PL214911B1 - Process for the preparation of 3�, 7?-dihydroxy-5?-androst-17-one - Google Patents

Description

The present invention relates to a process for the preparation of 33,7a-dihydroxy-5a-androst-17-one (7a-hydroxy-EpiA), of the formula 2 shown in the drawing.

This compound may find application in the pharmaceutical industry.

Dehydroepiandrosterone (DHEA), synthesized in the brain tissues of rodents and humans, is classified as a neurosteroid (R. C. Bron, C. Jasio, V. Papadopoulos, J. Neurochem. 2000, 74, pp. 847-59). The results of many studies indicate that the neuroprotector effect is not exerted by DHEA, but by its metabolites, mainly hydroxy derivatives at C-7 (A. Pringle, W. Schmidt, J. Deans, E. Wulfert, K. Reymann, L. Sundstrom, Eur. J Neurosci, 2003, 18, pp. 117-24).

In vivo studies have shown the neuroprotector effect of 7α-hydroxy-DHEA and both 7-hydroxy derivatives of 3β-hydroxy-5α-androstane-17-one (epiandrosterone, EpiA): 3β, 7α-dihydroxy-5α-androst-17-one ^ -hydroxy- EpiA) and 3β, 7β-dihydroxy-5α-androst-17-one (7β-hydroxy-EpiA) (B. Dudas, M. Rose, E. Wulfert, Neurobiol. Dis. 2004, 15, pp. 262-268) .

Known microbial hydroxylations, most often carried out using cultures of filamentous fungi, are an effective method of introducing a hydroxyl group into an inactive position of the substrate (P. Fernandes, A. Gruz, B. Angelova, HM Pinheiro, JMS Carbal, Enzyme Microbial Technology, 2003, 32, pp. 688-705).

In the examples of microbial hydroxylation of epiandrosterone known from the literature, low yields of 7α-hydroxyepiandrosterone were obtained. The share of 7α-hydroxy-EpiA in the agitation, after 8 days transformation of EpiA by the strain Cephalosporium aphidicola, was 4% (C. A. Bensasson, J. R. Hanson, A. C. Hunter, Phytochemistry, 1998, 40, pp. 2355-58). However, the content of 7α-hydroxy-EpiA, after incubation of this substrate in Rhizopus nigricans culture, did not exceed 20% (VEM Chambers, WA Denny, JM Evans, ERH Jones, A. Kasal, GD Meakins, J. Pargnell, J. Chem. Soc. ., Perkin Trans I, 1973, pp. 1500-11; S. Chalbot, C. Trap, JP. Monin, R. Morfin, Steroids, 2002, 67, pp. 1121-27).

The invention relates to a process for the preparation of 3β, 7α-dihydroxy-5α-androst-17-one of the formula 2 by microbial hydroxylation of 3β-hydroxy-5α-androst-17-one (epiandrosterone, EpiA).

The essence of the invention is that epiandrosterone (EpiA) is transformed into 3β, 7α-dihydroxy -andrest-K-one -hydroxy-EpiA) by the culture of the strain Mortierella isabellina AM212.

It is advantageous if the transformation process is carried out with an aqueous culture of the strain, with continuous shaking of the reactants, at a temperature of 293-300K.

In accordance with the invention, 3β-hydroxy-5α-androst-17-one is converted into 3β, 7α-dihydroxy-5α-androst-17-one by the action of the enzyme system contained in the cells of the fungus Mortierella isabellina AM212. The product obtained in this way is separated from the aqueous culture of the microorganism by a known method by extraction with chloroform.

The main advantage of the invention is the preparation of 3β, 7α-dihydroxy-5α-androst-17-one with a yield of 73%, at room temperature and near neutral pH.

The invention is explained in more detail using an exemplary embodiment.

P r z k ł a d

To the Erlenmeyer flask with a capacity of 300 cm ^3, which is 100 cm ^{3 of} sterile medium ₃ containing 3 g of glucose and 1 g aminobaku introduced into a 1 cm ³ cell suspension Mortierella isabellina AM212 that grow for three days at the substrate, constant shaking at a temperature of 295K. After three days of growth, 20 mg of 3β-hydroxy-5α-androst-17-one, formula I, dissolved in 1 cm ^{3 of} acetone, are added. The transformation is carried out under continuous shaking for a further 21 hours under culturing conditions for the microorganism. Then, the resulting transformation solutions were extracted three times with chloroform, dried with anhydrous magnesium sulfate, and the solvent was evaporated. 26 mg of crude product are obtained, which product is purified by chromatography using hexane: acetone: chloroform (1: 1.5: 1.25) as the eluent.

In this way, 15.4 mg of 3β, 7α-dihydroxy-5α-androst-17-one are obtained (73% yield).

The obtained product is characterized by the following spectral data:

¹ H NMR: δ (ppm): 0.81 (s, 3H, 19-CH3); 0.83 (s, 3H, 18-CH3); 3.61 (m, 1H, Wh = 31.2 Hz 3αΉ); 3.95 (m, 1H, Wh = 2.4 Hz, 7β-Η).

¹³ C NMR: δ (ppm): 221.1 (C-17), 70.9 (C-3), 66.7 (C-7), 47.5 (C-13), 46.1 (C -9), 45.8 (C-14), 37.5 (C-4), 39.1 (C-8), 37.0 (C-5), 36.6 (C-1), 36 , 6 (C-16), 36.0 (C-10), 35.7 (C-6), 31.4 (C-2), 31.3 (C-12), 21.3 (C- 15), 20.3 (C-11), 13.5 (C-18), 11.2 (C-19).

IR u _max (cm ^-1 ) (CHCl 3) 3625: 1743.

Claims (3)

1. A method for the preparation of 33,7a-dihydroxy-5a-androst-17-one (7a-hydroxy-EpiA), of formula 2, by means of microbial transformation, wherein the substrate is 33-hydroxy-5a-androst-17- on (epiandrosterone, EpiA), characterized in that the substrate is transformed with the enzyme system of a strain of the filamentous fungus Mortierella isabellina AM212. 2. The method according to p. The method of claim 1, wherein the substrate biotransformation process is carried out in an aqueous culture of the strain with continuous shaking of the reactants. 3. The method according to p. The method of claim 1, characterized in that the substrate biotransformation process is carried out at the temperature of 293-300K.