RU2377309C1 - MICROBIOLOGICAL METHOD FOR PREPARATION OF 7α-HYDROXYANDROSTENES - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method of selective preparation of 7α-hydroxyandrostens with the help of floccus, includes transformation of Δ⁵ -androstenes with the help of mycelium Curvularia lunata VKPM F-981 separated and washed from medium. At the same time substrate is used in the form of microchips, or in the complex with cyclodextrins.

EFFECT: yield of end 7-hydroxyproducts - 60-90%.

2 cl, 5 ex

Description

The present invention relates to the field of biotechnology. The invention relates to methods for producing steroidal drugs using microorganisms, and more particularly to a method for producing 7α-hydroxy derivatives of steroids of a number of androstan, in particular androstenolone (dehydroepiandrosterone - DEA) and its 17β-hydroxy analogue.

From literature it is known that 7-hydroxy derivatives of steroidal 5-olefins have unique biological properties. They found the presence of antitumor, immunostimulating, anticholesterolemic, nootropic, and radioprotective activities [1-9].

The versatile and high biological activity of Δ ⁵ -7-hydroxysteroids makes the problem of their synthesis extremely urgent and explains the great interest of researchers in the C7-hydroxylation reaction. However, there is currently no promising way to obtain 7-hydroxy derivatives of steroidal 5-olefins, which is necessary to create a number of new drugs.

Only chemical methods for the synthesis of such compounds are described, since allylic oxidation refers to the fundamental reactions of organic chemistry [10-14]. However, the presence of such shortcomings as low yields associated with the complexity of the process of oxidation and isolation of reaction products, as well as the use of expensive or harmful to health and ecology substances, lead to the inefficiency of these methods for producing 7-hydroxysteroids.

The hexavalent chromium compounds in the form of a complex of chromic anhydride with pyridine, chlorochromate and pyridinium dichromate, potassium or sodium dichromates in acetic acid are most widely used for the 7-hydroxylation reaction [10, 12]. Among other reagents, manganese compounds (potassium dioxide and permanganate) and ruthenium and selenium oxides are used [13].

An alternative to the chemical synthesis of steroid Δ ⁵ -7-hydroxy-olefins is microbiological hydroxylation, which is distinguished by stereo- and regiospecificity, inaccessible to chemical methods.

The ability to C7-hydroxylation of a steroid molecule was found in some representatives of higher and lower fungi. Examples of applications for C7-hydroxylation of steroids of ascomycetes (Diapothe and Gibberella), deuteromycetes (representatives of the genera Cephalosporium, Diplodia, Fusarium) and zygomycetes (Absidia, Cunningamella, Mucor, Rhizopus) are known [1-5, 15-21]. However, with the participation of these microorganisms, only Δ ⁴ -3-ketosteroids are described. Moreover, the products of these reactions, as a rule, are dihydroxy derivatives, and the target 7-monohydroxy compounds are formed in small quantities.

The closest analogue to the claimed method is the transformation of DEA and pregnenolone by the culture of the fungus Fusarium moniliforme [5] with the formation of the corresponding 7-monohydroxy derivatives - 86% 7α-hydroxy-DEA (GDA) and 65% 7α-hydroxypregnenolone. To obtain the indicated 7-hydroxy compounds, the mycelium Fusarium moniliforme [5] was contacted with DEA for 18 hours to activate 7α-hydroxylase. Then the biomass of the fungus in the amount of 10 g / l is transferred to a fresh nutrient medium and the DEA or pregnenolone is transformed at a load of 0.4 g / l for 24 hours under aerobic conditions at a temperature of 27 ° C. The low content in the environment of substrates transformed with Fusarium moniliforme, the need for activation of 7α-hydroxylase, and the low rate of hydroxylation are the main disadvantages of this method for producing GEA.

The low concentration of steroid compounds in the reaction medium, associated with their very low solubility in aqueous media, is a feature of processes carried out using living microorganisms. This factor, along with the insufficient availability of steroids for microbial cells, are usually the reason for the low degree of conversion of steroid substrates and represent a serious obstacle to obtaining steroid drugs, especially on an industrial scale. To increase the load of steroid substrates, increase their bioavailability and degree of conversion, preliminary mechanical grinding of the steroid to a microcrystalline state is used, or it is applied in the form of a finely dispersed emulsion in organic solvents, and sometimes a reaction is carried out in the presence of a solvent miscible with water [20]. However, organic solvents have a toxic effect on microorganisms. There are also known methods of introducing steroid substrates in the form of complexes with β-cyclodextrin (CD) [22] or chemically modified CD (CMCD), which significantly increase the solubility of steroid substrates and the reaction rate. However, the effectiveness of HMCD has been demonstrated only in processes carried out using bacteria [23]. For the transformation of steroids with the help of fungi, HMCD was not previously used.

The problem to which the invention is directed, is to develop a method for the selective 7α-hydroxylation of steroidal Δ-5-olefins of the androstane series.

The technical result that can be achieved by carrying out the invention is to increase the selectivity of the process, increase the load of the steroid substrate and increase its bioavailability.

The essence of the method is to use for the microbiological process of hydroxylation of Δ ⁵ -androstenes of the mold fungus Curvularia lunata VKPM F-981, and the substrate itself in a crystalline form or in the form of an inclusion complex with HMCD.

Examples of the use of the mold fungus Curvularia lunata VKPM F-981 for hydroxylation of steroids show that HMCD significantly increase the bioavailability of the substrate, as evidenced by the high rate of modification of the steroid molecule.

Unlike the growing culture of the fungus Fusarium moniliforme, the mycelium Curvularia lunata VKPM F-981 carries out selective 7α-hydroxylation of steroids in the absence of nutrient components. The accumulation of 7α-hydroxyproducts is carried out by mycelium in a buffer solution containing up to 10 g / l of steroid substrate. For transformation, steroids are used either in a crystalline form or in the form of complexes with HMCD. In this case, the hydroxylation of 5-androstenes proceeds at a high rate — no more than 2 days, and the stage of preliminary activation of the enzyme is not required for 7α-hydroxylation. Conversion of steroids by the fungus Curvularia lunata is not accompanied by the destruction of the substrate and product, as illustrated by the following examples.

Example 1. Obtaining 3β, 7α, 17β-trihydroxyandrost-5-ene from 3β, 17β-dihydroxyandrost-5-ene.

Spores of Curvularia lunata VKPM F-981 were transferred from an agglomerated medium of the following composition, g / l: yeast extract - 4.0, malt extract - 10.0, glucose - 4.0, agar - 25; pH 6.2-6.9, in a liquid medium containing in g / l: sucrose - 30, yeast autolysate - 2.5, NaNO ₃ - 2.0, K ₂ NRA ₄ - 1.0, (NH ₄ ) H ₂ PO ₄ - 3.0, KCl - 0.5, MgSO ₄ × 7H ₂ O - 0.5, pH 6.0-6.2. The fungus culture was grown for 72 h in 100 ml of the indicated medium in 750 ml conical rocking flasks at 28 ° С and a stirring speed of 220 rpm. The inoculum was transferred to the same medium and incubated under the same conditions for 24 hours. Then, the mycelium was separated from the medium and suspended in phosphate buffer, pH 6.0-6.3, containing micronized 3β, 17β-dihydroxyandrost-5-ene in an amount of 2 g / L. Incubated on a shaker under the above conditions for 20-24 hours until the disappearance of the original substrate.

The mycelium was separated from the buffer, after which the aqueous phase was extracted three times with chloroform. The extract was evaporated, dried at 60 ° C, and crystalline 3β, 7α, 17β-trihydroxyandrost-5-ene was obtained with a yield of 58-62%. The technical product was washed with acetone, dried at 60 ° C, crystallized from methanol and a triol was obtained with mp. 252-256 ° C. PMR spectrum (δ, ppm), (D-DMSO): 0.63 s (18 Me); 0.87 s (19 Me); 3.27 m (3H _α ); 3.42 m (17H _α ); 3.57 t (7β); 4.03 d (7-OH _α ) ³ J _{7-OH, 7Hb} = 6.66 Hz; 4.38 d (17-OH) ³ J _{17-OH, 17-Hα} = 4.78 Hz; 5.40 dd (6H) J _6.7 = 5.37 Hz.

Example 2. Obtaining 3β, 7α, 17β-trihydroxyandrost-5-ene from 3β, 17β-dihydroxyandrost-5-ene in the presence of methyl β-cyclodextrin.

The transformation of 3β, 17β-diol was carried out analogously to example 1, but the convertible substrate was introduced at a load of 5 g / l into a phosphate buffer containing methyl β-cyclodextrin in the ratio to steroid 1: 1 (mol / mol). Incubation was carried out for 48 hours. The indicated triol was obtained with a yield of 70-75%.

Example 3. Obtaining 7α-hydroxy-DEA from acetate DEA. The transformation of DEA acetate was carried out analogously to Example 2, but DEA acetate was used for hydroxylation at a load of 10 g / L, which was incubated for 44-48 hours. The aqueous phase obtained after separation of the mycelium was extracted with ethyl acetate, the extract was evaporated and 7a-hydroxy- was obtained in quantitative yield. DEA in the form of a very thick (viscous) oil. After treating the oil with aqueous acetone, a crystalline product was obtained with mp. 181-184 ° C. Lit. data 181.5-183.5 ° С [15]. Mass spectrum (m / z): 304 (M ⁺ ), 286, 271, 253. Spectrum ¹ PMP (CDCl ₃ ) (δ, ppm): 0.86 s (18 Me)); 0.99 s (19 Me); 3.55 m ( ³ J _3.2 = 11.1, 3H _α ); 3.94 m ( ³ J _6.7 = 5.3 Hz, 7H _β ); 5.61 q (6H).

Example 4. Obtaining 7α-hydroxy-DEA from DEA.

The hydroxylation of DEA at a load of 4 g / L was carried out analogously to Example 2. The transformation of DEA was completed after 19 h with a yield of 7a-hydroxy-DEA 85%.

Example 5. Obtaining 7α-hydroxy-DEA from acetate DEA in the presence of CD and hydroxypropyl-CD.

Hydroxylation of DEA acetate was carried out analogously to Example 3, but instead of methyl-β-cyclodextrin, β-cyclodextrin or hydroxypropyl-CD was used as a solubilizer. Transformation of DEA acetate was carried out for 48 hours. After processing analogously to Example 3, 60-76% of 7α-hydroxy-DEA identical to that obtained in Example 3 was obtained.

Literature

1. Femandes P., Cruz A., Angelova B., Pinheiro H.M., Cabral J.M.S. // Enzyme and Microbial Technol. V.32. P.688-705. 2003.

2. Lathe R., Rose K.A., Seckl J.R., Best R., Yau J.L., Leckie CM. // WO 9737664, 1997.

3. Morfin R., Courchay G. // J. Steroid Biochem. Mol. Biol. V. 50, p. 91-100, 1994.

4. Cotillon A.C., Morfin R. // J. Steroid Biochem. Mol. Biol. V.68. P.229-237, 1999.

5. Dray F.J., Cotillon A.C. // FR Pat. 2771105, 1999.

6. Sebastien W-E., David J-P., Sasdovitch V., Liere P., Schumacher M.

7. Delacourte A. Baulieu E.E., Akwa Y. // Brain Res. V.969, p.117-125, 2003.

8. Shi J., Schulze S., Lardy H.A. // Steroids. V.65. P.124-129. 2000.

9. Loria R.M. // Steroids. V.67, p.953-966, 2002.

10. Ausi D.L., Ahlem C., Li M. et al. // Mod. Asp. Immunol. V.3, p. 64-70, 2003.

11. Lathe R. // Steroids. V.67, p.967-977, 2002.

12. Kumar V., Amann A., Ourisson G., Luu B. // Synthetic commun. V.17, p.1279-1286, 1997.

13. Graham A.W. // WO 97/48367.

14. Marwah P., Marwah A., Kneer N., Lardy H. // Steroids. V.66. P.581-595, 2001.

15. Loria R.M. // US Patent 5,277,907.

16. Akhrem A.A., Titov Yu.A. // Steroids and microorganisms. "The science". 1970.

17. Mahato S. B., Banerjee S., Podder S. // Phytochemistry. V.28. P.7-40. 1989.

18. Kolek T.J. // Steroid Biochem. Mol. Biol. V.71, p. 83-90, 1999.

19. Wilson M.R., Gallimore W.A., Reese P.B. // Steroids. V.64. No.12. P.834-843. 1999.

20. Sedlaczek L. // Crit. Rev. Biotechnol. V.7. No.3. P.187-236. 1988.

21. Choudhary M.I., Ali Shah S.A., Musharrah S.G., Shaheen F., Ur-Rahman A. // Nat. Prod. Res. V.17. No.3. P.215-220. 2003.

22. Nagy E.U., Bartho I., Hantos G., et all. // GB Patent 2108965.1983.

23. Donova M.V., Dovbnya D.V., Kalinichenko A.N. et al. // RF Patent No. 940017640. 1996.

Claims (2)

1. Microbiological method for producing 7α-hydroxyandrostenes using mushroom mycelium, characterized in that the transformation of Δ ⁵ -androstenes is carried out using Curvularia lunata VKPM F-981 separated and washed from the mycelium, and the substrate is used in the form of microcrystals or in the form of a complex with cyclodextrins . 2. The method according to claim 1, characterized in that β-cyclodextrin or its chemically modified derivatives, such as methylcyclodextrin and hydroxypropyl-cyclodextrin, are used as cyclodextrins.