Chemistry

N. N. Suvorov, L. V. Sokolova, V. M. Ryzhkova, G. G. Dvoryantseva

Microbiological \(20\alpha\)-Reduction of Ketosteroids by Means of Bacillus megatherium

(Presented by Academician M. M. Shemyakin, March 27, 1963)

It had previously been established that the strain VNIHFI-1 isolated by us is capable of carrying out selective deacetylation of 21-acetates of corticosteroids \((^1)\). A thorough morphological and biochemical study of this microorganism showed that, systematically, it is indeed Bacillus megatherium. Since there are indications in the literature of the ability of various strains of the latter to carry out 15 \(\beta\)-hydroxylation of steroids \((^{2,3})\), we decided to study the relation of strain VNIHFI-1 to steroids that do not contain a 21-acetoxy group. This was all the more interesting because even in the case of the latter, when a nutrient medium rich in nitrogen was used, formation of more polar substances was observed alongside deacetylation, according to paper-chromatography data. As the object of the study, \(16\alpha,17\alpha\)-oxidoprogesterone (I) was chosen, giving the maximum yield of the biotransformation product. Upon incubation of (I) with a growing culture of Bac. megatherium VNIHFI-1, it is possible to obtain substance (II) of composition \(\mathrm{C}_{21}\mathrm{H}_{30}\mathrm{O}_3\), forming a monoacetate (IIa). The ultraviolet spectrum of (II) showed that the \(\Delta^4\)-3-keto unsaturated grouping in ring A remained unaffected. The data of quantitative infrared spectroscopy (determination of the frequencies and integral intensities of the carbonyl and hydroxyl groups of II and IIa \((^4)\)), as well as the presence in the spectra of II and IIa of frequencies characteristic of the \(\Delta^4\)-3-keto unsaturated grouping and of the \(\alpha\)-oxide ring, clearly showed that in the course of the microbiological transformation of (I) reduction of the 20-keto group occurred (see Table 1), which was also confirmed by reverse oxidation of (II) with chromic acid to the starting oxidoprogesterone (I). It remained to settle the question of the configuration of the

Table 1

20-hydroxyl group (II). Substance (II) differed in its constants from \(16\alpha,17\alpha\)-oxido-\(\Delta^4\)-pregnenol-20\(\beta\)-one-3 (III), obtained according to the data of Camerino et al. \((^5)\) by reduction of oxidoprogesterone (I) with sodium borohydride. The acetates of these substances IIa and IIIa are also different.

Thus, Bac. megatherium VNIKhFI-1 carries out the microbiological reduction of 16α,17α-oxidoprogesterone (I) to 16α,17α-oxido-Δ⁴-pregnenol-20α-ol-3 (II):

\[ \begin{array}{ccc} \text{(III)} & \xleftarrow{\ \mathrm{NaBH_4}\ } & \text{(I)} \ \xrightleftharpoons[\mathrm{CrO_3}]{\textit{B. megath.}}\ \text{(II)} \end{array} \]

The possibility of microbiological 20α-reduction is of great interest, since, as a rule, in processes of microbiological transformation 20β-hydroxy compounds are formed. 20α-Reduction has apparently been observed only in the case of the yeast Rhodotorula longissima \((^{6})\), although it is quite characteristic of enzyme systems of higher animals \((^{7})\).

Experimental part

IR spectra were obtained with a UR-10 instrument; UV spectra (in alcohol) with an SF-4 spectrophotometer.

16α,17α-Oxido-Δ⁴-pregnenol-20α-ol-3 (II). A culture of Bac. megatherium VNIKhFI-1 is grown on corn-glucose agar for 2 days at 37°. The fermentation process is conducted for 48 h in a fermenter (stirring rate, 260 rpm; air supply, 1 liter per 1 liter of medium per minute; temperature, 37°) in a medium containing 0.2% corn extract, 0.2% ammonium succinate, and 1.5% glucose. The starting 16α,17α-oxidoprogesterone is introduced as a solution of 1 g of (I) in 50 ml of acetone into 8 liters of culture medium. The process is monitored by paper chromatography in the formamide—benzene system. The transformation product is extracted with chloroform. Yield of (II), 0.7 g. M.p. 242.5–245° (from isopropyl alcohol); \(R_f\ 0.53\); \([\alpha]_{D}^{20} +114^\circ\) \((C = 1,\ \mathrm{CHCl_3})\); \(\lambda_{\max} = 240\ \mathrm{m}\mu\) \((\log \varepsilon = 4.22)\).

\[ \begin{aligned} &\text{Found, \%: } &&\mathrm{C}\ 76.42;\ \mathrm{H}\ 9.40\\ &\mathrm{C}_{21}\mathrm{H}_{30}\mathrm{O}_{3}.\ \text{Calculated, \%: } &&\mathrm{C}\ 76.33;\ \mathrm{H}\ 9.15 \end{aligned} \]

To a solution of 0.10 g of II in 5 ml of acetic acid, 0.05 g of \(\mathrm{CrO_3}\) in 1 ml of the same acid is added. After 12 h the excess oxidant is destroyed with \(\mathrm{Na_2SO_3}\). As a result of the usual workup, 0.07 g of 16α,17α-oxidoprogesterone (I), m.p. 202–203°, is obtained.

Acetate of 16α,17α-oxido-Δ⁴-pregnenol-20α-ol-3 (IIa) is obtained by acetylating 0.59 g of II with acetic anhydride (1.2 ml) in pyridine (6 ml) for 16 h at room temperature. Yield 0.52 g, m.p. 228.5–229.5° (from \(\mathrm{CH_3OH}\)), \([\alpha]_{D}^{20} +78^\circ\) \((C = 1,\ \mathrm{CHCl_3})\); \(\lambda_{\max} = 240\ \mathrm{m}\mu\) \((\log \varepsilon = 4.17)\).

\[ \begin{aligned} &\text{Found, \%: } &&\mathrm{C}\ 74.27;\ \mathrm{H}\ 8.47\\ &\mathrm{C}_{23}\mathrm{H}_{32}\mathrm{O}_{4}.\ \text{Calculated, \%: } &&\mathrm{C}\ 74.13;\ \mathrm{H}\ 8.66 \end{aligned} \]

All-Union Scientific Research
Chemical-Pharmaceutical Institute
named after S. Ordzhonikidze

Institute of Chemistry of Natural Compounds
Academy of Sciences of the USSR

Received
26 III 1963

References

1. N. N. Suvorov, L. V. Sokolova et al., DAN, 132, 1325 (1960).
2. W. McAleer, T. Jacosb et al., Arch. Biochem. and Biophys., 73, 127 (1958).
3. H. Herzog, M. Gentles et al., J. Org. Chem., 24, 691 (1959).
4. G. G. Dvoryantseva, Yu. N. Sheinker, ZhAKh, 7, 883 (1962); F. Meda, Gazz. chim. ital., 87, 52 (1957).
5. B. Camerino, C. Alberti, Gazz. chim. ital., 85, 51 (1955).
6. F. Carvajal, O. Vitale et al., J. Org. Chem., 24, 695 (1959).
7. L. Fieser, M. Fieser, Steroids, N. Y., 1959, p. 569.