Phytochemistry 63 (2003) 471–474 www.elsevier.com/locate/phytochem Prenylated isoflavonoids from Millettia pervilleana§ Giovanna Palazzinoa,*, Philippe Rasoanaivob, Elena Federicia, Marcello Nicolettic, Corrado Galeffia a Laboratorio di Chimica del Farmaco, Istituto Superiore di Sanità, V. le Regina Elena 299, I-00161 Rome, Italy Institut Malgache de Recherches Appliquées, Laboratoire de Phytochimie-Pharmacologie, B.P.3833, 101 Antananarivo, Madagascar c Dipartimento di Farmacologia delle Sostanze Naturali e Fisiologia Generale, Università ‘‘La Sapienza’’, P. le A. Moro 5, I-00185 Rome, Italy b Received 5 April 2002; received in revised form 25 September 2002 Abstract From the root bark of Millettia pervilleana, which had shown significant cytotoxic activity, a 3-phenylcoumarin, named pervilleanine, two new pterocarpans, pervilline and pervillinine, and one known, emoroidocarpan, were isolated in addition to rotenone and 3a-hydroxyrotenone. The anticancer activity of two previously isolated isoflavanones, pervilleanone and 30 -O-demethylpervilleanone is reported. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Millettia pervilleana; Leguminosae; Coumarins; Pervilleanine; Pterocarpans; Pervilline; Pervillinine; Cytotoxic and anti-cancer activity 1. Introduction The genus Millettia (Leguminosae) includes about 150 subtropical species. Phytochemical research has revealed isoflavonoids as the main constituents of the genus. Our previous study on the chloroform extract of root bark of Millettia pervilleana Viguier of Madagascar (IC50 0.12 mg ml1 on KB cells, Galeffi et al., 1997) resulted in the isolation of two prenylated isoflavanones, pervilleanone (1) and 30 -O-demethylpervilleanone (2) from fraction D endowed with mild cytotoxicity (IC50 0.57 mg ml1). The anticancer activity of 1 and 2, performed by NCI on human tumor cell lines of lung, breast and CNS, is now reported (Table 1). From two more active fractions of the same extract, further isoflavonoids, namely a 3-phenylcoumarin, pervilleanine (3), two new pterocarpans, pervilline (4) and pervillinine (5), and one known pterocarpan, emor- § Presented at the 10th Congresso Nazionale della Società Italiana di Fitochimica, Florence, Italy, 7–10 May 2000. * Corresponding author. Tel.: +39-06-4990-2550; fax: +39-064938-7100. E-mail address: palazzin@iss.it (G. Palazzino). oidocarpan (6) (Machocho et al., 1995), besides rotenone and 3a-hydroxyrotenone, were isolated. 2. Results and discussion 2.1. Characterization of the constituents Fraction B (IC50 0.047 mg ml1 on KB cells) obtained from the chloroform extract of M. pervilleana (Galeffi et al., 1997) was submitted to counter-current distribution (CCD) and two main substances, 3 and 6, were thus isolated. The less polar one, pervilleanine (3), C22H18O6 (HR–EI–MS, m/z 378.1124 [M]+, calc. 378.1103), had an UV spectrum (see Section 3.2.1) showing extended conjugation. Its 1H and 13C NMR spectra obtained by HETCOR and DEPT experiments showed the presence of a methylenedioxy group (
H 5.97, s) and an ortho,ortho disubstituted methoxy group (
H 3.54,
C 60.9). The fourth oxygen atom of the molecule belonged to a a,adimethylpyrane ring (
H 1.43, s, 2 Me; 5.69 and 6.89, both d, J=10.2 Hz) originated by cyclisation of a prenyl group, whereas the last two oxygen atoms were those of a coumarin unit (
CO 163.6). The presence among the 13 C NMR signals of 3 of an ortho,ortho-dioxygen substituted oxygen-bearing carbon,
C 132.5, and of two ortho hydrogens,
H 7.55 and 6.71, d, J=8.2 Hz (the latter shif- 0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(02)00489-2 472 G. Palazzino et al. / Phytochemistry 63 (2003) 471–474 ted upfield as well the corresponding carbon,
C 113.1) accounted for a 2-methoxy-3,4-methylenedioxyphenyl ring linked in position 3 to the coumarin system. Two of the three remaining aromatic hydrogens,
H 6.92, d, and 6.86, d, in the peri position, at C-4 and C-5, were mutually coupled (J=1.7 Hz) whereas the last hydrogen,
H 6.84, s, was attached to C-8,
C 108.2, which is ortho,ortho-dioxygen substituted. The structure of pervilleanine, 3, was thus fully established. Substance 6, C21H18O5 (HR–EI–MS, m/z 350.1093 [M]+, calc. 350.1154), was emoroidocarpan, a pterocarpan isolated from Tephrosia emeroides A. Rich. (Machocho et al., 1995). The complete assignment of its 13 C NMR frequencies is reported in Table 2. The absolute R configuration of both chiral centres 6a and 11a (which was earlier not reported, as well the specific optical rotation) can be assigned on the basis of cisdiaxial relationship of their hydrogens (J=6.8 Hz) and the negative Cotton effect of the CD curve (Baruah et al., 1984). Fraction C (IC50 0.08 mg ml1 on KB cells) was likewise submitted to CCD and column chromatography and four substances were isolated in order of increasing polarity, two new pterocarpans, pervilline (4) and pervillinine (5), rotenone and 3a-hydroxyrotenone. Pervilline (4), C21H20O5 (HR–EI–MS, m/z 352.1263 [M]+, calc. 352.1311), was a pterocarpan as demonstrated by the sequence O–CH2–CH(Ar)–CH–O (CH2, Table 1 3-Cell line assay for the primary anticancer screening of 1 and 2 at conc. 104 M Compound Growth percentages of the treated cell lines Pervilleanone, 1 30 -O-Demethylpervilleanone, 2 (Lung) NCI-H460 (Breast) MCF7 (CNS) SF-268 36 2 38 56 27 42 Negative values of the growth percentage correspond to Ti <Tz, where Tz is the cell population at time zero and Ti is the cell population in the presence of the drug after 48 h. Table 2 13 C and 1H NMR spectral data of pterocarpans 4, 5 and 6 Position 4 (CDCl3)
C 1 1a 2 3 4 4a 6 6a 6b 7 8 9 10 10a 11a 10 20 30 40 50 OMe OCH2O 5 (CDCl3)
H (J in Hz) 126.8 7.33, 111.7 120.7 161.2 98.1 6.35, 156.0 66.5 3.60, 40.1 3.50, 121.7 114.7 6.72, 103.6 6.42, 148.0 130.6 146.1 79.8 5.52, 33.8 2.97, 86.6 5.16, 143.8 17.1 1.76, 112.1 5.05; 56.4 3.84,
C
H (J in Hz) 126.3 7.33, n.o. 120.8 161.2 s 98.2 6.35, 156.1 dd (10.8; 10.6); 4.19, dd (10.8; 4.9) 66.6 3.60, m (10.6; 6.6;4.9) 40.3 3.50, 118.1 d (8.0) 110.4 6.79, d (8.0) 139.9 146.9 94.7 6.41, 152.8 d (6.6) 78.7 5.52, dd (15.8; 7.6); 3.26, dd (15.8; 9.8) 33.9 2.97, dd (9.8; 7.6) 86.7 5.16, 143.8 s 17.2 1.76, 4.88, br s 112.1 5.05; s 56.2 3.84, s 6 (CDCl3) dC
H (J in Hz) 126.2 7.20, 111.8 120.8 161.4 s 98.2 6.38, 156.3 t (11.0); 4.19, dd (11.0; 5.0) 66.5 3.55, m (11.0; 6.7; 5.0) 40.1 3.40, 118.1* s 104.7 6.67, 141.8 147.5 s 93.7* 6.33, 154.1* d (6.7) 79.0 5.40, dd (15.7; 7.7); 3.26, dd (15.7; 9.8) 33.8 2.94, dd (9.8; 7.7) 86.6 5.12, 143.7 s 17.1 1.70, 4.88, br s 112.1 5.01; s 101.2 5.83; s * The assignments of Machocho et al. (1995) for these carbons are not correct. s s t (11.4); 4.16, dd (11.4; 5.1) m (11.4; 6.8; 5.1) s s d (6.8) dd (15.6; 8.0); 3.23, dd (15.6; 9.8) dd (9.8; 8.0) s 4.85, d (1.1) 5.89, d (2.5) G. Palazzino et al. / Phytochemistry 63 (2003) 471–474
H 3.60, dd, J=10.8; 10.6 Hz, and 4.19, dd, J=10.8; 4.9 Hz; CH–Ar, 3.50, m, J=10.6; 6.6; 4.9 Hz; CH–O 5.52, d, J=6.6 Hz). The substitution of the benzofuran ring C was shown by the COLOC connectivities of C-10a (
C 146.1) and C-9 (
C 148.0) with the hydrogen at
H 6.72 (d, J=8.0 Hz) and by the connectivity of C-6b (
C 121.7) with the hydrogen at
H 6.42 (d, J=8.0 Hz). These two ortho hydrogens are in position 7 and 8, respectively. The connectivity of C-9 with the methoxy group at
H 3.84 and the 13C resonance of the latter (
C 56.4) accounted for its mono-ortho substituted position 9. The remaining hydroxy group was thus located in position 10 in agreement with the upfield resonance of C-10 (
C 130.6) due to the ortho,ortho-dioxygen substitution. Moreover in 4 a prenyl group was linked to ring B to give a methylethenyldihydrofuran-fused ring (H2-10 ,
H 2.97, dd, and 3.26, dd; H-20 , 5.16, dd; H-40 , 1.76, s; H2-50 , 4.88 and 5.05, br s). The latter is located as in emoroidocarpan in agreement with the presence of an ortho,ortho-dioxygen substituted methine (
H 6.35, s,
C 98.1) in position 4. The absolute configuration of 4 is identical to that of emoroidocarpan on account of the coupling constant J6a/11a (6.6 Hz) and the negative Cotton effect. Pervillinine (5), C21H20O5 (HR–EI–MS, m/z 352.1251 [M]+, calc. 352.1311), differed from pervilline on the 473 substitution in ring C (see Table 2). In the former, the two hydrogens at
H 6.79, s and
H 6.41, s were located in the para positions 7 and 10, respectively, and the methoxy group (
H 3.84) in position 9 inasmuch its irradiation by NOE experiments enhanced the signal of the more shielded H-10 (
H 6.41). The hydroxy group was thus located in position 8. 2.2. In vitro anticancer activity Isoflavonoids have shown activity in the search for antiviral and antineoplastic or cancer chemopreventive agents of plant origin. Thus, some isoflavonoids from genus Millettia showed inhibitory effects against Epstein–Barr virus (Ito et al., 2000), whereas those of M. pachycarpa inhibited the activities of murine retroviral reverse transcriptase and human DNApolymerases (Ono et al., 1989). Rotenone and 3a-hydroxyrotenone, which were found in the cytotoxic fraction C of M. pervilleana, showed inhibition of TPA-induced ornithine decarboxylase at the level of its mRNA expression and were therefore regarded as promising cancer chemopreventive agents (Gerhauser et al., 1995). Pervilleanone (1) and 30 -O-demethylpervilleanone (2), isolated from fraction D of M. pervilleana endowed with mild cytotoxicity, showed growth inhibition of the 474 G. Palazzino et al. / Phytochemistry 63 (2003) 471–474 human cancer cell lines of lung, breast and CNS (Table 1) used in the NCI antitumor prescreen. 3. Experimental 3.1. General A Craig-Post apparatus (200 stages, 10:10 ml, upper and lower phase) was used for the separation by CCD. TLC: silica gel F254, cyclohexane–EtOAc (1:1). CD: Jasco 710. 1H and 13C NMR: 500.13 and 125.77 MHz, respectively, Bruker AM 500. Chemical shifts are given in
(ppm) from internal TMS. EI–MS: 70 eV, HP 5989A. HR–EI–MS: VG 7070 EQ-HF. The bioassay tests on lung, breast and CNS cells were performed by the NCI, Bethesda (USA). 3.2. Separation Fraction B (1.4 g) was submitted to CCD with solvent system H2O–Me2CO-EtOH–cyclohexane (2:6:2:8). Two chromatographically pure fractions, pervilleanine (3, 39 mg) and emoroidocarpan (6, 68 mg) were thus obtained. Fraction C (1.4 g) was submitted to CCD with solvent system H2O–Me2CO–EtOH–cyclohexane (5:4:4:10) and four fractions were obtained. Each fraction was subsequently purified by column chromatography on silica gel 60 (70–230 mesh), eluent cyclohexane–EtOAc (8:2) and thus pervilline (4, 26 mg), pervillinine (5, 31 mg), rotenone (89 mg) and 3a-hydroxyrotenone (56 mg) were obtained in order of increasing polarity. The last two substances were identified by comparison of their physico-chemical data. 3.2.1. Pervilleanine (3) Pale crystals from n-hexane, mp 177–179  C. UV (MeOH): lmax nm (log e): 324 (4.09), 239 (4.02). 1H NMR (CDCl3):
1.43 (6H, s, 2 Me-400 ); 3.54 (3H, s, OMe); 5.69 (1H, d, J=10.2 Hz, H-200 ); 5.97 (2H, s, O– CH2–O); 6.71 (1H, d, J=8.2 Hz, H-50 ); 6.84 (1H, s, H8); 6.86 (1H, d, J=1.7 Hz, H-5); 6.89 (1H, d, J=10.2 Hz, H-100 ); 6.92 (1H, d, J=1.7 Hz, H-4); 7.55 (1H, d, J=8.2 Hz, H-60 ). 13C NMR (CDCl3):
28.0 (2 Me, C400 ); 60.9 (OMe); 77.4 (C-300 ); 101.2 (O–CH2–O); 108.1 (C-4a); 108.2 (C-8); 108.8 (C-6); 110.9 (C-10 ); 111.4 (C4); 113.1 (C-50 ) 115.4 (C-100 ); 123.8 (C-60 ); 124.7 (C-5); 125.9 (C-3); 130.3 (C-200 ); 132.5 (C-30 ); 147.5 (C-7); 147.6 (C-40 ); 148.4 (C-20 ); 156.2 (C-8a); 163.6 (C-2). EI– MS, m/z (rel. int.): 378 ([M]+, 44), 363 (100), 320 (17), 187 (13), 159 (15), 145 (10). Molecular formula C22H18O6, HR–EI–MS, m/z: 378.1124 [M]+, calc. 378.1103. 3.2.2. Pervilline (4) White powder from n-hexane, mp 112–114  C. [a]20 D 192 (CHCl3; c 0.12). CD: [y]240 44 600 (MeOH; c 0.013). UV (MeOH): lmax nm (log e): 298 (3.9), 220 (4.1). 1H and 13C NMR data in Table 2. EI–MS, m/z (rel. int.): 352 ([M]+, 22), 350 (24), 337 (22), 162 (37), 149 (100). Molecular formula C21H20O5, HR–EI–MS, m/z: 352.1263 [M]+, calc. 352.1311. 3.2.3. Pervillinine (5) Yellow powder from n-hexane, mp 152–154  C. [a]20 D 189 (CHCl3; c 0.07). CD: [y]240 46 800 (MeOH; c 0.011). UV (MeOH): lmax nm (log e): 298 (3.9); 220 (4.1). 1H and 13CNMR data in Table 2. EI–MS, m/z (rel. int.): 352 ([M]+, 100), 337 (58), 322 (18). Molecular formula C21H20O5, HR–EI–MS, m/z: 352.1251 [M]+, calc. 352.1311. 3.2.4. Emoroidocarpan (6) White crystals from n-hexane, mp 189–190  C. [a]20 D 268 (CHCl3; c 0.11). CD: [y]240 45 200 (MeOH; c 0.012). UV (MeOH): lmax nm (log e): 300 (2.9); 220 (3.9). 1 H and 13C NMR data in Table 2. EI–MS, m/z (rel.int.): 350 ([M]+, 100), 335 (87). Molecular formula C21H18O5, HR–EI–MS, m/z: 350.1093 [M]+, calc. 350.1154. Acknowledgements The authors are greatly indebted to the National Cancer Institute, Bethesda (USA), for the anticancer assays. References Baruah, P., Barua, N.C., Sharma, R.P., Baruah, J.N., Kulanthaivel, P., Herz, W., 1984. Flavonoids from Millettia pulchra. Phytochemistry 23, 443–447. Galeffi, C., Rasoanaivo, P., Federici, E., Palazzino, G., Nicoletti, M., Rasolondratovo, B., 1997. Two prenylated isoflavanones from Millettia pervilleana. Phytochemistry 45, 189–192. Gerhauser, C., Mar, W., Lee, S.K., Suh, N., Luo, Y., Kosmeder, J., Luyengi, L., Fong, H.H.S., King-Horn, A.D., Moriarty, R.M., Mehta, R.G., Constantinou, A., Moon, R.C., Pezzuto, J.M., 1995. Rotenoids mediate potent cancer chemopreventive activity through transcriptional regulation of ornithine decarboxylase. Nature Medicine 1, 260–266. Ito, C., Itoigawa, M., Tan, H.T.W., Tokuda, H., Mou, X.Y., Mukainaka, T., Ishikawa, T., Nishino, H., Furukawa, H., 2000. Antitumor-promoting effects of isoflavonoids on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Cancer Letters 152, 187–192. Machocho, A.K., Lwande, W., Jondiko, J.I., Moreka, L.V.C., Hassanali, A., 1995. Three new flavonoids from the root of Tephrosia emoroides and their antifeedant activity against the larvae of the spotted stalk borer Chilo partellus Swinhoe. International Journal of Pharmacognosy 33, 222–227. Ono, K., Nakane, H., Meng, Z.M., Ose, Y., Sakai, Y., Mizuno, M., 1989. Differential inhibitory effects of various herb extracts on the activities of reverse transcriptase and various deoxyribonucleic acid (DNA) polymerase. Chemical and Pharmaceutical Bulletin 37, 1810–1812.