DD277697A1 - PROCESS FOR THE PREPARATION OF 2 ALPHA, 3 ALPHA, 6 ALPHA-TRIHYDROXY-5 ALPHA ANDROSTAN-17-ON - Google Patents

Abstract

The invention relates to a process of 2a, 3a, 6a-trihydroxy-5a-androstane-17-one by microbial degradation of 2a, 3a-dihydroxy-5a-cholestan-6-one. According to the invention, the substrate is added in finely divided form to a culture of a bacterial microorganism capable of sterol side chain degradation, preferably of the genus Mycobacterium, and fermented to give 2a, 3a, 6a-trihydroxy-5a-androstan-17-one. It has been found that the method is particularly effective when using selected strains of the species Mycobacterium vaccae and Mycobacterium fortuitum. The compound prepared according to the invention serves as an intermediate for the preparation of steroidal drugs.

Description

Field of application of the invention

The invention relates to a process for the preparation of 2α, 3α, 6α-trihydroxy-5α-androstan-17-one. 2a, 3a, 6a-trihydroxy-5aandrostan-17-one is a hitherto undescribed steroid, which can be used as an intermediate for the production of steroid drugs due to various reactive centers in the molecule

 The field of application of the invention lies in the pharmaceutical chemical research and industry.

Characteristic of the known state of the art

It is known that sterols, such as ζ. As cholesterol, sitosterol, stigmasterol, campesterol and ergosterol, by microorganisms from numerous genera, in particular with enzyme defect mutants of mycobacteria, to Ci ₉ steroids, such as. As 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione, can be degraded (see for example the patent JP 7973191 or JP 80148 083). By the microbial hydroxylation of 4-androstene-3,17-dione with penicillin sp. ATCC 12556 was obtained in low yields as a by-product of the 2β-hydroxy compound of 4-androstene-3,17-dione (see US Pat. No. 2,805,231). Processes for the preparation of 2a, 3a, 6a-trihydroxy-5a-androstane-17-one are hitherto unknown.

Object of the invention

The aim of the present invention is the preparation of 2α, 3α, 6α-trihydroxy-5α-androstan-17-one.

Explanation of the invention of the invention

The invention is based on the object, oin mil mhiollos Verfahron for the preparation of 2 ', 3a, 6a-trihydroxy-5aondrostan-17 · on from 2a, 3a-dihydioxy-5a-cholostan-6-one to boschroibon.

According to the invention, Clio Auigabo is achieved by formulating 2a, 3a-dihydroxy-5a-cholestan-6-one with bacterial microorganisms capable of solubilizing soyotone toto of Storolen, for example from the genus Mycobacterium. It is formontiort in Modion, the carbon quollone, such as glucose, sucrose, glycerol, and nitrogen quartet, wio boispiolswoiso Hofuoxtrakt, urea, ammonium salts and Minoralsalzo various Zusammungssotzung onthalton. Dio Formontaüon orstreckt over 24 hours to 72 hours and is at 2O ⁰ C to 37 ° C,

preferably at 28 ° C at pH values in the range of pH 6 to pH 8 under aerobic conditions. After the fermentation has ended, the fermentation medium is extracted with an organic solvent, preferably chloroform, and the crude extract is brought to dryness by distillation. Subsequently, the desired 2a, 3a, 6a-trihydroxy-5a-androstan-17-one is isolated from the product mixture by known separation methods such as column chromatography and / or preparative thin-layer chromatography and further purified by known process steps, for example recrystallization.

In the further development of the method, microorganisms of the species Mycobacterium vaccae or Mycobacterium fortuitum are used for fermentation.

The process is advantageously carried out as follows:

The starting material used for the process can be prepared according to M.Kondo and K. Mori (see Agric Biol.Chem.47.97 [1983]). A bacterial strain of the genus Mycobacterium, preferably from Mycobacterium vaccae LA 12 or Mycobacterium fortuitum N3 / 138 is on agar slants (20 g glycerol, 5 g Bacto-peptone, 3g of meat extract, 15 g agar ad 1000ml distilled .; pH7) in culture tubes at 28 ⁰ C cultivated for 48 hours to 96 hours.

A tube of this preculture is used as inoculum for each a standard flask with 50ml of a nutrient solution of the following composition: 10g glucose, 10g yeast extract, 0.5g KH ₂ PO ₄ , 0.5g K ₂ HPO ₄ , 1.5g (NH ₄ I ₂ HPO _4, 0.2g MgSO ₄ · / H ₂ O, 0.01 g FeSO ₄ · 7 H ₂ 0,0,002g ZnSO ₄ · 7 H ₂ O, distilled water to make 1000 ml;. pH 7. After shaking at 28 ⁰ C + 3 ° C after 24 hours to 36 hours with 5% to 10% of the submerged culture so obtained more prior pistons receive the same nutrient medium inoculated. these prior flask after 24 hours shaking at 28 ⁰ C ± 3 ° C, the substrate (2a, 3a-dihydroxy-5a-cholestan-6-one) in a concentration of 0.05 g / l to 1 g / l, dissolved in a little methanol, ethanol, dimethylformamide, etc. added and further fermented until a maximum substrate conversion is achievable After completion of the fermentation, the culture medium is treated with an organic solvent such as, for example, chloroform, methylene chloride, ether, ethyl acetate, butyl acetate and the like

extracted and the crude extract by distillation to dryness and chromatographed on silica gel using cnloroform / methanol or ethyl acetate / gasoline as the eluent.

In connection with production processes of androst-4-ene-3,17-dione and 9a-hydroxy-androst-4-ene-3,17-dione, in earlier applications of the central institute in the depository for microorganisms of the GDR - ZIMET, Beutenbergstr. 11, Jena, DDR-6900 under the registration numbers the microorganism strains Mycobacterium vaccae LA 12 ZIMET 11094 and Mycobacterium fortuitum N3 / 138 ZIMET10850 deposited.

2a, 3a, 6a-trihydroxy-5a-androstan-17-one possesses an aromatase inhibitor activity of the human placenta. Furthermore, it is a favorable starting material for brassinosteroids and their analogues, which are currently being tested as anti-stress factors in agriculture.

embodiments example 1

Mycobacterium vaccae LA 12 is cultured for 48 hours to 96 hours at 28 ° C. on slants of the following composition:

20g glycerol, 5g Bacto-peptone, 3g meat extract, 15g agar-agar ad 1000ml aqua dest .; pH 7.0. With the washed-out cells of each tube, inoculate 5 pieces of 500 ml culture flasks with 50 ml nutrient solution each of the following composition:

10g glucose, 10g yeast extract, 0.5 g KH ₂ PO _4, 0.5g K ₂ HPO _4, 1.5 g (NH ₄ I ₂ HPO _4, 0.2 g MgSO ₄ .7H ₂ 0,0,01 g FeSO ₄ · 7H ₂ O, 0,002g ZnSO ₄ · 7H ₂ O, distilled water ad 1000ml;. pH 7.0 After 36stündigem shaking at 28 ⁰ C it is submerged in a In'pfverhältnisvon 1:10 to inoculate another 20 Version flasks each. used 100 ml of nutrient solution. these 20 stand piston ^r C 200mg 2a, Sa-dihydroxy-OEA cholestane-e-one, dissolved in 20 ml of methanol, distributed uniformly be added after 24 hours of shaking at 28. after a further 72 hours shaking the combined culture solution is washed 3 times extracted with chloroform, the solvent was distilled off and brought the concentrated crude extract in vacuo to dryness.

The residue is chromatographed on 25 g of silica gel G 60 using CHCl ₃ / CH ₃ OH (9: 1).

Elution with chloroform / methanol (9: 1) affords 2a, 3a, 6a-trihydroxy-androstane-17-one.

Yield: 50 mg (40%), all ⁹ + 44.6 "(CHCl ₃ , c = 1.5)

I _R γ CDCl _{3 cm} 1-3350 (OH); 1740 (five ring ketone); Max

ORD: / 0 / _275-5098 ° / 0 / _3OO ^o O, / 0 / + 67O8 _32O °, a = + 161; ¹ H-NMR (200MHz, Py-d5-TMS): 50.778 (s, H ₃ -18); 0.919 (s, H ₃ -19); 2,982

(ddd.J = 13.9, 3.4 and 2 ~, 6Hz (H, -4), 3.715 (ddd, J = 10.7, 10.7 and 4.5Hz H, -6β), 4.125 (ddd , J = 11.4, 5.2 and 5.5Hz H, -2β); 4.466 (dd, J = 5.5 and 2.6Hz H, -3β);

MS m / z (%): 322 (M *, 61), 304 (72), 286 (80), 260 (100), 229 (20), 216 (48), 197 (18); HA-MS: 322.2150 (C ₁₉ H ₃₀ O ₄ ); 304, 2032 (C ₁₉ H ₂₈ O ₃ ), 286.1924 (C ₁₃ H ₂₆ O ₂ ), 260.1768 (C ₁₇ H ₂₄ O ₂ ), 216.1514 (C ₁₅ H ₂₀ O).

Example 2

When using 1 mg of 2a, 3a-dihydroxy-5a-cholestan-6-one in 10 ml of nutrient solution in a 100 ml Stoilbrustflasche, but with dom strain Mycobacterium fortuitum N3 / 138 described under Boispiol 1 Formentations- and extraction conditions by thin-layer chromatography dio formation of 2a, 3a, 6a-trihydroxy-5a-androstan-17-one are detected.

Claims (7)

claims: 1. A process for the preparation of 2a, 3a, 6a-trihydroxy-5a-androstan-17-one _/ characterized in that 2a, 3a-dihydroxy-5a-cholestan-6-one in finely divided form in concentrations up to 10 g / l of a fermentation medium inoculated with a bacterial microorganism capable of sterol side-chain degradation and containing carbon and nitrogen sources, - This approach fermented at temperatures of 20 ⁰ C to 37 ° C for a period of 24 hours to 72 hours at pH6 to pH8 under aerobic conditions and - From the culture broth, the riveted compound is obtained by extractive means. 2. The method according to claim 1, characterized in that a microorganism of the genus Mycobacterium is used for fermentation. 3. The method according to claim 1 and 2, characterized in that a microorganism of the species Mycobacterium vaccae is used for fermentation. 4. The method according to claim Ί and 2, characterized in that a microorganism of the species Mycobacterium fortuitum is used for fermentation. 5. The method according to claim 3, characterized in that the strain Mycobacterium vaccae L12 is used for fermentation. 6. The method according to claim 4, characterized in that the strain Mycobacterium fortuitum N 3/138 is used for fermentation. 7. The method according to claim 1 to 6, characterized in that 2a, 3a-dihydroxy-5a-cholestan-6-one as a solution in an organic solvent, as ultrasound suspension, as a preparation prepared by wet grinding, as prepared by micronization - As a preparation prepared by spray drying
is added.