Letters Planta Med 67 (2001) 191 Crinane and Lycorane Type Alkaloids from Zephyranthes citrina 1 1 2 María R. Herrera , Alex K. Machocho , Reto Brun , 1 1 1, Francesc Viladomat , Carles Codina , Jaume Bastida * 1 Departament de Productes Naturals, Facultat de Farmàcia, 2 Swiss Tropical Institute, Protozoology Laboratory, Basel, Universitat de Barcelona, Barcelona, Spain Switzerland Received: March 30, 2000; Accepted: May 13, 2000 Abstract: Eight alkaloids have been isolated from Zephyranthes citrina (Amaryllidaceae). The alkaloid oxomaritidine is reported here for the first time from a natural source. The structure and stereochemistry of the alkaloids were determined by physical Baker (Amaryllidaceae, tribe Hippeastreae, subtribe Zephyranthinae) (1) commonly known as ªbrujita amarillaº (yellow little witch) in Cuba, inhabits humid places and appears immediately after the start of rainy season (2). The bulbs, leaves, or whole plant of closely related species are used in traditional medicine for treatment of various diseases. The decoction of leaves of Herb. has been used in South Africa as a remedy for diabetes mellitus (3) and is reported in Per‚ for treating tumours (4). alkaloids are reported to have cytotoxic activities (5). Previous phytochemical analysis of led to the isolation of four alkaloids (galanthine, haemanthamine, lycorine and lycorenine) (6), which were isolated in the present study except lycorenine. Additionally, further five alkaloids were isolated and oxomaritidine (1) is being reported here for the first time. Haemanthamine, the main constituent alkaloid was reported to have inhibitory activity on the growth of HeLa cells and protein synthesis (7) and as cytotoxic agent against tumoral cells (MOLT 4) (8). Haemanthidine, the other principal constituent, was reported as a cytotoxic agent against various human tumoral cell lines (9). Galanthine showed a high inhibitory capacity of ascorbic acid biosynthesis in potatoes (10), while maritidine exhibited antineoplastic activity (11). Zephyranthes citrina Z. candida Z. parulla Z. carinata Z. citrina The absolute configuration of the crinane type alkaloids was determined by circular dichroism (CD). The curves of haemanthamine, haemanthidine, vittatine, maritidine and oxomaritidine (1) showed a minimum around 250 nm and a maximum around 290 nm, which was in agreement with alkaloids with the 5,10b-ethano bridge in an a orientation (12). Oxomaritidine (1) has been reported as a synthetic intermediate compound in the synthesis of maritidine and in the conversion of the latter into epimaritidine (13). The IR spectra of compound 1 displayed an absorption band around 1680 cm±1 suggesting the presence of a carbonyl group. Its EIMS showed the base peak at 285 which represented the molecular ion. The fragments closely compared with those of oxocrinine m/z Planta Med 67 (2001) 191±193  Georg Thieme Verlag Stuttgart New York ISSN: 0032-0943 · (14) and were in agreement with the fragmentation patterns of crinane type alkaloids with no substitution on the 5,10bethano bridge and an unsaturation between C-1 and C-2. The 1H-NMR spectrum of 1 was similar to the one of oxocrinine (14), with the only notable difference being that spectrum of compound 1 lacked the signals associated with methylenedioxy group. However, the spectrum showed the presence of two aromatic methoxy groups at d 3.89 and 3.82, which were bonded to C-9 and C-8. The spectrum displayed two singlets at d 6.89 and 6.54 assignable to H-10 and H-7, respectively, where H-10 showed contours with C-10b and H-7 showed with C-6, in the HMBC spectrum (15). Further support was noted in the ROESY experiment, where H-10 showed correlations with H-1 and the protons of the methoxy group at C-9. On the other hand, the H-7 proton showed correlations with both H6 protons as well as the methoxy group assigned at C-8. Two other significant signals at d 7.68 (d) and 6.10 (dd) were assigned to the olefinic protons H-1 and H-2 respectively, where H-1 correlated with C-3, C-4a, C-10a and C-10b and H-2 with C-4 and C-10b in the HMBC experiment (Table 1). A long range W coupling ( = 1 Hz) between H-2 and H-4b was also noted. A typical AB system is observed at d 4.43 and 3.82 corresponding to H-6b and H-6a respectively, as the former appears on the same side with the lone pair of electrons of nitrogen. The signal centred at d 2.48 appearing as dd with large J Downloaded by: Universidad de Barcelona. Copyrighted material. and spectroscopic methods. Planta Med 67 (2001) Letters coupling constants was assigned to H-4a due to its -diaxial orientation with H-4a. The ROESY contours between H4a and H-12 exo (d 3.55) and H-11 exo (d 2.18) assisted in the assignment of the protons of the bridge. haemanthidine showed no activity. All tested compounds showed no activity against The 13C-NMR spectrum of compound 1 displayed seventeen carbon atoms, which was consistent with the proposed structure, and closely compared with the spectrum of oxocrinine except for the two signals at d 55.9 and 56.1 assignable to aromatic methoxy groups at C-8 and C-9 respectively (Table 1). The aliphatic shift range consisted of one singlet (C-10b), one doublet (C-4a) and four triplets (C-4, C-6, C-11 and C-12). The four lowfield doublets were assigned to the aromatic (C-7 and C-10) and the two olefinic carbons (C-1 and C-2). The five lowfield singlets were assigned by HMBC to the carbonyl group at C-3 (d 198.1) and the four quaternary carbons of ring A. M.p.©s were uncorrected. Optical rotations: Perkin-Elmer 241 Polarimeter. Elemental analysis: Carlo Erba-Fisons EA 1108. IR spectra: Perkin-Elmer 1600 FTIR series Spectrometer in dry film on NaCl cell. CD: Jasco J-700 Spectropolarimeter. EIMS: Hewlett Packard 5989A Mass Spectrometer at 70 eV. 1H-NMR, 13C-NMR, DEPT, 1H COSY, HMQC, HMBC (60 and 110 ms) and ROESY (300 ms) spectra: Varian VXR 500, using CDCl3 (except for lycorine where CD3OD was used) and TMS as internal standard. Chemical shifts are reported in d units (ppm) and coupling constants ( ) in Hz. Silica gel SDS silice 60 A CC (6 ± 35 mm) were used for VLC. Silica gel 60 F254 Macherey-Nagel for analytic and prep. TLC. Spots on chromatograms were detected under UV light (254 nm) and by Dragendorff©s reagent. trans Haemanthidine showed some biological activity by assays against (strain STIB900, stage trypomastigotes) with an IC50 of 1.1 mg/ml. Melarsoprol was used as the standard (IC50 of 0.002 mg/ml). Galanthine and compound 1 showed mild activity with IC50 values of 3.1 and 2.8 mg/ml, respectively. Maritidine, which was also tested, showed no activity. Haemanthidine also showed some activity against (strain Tulahuen C4, stage trypomastigotes) with an IC50 of 1.4 mg/ml. The other compounds that were screened showed no activity. Benznidazole was used as standard (IC50 of 0.6 mg/ml). A mild activity was observed in galanthine against (strain K1, stage IEF) with an IC50 of 0.2 mg/ml. Chloroquine was used as standard (IC50 of 0.04 mg/ml). Compound 1, maritidine and in vitro Trypanosoma brucei rhodesiense Trypanosoma cruzi Plasmodium falciparum Table 1 1 H-NMR, HMQC and HMBC data of compound Leishmania donovani. Materials and Methods J Plants of were collected in the spring of 1998 in Camagüey (Cuba), and a voucher specimen (No. 1044) has been deposited in the Herbarium of the Instituto Superior Pedagógico JosØ Martí, Camagüey. Z. citrina Dry whole plant (aerial part and bulbs) of (193 g) was crushed and macerated with EtOH for 48 h (3 ” 1.5 L). The extract was evaporated under reduced pressure, the residue dissolved in water (100 mL) and acidified with 5 % H2SO4 to pH 3 ±4. After removing the neutral material with Et2O (3 ” 50 mL), the acidic solution was basified with 10% aqueous NH4OH to pH 8 ± 9. The solution was extracted with EtOAc (5 ” 50 mL) and finally, with EtOAc-MeOH (9 :1) (3 ” 50 mL). AfZ. citrina 1 Correlated C-atom Proton d H-1 7.68 d (10.0) 149.5 d C-3, C-4a, C-10a, C-10b H-2 6.10 dd (10.0; 1.0) 128.7 d C-4, C-10b a H-4b H HMQC HMBC 198.1 s (C-3) 2.48 dd (17.0; 13.0) 40.2 t C-3, C-4a, C-10b 2.69 ddd (17.0; 5.5, 1.0) 40.2 t C-2, C-3, C-4a, C-10b, C-11 H-4a 3.66 dd (13.0, 5.5) 68.9 d C-4, C-6, C-10a, C-11, C-12 H-6 3.82 d (16.0) 61.5 t C-4a, C-6a, C-7, C-10a, C-12 4.43 d (16.0) 61.5 t C-6a, C-7, C-10a, C-11, C-12 H-4 a H-6b 125.0 s (C-6a) H-7 6.54 s 110.0 d C-6, C-6a, C-8, C-9, C-10, C-10a 147.9 s (C-8) 147.6 s (C-9) H-10 6.89 s 105.4 d C-6a, C-7, C-8, C-9, C-10a, C-10b 134.7 s (C-10a) 44.4 s (C-10b) H-11 endo 2.39 ddd (12.5; 9.0; 3.5) 44.8 t H-11 exo 2.18 ddd (12.5; 10.5; 6.5) 44.8 t C-4a, C-10a, C-10b C-1, C-10a, C-10b, C-12 H-12 endo 3.01 ddd (13.0, 9.0; 6.5) 54.1 t C-4a, C-6, C-11 H-12 exo 3.55 ddd (13.0; 10.5; 3.5) 54.1 t C-6 8-OMe 3.82 s 55.9 q C-8 9-OMe 3.89 s 56.1 q C-9 Chemical shifts in ppm rel. to TMS. Coupling constants ( J) in Hz. C-multiplicities were determined by DEPT data. Downloaded by: Universidad de Barcelona. Copyrighted material. 192 Letters (1): Found: C, 71.23; H, 6.87; N, 4.96. C17H19NO3 requires: C, 71.54; H, 6.71; N, 4.91 %. M.p. 140± 142 8C. [a]D20: +558 (MeOH, c 0.29). CD [Q]294 + 7253, [Q]246 ± 715. IR: nmax = 2925, 2852, 1680 (CO), 1513, 1260, 1219, 1133, 1037, 761 cm±1. EIMS (70 eV): m/z (rel. int.) = 285 [M]+ (100), 284 (12), 257 (18), 256 (27), 242 (16), 228 (25), 216 (17), 202 (59), 187 (53), 144 (19). 1H-NMR (500 MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) see Table 1. Oxomaritidine [a]D20: ± 958 (CHCl3, c 0.69), m.p. 160± 162 8C; (17), (18), [a]D20: +388 (CHCl3, c 0.45), m.p. 195 ± 198 8C; haemanthidine (18), [a]D20: ± 148 (CHCl3, c 0.28), m.p. 176 ± 179 8C; maritidine (15), [a]D20: +278 (MeOH, c 0.78), m.p. 250± 252 8C; lycorine (19), [a]D20: ± 678 (EtOH, c 0.1), m.p. 260 ±262 8C; vittatine (17), (18), [a]D20: +268 (MeOH, c 0.25), m.p. 206 ±208 8C; narcissidine (20), [a]D20: ± 298 (CHCl3, c 0.32), m.p. 203 ± 205 8C, were identified by comparison of their properties (TLC, mp, [a]D, 1H and 13C NMR) with those of authentic samples obtained from other plant sources. Galanthine(16), haemanthamine Acknowledgements Part of this work was financially supported by CIRIT-CICYT project (QFN95-4711) and the Comissionat per a Universitats i Recerca, Generalitat de Catalunya. MRH and AKM are grateful to the Ministerio de Educación y Ciencia for doctoral fellowships. The authors are very much indebted to both D. Odel Carrazana and Ms. Rafael MilanØs for collection and identification of plant material, respectively. References Müller-Doblies D, Müller-Doblies U. Tribes, subtribes and some species combinations in Amaryllidaceae. Feddes Repertorium 1996; 107: 1 ±9 2 León Hno. Contribuciones Ocasionales del Museo de Historia Natural de Salle. In: Flora de Cuba I. La Habana, Cuba: 1946: 441 3 Watt JM, Breyer-Brandwijk MG. The medicinal and poisonous plants of southern and eastern Africa. Edinburgh, London: E. and S. Livingston, Ltd., 1962 4 Trimiæo Z, Castillo M, Spenglers I. Estudio Preliminar de Zephyranthes eggersiana Urban. Revista Cubana de Farmacia 1989; 23: 147 ±50 5 Keisuke K, Motoh M, Makoto I, Yukio O. Two alkaloids from Zephyranthes carinata. Phytochemistry 1998; 48: 1199 ±202 1 HG, Döpke W, Stender W. Alkaloide aus Crinum, Zephyrund Clivia Arten. Chemische Berichte 1957; 90: 2203± 6 7 JimØnez A, Santos A, Alonso G, Vµzquez D. Inhibitors of protein synthesis in eukaryotic cells. 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Copyrighted material. ter combining the extracts and drying in vacuo, the brown gummy residue (2 g) was subjected to VLC (silica gel 35 g) eluting with hexane (2 L), increasing the polarity with EtOAc (1 L per each eluent at intervals of 10%) and later up to EtOAcMeOH (8 :2) (1 L per each eluent at intervals of 5%), where three fractions containing alkaloids were obtained. Fr. I (430 mg) was subjected to preparative TLC using EtOAc in an NH3 atmosphere to afford compound 1 (5 mg, Rf 0.46), galanthine (34 mg, Rf 0.76), haemanthamine (111 mg, Rf 0.57), haemanthidine (107 mg, Rf 0.38) and lycorine (7 mg, Rf 0.30). Maritidine (23 mg) crystallised out in methanol from fr. II (130 mg). The alkaloids in the solution were separated by preparative TLC as fr. I, using EtOAc-MeOH (1 :1) as eluent yielded more maritidine (26 mg, Rf 0.45), narcissidine (4 mg, Rf 0.51) and vittatine (5 mg, Rf 0.63). Fr. III (50 mg) was treated as fr. II and afforded more maritidine (5 mg) and vittatine (2 mg). Planta Med 67 (2001)