Rearrangements of Exogenus 17-Hydroxy- 17r-methylandrosta-1,4-dien-3-one in Cultures of the Green Alga T76 Scenedesmus quadricauda Marina Della Greca, † Antonio Fiorentino, † Gabriele Pinto, ‡ Antonino Pollio, ‡ and Lucio Previtera* ,† Dipartimento di Chimica Organica & Biologica, Universita ` Federico II, via Mezzocannone 16, I-80134 Napoli, Italy, and Dipartimento di Biologia Vegetale, Universita ` Federico II, via Foria 223, I-80139 Napoli, Italy Received February 21, 1996 The transformation of exogenus substrates either by higher plant cells or by microorganisms has been re- ported in numerous studies, 1,2 while the use of microalgae in biotransformations has been only occasionally inves- tigated. 3 In a systematic study of the potential use of unicellular freshwater algae as bioreactors, we found that 5R- androsta-3,17-dione (1) and progesterone (2) were biotrans- formed by algae belonging to Rhodophyta and Chloro- phyta. 4,5 The regio- and stereoselective reduction or hydroxylation of the substrates was the main reaction, and noteworthy in cultures of T76 Scenedesmus quadri- cauda, progesterone was transformed into the 9,10-seco derivative 3 in high yield. 5 In an extension of our investigations, we have incu- bated 17-hydroxy-17R-methylandrosta-1,4-dien-3-one (4) with S. quadricauda, and the main bioproducts isolated from the cultures were two new sterols identified as 1,17R-dimethyl-1,3,5(10)-estratriene-3,17-diol (5) and (3aR,8aR,8b)-dodecahydro-5a,6R-dimethyl-6-hydroxy- 2′-methyl-as-indacene-3(R)-spiro-4′-(2′,5′-cyclohexadi- enone) (6), 6 respectively. Compound 5, mp 118 -121 °C, has a molecular formula C 20 H 28 O 2 , as suggested by the HRMS and 13 C-NMR data. In the 1 H-NMR spectrum, the aromatic H-2 and H-4 protons appear as two meta coupled doublets (J ) 2.5 Hz) at δ 6.49 and 6.42, the H-6R and H-6 protons as two multiplets at δ 2.86 and 2.66, respectively, and the H-18, H-19, and H-20 methyl protons as singlets at δ 0.95, 2.31 and 1.26, while the remaining protons are overlapped signals in the upfield region of the spectrum. In the 13 C-NMR spectrum the protonated carbons C-2 and C-4 are at δ 116.0 and 113.2, the other aromatic C-1, C-3, C-5, and C-10 carbons at δ 138.7, 152.8, 140.0, and 131.1, and the C-18, C-19, and C-20 carbons at δ 14.6, 22.3, and 26.0. The 1 H- 1 H one-bond COSY and 1 H- 13 C one-bond COSY experiments have also identified the H-8 and H-14 protons, overlapped at δ 1.56 and correlated to the carbons at δ 42.4 and 49.9; the H-9 proton at δ 2.31, correlated to the carbon at δ 46.4; and the H-11 meth- ylene protons at δ 1.32 and 2.42, correlated to the carbon at δ 27.6. In the 1 H- 13 C long-range COSY, the H-2 proton is correlated to the C-1, C-3, C-4, C-10, and C-19 carbons while the H-4 proton is correlated to the C-2, C-3, C-5 and C-10 carbons. The correlations of the signal at δ 2.31 with the C-1, C-5, and C-10 carbons may be attributable to the H-19 as well as to the H-9 protons, owing to their overlapping, while the correlation with the C-2 carbon is confidentially attributable to the H-19 protons. Finally the C-10 carbon is heterocorrelated to the H-8 proton while the C-5 carbon gives cross peaks with the H-6 protons. In a NOESY experiment, NOE interactions are evidenced between the H-4 and the H-6 protons, the H-19 and the H-2 protons, and the H-19 and the H-11 protons. The spirocompound 6, mp 138-140 °C, has the molec- ular formula C 20 H 28 O 2 , in agreement with the HRMS and 13 C-NMR data. The 1 H-NMR spectrum showes the H-1 and H-2 protons as a doublet (J ) 10.2 Hz) and a double doublet (J ) 10.2 and 1.9 Hz) at δ 6.88 and 6.20, the H-4 proton as a doublet (J ) 1.9 Hz) at δ 6.18, and the H-18, H-19, and H-20 methyls as singlets at δ 0.85, 1.97, and 1.23, respectively. In the 13 C-NMR spectrum, the olefinic C-1, C-2, C-4, and C-5 carbons are at δ 154.7, 126.8, 128.7, and 162.8, and the C-3 carbonyl carbon is at δ 186.3, the quaternary C-10 carbon at δ 52.2, and the C-19 methyl carbon at δ 19.6. On the basis of the one-bond homo- and heterocorrelations, the H-6 protons at δ 2.03 and 1.35 are correlated to the carbon at δ 34.9, the H-8 and H-9 protons at δ 1.51 and 1.91 are correlated to the carbons at δ 56.4 and 43.3, respectively, and the H-11 protons at δ 1.27 are correlated to the the methylene carbon at δ 21.6. In the 1 H- 13 C long-range COSY, the H-1 proton gives cross peaks with the C-2, C-3, C-5, C-9, and C-10 carbons, the H-2 proton is heterocorrelated to the C-1, C-3, C-4, and C-10 carbon, the H-4 proton to the C-2, C-3, C-5, C-10, and C-19 carbons, and finally the H-19 protons give cross peaks with C-1, C-4, C-5, and C-10. In the NOESY spectrum, NOE effects are present between the H-1 and the H-9 protons and among the † Dipartimento di Chimica Organica & Biologica. ‡ Dipartimento di Biologia Vegetale. (1) Suga, T.; Hirata, T. Phytochemistry 1990, 29, 2393-2406. (2) Mahato, S. B.; Majumdar, I. Phytochemistry 1993, 34, 883-898. (3) Abdul-Hajj, Y. J., Qian, X. J. Nat. Prod. 1986, 49, 244-248. (4) Fiorentino, A., Pinto, G., Pollio, A., Previtera, L. Biomed. Chem. Lett. 1991, 1, 673-674. (5) Pollio, A., Pinto, G., Della Greca, M., De Maio, A., Fiorentino, A., Previtera, L. Phytochemistry 1994, 37, 1269-1272. (6) The numbering of structure 6 in the text is referred to the parent steroid 4. 7178 J. Org. Chem. 1996, 61, 7178-7179 S0022-3263(96)00366-0 CCC: $12.00 © 1996 American Chemical Society