PL228764B1 - Method for producing 6β-hydroxyandrost-4-en-3,11,17-trione - Google Patents

Description

REPUBLIC

POLAND

Patent Office of the Republic of Poland (54) (12) PATENT DESCRIPTION ₍ i ₉₎ PL ₍ n) 228 764 (13) B1 (21) Application number: 417205 (22) Application date: 13.05.2016 (51) Int.CI.

C12P 33/06 (2006.01) C07J 1/00 (2006.01) C12R 1/645 (2006.01)

Method for the production of 6β-hydroxyandrost-4-ene-3,11,17-trion (73).

UNIVERSITY OF NATURE IN WROCŁAW, Wrocław, PL (43) Application was announced:

10.04.2017 BUP 08/17 (45) The following was announced about the grant of the patent:

30.05.2018 WUP 05/18 (72) Inventor (s):

EWA KOZŁOWSKA, Wrocław, PL MONIKA DYMARSKA, Wrocław, PL EDYTA KOSTRZEWA-SUSŁOW, Wrocław, PL JAKUB GRZESZCZUK, Opole, PL ELŻBIETA PLĄSKOWSKA, Wrocław, PL TOMASZ JANECZKO, Wrocław, PL (74) Plenipotentiary:

thing, pat. Anna Olszewska 'st co rco

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PL 228 764 B1

Description of the invention

The present invention relates to a process for the preparation of 63-hydroxyandrost-4-ene-3,11,17-trione.

The method according to the invention can be used in the pharmaceutical industry for the production of drugs used during the malfunctioning of muscle tissue (Cavaco JEB, Van Blijswijk B, Leatherland JF, Goos HJT, Schulz RW. 1999, Androgen-induced changes in Leydig cell ultrastructure and steroidogenesis in juvenile African catfish Clarias gariepinus. Cell Tissue Res. 297, 291).

A significant proportion of steroid compounds used in pharmaceuticals as anti-inflammatory, anabolic, contraceptive, diuretic or anti-cancer agents is difficult to obtain by chemical methods (Mahato SB, Garai S. 1997. Advancesin Microbial Steroid Biotransformation. Steroids: 61, 33; Ko D., Heiman) AS, Chen M., Lee HJ 2000. New steroidal anti-inflammatory antedrugs: methyl 21-desoxy-21-chloro-11,17-dihydroxy-3,20-dioxo-1,4-preg-nadiene-16-carboxylate, methyl 21-desoxy-21-chloro-11-hydroxy-3,20-dioxo-1,4-pregnadiene-16-carboxylate, and their 9-fluoro derivatives. Steroids: 65, 210).

The most frequently observed transformation of steroid compounds in biocatalyst cells is hydroxylation (Romano A., Romano D., Ragg E., Costantino F., Lenna R., Gandolfi R. 2006. Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum linear. Steroids: 71,429).

From the description of the application of the invention P.416996 the strain Isaria fumosorosea KCh J2 is known.

There is a known microbiological method of obtaining 63-hydroxyandrost-4-ene-3,11,17-trion from androst-4-ene-3,11,17-trione (adrenosterone) in the yield of 4,2%, by using the Cunninghamella strain culture elegans (Choudhary MI, Khan NT, Musharraf SG, Anjum S., Atta-urRahman, 2007, Biotransformation of adrenosterone by filamentous fungus, Cunninghamella elegans. steroids 7, 923).

The essence of the invention consists in introducing the Isaria fumosorosea KCh J2 strain into a medium suitable for filamentous fungi. After at least 48 hours, the substrate is introduced into the culture, which is androst-4-ene-3,11,17-trione of formula 1, dissolved in a water-miscible organic solvent. The transformation is carried out at a temperature of 20 to 30 degrees Celsius with continuous shaking for at least 72 hours. Subsequently, the product is extracted with a water-immiscible organic solvent and purified by chromatography.

As a result of regioselective introduction of the hydroxyl group in the substrate molecule, which is androst-4-ene-3,11,17-trione (adrenosterone), 63-hydroxyandrost-4-en-3,11, -17-trione is obtained, and the reaction is carried out in an aqueous culture of the strain Isaria fumosorosea KCh J2.

Preferably, the ratio of weight of the substrate added to the culture volume to be added is 0.1 mg: 1 mL.

It is also preferred that the process is carried out at a temperature of 25 degrees Celsius.

Additionally, it is preferred that the transformation is carried out for 72 hours.

In accordance with the invention, as a result of the action of the enzyme system contained in the cells of the Isaria fumosorosea KCh J2 strain, hydroxylation takes place at an unactivated C-6 carbon atom. The product obtained in this way is separated from the aqueous culture of the microorganism by a known method by extraction with a water-immiscible organic solvent (chloroform).

The main advantage of the invention is the preparation of 63-hydroxyandrost-4-ene-3,11,17-trione with an isolated yield of 33% (substrate conversion according to GC> 99%), at room temperature and pH natural for the strain.

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

Example d. To the Erlenmeyer flask with a capacity of 2,000 cm ^3, which is 500 cm ^{3 of} a sterile medium containing 10 g aminobaku and 30 g of glucose, the strain is introduced Isaria fumosorosea SDS J2. After 72 hours of its growth, 100 mg of androst-4-ene-3,11,17-trione of formula 1, dissolved in 1 cm ^{3 of} tetrahydrofuran, are added. The transformation is carried out at 25 degrees Celsius with continuous shaking for 72 hours. The reaction mixture was then extracted three times with chloroform, dried with anhydrous magnesium sulfate, and the solvent was evaporated. The extract obtained is purified by chromatography using a 2: 1 v / v mixture of hexane and acetone as eluent.

In this way, 33 mg of 63-hydroxyandrost-4-ene-3,11,17-trione are obtained (yield 33%, substrate conversion> 99%).

The obtained product is characterized by the following spectral data:

¹ H NMR (600 MHz) (ppm) (CDCl 3) δ: 0.89 (s, 3H, 18-H); 1.50 (ddd, 1H, J = 14.1, 11.6, 2.6 Hz,

7-Ha); 1.60 (s, 3H, 19-H); 1.62 (td, 1H, J = 14.5, 4.0 Hz, 1-Ha); 1.73 (ddd, 1H, J = 21.4, 12.1, 9.4 Hz,

15-H3); 1.86 (d, 1H, J = 11.5 Hz, 9-H); 1.89 (td, 1H, J = 11.8, 5.7 Hz, 14-H); 2.12-2.18 (m, 1H,

15-Ha); 2.20-2.37 (m, 4H, 2-Ha, 7-Hp, 12-Ha, 16-Ha); 2.44-2.60 (m, 4H, 2-Hp, 8-H, 12-Hp, 16-Hp);

2.80 (dm, 1H, J = 13.4Hz, 1-Hp); 4.39 (br s, 1H, 6-Ha); 5.79 (brs, 1H, 4-H).

¹³ C NMR (151 MHz) (ppm) (CDCl 3) δ: 14.91 (C-18), 19.32 (C-19), 21.69 (C-15), 30.65 (C-8), 34.11 (C-2), 36.03 (C-16), 36.10 (C-1), 37.68 (C-10), 38.10 (C-7), 49.71 (C-14), 50.44 (C-13), 50.50 (C-12), 63.13 (C-9), 72.13 (C-6), 127.17 (C-4), 166.61 (C-5), 200.67 (C-3), 207.41 (C-11), 217.24 (C-17).

Claims (4)

Patent claims Method for the production of 63-hydroxyandrost-4-ene-3,11,17-trion, characterized in that the Isaria fumosorosea KCh J2 strain is introduced into a substrate suitable for filamentous fungi, then, after at least 48 hours, the substrate is introduced into the culture which is androst-4-en-3,11,17-trione of formula 1, dissolved in a water-miscible organic solvent, the transformation is carried out at a temperature of 20 to 30 degrees Celsius, with continuous shaking, at least 72 hours, the product of formula II is then extracted with a water-immiscible organic solvent and purified by chromatography. 2. The method according to p. The method of claim 1, wherein the ratio of the weight of the added substrate to the culture volume is 0.1 mg: 1 mL. 3. The method according to p. 1., characterized in that the process is carried out at a temperature of 25 degrees Celsius. 4. The method according to claim 1, characterized in that the transformation is carried out for 72 hours.