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
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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.
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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.
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193
6 Boit
anthes, Leucojum
Dr. Jaume Bastida
Departament de Productes Naturals
Facultat de Farmàcia
Universitat de Barcelona
Avda. Diagonal 643
08028-Barcelona
Spain
E-mail: bastida@farmacia.far.ub.es
Fax: 34-93.402.90.43
Tel.: 34-93.402.44.93
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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)