina VA, Stetsenko T.S. go active. Biochem. Microbiol., 2008, 44(1), 56-62] and 9α-hydroxyandrost-4-ene-3,17-dione (9-OH-BP) [US 4035236, 1977; EN 2077590, 1997; birthplace NV, Andryushina VA, Stetsenko T.S., Turov, ETC, Butlerov W., Panteleyev, A., Voisw the DLO N.E. Go active. Biochem. Microbiol., 2009, 45(4), 439-445]derived from natural sterols by microbial transformation.

Introduction to steroid nucleus is functionally significant deputies carried out, usually by chemical synthesis. The reaction of the 1,2-dehydrogenation, amplifying the physiological activity of the target steroid compounds, conduct chemical or microbiological method.

The totality of the known disadvantages of the chemical method of producing 1,2-dehydrogenated products is well known. This use of expensive reagents, a lot of stages of chemical reactions with the introduction of double bonds in the ring And steroid nucleus and subsequent selection of product, stringent conditions for reactions (fever, use of toxic compounds, some of which are expressed to be carcinogenic and pose a real threat to the health of operating personnel), the use of column chromatography for the purification of the product and, consequently, its low output. The chemical industry requires solutions to many environmental problems.

Microbiological methods the process of 1,2-dehydrogenation of steroids, including steroid androstenone series, are an alternative. The use of biocatalysts lets in soft conditions OS which may serve as a basis in one technological stage multienzyme complex processes with high yield. High selectivity of enzymatic reactions, relative low cost, environmental friendliness 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 S.K., Johri B.N. Biotechnol. Adv., 1992, 10(1), 1-67; EN 2039824, 1997; EN 2297455, 2007; US 6071714, 2000; US 7259005 B2, 2007]. However, the microbiological method of obtaining ADD from phytosterols source load above 1 g/l requires the fulfillment of certain conditions for the efficient conversion of substrate into product (high reaction rate, prevention of degradation of the product, the selectivity of the reaction), for example the use of cyclodextrins [EN 2039824, 1997], liquid polymers and oils [Carvalho F., Marques MR, C.C. de Carvalho, J.M. Cabral, P. Fernandes Bioresour. Technol., 2009, 100(17), 4050-4053; Marques MRS, Carvalho F., de Carvalho ...R., J.M.S. Cabral, P. Fernandes Food Bioprod. Process., 2010, 88(1), 12-20; Stefanov, S., D. Yankov, V. Beschkov Chem. Biochem. Eng. Q., 2006, 20, 421-427] synthetic polymers of the class of N-vinylamide [EN 2042687, 1995], adsorption resins [DD 291341, 1991], and so an Alternative method of synthesis of ADD is 1,2-dehydrogenation HELL natural, mutant and recombinant strains of microorganisms, with 3-CFC [Yamane T., Nakatani H., Sada E., Omata T., Tanaka A., Fukui S. Biotechnol. Bioeng., 1979, 21, 2133-2145; US 4684610, 1987; GB 2131811 A, 1984; US 4524134, 1985; US 4749649, 1988; EN 2156302, 2000; US 7416866 B2, 2008]. Unlike HELL, 9-HE-AD cannot be used in Mick is biologicheskii process 1,2-dehydrogenation directly due to spontaneous aromatization of the ring And steroid molecules and split ring with the formation of 9,10-scottoline derivative - 3-hydroxy-9,10-secondrate-1,3,5(10)-triene-3,17-dione and its subsequent degradation to CO2and water [Dodson R., R.D. Muir J. Am. Chem. Soc., 1961, 83, 4627-4631; Sih C.J., Wang, K.C., Gibson D.T. J. Am. Chem. Soc., 1967, 87, 1386-1388]. Therefore, conduct preliminary dehydration of the 9α-hydroxy group with the formation of Δ9(11)-derived chemical methods HELL [US 3065146, 1962; US RE33364, 1990].

As biocatalysts process 1,2-dehydrogenation use culture with 3-ketosteroid-Δ1-dehydrogenase (EU 1.3.99.4) genera: Arthrobacter, Alternaria, Alcaligenes, Calonectria, Nauchnye, Corynebacterium, Bacillus, Nocardia, Streptomyces, Bacterium, Mycobacterium, Fusarium, Cylindrocarpon, Pseudomonas, Protaminobacter, Septomyxa, Didymella, Rhodococcus and other [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. P. J. Basic. Environ., 1990, 30(6), 415-423; Fujui Ch., Morii, S., Kadode M., Sawamoto, S., Iwami, M., Itagaki, E. J. Biochem., 1999, 126, 662-667]. Most often the process of 1,2-dehydrogenation of steroid compounds of the number pregnane and androstane are strains of the species Arthrobacter simplex.

The bioconversion carried out using the culture fluid, natural native and mutant cells (60-85% humidity) [US 4749649, 1988; Falero A., Llanes N., Perez C., Hung B.R., Aguila Century, Forseca M., Herve E. Revista CENIC Ciencias Biologicas., 2004, 35(1), 41-43; K.P. Choi, I. Molnar, Murooka Y. Appl. Environ. Biotechnol, 1995, 43, 1044; US7259005, 2007], the dried cells obtained by treatment with acetone, dried in vacuum or in air when heated [US 4749649, 1988; US 4704358, 1987], in terms of leofiles the tion, as well as using cell-free extracts obtained by ultrasound destruction or destroyed by passing the frozen cells through a Spinneret under pressure [EP 0350488, 1993]. In order to eliminate additional processing steps, preferably using native cells.

The main problem of obtaining modified the structure of 1,2-dehydrogenated derivatives of steroids several androstane microbiological method in the aquatic environment is the low efficiency of the process due to the low solubility of the substrates, the limited availability of substrate enzyme systems of microorganisms, low reaction speed, the possible inhibitory effect of substrate or product in the activity of the cells of microorganisms, degradation product [Klibanov A.M. Nature, 2001, 409(11), 241-246]. When the content of the substrate in an environment that exceeds the value of the solubility, the bulk of the substrate and product are in the solid phase. In this case, the speed of 1,2-dehydrogenation is limited by the dissolution rate of the particles of the substrate. In addition, there is cocrystallization product with the substrate. In the resulting crystal structures of the substrate becomes unavailable to the enzyme systems of the organism (Kondo F., Masuo, M. J. Gen. Appl. Environ., 1961, 7, 113-117). In this case, the residual substrate-the m target product can reach 15-20%, what defines a significant loss of product during its purification from residual substrate.

It is known that to increase the solubility of steroid substrates in the aquatic environment using α-, β - or γ-cyclodextrins, and derivatives of β-cyclodextrin (β-CD) - methyl-β 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 J. J. 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 water cyclodextrines solutions can be increased at 2-1200 times compared to water [Habon I., Stadler-A. Szoke, Szejtli J. Acta Biochim. Biophys. Hung., 1984, 19(1/2), 86]. In particular, there is a method for producing 1,2-dehydrofreezing Δ4-3-ketosteroids by transformation Δ4-3-ketosteroids using microorganism Arthrobacter globiformis 193 in the presence of cyclodextrin, characterized in that the cyclodextrin used is a water-soluble chemically modified derivatives of β-cyclodextrin [EN 2156302, 2000]. According to this method, the use of methyl-β-CD allows the 1,2-dehydrogenation of the substrate at a load of 20 g/L. When the weight ratio of methyl-β-CD/substrate 7/1 full conversion of the substrate is completed within 6 hours. The content of 1,2-negidrirovannogo product in the culture fluid status is made by 95%. However, the practical significance of this way of 1,2-dehydrogenation largely limited to 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 how to obtain 1,2-dehydrogenated derivatives of 17α-methyltestosterone, testosterone, 6-methylenecholesterol, 6-medienanstalt-4-ene-3,17-dione and androsta-4,9(11)-diene-3,17-dione.

1,2-Dehydrogenation of 17α-methyltestosterone cells Pimelobacter simplex PMBC Ac-1632 with the use of the modified derivatives of cyclodextrins described Druzhinina et al. [ALEXANDER Druzhinin, Andryushina VA, Stetsenko T.S., Voishvillo N.E. go active. Biochem. Microbiol., 2008, 44(6), 642-646]. In a medium containing methyl-β-CD (pH 7.2) and 7% methanol in aerated conditions at 28°C. complete conversion of the substrate (20 g/l) at a weight ratio of substrate to methyl-β-CD = 1:5, ran for 15 hours. When replacing methyl-β-CD on hydroxypropyl-β-CD at the same concentration of unfrozen cells of P. simplex (4.0 g/l, based on the weight of dry biomass) full conversion decreased to 4.5 hours. Given the inhibitory effects of substrate and 1,2-negidrirovannogo product - methandrostenolone - for the enzymatic activity of bacterial microorganisms, the use of derivatives of β-CD, is able to form inclusion complexes with steroid substrates and the product is and allowed the authors to effectively implement the 1,2-dehydrogenation of 17α-methyltestosterone in high concentrations. However, the high cost of modified derivatives of β-CD in the absence in Russia of their production is the main disadvantage of this method that limits its industrial application.

Along with the use of cyclodextrins to increase the concentration and availability for microorganisms steroid substrates can be used synthetic polymers of the class of N-vinylamide - polyvinylpyrrolidone (PVP), polyvinylcaprolactam (PVA), polyvinyl alcohol (PVA) and copolymers of N-vinylcaprolactam. So, there is a method of obtaining dehydrolinalool steroids, including microbiological transformation of the corresponding Δ4-3-ketosteroids in the presence of a polymer material, followed by separation from the reaction medium of the target product, characterized in that the polymer material used Homo - or copolymer of N-vinylcaprolactam with 10 mm4-106Yes or copolymers of N-vinylcaprolactam with vinyl alcohol, vinyl acetate or vinylethylene with the contents thereof to 46%, 45% and 15%, respectively [EN 2042687, 1995]. However, the known method declared in the General form and the invention is not illustrated by examples.

Druzhinina et al. [ALEXANDER Druzhinin, Andryushina VA, AND isbasarova, A., Going to America I.I., Savinova T.S., Stetsenko T.S., Voishvillo N.E. Biotechnology, 2008, 1, 46-50] described the results of a comparative study of the influence of PVP, PVA and PVA on the process of 1,2-dehydrogenation of 17α-methyltestosterone cells of R. simplex PMBC As-1632, whereby the selectivity of the formation of methandrostenolone of 17α-methyltestosterone at substrate concentration of 4 g/l in the best case (solubilization PVP with mm 500 kDa or STC with 900-2000 mm kDa) for 8 hours transformation was only 70%.

Also known is a method of obtaining methandrostenolone, including microbiological transformation of 17α-methyltestosterone (MTS) strains of microorganisms, with 3-CFC activity in the presence of the polymeric material and the subsequent allocation from the culture fluid of the target product, characterized in that the polymer material used polyvinylpyrrolidone. As microorganisms with 3-CFC activity, use Arthrobacter globiformis, Pimelobacter simplex, Rhodococcus erythropolis [EN 2236464, 2002]. Application of PVP with mm 500 kDa allowed to 1,2-dehydrogenation MTS at a concentration of 5-6 g/L. the Degree of conversion of MTS under conditions of aeration and at 28°C, according to HPLC, was 95-96% after 48 hours of conversion for the culture of P. simplex PMBC As-1632, 95% through 50-52 hours of incubation for the culture of A. globiformis 193 and 94-95% after 50-54 hours for Rh. erythropolis PMBC Ac-1014.

Low efficiency% the SSA, due to the use of the substrate in a concentration of not more than 6.0 g/l, is the main disadvantage of this method of obtaining methandrostenolone. In addition, the method is limited to using only a single substrate and are not considered other steroid compounds of the number of androstane.

In order to intensify the process of 1,2-dehydrogenation use of organic solvents and exogenous electron acceptors [E. Antonini, Carrea G. and P. Cremonesi Enzyme Microb. Tech., 1981, 3(4), 291-296; US 4749649, 1988]. It is known that exogenous electron acceptors - finishingtouches (FMS), menadione (2-methyl-1,4-naphthoquinone), 1,4-naphthoquinone, menadione bisulphite is used not only to promote the process of 1,2-dehydrogenation of steroid compounds, but also to inhibit the destructive activity of the cells of microorganisms and adverse reactions remedial action occurring in the presence of restored cofactors. The formation of by-products, destruction of the target product reduces 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 environment. Microorganisms or enzymes are usually localized in the aqueous phase and the organic phase concentrated hydrophobic substrate and product. Thus, rest the area of the substrate, eliminates the inhibition of substrate or product, prevents destruction of the product, and is achieved by direct extraction of the product from the aqueous phase [Wang Z. Appl. Environ. Biotechnol., 2007, 75, 1-10].

So, there is a method for producing 1,2-degidro-3-ketosteroid, which is that the 1,2-saturated 3-ketosteroid subjected to interaction with A. simplex or Bacterium cyclooxydans in the presence of exogenous carrier electrons and water-immiscible solvents, including aromatic hydrocarbons [GB 2131811, 1984]. As substrates mentioned androst-4-ene-3,17-dione (AD), 11β-hydroxy-HELL, androsta-4,9(11)-diene-3,17-dione and 6α-fluoro-, 6α-methyl and 16β-methylpropane. However, only describes the transformation of androsta-4,9(11)-diene-3,17-dione cell culture of A. simplex. According to this method (example 2) the process of transformation of a substrate with a load of up to 8 g/l conducting cells of A. simplex in the presence of menadione in the environment of phosphate buffer solution (pH 7.5)containing 10% toluene in 4 days. Product androsta-1,4,9(11)-triene-3,17-dione is obtained in yield of 100%. Without toluene under the same conditions, the yield of product amounted to 82.3%.

Also known similar way by the same author - the way of transformation of 1,2-saturated steroid 1.2-dehydrothermal, which includes the interaction of 1,2-saturated steroid with A. simplex or Century cyclooxydans in the presence of exogenous electron carrier is in, water-immiscible aromatic hydrocarbon solvent, where the oxygen concentration is maintained at a minimum concentration of oxygen required for oxidation, or where the reaction is carried out below the flash temperature of the mixture provided that the 1,2-saturated steroid has a solubility in water-immiscible 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, in the presence of exogenous electron acceptors selected from the group consisting of menadione, menadione bisulphite, 1,4-naphthoquinone or other compounds similar to vitamin K. as substrates mentioned androst-4-ene-3,17-dione (AD), androsta-4,9(11)-diene-3,17-dione and 6α-fluoro-, 6α-methyl and 16β-methylpropane. However, as in the patent [GB 2131811, 1984]described only a transformation of androsta-4,9(11)-diene-3,17-dione cell culture A. simplex (example 2) in the presence of menadione. In a medium containing from 7.4 to 50% vol. toluene for 4 days transformation androsta-4,9(11)-diene-3,17-dione in a weight ratio of substrate/cell equal to 1:1 in phosphate buffer solution (pH 7.5) is formed to 100% of the product of androsta-1,4,9(11)-triene-3,17-dione according to the analysis of the extracts.

The main disadvantage of this method is perceived by the Oia 1,2-negidrirovannogo derivative - androsta-1,4,9(11)-triene-3,17-dione is low substrate concentration (up to 8 g/l) and the lack of information on the allocation method, and the yield and quality of the selected product.

A method of obtaining 6-medienanstalt-1,4-diene-3,17-dione, which consists in contacting 6-medienanstalt-4-ene-3,17-dione with Δ1-dehydrating enzymes A. simplex in the presence of water-immiscible organic solvent and exogenous electron acceptor [WO 0104342, 2001]. According to the method for carrying out the process of the 1,2-dehydrogenation of 6-methylene-AD using whole cells or cell-free extract of A. simplex ATCC 6946, with the use of whole cells is preferable. 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 chemical exogenous electron acceptor (4% of menadione in relation to the weight of the substrate) and the enzyme catalase. Although the authors claimed that the substrate 6-medienanstalt-4-ene-3,17-dione is present in the environment at concentrations from 10 to 125 g/l (p and 19 formula), the example And illustrating the method, the concentration of the primary substrate is of 55.6 g/L. Thus, for the process 6-medienanstalt-4-ene-3,17-dione (50 g) and menage is n (2.0 g) is mixed with toluene (800 ml). The aqueous phase (pH 8.7-8.9), prepared as a mixture of catalase, discalificata, cellular concentrate A. simplex (50 ml) and water (50 ml), add to intensively mix the mixture in toluene. The process is carried out at 30°C, the reaction mixture is blown adjustable mixture of air and nitrogen and air (0.1% SCHF) and nitrogen (0.3% SCHF). If necessary during the reaction type cells A. simplex to complete the process. Moreover, the number of added advanced cell concentrate is not specified. After the process is complete, the organic phase is separated, the aqueous phase is extracted with toluene. After filtration and clarification toluene solution is concentrated and to the residue add a 2.5-fold volume number octane crystalline product is filtered off. The main disadvantage is described in WO 0104342 process is the use of the enzyme catalase in the amount of 0.03% by weight of the substrate, which increases the technological process and limits its industrial application.

The described method of 1,2-dehydrogenation of 17α-methyltestosterone (10 g/l) cells of A. simplex AS 1.94 in biphasic system (70% vol./vol.), consisting of carbon tetrachloride containing tween-80 at a concentration of 0.5 g/l (30% vol./vol.), and potassium phosphate buffer solution (pH 7.0) [E. Antonini, Carrea G., P. Cremonesi 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]. As the electron acceptor, the authors used menadione at a concentration of 0.2 g/L. In the conditions of aeration 95% conversion of the substrate in 1,2-digidrirovannoe derived happened for 48 hours at 33°C.

General shortcomings of ways 1,2-dehydrogenation in a two-phase medium with the use of water-immiscible organic solvents are:

- the use of large amounts of flammable organic solvents in the transformation stage, which requires a special fermentation equipment and the special rules of fire safety; requiring the use of special technological equipment virtually eliminates the practical implementation of these methods in terms of industrial production;

the difficulty of carrying out the process in terms of the necessary regulation of the composition of the gas mixture supplied into the reaction environment: the need to create and maintain strict minimum allowable concentration of dissolved oxygen in the environment to avoid the flash vapor gas mixture and at the same time to provide optimum conditions for oxidation of the substrate;

- the use of toxic organic compounds poses a threat to the health professionals involved in the production. Used aromatic hydrocarbons (benzene, the Wal, xylene) are potent poisons that affect the function of blood in the body. So, for example, toluene causes cyanosis, hypoxia, Central nervous system, as well as toluene substance abuse, which has carcinogenic effects. Carbon tetrachloride (CCL4), despite the fact that it is non-flammable and pozharovzryvobezopasen is poisonous and is potentially carcinogenic to humans agents. Inhalation of vapors, ingestion via the gastrointestinal tract or absorption through the skin and mucous membranes, it causes liver damage and kidney failure and malfunction of the Central nervous system. MAC vapors in the air of working zone is 20 mg/m3,

- the need to solve environmental problems: CCL4as a number of other halocarbons, destroy the ozone layer, and in 1991 was included in the list of prohibited products in accordance with the amendments to the Montreal Protocol (1987), which established the economic constraints on its production and use.

In the processes of microbiological transformation of steroid compounds, along with the use of organic solvents not miscible with water, use miscible with water and organic solvents, such as dimethylsulfoxide, dimethylformamide, methanol, ethanol, acetone etc. However, they were used solely as an aid in ensuring the inclusion of the source substrate and/or the electron acceptor in dissolved form in the environment for transformation, and the concentration of this solvent was limited to 5% vol. from the final volume of the reaction medium.

So, there is a method of transformation of 1,2-saturated 3-keto steroids through 1.2-degidro-3-ketosteroids, which is that the 1,2-saturated 3-ketosteroids is subjected to interaction with the tool, with steroid-1,2-dehydrogenase activity A. simplex or Century cyclooxydans, in the presence of exogenous carrier electrons and one or more additional scavenger of toxic oxygen selected from the group consisting of catalase, superoxide dismutase or platinum [US 4749649, 1988]. While 1,2-saturated 3-ketosteroid is 1,2-saturated 3-ketoandrosterone selected from the group comprising HELL, androsta-4,9(11)-diene-3,17-dione and 6α-fluoro-, 6α-methyl and 16β-methylpropane; 11β-hydroxyandrost-4-ene-3,17-dione and 6α-fluoro-, 6α-methyl-, 16β-methyl-derivatives, 11α-hydroxyandrost-4-ene-3,17-dione and androst-4-ene-3,11,17-Trion. According to this method steroid substrate can be made in the form of powder, aqueous paste or in the form of a solution (or suspension) in miscible with water, an organic solvent, such as DMF, DMSO, ethanol, methanol, acetone, number is the number 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 making of the substrate used (load substrate androsta-4,9(11)-diene-3,17-dione 10 g/l). Consider the way [US 4749649, 1988] proposes the use of exogenous enzyme catalase. Adding catalase in an amount of 10 mg/l contributes to the active transformation of the substrate and increases its load to 10 g/l (example 1). For 22 hours in the presence of menadione (5·10-4M) and catalase (10 mg/l) degree of conversion of the substrate is 83%for 46 hours - 95%. In addition, in the presence of catalase and of menadione is complete suppression of destructive activity.

Similar conditions are conversion androst-4-ene-3,17-dione (example 4) with a load of 10 g/l and with a degree of conversion of the substrate 93% for 24 hours Method of making substrate is also not specified.

However, the appreciation of the technology due to the need to add to the environment of the absorber (enzyme catalase, or superoxide dismutase, or platinum)that eliminates the inhibitory effect of singlet oxygen on the activity of dehydrogenase, defines the main disadvantage of this method, which therefore cannot be recommended for industrial use. In addition, in a known manner not described obtaining 1,2-dehydrofreezing test is sterone, 6-methylenecholesterol, 17α-methyltestosterone and 6-medienanstalt-4-ene-3,17-dione.

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

In the patent [US 3091575, 1963] described a method of enhancing fermentation, containing 1,2-dehydrating microbial system for producing 1,2-dihydroceramides, in which the destruction of Δ1,4-3-ketosteroids ingibirovany, including adding as an inhibitor of a substance selected from the group consisting of chromium, molybdenum and tungsten, and compounds containing ortho - and para-quinoid structure in the oxidized or the reduced form, and this compound can be oxidized or the reduced form of a quinone, phenazine, thiazine, oxazine, etc. According to this method the transformation is carried out in a medium containing methanol, in the presence of compounds quinoid structure, which is phenazine methosulfate, and Δ1,4-3-ketosteroid - Δ1-Androstenedione (18 p. formulas). However, the transformation HELL in ADD-growing cells N. corallina shown in medium containing 10-3M potassium dichromate (example 8). The concentration of methanol is 2% of the volume, although after 24 h of transformation degree of conversion was 98%, the load of the substrate was only 0.02 g/L. in Addition, the patent does not consider the handling of such substrates, as testosterone, 17α-methyltestosterone, 6-medienanstalt-4-ene-3,17-dione, 6-methylenecholesterol and androsta-4,9(11)-diene-3,17-dione.

In the patent [US 4524134, 1985] describes a method for 1,2-degidro-Δ4-3-ketosteroid, which is that the corresponding 1,2-saturated Δ4-3-ketosteroid subjected to interaction with air-dried or heat cells of A. simplex containing from 1 to 10% moisture, where the cells are dried in the absence of organic solvent. According to the method of (example 2b) a suspension of micronized androsta-4,9(11)-diene-3,17-dione in DMF contribute in phosphate buffer solution (pH 7.5)containing a suspension of dried cells of A simplex ATCC 6946 (10 g/l) and menadione (86 mg/l). The concentration of DMF in the environment is 2%, and the load of the substrate is from 2.5 g/l (example 2b) to 10 g/l (example 4). The transformation should be performed within 24 h at a temperature of 31°C. as the substrate for the 1,2-dehydrogenation in the conditions of example 2b mentioned HELL and 6α-fluoro-, 6α-methyl and 16β-methylpropane of androsta-4,9(11)-diene-3,17-dione. However, the description of examples of the use of these substrates in the patent [US 4524134, 1985] no. In addition, in this patent are not considered such substrates as testosterone, 17α-methyltestosterone, 6-medienanstalt-4-ene-3,17-dione, 6-methylenecholesterol.

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

Thus, the application of miscible with water and organic solvents at low concentration of 2 vol.% not significantly increase the solubility of the substrate in the aquatic environment and its availability to the cells of the microorganism and does not allow to increase the efficiency of the process. Process intensification using miscible with water and organic solvents in a concentration of more than 5% vol. can be achieved by applying additional modified polysaccharides. Potential use of methanol in concentrations up to 7% vol. mentioned previously in the process of 1,2-dehydrogenation of 17α-methyltestosterone with a load of 20 g/l in medium containing methyl-β-CD or hydroxypropyl-β-CD in concentrations up to 100 g/l [V AV, Andryushina VA, Stetsenko T.S., Voishvillo N.E. go active. Biochem. Microbiol., 2008, 44(6), 642-646].

The technical task of the invention is to improve the efficiency, maintainability and safety of 1,2-dehydrogenation and creating an effective microbiological method for producing 1,2-dehydrogenated Δ4-3-ketosteroids number of androstane devoid of these shortcomings.

The technical problem is solved by a method for producing 1,2-dehydrogenated derivatives Δ4-3-ketosteroids number of androstane total fo the formula (I)

where R1=R2=H or together CH2group; R3=R4=H or together a double bond; R5HE =and R6=N or CH3group; R5and R6together ketogroup,

microbiological transformation Δ4-3-ketosteroids General formula (II)

where R1=R2=H or together CH2group; R3=R4=H or together a double bond; R5HE =and R6=N or CH3group; R5and R6together ketogroup,

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

Thus, the essence of Savannah the invention is the introduction of the 1,2-double bond in the ring And steroid nucleus Δ4-3-ketosteroids number of androstane carry out microbiological reaction of the 1,2-dehydrogenation in an aqueous medium containing from 8 to 40 vol.% aprotic solvent, miscible with water, and exogenous electron acceptor, with the participation of the microorganism cells with 3-ketosteroid-1-dehydrogenase activity.

Besides, the 1,2-dehydrogenation order to obtain 1,2-dehydrofreezing Δ4-3-ketosteroids number of androstane carried out using bacterial cells Nocardioides simplex VKM AC-D.

In addition, as the aprotic organic solvent in the conduct of the 1,2-dehydrogenation using dimethylformamide or dimethylsulfoxide, preferably dimethyl sulfoxide. The concentration of DMF in the aquatic environment is not less than 8% vol., as the concentration of DMSO in the aquatic environment can reach 40%.

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

The technical result of the proposed method microbiological 1,2-dehydrogenation Δ4-3-ketosteroids number of androstane is as follows:

the process is conducted under high load of the substrate (up to 100 g/l) under mild conditions;

in the process, not using flammable and toxic organic RA the founders;

the method is environmentally safe because as the preferred miscible with water solvent serves DMSO - high-boiling liquid with low toxicity and a flash point of 89°C (closed Cup), used in medicine in the form of aqueous solutions with a concentration of from 10 to 90% as drug anti-inflammatory and analgesic actions (trade name "Dimexidum") for external use;

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

the method eliminates the use of the enzyme catalase and any other scavengers of singlet oxygen;

the method can be implemented on standard processing equipment;

the selectivity of the formation of 1,2-dehydrogenated Δ4-3-ketosteroids on stage microbiological 1,2-dehydrogenation is 85-98%.

Below cited as an example of a detailed description of the invention.

Δ4-3-Ketosteroid number of androstane General formula (II)

where R1=R2=H or together CH2group; R3=R4=H or together a double bond; R5HE =and R6 =N or CH3group; R5and R6together ketogroup,

turn in Δ1,4-3-ketosteroid number of androstane General formula (I)

where R1=R2=H or together CH2group; R3=R4=H or together a double bond; R5HE =and R6=N or CH3group; R5and R6together ketogroup,

the introduction of the 1,2-double bond method microbiological dehydrogenation involving cells of the microorganisms, with 3-ketosteroid-Δ1-dehydrogenase activity (preferably cells of bacteria N. simplex VKM AC-D), and the load of the source substrate is from 5 to 100 g/l, and the length of fermentation - from 24 to 52 hours and the process is carried out in an aqueous medium containing exogenous electron acceptor and miscible with water aprotic solvent is DMF or DMSO, preferably DMSO).

The use of DMF or DMSO in the process of 1,2-dehydrogenation provides translation of steroid substrate or a greater part in soluble form, available for enzyme systems of the microorganism cells, and the process at high (up to 100 g/l) loads substrate greatly reduces the effect of cocrystallization substrate and product and formation of mixed crystals. In the literature there is no information about the use of the high concentrations (more than 5 vol.%, specifically from 8 to 40 vol.%) miscible with water and organic solvents in the processes of microbiological 1,2-dehydrogenation of steroid substrates.

Analysis of the products of microbial transformation carried out by thin layer chromatography (TLC) and HPLC. Determination of the contents of the products by HPLC performed on the chromatograph Agilent 1100/1200 (Agilent, Germany) with precolonial Symmetry C18, 5 μm, of 3.9×20 mm and column Symmetry C18, 5 μm, a 4.6×250 mm (Waters, Ireland); in the system with mobile phase (v/v): 60% acetonitrile, 10% isopropanol, in 0.01% acetic acid and water to 100%, at a flow rate of 1 ml/min, 50°C and detection by absorption at 250 nm; typical retention times:

Steroid compoundThe retention time, min
6-Medienanstalt-4-ene-3,17-dione(6-methylene-AD)4.9
6-Medienanstalt-1,4-diene-3,17-dione(exemestane, 6-methylene-ADD)4.3
Androst-4-ene-3,17-dione(AD)8.63
Androsta-1,4-diene-3,17-dione(ADD)6.59
17β-hydroxyandrost-4-EN-3-one(testosterone)7.06
17β-hydroxyandrost-1,4-Dien-3-one(boldenone)5.54
Androsta-4,9(11)-diene-3,17-dione8.3
Androsta-1,4,9(11)-triene-3,17-dione6.4
6-Methylene-17β-hydroxyandrost-4-EN-3-one(6-methylenecholesterol)4.54
17β-Hydroxy-17-methylandrosta-4-EN-3-one(17?-methyltestosterone)4.42
17β-Hydroxy-17-methylandrosta-1,4-Dien-3-one(methandrostenolone)3.81

The allocation of 1.2-dehydrogenated Δ4-3-ketosteroids from the culture fluid can be carried out by extraction of water-immiscible solvent or extraction of steroid pasta biomass in any suitable solvent, after preliminary separation from the aqueous phase. To extract steroid can also be applied, for example, sorption method of extraction, as well as any other effective method.

The claimed group of inventions is illustrated by the following examples without limiting it.

Microbiological 1,2-dehydrogenation in the implementation of the present invention, the method for producing 1,2-dehydrogenated derivatives Δ4-3-ketosteroids number of androstane in the but-organic media - can be done resting (washed) cells of bacteria of the genus Nocardioides, specifically Nocardioides simplex VKM AC-D.

As a component of the environment during implementation of the invention is a method for producing 1,2-dehydrogenated derivatives Δ4-3-ketosteroids number of androstane in aqueous-organic media can be used mixed with water organic aprotic solvents such as DMF or DMSO, preferably DMSO.

Steroid substrates and electron acceptors in the implementation of the present invention, the method for producing 1,2-dehydrogenated derivatives Δ4-3-ketosteroids number of androstane - can be made in the form of powder, aqueous paste, or in the form of a solution (or suspension) in miscible with water, an organic solvent, such as DMF, DMSO, ethanol, methanol, acetone, etc.

A sample of the culture fluid in the implementation of the present invention, the method for producing 1,2-dehydrogenated derivatives Δ4-3-ketosteroids number of androstane - in process and at the end of the transformation process is extracted with ethyl acetate.

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

Examples

In use, the strain Nocardioides simplex VKM AC-D, which is supported on agar medium of the following composition (g/l): glucose-10, soy peptone-4, yeast extract, MgSO4-0.5, KN2RHO4-2, K2NRA4-4, agar-20, distilled water, pH 7.0-7.2. The cultivation is carried out in the next 3-4 days at 30°C.

The cultivation of the 1st inoculum

Washing the culture with the surface of the agar medium in 5 ml of saline solution to make a 750 ml Erlenmeyer flask with 100 ml of medium (g/l): glucose-15, soy peptone-6, yeast extract-6, MgSO4-0.5 KN2RHO4-2, K2NRA4-4, and cultured at 28-30°C for 24-26 hours.

The cultivation of transforming culture

5 ml of the culture fluid at 24-26 hours growth 1 inoculum make 100 ml of medium of the same composition and spend growing in similar conditions for 10-12 hours, then make the inducer of the synthesis of steroid-1-dehydrogenase - acetate cortisone in the form of an ethanol solution (20 mg in 1 ml ethanol). The induction of the synthesis of 1,2-dehydrogenase spend over the next 20-24 hours. Cells of microorganisms precipitated by centrifugation at 5000 g for 30 min and used for the process of 1,2-dehydrogenation or stored at -18°C for one month before using.

Example 1. Getting androsta-1,4,9(11)-triene-3,17-dione

2.5 g of Androsta-4,9(11)-diene-3,17-dione (in recalculation on 100% of the basic substance content) transform in flasks with a volume of 750 ml in a medium containing 15 ml M Na-phosphate buffer solution (pH 8.0), 10 ml of DMSO, 0.1 g of menadione I g biomass (dry weight biomass). The bioconversion is carried out in aerated conditions at 30°C on rotary shaker at 200 rpm, the Process stopped at 24-30 hours of cultivation at achieving content androsta-1,4,9(11)-triene-3,17-dione 94-95%.

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

1.25 G. of Androst-4-ene-3,17-dione (AD) transform in flasks 750 ml containing 15 ml M Na-phosphate buffer solution (pH 8.0), 10 ml of DMSO, 0.05 g of menadione and 2.0 g of biomass (dry weight biomass). The bioconversion is carried out in aerated conditions at 25°C on a rotary shaker at 200 rpm Process stop for 22 hours cultivation at achievement of contents ADD 96%.

Example 3. Receiving 17β-hydroxyandrost-1,4-Dien-3-one (boldenone)

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

Example 4. Receiving 17β-hydroxy-17-methylandrosta-1,4-Dien-3-one (methandrostenolone)

0.25 g of 17β-Hydroxy-17-methylandrosta-4-EN-3-one(17?-methyltestosterone) transform in flasks 750 ml, containing 22 ml M Na-phosphate buffer solution (pH 8.0), 3 ml of LCA is, 0.02 g of finishingtouches (FMS) and 0.4 g of biomass (dry weight biomass). The bioconversion is carried out in aerated conditions at 30°C on rotary shaker at 200 rpm, the Process is stopped for 42-48 hours of cultivation at achieving content methandrostenolone 92-95%.

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

Option 1

0.125 g of 6-Medienanstalt-4-ene-3,17-dione(6-methylene-AD) transform in flasks of 250 ml containing 23 ml of 0.02 M Na-phosphate buffer solution (pH 8.0), 2 ml of DMSO. 0.005 g of menadione and 0.2 g of biomass (dry weight biomass). The bioconversion is carried out in aerated conditions at 30°C on rotary shaker at 200 rpm, the Process stopped at 48 hours of cultivation at achievement of contents 6-methylene-ADD 94-96%.

Option 2

The method is carried out for option 1, but the reaction medium presents 0.02 M Na-phosphate buffer solution, pH 8.0 (23 ml) and DMF (2 ml).

Option 3

The method is carried out for option 1, but the reaction medium contains 2.5 g of 6-methylene-HELL, 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 stopped at 52 hours of cultivation at achievement of contents 6-methylene-ADD 95-97%.

Option 4

0.2 g of 6-Methylenecholesterol transform in flasks 750 ml, containing 18 ml of 0.02 M Na-phosphate buffer is of astora (pH 8.0), 2 ml of DMSO, 0.008 g of menadione and 40 g of biomass (dry weight biomass). The bioconversion is carried out in aerated conditions at 30°C on rotary shaker at 200 rpm, the Process stopped at 120 hours of cultivation at achievement of contents 6-methylene-ADD 97-98%.

1. Method for producing 1,2-dehydrogenated derivatives Δ4-3-ketosteroids number of androstane General formula (I)

where R1=R2=H or together CH2group; R3=R4=H or together a double bond; R5=OH and R6=H or CH3group; R5and R6together ketogroup,
microbiological transformation Δ4-3-ketosteroids General formula (II)

where R1=R2=H or together CH2group; R3=R4=H or together a double bond; R5=OH and R6=H or CH3group; R5and R6together ketogroup in an aqueous medium containing exogenous electron acceptor, with the use of cells of the microorganism with 3-ketosteroid-Δ1-dehydrogenase activity, using an initial substrate concentrations from 5 to 100 g/l and the selection of the target product from the culture fluid, characterized in that as component of the environment using miscible with water aprotic organic solvent in concentric and more than 5 vol.%, specifically from 8 to 40%vol.

2. The method according to claim 1, characterized in that the microorganism with 3-ketosteroid-Δ1-dehydrogenase activity using Nocardioides simplex VKM AC-D.

3. The method according to claim 1, characterized in that as an exogenous electron acceptor use menadione or FMS in an effective amount.

4. The method according to claim 1, characterized in that as the aprotic miscible with water, the organic solvent used is dimethylformamide or dimethylsulfoxide, preferably dimethyl sulfoxide.



 

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5 dwg, 2 tbl, 2 ex

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 Δ4-3-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

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to process of crystallisation, obtaining and isolation of novel crystalline form of fusidic acid, to application of said processes in production of pharmaceutical composition or medication, and to application of said form of crystalline fusidic acid in treatment of bacterial infections.

EFFECT: obtaining pharmaceutical composition for treatment of bacterial infections.

11 cl, 10 ex, 1 tbl, 10 dwg

FIELD: chemistry.

SUBSTANCE: invention enables synthesis of 3-methoxy-2-fluoro-18ethyl-8α-gona-1,3,5(10)-trienes, having osteoprotector and hypocholesteremic activity.

EFFECT: invention has osteoprotector and hypocholesteremic activity and can be used in medicine for hormonal replacement therapy.

1 cl, 3 ex, 1 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: claimed invention relates to application of steroid compounds, preferably 17 α-ethinyl derivatives of androstane.

EFFECT: insuring treatment of inflammatory or autoimmune disease in subject and/or infection prevention.

7 cl, 12 ex, 18 dwg

FIELD: medicine.

SUBSTANCE: invention provides administration of triterpene glycosides of holothurian Cucumaria okhotensis chosen from a group consisting of Frondoside A1, Ochotoside B1, Ochotoside A1-1, Ochotoside A2-1 or Cucumarioside A2-5 or their mixtures, as an agent stimulating cell-mediated immunity in mammals, as well as for preparing a pharmaceutical composition stimulating cell-mediated immunity in mammals.

EFFECT: extended range of the agents stimulating cell-mediated immune reaction in mammals.

4 dwg, 1 tbl, 2 cl

FIELD: medicine.

SUBSTANCE: invention refers to a method of Δ4-3-ketosteroids 11 β-hydroxylation by a biomass Curvularia lunata strain mycelium, RNCIM No. F-988. For the transformation, Curvularia lunata strain mycelium, RNCIM No. F-988 not older than 30 h and washed of nutrient medium is used. Mycelium is taken in such amount that the relation of the biomass to the transformed steroid makes 1.5-2.5:1. The transformation is performed in a buffer solution, and a steroid substratum is added as a microcrystal suspension, or as a water-soluble methyl - β-cyclodextrine complex with steroid related thereto as 1:1-0.6:1 (mol/mol). The yield of 11β-hydroxyderivatives is 50-80 %.

EFFECT: offered invention allows for higher selectivity of 11β-hydroxylation process, concentration of the transformed steroid substratum up to 20 g/l and reduced reaction period to 24-50 h.

1 tbl, 10 ex, 2 cl

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