0031-9422(94)00639-3 Pergamon Phytochemistry, Vol. 38, No. 2, pp. 549-551, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 A L K A L O I D S F R O M NARCISSUS TORTUOSUS* JAUME BASTIDA,JUAN MARCOS FERNANDEZ, FRANCESCVILADOMAT,CARLESCODINA and GABRIELDE LA FUENTE~: Departament de Productes Naturals, Facultat de Farmficia, Universitat de Barcelona, E-08028 Barcelona, Spain; tInstituto de Productos Naturales y Agrobiologia, CPNO 'Antonio Gonzfilez', CSIC-Universidad de La Laguna, La Laguna, Tenerife, Spain (Received 7 March 1994) Key Word Index--Narcissus tortuosus; Amaryllidaceae; alkaloids; tortuosine; lycorine; 2 D N M R spectroscopy. Abstract--Whole plants of Narcissus tortuosus were found to contain tortuosine and lycorine. Tortuosirm is reported for the first time and its structure was established by spectroscopic methods. INTRODUCTION OMe Narcissus tortuosus, a species belonging to the Pseudonarcissus section, is notable for its pale and beautifully 2 symmetrical flowers, regularly twisted. Morphologically, it is separable from N. moschatus by its stronger growth and broader foliage, and by its larger and less concolorous flowers 1"21. Histological and caryological studies of N. tortuosus have been made recently I-3,4]. In the present paper, we describe the isolation and characterization of tortuosine (1) and lycorine (2) from whole plants of this species. ,o I,o 114 11 12 7 6 RESULTSAND DISCUSSION The alkaloids were extracted by routine acid-base work-up procedures and after CC (see Experimental) 1 was isolated as yellow crystals. Its HR mass spectrum ([M] + m/z 296.1292) indicated the molecular formula ClsH~sNO 3 which was consistent with a phenantridinium structure. Support for this assumption was obtained by comparison with spectroscopic data of the anhydrolycorinium ion, one of the biosynthetic products of Amaryllis belladonna most inhibitory to murine 3PS leukaemia [51, and vasconine, a quaternary alkaloid isolated from N. vasconicus [61. The ~H and ~3CNMR spectra were assigned taking into account the correlations observed in 1HCOSY, H M Q C [71, HMBC 181 and ROESY [91 experiments. The most characteristic signals of the ~H N M R spectrum (Table 1) were a one proton doublet at 58.13 (J = 1.4 Hz) assigned to H-1 and one broad singlet at 57.59 assigned to H-3, since they showed scalar coupling in the 1H COSY spectrum. Also, the ROESY showed a spacial correlation between H-1 and the one-proton signal at OH HO...~ 2 *Part 21 in the series 'Narcissus alkaloids'. For Part 20 see ref. [1]. 68.34, and between H-1 and the methoxy group at 54.20, allowing us to assign those signals to H-10 and H-2', respectively. Moreover, in the 1H COSY spectrum, the H10 signal showed zig-zag coupling to H-6 at 69.55 1"101 and a NOE contour with the methoxy group at 34.33 (H9') in the ROESY spectrum. After that, the signals at 549 PRY 38-2-5 550 J. BASTIDAet al. Table 1. 1H-~H scalar and spatial correlations of tortuosine Proton fi COSY ROESY 1 8.13 d (1.4) 3 6 7 10 ll 12 2' 8' 9' 7.59 brs 9.55 s 7.92 s 8.34 s 3.88 t (7.2) 5.35 t (7.2) 4.20 s 4.17s 4.33 s H-3 H-11, H-1 H-12, H-10* -H-6* H-12 H-11 ---- H-10, H-2' ---H-l, H-9' -H-1 H-10 Coupling constants in parentheses are in Hz. *Zig-zag coupling. Table 2. 13C NMR, HMQC and HMBC data of tortuosine Correlated carbon Proton 1 3 6 7 10 11 12 2' 8' 9' HMQC HMBC 102.2 d 164.5 s (C-2) 118.0 d 139.7 s (C-4) 133.0 s (C-4a) 142.6 d 122.9 s (C-6a) Ill.0 d 153.1 s (C-8) 159.1 s (C-9) 104.6 d 131.4 s (C-10a) 126.1 s (C-10b) 27.8 t 57.0 t 57.4 q 57.0 q 57.8 q C-2, C-3, C.4a, C-10, C-10a C-l, C-4a, C-11 C-6, C-9, C-10a C-6a, C-8, C-9, C-10b C-4 C-2 C-8 C-9 Carbon multiplicities were established by DEPT pulse sequence. ,~3.88 (2H, t, J = 7.2 Hz), 5.35 (2H, t, J = 7.2 Hz) and 4.17 (3H, s) were easily assigned to H-11, H-12 and H-8', respectively. The l a C N M R signals of 1 were assigned considering the connectivities from H M Q C and H M B C spectra (Table 2). The carbon singlets at ~ 164.5, 159.1 and 153.1 were assigned to C-2, C-9 and C-8, respectively, because of their three-bond correlation with their corresponding methoxy protons in the H M B C spectrum. The carbon singlet at 6133.0 was ascribed to C-4a due to its three-bond connectivities to H-1 and H-3. H-1 and H-7 also gave a three-bond correlation with the carbon singlet at fi 131.4, allowing us to assign this signal to C-10a. The carbon singlets at 6126.1 and 122.9 were tentatively assigned to C-10b and C-6a, according to their threebond correlation with H-10 shown in the H M B C experi- ment. Finally, the two-proton triplet at 6 3.88 (H-11) gave a two-bond connectivity with the carbon singlet at t5139.7 for the C-4 carbon resonance. EXPERIMENTAL General. Mps uncorr. IR were recorded in KBr discs. tH, 13CNMR, D E P T and 1HCOSY spectra were recorded in a Varian VXR 500 and the H M Q C , H M B C (J = 7 Hz) and ROESY (spin lock 1000 msec) spectra in a Bruker AMX 400 using C D 3 O D as solvent with T M S as int. standard. Chemical shifts are reported in ~ units (ppm). Silica gel Merck (70-230 mesh) and silica gel SDS Chromagel 60 A CC (230-400 mesh) were used for CC and flash CC, respectively. Silica gel 60 F 254 (Merck) was used for analytical (0.25 mm) and prep. (1 mm) TLC. Spots on chromatograms were detected under UV light (254 nm) and by Dragendorff's reagent. Plant material. Narcissus tortuosus Haworth was collected in May 1989 in Pefia Mayor, Asturias, Spain. Samples were authenticated by Dr Herminio Nava, Facultad de Biologia, Universidad de Oviedo, and a voucher specimen (No. 890 183) is deposited at the Jardl Bot~inic de Barcelona. Extraction and isolation of alkaloids. F. whole plants (aerial parts and bulbs 1.2 kg) were extracted with EtOH in a Soxhlet apparatus for 10 hr. The EtOH soln was evapd under red. pres. and the residue acidified with 2% HCI. The filtered acidic sotn was extracted with Et20 to remove neutral materials and was made basic (pH 8-9) with Na2CO 3 and extracted with CH2C12. The CH2CI 2 soln was concd in vacuo and taken to dryness to yield extract C (1.68 g). The former aq. phase was then extracted with C H 2 C I 2 - E t O H (3:2) and evapd to provide extract D (0.79 g). The aq. phase was found to be free of alkaloids. Extract C was first chromatographed by flash CC eluting with C H 2 C I 2 - M e O H (19: 1), increasing the gradient for the last steps until (4"1) and 2 frs were obtained. Fr. 1 yielded 2 by direct recrystallization (327 mg). Ft. 2 was chromatographed by CC on silica gel, eluting with CH2C12-MeOH (19." 1), giving 2 (121 mg) and 1 (95 mg), after purifying the last one by prep. TLC eluting twice with CHECI2-MeOH (9:1). Additional 2 (59 mg) was obtained from extract D. Tortuosine (l). H R M S m/z 296.1292 (calcd 296.1287 for ClsHlsNO3). Mp 242-243 °. IR Vma~ cm-1: 3450, 2923, 1630, 1515, 1266. EIMS 70 eV m/z (rel. int.): 296 [M] + (4), 294 (8), 280 (35), 267 (18), 239 (12), 180 (22), 167 (23), 58 (21), 44 (34), 43 (100). 1H and ~3CNMR: Tables 1 and 2, respectively. ( - )-Lycorine (2). This alkaloid was identified by direct comparison of its chromatographic and spectroscopic data with those of an authentic sample [ l l ] . Acknowledgements--This work was financially supported by CIRIT-CICYT (project QFN91-4205). J.M.F. thanks the Generalitat de Catalunya for the provision of research Alkaloids from N. tortuosus fellowships. The cooperation of Dr Herminio Nava in identifying the plant material is very gratefully acknowledged. REFERENCES 1. Bastida, J., Bergofi6n, S., Viladomat, F. and Codina, C. (1994) Planta Med. 60, 95. 2. Pugsley, H. W. (1933) J. Roy. Hort. Soc. 5fl, 17. 3. Dorda, E. and Fernhndez-Casas, J. (1990) Fontqueria 30, 235. 4. Fernandes, A. (1991) Fontqueria 31, 141. 5. Pettit, G. R., Gaddamidi, V., Goswani, A. and Cragg, G. M. (1984) J. Nat. 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