Alkaloids from Nerine filifolia

Article abstract of DOI:10.1016/j.phytochem.2004.12.009

The novel compounds N-demethylbelladine, 6α-methoxybuphanidrine and filifoline, in addition to five known alkaloids, and phenol have been isolated from fresh bulbs of Nerine filifolia (Amaryllidaceae). The structure and stereochemistry of the compounds we

Full text of DOI:10.1016/j.phytochem.2004.12.009

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 373–382
www.elsevier.com/locate/phytochem
q
Alkaloids from Nerine filifolia
a
b
c
Jerald J. Nair , William E. Campbell , Reto Brun , Francesc Viladomat ,
a
a
Carles Codina , Jaume Bastida
a,
*
a
`
Departament de Productes Naturals, Facultat de Farmacia, Universitat de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain
b
Pharmacology Division, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
c
Protozoology Laboratory, Swiss Tropical Institute, Socinstraße 57, CH-4002 Basel, Switzerland
Received 26 April 2004; received in revised form 7 December 2004
Abstract
The novel compounds N-demethylbelladine, 6a-methoxybuphanidrine and filifoline, in addition to five known alkaloids, and
phenol have been isolated from fresh bulbs of Nerine filifolia (Amaryllidaceae). The structure and stereochemistry of the compounds
were determined by physical and spectroscopic methods, including 1D and 2D NMR and mass spectroscopic techniques. An unu-
sual circular dichroism response from filifoline has required a semi-synthetic derivatisation strategy towards key C-11endo analogues
of the b-crinane representative ambelline in which the nature of substituents was observed to have a profound effect on molecular
ellipticity. Filifoline was not cytotoxic to myoblast (L6) cells and exhibited no anti-protozoal activity in an in vitro screen against
four different parasitic protozoa.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Nerine filifolia; Amaryllidaceae; Bulbs; Alkaloids; Phenol; Belladine; N-Demethylbelladine; 6a-Hydroxybuphanidrine; 6a-Methoxybuph-
anidrine; Filifoline; Ambelline; 11-O-Acetylambelline; Undulatine; Circular dichroism
1. Introduction
winters. Most Nerine species have been cultivated for
their elegant flowers, presenting a source of innumerable
horticultural hybrids (Snijman and Linder, 1996; Dyer,
1976). The exploitation of plants of this genus in the tra-
ditional medicinal practices of the indigenous people of
southern Africa has been documented (Watt and
Breyer-Brandwijk, 1962). In particular, the southern
Sotho and Zulu tribes have made use of decoctions of
bulbs to treat coughs and colds, in renal and hepatic
conditions, for the relief of back pain and as a remedy
for infertility. In continuation of phytochemical analy-
ses of the South African Amaryllidaceae (Viladomat et
al., 1997), Nerine filifolia Baker was examined for alka-
loidal constituents. This evergreen plant, with small
bulbs and thin thread-like leaves, flowers between
September and November and occurs in the Eastern
Cape, Transkei, Orange Free State, Swaziland and
The genus Nerine Herbert (Amaryllidaceae), a mem-
ber of the tribe Amaryllideae, is one of seven represen-
tatives of the subtribe Amaryllidinae which is confined
to the temperate regions of southern Africa (Snijman
and Linder, 1996). This genus, the second largest within
the Amaryllideae with ca. 23 species, is an autumn-flow-
ering perennial bulbous plant group, whose species in-
habit areas with summer rainfall and cool, dry
q
Part XII in the series ‘‘Alkaloids from South African Amaryllid-
aceae’’. For Part XI see Brine et al., 2002. A dinitrogenous alkaloid
from Cyrtanthus obliquus. Phytochemistry 61, 443–447.
*
Corresponding author. Tel.: +34 93 402 4493; fax: +34 93 402
9043.
E-mail address: jaumebastida@ub.edu (J. Bastida).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.12.009

374
J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
Mpumalanga. Following the usual extractive and chro-
matographic procedures (see Section 4), eight alkaloids
as well as phenol were isolated. The known compounds
belladine (1), 11-O-acetylambelline (6) and undulatine
(8) were isolated as the principal constituents in addi-
tion to the minor components ambelline (5) and 6a-
hydroxybuphanidrine (3), while N-demethylbelladine
(2), 6a-methoxybuphanidrine (4) and filifoline (7), the
11-O-nicotinyl analogue of ambelline, are reported here
for the first time as natural products. A previous phyto-
chemical analysis of Nerine filifolia has led to the detec-
tion of small quantities of galanthamine (4 · 10ꢀ3 % dry
wt) by radioimmunoassay techniques (Tanahashi et al.,
1990). Interestingly, the crinane-related compounds
reported here all exhibit a b-5,10b-ethano bridge orien-
tation and have in common methoxyl substituents at
C-3a and C-7. An unusual circular dichroism (CD) re-
sponse from filifoline (7) has led to the novel discovery
that substituent effects at C-11endo in crinane alkaloids
play a critical role in molecular ellipticity as expressed
by the shape of the CD curve, which is indicative of
the stereochemistry of the dominant C-10b benzylic ring
junction and hence the orientation of the 5,10b-ethano
bridge. Synthetic 11-O-benzoyl and 11-O-methanesul-
phonyl analogues of ambelline (5) (see Section 4) collec-
tively provided further unequivocal evidence that, for
crinane alkaloids, functionalization at C-11endo is an
integral factor in molecular ellipticity.
(Ghosal et al., 1983, 1988). A complete assignment of
the proton signals, hitherto unreported, is afforded here
taking into account both homonuclear (COSY) and het-
1
eronuclear (HMQC) correlations. The H NMR spec-
trum had seven aromatic signals, three of which were
attributable to H-30, H-60 and H-70 of ring A while those
of ring B were an A2B2 system (H-4/H-8 and H-5/H-7)
(Table 1). Both 2H-10 (d 3.49, br s) and 2H-1 (d 2.60,
m) were assignable due to three-bond (HMBC) correla-
tions to the N-methyl carbon (d 42.2, q); and 2H-2 (d
2.77, m) by a COSY contour to 2H-1. Similarly, the
three methoxyl groups were assigned by HMBC correla-
tions. The 13C NMR data of belladine (Table 1) were in
accordance with a previous report (Ghosal et al., 1983).
Compound 2, C18H23NO3, isolated as a light-brown oil,
differed in molecular formular and mass from belladine
due to the absence of the N-methyl group. Its EIMS had
the [M]+ signal at m/z 301 as well as characteristic peaks
at m/z 180 [C10H14NO2]+, 151 [C9H11O2]+, 121
[C7H5O2]+ and 107 [C7H7O]+, while IR absorption
bands at 3225, 2833 and 1030 cmꢀ1 were indicative of
NH and C–O groups; and the UV spectrum was charac-
teristic of a N-benzylphenethylamine of the belladine
1
type (Ghosal et al., 1983). The H and 13C NMR data
of belladine and compound 2 (Table 1) were very similar
and the only significant differences were due to the effect
of N-substitution. Thus, in 2, both 2H-10 (d 3.74, br s)
and 2H-1 (d 2.87, m) were slightly deshielded
(Dd = +0.25 and +0.27, respectively) and C-10 (d 53.6,
t) and C-1 (d 50.6, t) markedly shielded (Dd = ꢀ8.4
and ꢀ8.5, respectively), in relation to the corresponding
signals in belladine. Three-bond HMBC correlations
facilitated assignment of the quartenary centres. Thus,
C-6 correlated to H-4, H-8 and the 6-methoxyl group
2. Results and discussion
Compound 1, C19H25NO3, was characterized as bell-
adine by its UV, IR, EIMS and NMR spectral data
Table 1
1H NMR and 13C NMR data for compounds 1 and 2 (J in Hz within parentheses)
Proton
1
2
1H
13C
1H
13C
2H-1
2H-2
2.60 m
2.77 m
–
59.1 t
32.9 t
2.87 m
2.77 m
–
50.6 t
35.3 t
–
132.5 s (C-3)
129.6 d
113.7 d
132.8 s (C-3)
129.6 d
113.8 d
4
7.10 d (8.7)
6.82 d (8.7)
–
7.12 d (8.7)
6.83 d (8.7)
–
5
–
157.8 s (C-6)
113.7 d
129.6 d
157.9 s (C-6)
113.8 d
129.6 d
7
6.82 d (8.7)
7.10 d (8.7)
3.49 br s
–
6.83 d (8.7)
7.12 d (8.7)
3.74 br s
–
8
2H-10
62.0 t
131.6 s (C-20)
53.6 t
131.9 s (C-20)
–
30
6.85 br s
–
110.6 d
6.82 br s
–
110.9 d
–
148.8 s (C-40)
147.9 s (C-50)
111.9 d
148.9 s (C-40)
147.9 s (C-50)
111.2 d
–
–
–
60
6.79 br s
6.79 br s
3.78 s
6.80 br s
6.80 br s
3.79 s
3.86 s
3.87 s
–
70
121.0 d
55.2 q
120.2 d
55.2 q
6-OCH3
40-OCH3
50-OCH3
N-CH3
3.86 s
3.87 s
2.28 s
55.8 q
55.8 q
42.2 q
55.8 q
55.8 q
–

J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
375
protons; C-3 to H-5, H-7 and 2H-1, while C-50 was dis-
tinguished from C-40 due to three-bond correlations to
H-30, H-70 and the 50-methoxyl group protons, leading
to the structure of compound 2 as N-demethylbelladine.
Incidentally, such compounds, which are fully O-meth-
ylated, have no biosynthetic significance since they can-
not be further oxidized (by phenolic coupling) into the
tetracyclic skeleton of the Amaryllidaceae alkaloids,
and occur merely as organic bases of the plant (Ghosal
et al., 1983).
The molecular ellipticities of the crinane-type alka-
loids (3, 4, 5, 6, 8) discussed herein all showed CD curves
which were qualitatively similar to those of the b-5,10b-
ethano bridge series, having a maximum around 250 nm
(Ali et al., 1984; Wagner et al., 1996), with the exception
of filifoline (7) whose CD curve (Fig. 1) was reminiscent
of a compound with an a-5,10b-bridge since a maximum
was detectable in the region of 290 nm. However, as we
have chemically proved (see Section 4) the ethano bridge
in filifoline is in fact b-orientated.
(Viladomat et al., 1995) and the only noteworthy differ-
ences were due to the effect of C-6 substitution in 3 and
4. The single aromatic proton resonance was ascribable
to H-10 due to three-bond HMBC correlations to C-6a,
C-8 and C-10b, in addition to a ROESY correlation to
H-1, allowing the aromatic methoxyl group to be placed
at C-7. The large trans diaxial coupling (J = 13.5 Hz) be-
tween H-4a and H-4b, as well as the small vicinal cou-
pling of both H-4a (J = 1.5 Hz, indicating a dihedral
angle ca. 90ꢁ) and H-4b (J = 4.0 Hz) with H-3, vindi-
cated the pseudoaxial disposition for the C-3 methoxyl
group. Both H-11exo and H-12exo were assignable
due to their spatial proximity to H-4b, in agreement
with the low field assignment of H-12exo (relative to
H-12endo) due to its syn disposition with the nitrogen
lone pair. ROESY correlation between H-12endo and
H-6b allowed us to assign the respective hydroxyl and
methoxyl groups at C-6 to the a-position. The one-pro-
ton doublets at d 3.95 (H-6b) and d 4.35 (H-6a) in buph-
anidrine (Viladomat et al., 1995) were replaced by
singlets at d 5.24 and 4.59, respectively, in compounds
3 and 4, in accordance with the hydroxyl and methoxyl
groups. The 13C NMR spectrum of both compounds
were confirmed by HMQC and HMBC techniques
(Tables 2 and 3), indicating for each a skeleton with
18 and 19 carbon atoms, respectively. The large deshiel-
ding of C-6 in compound 3 (d 85.6) and 4 (d 93.4) in
relation to that in buphanidrine (d 58.1, t) (Viladomat
et al., 1995) was indicative of oxy-substitution at this
carbon. The shielding (Dd = ꢀ6.4) of C-4a in both com-
pounds (relative to buphanidrine) can be explained in
terms of the Ôc-gaucheÕ effect as a result of the C-3 and
C-6 pseudoaxial substituents. Three-bond correlations
of H-6 to C-7 and C-10a, in addition to those described
above for H-10, allowed assignment of all quartenary
centres.
6a-Hydroxybuphanidrine (3) (C18H21NO5), previ-
ously isolated from Nerine bowdenii Watson (Slabaugh
and Wildman, 1971), and 6a-methoxybuphanidrine (4)
(C19H23NO5), described here for the first time from a
natural source, exhibited several similar spectral proper-
ties. Their EIMS fragmentations were consistent with
compounds of the crinine series with no bridge substitu-
ent, and having a double bond in the 1,2-position (Lon-
gevialle et al., 1973). The difference in mass between
them could be accounted for by the presence of the addi-
tional methoxyl group in 4. IR absorption bands for
methoxyl and methylenedioxy groups were detected in
both, as well as a hydroxyl group in 3. We present here
1
the complete assignment of H and 13C NMR spectra
(Table 2) of both compounds, previously not under-
taken. The spectra were similar to that of buphanidrine
Filifoline (7), isolated as a white powder, had a weak
molecular ion peak (1%) at m/z 436, correct for the
molecular formula C24H24N2O6. The IR spectrum had
absorption bands for ester carbonyl (1734 cmꢀ1), C–O
(1040 cmꢀ1) and methylenedioxy (934 cmꢀ1) groups.
Three-bond HMBC correlations (Table 4) to C-6a,
C-8 and C-10b, as well as the single ROESY contour
to H-1 (Table 5), allowed the aromatic proton singlet
at d 6.48 to be ascribed to H-10, thus allowing placement
of the aromatic methoxyl group at C-7 (as in com-
pounds 3 and 4). The pseudoaxial disposition for the
3-methoxyl group was arrived at by the large coupling
observed between H-4a and H-4b (J = 13.5 Hz, trans
diaxial interaction), H-4a and H-4b (J = 14.0 Hz, gemi-
nal) and smaller couplings of both H-4a and H-4b with
H-3, confirmed by a ROESY correlation between H-3
and H-4b. H-6a (d 4.34) was assignable to a lower field
than H-6b (d 3.90) due to its proximity to the nitrogen
lone pair, the same reason that H-12exo was further
downfield-shifted (relative to H-12endo), all confirmed
Fig. 1. CD curve of filifoline (7).

376
J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
Table 2
1H NMR and 13C NMR data for compounds 3 and 4 (J in Hz within parentheses)
Proton
3
4
1H
13C
1H
13C
1
6.52 d (10.0)
5.94 ddd (10.0, 5.0, 0.5)
131.9 d
125.8 d
6.53 d (10.0)
5.94 ddd (10.0, 5.0, 1.0)
132.6 d
125.5 d
2
3
3.79 ddd (5.0, 4.0, 1.5)
2.08 dddd (14.0, 4.0, 1.5, 0.5)
72.3 d
27.9 t
3.81 ddd (5.0, 4.0, 1.5)
1.94 dddd (14.0, 4.0, 1.5, 1.0)
72.4 d
28.9 t
4a
4b
4a
1.54 ddd (14.0, 13.5, 4.0)
3.84 ddd (13.5, 4.0, 0.5)
27.9 t
56.4 d
85.6 d
1.57 ddd (14.0, 13.5, 4.0)
3.65 ddd (13.5, 4.0, 0.5)
28.9 t
56.4 d
93.4 d
6b
–
5.24 s
–
4.59 s
–
119.2 s (C-6a)
142.5 s (C-7)
134.2 s (C-8)
149.3 s (C-9)
97.0 d
119.2 s (C-6a)
143.0 s (C-7)
134.7 s (C-8)
149.3 s (C-9)
97.0 d
–
–
–
–
–
–
–
10
–
6.55 s
–
6.54 s
–
–
139.8 s (C-10a)
44.2 s (C-10b)
40.8 t
–
140.2 s (C-10a)
44.2 s (C-10b)
41.3 t
–
–
–
11endo
11exo
12endo
12exo
OCH2O
3-OCH3
6-OCH3
7-OCH3
1.90 dddd (12.5, 9.0, 4.5, 0.5)
1.84 ddd (12.5, 10.5, 6.0)
2.79 ddd (13.0, 9.0, 6.0)
3.31 ddd (13.0, 10.5, 4.5)
5.84–5.87 2d (1.5)
3.32 s
1.87 dddd (12.5, 9.0, 4.5, 0.5)
1.81 ddd (12.5, 10.0, 6.5)
2.67 ddd (13.0, 9.0, 6.5)
3.27 ddd (13.0, 10.0, 4.5)
5.83–5.87 2d (1.5)
3.32 s
40.8 t
47.7 t
41.3 t
47.6 t
47.7 t
100.8 t
56.3 q
47.6 t
100.8 t
56.2 q
56.1 q
59.8 q
–
4.01 s
–
59.8 q
3.56 s
3.95 s
Table 3
COSY, ROESY and HMBC correlations for the protons of compound 4
H
COSY
ROESY
HMBC
1
H-2
H-1, H-3, H-4a
H-2, H-4a, H-4b
H-2, H-3, H-4b, H-4a
H-3, H-4a, H-4a
H-2, H-10
H-1, H-3
C-3, C-4a, C-10a
2
C-3, C-4, C-10b
C-1, C-2, 3-OCH3
C-2, C-4a, C-10b
C-4a, C-10b
3
H-2, H-4a, H-4b, 3-OCH3
H-3, H-4a, H-4b
H-3, H-4a, H-11exo, H-12exo
H-4a
H-12endo, 6-OCH3
4a
4b
4a
H-4a, H-4b
–
C-4, C-11, C-12
C-4a, C-6a, C-7, C-10a, C-12, 6-OCH3
6
10
–
H-11exo, H-12endo, H-12exo
H-1
H-11exo, H-12endo, H-12exo
C-6a, C-7, C-8, C-9, C-10b
C-4a, C-10a, C-10b
11endo
11exo
12endo
12exo
OCH2
H-11endo, H-12endo, H-12exo
H-11endo, H-11exo, H-12exo
H-11endo, H-11exo, H-12endo
H-4b, H-11endo, H-12endo, H-12exo
H-6b, H-11endo, H-11exo, H-12exo
H-4b, H-11endo, H-11exo, H-12endo
–
C-4a, C-10a, C-10b
C-4a, C-6, C-11
C-6
O
–
–
–
–
C-8, C-9
C-3
C-6
3-OCH3
6-OCH3
7-OCH3
H-3
H-6
–
C-7
by ROESY contours of H-6b with H-12endo; H-6a with
H-4a, as well as H-12exo with H-4b. Spatial proximity
was also established between H-4b and H-11exo (d
5.36), which suggested substitution at C-11endo. The
presence of the nicotinate ester at C-11endo was indi-
cated by the chemical shift, splitting pattern and cou-
pling constants of the four distinct protons arising
from the m-substituted pyridine ring: H-30 (d 8.74) and
H-50 (d 8.69), which are markedly deshielded by the vic-
inal nitrogen atom, as well as H-60 (d 7.27) and H-70 (d
7.93). The H-30, H-70 and H-11exo protons had three-
bond correlations to the ester carbonyl (d 165.0). The
use of HMQC and HMBC techniques facilitated assign-
ment of all carbon signals. The 24-carbon skeleton of fil-
ifoline was characterized by the presence of additional
aromatic signals (in comparison to the usual crinane
skeleton): two singlets (for C-20 and the carbonyl group)
and four doublets (attributable to C-30, C-50, C-60 and
C-70), all arising from the nicotinate ester. All quater-
nary centres were adequately revealed by three-bond
correlations of 2H-6 to C-10a and C-7, which was
shifted downfield to d 140.7 by the presence of the meth-
oxyl group, the methylenedioxy protons to C-8 and C-9,
those mentioned above for H-10, as well as H-60 to C-20,
in addition to the H-30/C-20 two-bond correlation. Filif-
oline (7) represents the first member of the crinane series

J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
377
Table 4
1H NMR, HMQC and HMBC data for filifoline (7)
H
d
HMQC
HMBC
1
6.58 d (10.0)
6.07 ddd (10.0, 5.0, 1.0)
131.3 d
126.8 d
C-3, C-4a, C-10a, C-10b
C-3, C-4, C-10b
2
3
3.87 ddd (5.0, 4.0, 2.0)
2.15 dddd (14.0, 4.5, 2.0, 1.0)
72.2 d
28.8 t
C-1, C-2, C-4a, 3-OCH3
C-4a, C-10b
4a
4b
4a
1.76 ddd (14.0, 13.5, 4.0)
3.48 ddd (13.5, 4.5, 1.0)
28.8 t
63.3 d
C-2, C-3, C-4a, C-10b
C-6, C-11, C-12
C-6a, C-7, C-10a, C-12
6a
6b
–
4.34 d (17.5)
3.90 d (17.5)
58.8 t
58.8 t
C-4a, C-6a, C-7, C-10a, C-12
–
–
117.3 s (C-6a)
140.7 s (C-7)
133.9 s (C-8)
147.9 s (C-9)
99.2 d
–
–
–
–
–
–
–
10
–
6.48 s
–
C-6a, C-8, C-9, C-10b
–
–
133.5 s (C-10a)
47.5 s (C-10b)
88.4 d
–
–
–
–
11exo
12endo
12exo
OCH2
5.36 dd (8.0, 4.0)
2.83 ddd (14.0, 4.0, 1.0)
3.88 dd (14.0, 8.0)
5.77–5.81 2d (1.5)
3.35 s
C-1, C-10a, C-10
C-4a, C-6, C-11
C-4a, C-6, C-11
C-8, C-9
59.8 t
59.8 t
O
100.5 t
56.6 q
3-OCH3
7-OCH3
C-3
C-7
3.97 s
–
59.2 q
–
165.0 s (C-10)
125.8 s (C-20)
150.7 d
–
–
–
–
30
50
60
70
8.74 ddd (2.0, 1.0, 0.5)
8.69 ddd (5.0, 2.0, 0.5)
7.27 ddd (8.0, 5.0, 1.0)
7.93 ddd (8.0, 2.0, 2.0)
C-10, C-20, C-50, C-70
C-30, C-60, C-70
C-20, C-50
C-10, C-30, C-50
153.4 d
123.2 d
136.9 d
Table 5
COSY and ROESY data for filifoline (7)
H
COSY
ROESY
1
H-2
H-1, H-3, H-4a
H-2, H-4a, H-4b
H-2, H-3, H-4b, H-4a
H-3, H-4a, H-4a
H-2, H-10
2
H-1, H-3, 3-OCH3
H-2, H-4a, H-4b, 3-OCH3
H-3, 3-OCH3, H-4b, H-4a
H-3, H-4a, H-11exo, H-12exo
H-4a, H-6a
3
4a
4b
4a
H-4a, H-4b, H-12endo
6a
6b
10
H-6b
H-6a
–
H-12endo, H-12exo
H-4a, H-6b, 7-OCH3
H-6a, 7-OCH3, H-12endo
H-1
11exo
12endo
12exo
H-4b, H-12endo, H-12exo
H-6b, H-11exo, H-12exo
H-4b, H-11exo, H-12endo
–
H-4a, H-11exo, H-12exo
H-11exo, H-12endo
OCH2
3-OCH3
O
–
–
H-2, H-3, H-4a
H-6a, H-6b
7-OCH3
–
30
50
60
70
H-50, H-70
H-30, H-60
H-50, H-70
H-30, H-60
–
H-60
H-50, H-70
H-60
to possess a nicotinate ester. Precedence for this heteroa-
romatic ester exists for the homolycorine analogues cliv-
iamartine, miniatine and clivimine as well as the unusual
degraded compound clivialine, all extractives of the
southern African Amaryllidaceae representative Clivia
miniata (Lindl.) Regel as reviewed by Viladomat et al.
(1997).
The b-orientation of the 5,10b-ethano bridge in filif-
oline (7) was unequivocally ascertained by cleavage of
the nicotinate ester (see Section 4) affording a synthetic
product which was unambiguously identified from its
chromatographic and spectroscopic properties as
ambelline (Viladomat et al., 1994, 1995), the molecular
ellipticity of which had a shape in the CD curve typical

378
J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
of a b-5,10b-ethano bridge compound (maximum
around 250 nm). Subsequent semi-synthetic derivatisa-
tion of ambelline (5) (see Section 4) provided unequiv-
ocal evidence that bulky substituents at C-11endo in
crinane alkaloids are responsible for reversing molecu-
lar ellipticity as expressed in the shape of CD curves.
Thus, both 11-O-acetylambelline (6) and synthetic 11-
O-methanesulphonylambelline (10), with relatively
small respective acyl and sulphonyl groups at C-
11endo, had CD curves (Figs. 2 and 4) with maxima
around 250 nm, as is expected of b-crinane structures,
antipodal to that of filifoline (7) and synthetic 11-O-
benzoylambelline (9) in which maxima were detectable
near 290 nm (Figs. 1 and 3). The shape of CD curves
of crinane alkaloids is determined by the configuration
of the benzylic B:C ring junction in conjunction with
Fig. 4. CD curve 11-O-methanesulphonylambelline (10).
the dominant methylenedioxyphenyl (MDP) chromo-
phore (Wagner et al., 1996). Since the C-10b terminus
of the ethano bridge is part of this junction, its abso-
lute stereochemistry is therefore qualitatively deter-
mined from the CD bands at ca. 290 and 250 nm
which, respectively, also correspond to the Ar(A1g ! B2u)
and Ar(A1g ! B1u) UV bands of the MDP chromophore
(DeAngelis, 1968). This has been proven theoretically
by sector rules, such as the quadrant rule for predicting
the absolute configuration of molecules possessing an
asymmetric centre adjacent to an aromatic moiety, as
in crinane compounds (DeAngelis and Wildman,
1969). While the effect of substituents on CD in crinane
derivatives remains mainly empirical (DeAngelis, 1968)
and unexamined, phenomena such as C-3 epimerisation
(Wagner et al., 1996) have been observed to have a
marked (but not drastic) effect on the amplitude and
magnitude of CD. However, no precedence exists for
the effect on CD by bulky, electron-rich substituents
which supersede the MDP chromophore. The nicotin-
ate ester at C-11endo in filifoline, as well as the benzo-
ate ester in compound 9, are both geometrically and
electronically predisposed so as to perturb the k290
and k250 CD bands emanating from the MDP chromo-
phore responsible for molecular ellipticity i.e., the aryl
esters are now the dominant chromophores. We sug-
gest that a combination of regio, stereo and electronic
factors introduced by the bulky aryl esters are respon-
sible for the anomalous reverse effect on molecular
ellipticity as expressed in the shape of the CD curves.
Filifoline was not cytotoxic to rat myoblast cells and
exhibited no activity in the in vitro screens against the
four different protozoan parasites Trypanosoma brucei
rhodesiense, Trypanosoma cruzi, Leishmania donovani
and Plasmodium falciparum.
Fig. 2. CD curve 11-O-acetylambelline (6).
Fig. 3. CD curve 11-O-benzoylambelline (9).

J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
379
6'
3'
2
H3CO
H3CO
R1
3
4
5'
4'
7'
2'
R
1
11
4
7
2
10 10b
10a
N
4a
N
3
8
9
8
5
6
O
O
1'
1
H
6
12
6a
OCH3
7
1 R = CH3
2 R = H
R3
R2
3
4
R1 = R3 = OCH3, R2 = OH
R1 = R2 = R3 = OCH3
OCH3
R
O
H
N
O
OCH3
O
OCH3
O
O
H
N
5
6
7
9
R = OH
R = OAc
R = ONic
R = OBz
OCH3
10 R = OMes
8
O
O
O
7'
6'
2'
H3CS
O
1'
O
O
O
5'
N
3'
OMes
(Methanesulphonate)
ONic (Nicotinate)
OBz (Benzoate)
3. Concluding remarks
run on a Hewlett–Packard 5989A Mass Spectrometer
fitted with a direct injection probe (DIP) with ioniza-
tion energy set at 70 eV. Elemental analysis was per-
formed on a Carlo Erba-Fisons EA1108 apparatus.
1H, 13C NMR, DEPT, 1H COSY, HMQC, HMBC
and ROESY spectra were recorded on a Varian VXR
500 or Gemini 300 spectrometer in CDCl3 with TMS
as internal standard. Chemical shifts are reported in
units of d (ppm) and coupling constants (J) are ex-
pressed in Hz. Silica gel SDS chromagel 60 A CC
(230–400 mesh) and silica gel Merck (70–230 mesh)
were used for flash CC and CC, respectively. Sephadex
LH-20 (Pharmacia) was used for gel filtration and sil-
ica gel SIL G/UV254 for analyt. and SIL G-25 UV254
for prep. TLC (both Macherey–Nagel). Spots on chro-
matograms were detected under UV light (254 nm) and
by DragendorffÕs reagent stain.
The present work contributes significantly to the
understanding of CD as related to crinane alkaloids;
we will report elsewhere in a more comprehensive man-
ner the implications of this unique phenomenon as ob-
served in filifoline.
4. Experimental
4.1. General
Mps are uncorr. and were measured on a Gallenk-
amp Melting Point apparatus. Optical rotations were
determined on a Perkin–Elmer 241 Polarimeter in-
stalled with a k589 sodium lamp. A Jasco J-700 Spectro-
polarimeter was utilized to run CD spectra, all
recorded in MeOH. UV absorption spectra were deter-
mined on a Varian Cary IE UV–Vis spectrophotome-
ter. IR spectra were measured on a Perkin–Elmer
1600 FTIR series spectrometer in dry film. EIMS were
4.2. Plant material
Bulbs of Nerine filifolia Baker were purchased at the
van den Berg Nurseries in Durbanville, Cape Province

380
J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
(South Africa) and were authenticated by the proprietor
Mrs. J. van den Berg.
NMR (75 MHz, CDCl3) spectral data are tabulated in
Table 1.
4.6. 6a-Hydroxybuphanidrine (3)
4.3. Extraction and isolation of alkaloids
Found: C, 64.54; H, 6.44; N, 4.11. Calc. for
C18H21NO5: C, 65.24; H, 6.39; N, 4.23%.
Fresh bulbs (2.08 kg) were crushed and macerated
with EtOH for 48 h after which the solvent was re-
moved under reduced pressure, the residue (179 g) dis-
solved in H2O (200 ml) and acidified to pH 4 with
dilute HCl. After removing neutral material with
Et2O, the acidic soln. was extracted with CHCl3 to pro-
vide extract A (1.2 g). Basifying the soln. to pH 8–9
with aq. NH4OH and extracting again with CHCl3
afforded extract B (3.9 g). Finally, a CHCl3–MeOH
(3:2) extraction of the basic soln. gave extract C (0.65
g). Extracts A, B and C were combined (5.75 g) after
TLC [EtOAc–MeOH (1:1)] indicated that they con-
tained the same alkaloids, and subjected to flash CC
on silica gel by gradient elution with n-hexane, n-hex-
ane-EtOAc, EtOAc, EtOAc–MeOH and finally MeOH.
Four fractions were generated in this manner; PhOH
(15 mg) was obtained directly from fr. I by recrystalli-
zation with MeOH. Fr. II was rechromatographed by
CC using an EtOAc–MeOH step gradient, followed
by prep. TLC (in Me2CO) from which belladine (70
mg), 11-O-acetylambelline (10 mg) and 6a-hydrox-
ybuphanidrine (5 mg) were obtained. Similarly, fr. III
yielded further quantities of belladine (5 mg) and 11-
O-acetylambelline (11 mg) and fr. IV, belladine (8
mg), 6a-hydroxybuphanidrine (16 mg), 6a-meth-
oxybuphanidrine (10 mg), undulatine (27 mg) and
ambelline (9 mg), together with N-demethylbelladine
(7 mg) and filifoline (17 mg) by gel filtration on Sepha-
dex LH-20.
20
20
½aꢁ þ19 ðMeOH; c 0:80Þ; CD½hꢁk : ½hꢁ
þ 2141, [h]288
D
250
ꢀ1337 (MeOH; c 0.163). IR mmax cmꢀ1: 3500–3300,
2933, 2893, 1733, 1618, 1479, 1286, 1241, 1209, 1084,
1043 (C–O), 937 (OCH2O), 754. EIMS 70 eV, m/z (rel.
int.): 331 [M]+ (54), 299 (22), 287 (49), 276 (100), 261
(37), 255 (37), 243 (20), 217 (34), 203 (20), 128 (23),
115 (38), 91 (21), 77 (36), 63 (22), 56 (75). For 1H
NMR (500 MHz, CDCl3) and 13C NMR (75 MHz,
CDCl3) spectral data see Table 2.
4.7. 6a-Methoxybuphanidrine (4)
Found: C, 65.48; H, 6.87; N, 3.99. C19H23NO5 re-
C,
quires:
66.07;
H,
6.71;
N,
4.06%.
20
D
20
½aꢁ þ34:6 ðCHCl3; c 0:13Þ; CD½hꢁk : ½hꢁ
þ 2883, [h]285
250
ꢀ1863 (MeOH; c 0.1). IR mmax cmꢀ1: 2924, 2853,
1736, 1618, 1477, 1403, 1366, 1329, 1286, 1241,
1206, 1080, 1045 (C–O), 976, 935 (OCH2O), 829,
757. EIMS 70 eV, m/z (rel. int.): 345 (65), 330
[M ꢀ CH3]+ (32), 315 [M ꢀ 2CH3]+ (27), 314 (100),
290 (33), 287 (41), 259 (88), 227 (30), 129 (24), 97
(26), 81 (44), 69 (97). 1H NMR (500 MHz, CDCl3)
and 13C NMR (75 MHz, CDCl3) spectral data are
tabulated in Table 2.
4.8. Filifoline (7)
Found: C, 65.55; H, 5.62; N, 6.48. C24H24N2O6 re-
quires: C, 66.05; H, 5.54; N, 6.42%. Mp 191–193 ꢁC.
4.4. Belladine (1)
20
nm (log ꢀ): 280 (5.93). ½aꢁ þ12
MeOH
max
UV
k
D
20
245
0.171). IR mmax cmꢀ1
Found: C, 71.83; H, 8.02; N, 4.38. Calc. for
C19H25NO3: C, 72.35; H, 7.99; N, 4.44%. UV, IR and
EIMS data were in agreement with those reported previ-
ously (Ghosal et al., 1983, 1988). For H NMR (500
MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) spectral
data see Table 1.
ðMeOH; c 0:85Þ; CD½hꢁk : ½hꢁ
ꢀ 2391,
[h]290 +419
2930, 1734
(MeOH;
c
:
(>C@O), 1617, 1477, 1401, 1317, 1279, 1216, 1131,
1095, 1073, 1040 (C–O), 979, 934 (OCH2O), 755.
EIMS 70 eV, m/z (rel. int.): 436 [M]+ (1), 296 (1),
185 (2), 165 (3), 145 (2), 122 [C6H4NO2]+ (3), 110
(3), 106 [C6H4NO]+ (45), 99 (7), 91 (26), 79
1
1
[C5H5N]+ (100), 69 (25), 55 (63). For H NMR (500
4.5. N-Demethylbelladine (2)
MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) spec-
tral data see Table 4.
Found: C, 71.25; H, 7.76; N, 4.79. C18H23NO3 re-
quires: C, 71.73; H, 7.69; N, 4.65%. UV k
MeOH
max
nm
4.9. Hydrolysis of filifoline (7)
(logꢀ): 220 (14.63), 240 (5.95), 260 (3.82), 280 (5.11).
IR mmax cmꢀ1: 3225 (NH), 2926, 2833 (C–O), 1733,
1611, 1512, 1462, 1243, 1157, 1030 (C–O), 811. EIMS
70 eV, m/z (rel. int.): 301 [M]+ (2), 299 (4), 273 (2),
195 (5), 180 [C10H14NO2]+ (16), 151 [C9H11O2]+ (100),
138 (5), 128 (1), 121 [C7H5O2]+ (9), 107 C7H7O+ (7),
Filifoline (8 mg) was dissolved in 2 ml of an aqueous
methanolic solution of K2CO3 and the mixture warmed
to gentle reflux. After TLC [MeOH/EtOAc (2:3)] indi-
cated the reaction to be complete (6 h), the solution
was neutralized with 1 M HCl, diluted with water (5
1
91 (6), 77 (7). H NMR (500 MHz, CDCl3) and 13C

J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
381
ml) and extracted with EtOAc (3 · 5 ml). The combined
organic fractions were then dried over anhydrous
MgSO4, filtered and the solvent removed under reduced
pressure to yield a crude gum (15 mg) which was purified
by prep. TLC [MeOH/EtOAc (2:3)] to give ambelline
(5.8 mg, 95%) as white crystals from Me2CO (Viladomat
et al., 1994, 1995).
4.11. Synthesis of 11-O-methanesulphonylambelline (10)
Ambelline (20 mg, 60.4 lmol) and 4-N,N-dimethyla-
minopyridine (DMAP) (0.74 mg, 6.04 lmol) were dis-
solved at rt in CH2Cl2 (1 ml) to which pyridine (9.8
ll, 120.8 lmol) was added. After 15 min methanesul-
phonyl chloride (9.8 ll, 120.8 lmol) was introduced
and the solution stirred at 40 ꢁC. After TLC [MeOH/
EtOAc (2:3)] indicated the reaction to be complete (4
h) the mixture was diluted with water (5 ml) and ex-
tracted with CH2Cl2 (3 · 5 ml). The combined organic
fractions were then dried over anhydrous MgSO4, fil-
tered and the solvent removed under reduced pressure
to yield a crude gum (30 mg) which was purified by
prep. TLC [MeOH/EtOAc (2:3)] to give 11-O-methane-
sulphonylambelline (10) (23.7 mg, 96%) as white crys-
tals from Me2CO. Found: C, 55.47; H, 5.41; N, 3.32.
C19H23NSO7 requires: C, 55.73; H, 5.66; N, 3.42%.
4.10. Synthesis of 11-O-benzoylambelline (9)
Sodium hydride (60% suspension in mineral oil) (4.8
mg, 120.8 lmol) was added to a solution of ambelline
(20 mg, 60.4 lmol) in 1 ml DMF and the mixture stirred
at rt for 20 min, then cooled to 0 ꢁC and benzoyl chlo-
ride (17.0 mg, 120.8 lmol) introduced and the solution
allowed to warm to rt. After TLC [MeOH/EtOAc
(2:3)] indicated the reaction to be complete (6 h) the mix-
ture was diluted with water (5 ml) and extracted with
CH2Cl2 (3 · 5 ml). The combined organic fractions were
then dried over anhydrous MgSO4, filtered and the sol-
vent removed under reduced pressure to yield a crude
gum (34 mg) which was purified by prep. TLC
[MeOH/EtOAc (2:3)] to give 11-O-benzoylambelline
(9) (25.5 mg, 97%) as white crystals from Me2CO.
Found: C, 68.36; H, 5.58; N, 2.99. C25H25NO6
requires: C, 68.95; H, 5.79; N, 3.22%. Mp 118–120 ꢁC.
20
20
Mp 98–100 ꢁC. ½aꢁ þ4:1 ðCHCl3; c 0:565Þ; CD½hꢁk :
D
½hꢁ
þ603, [h]286 –631 (MeOH; c 0.05). IR mmax
253
cmꢀ1: 2925, 1618, 1500, 1479, 1402, 1356 (O@S@O),
1282, 1215, 1176, 1130, 1086, 1045 (C–O), 953, 934
(OCH2O), 757. EIMS 70 eV, m/z (rel. int.): 409 [M]+
(34), 394 [M ꢀ CH3]+ (1), 378 [M ꢀ CH3–O]+ (2), 330
[M ꢀ CH3–SO2]+ (41), 313 (100), 298 (41), 282 (48),
1
270 (80), 254 (51), 240 (53), 215 (24). H NMR spectral
data (500 MHz, CDCl3): d 1.67 (1H, ddd, J = 14.0,
13.6, 4.0 Hz, H-4b), 2.21 (1H, ddd, J = 14.0, 4.0, 2.0
Hz, H-4a), 2.88 (3H, s, S-CH3), 2.91 (1H, dd,
J = 14.0, 4.0 Hz, H-12endo), 3.35 (3H, s, 3-OCH3),
3.49 (1H, dd, J = 13.6, 4.0 Hz, H-4a), 3.85 (1H, ddd,
J = 5.6, 4.0, 2.0 Hz, H-3), 3.86 (1H, dd, J = 14.0, 8.4
Hz, H-12exo), 3.93 (1H, d, J = 17.6 Hz, H-6b), 3.99
(3H, s, 7-OCH3), 4.36 (1H, d, J = 17.6 Hz, H-6a),
5.14 (1H, dd, J = 8.4, 4.0 Hz, H-11exo), 5.87-5.90
(2H, 2d, J = 1.4 Hz, OCH2O), 6.10 (1H, dd, J = 10.4,
5.6 Hz, H-2), 6.57 (1H, s, H-10), 6.58 (1H, d,
J = 10.4 Hz, H-1). 13C NMR spectral data (75 MHz,
CDCl3): d 28.8 (t, C-4), 38.4 (q, S-CH3), 47.0 (s, C-
10b), 56.9 (q, 3-OCH3), 59.3 (q, 7-OCH3), 59.4 (t, C-
6), 59.5 (t, C-12), 63.5 (d, C-4a), 72.1 (d, C-3), 91.5
(d, C-11), 100.2 (d, C-10), 101.0 (t, OCH2O), 117.0 (s,
C-6a), 127.8 (d, C-2), 130.3 (d, C-1), 132.3 (s, C-10a),
134.4 (s, C-8), 140.8 (s, C-7), 148.3 (s, C-9).
2 0
2 0
½aꢁ þ25:8 ðCHCl3; c 0:295Þ; CD½hꢁk : ½hꢁ
ꢀ 6746,
D
2 47
[h]286 +2024 (MeOH; c 0.02). IR mmax cmꢀ1: 2925, 1716
(>C@O), 1617, 1477, 1401, 1315, 1277, 1217, 1086,
1072, 1046 (C–O), 978, 933 (OCH2O), 756. EIMS 70
eV, m/z (rel. int.): 435 [M]+ (23), 420 [M ꢀ CH3]+ (2),
330 [M ꢀ PhCO]+ (4), 313 (54), 298 (21), 282 (21), 270
(8), 254 (30), 240 (22), 227 (6), 211 (11), 105 [PhCO]+
(100), 77 [C6H5]+ (57). 1H NMR spectral data (500
MHz, CDCl3): d 1.82 (1H, ddd, J = 14.0, 13.6, 4.0 Hz,
H-4b), 2.42 (1H, ddd, J = 14.0, 4.0, 2.0 Hz, H-4a), 2.92
(1H, dd, J = 14.0, 4.0 Hz, H-12endo), 3.38 (3H, s, 3-
OCH3), 3.65 (1H, dd, J = 13.6, 4.0 Hz, H-4a), 3.90
(1H, ddd, J = 5.0, 4.0, 2.0 Hz, H-3), 4.01 (3H, s, 7-
OCH3), 4.03 (1H, d, J = 17.2 Hz, H-6b), 4.08 (1H, dd,
J = 14.0, 8.0 Hz, H-12exo), 4.47 (1H, d, J = 17.2 Hz,
H-6a), 5.37 (1H, dd, J = 8.0, 4.0 Hz, H-11exo), 5.81–
5.86 (2H, 2d, J = 1.2 Hz, OCH2O), 6.11 (1H, dd,
J = 10.0, 5.0 Hz, H-2), 6.55 (1H, s, H-10), 6.58 (1H, d,
J = 10.0 Hz, H-1), 7.35 (2H, d, J = 7.6 Hz, H-40/H-60),
7.51 (1H, t, J = 7.6, 7.2 Hz, H-50), 7.66 (2H, d, J = 7.6
Hz, H-30/H-70). 13C NMR spectral data (75 MHz,
CDCl3): d 28.3 (t, C-4), 47.8 (s, C-10b), 57.0 (q, 3-
OCH3), 58.5 (t, C-6), 59.4 (t, C-12), 59.5 (q, 7-OCH3),
63.8 (d, C-4a), 72.0 (d, C-3), 86.5 (d, C-11), 99.9 (d, C-
10), 101.0 (t, OCH2O), 115.7 (s, C-6a), 124.8 (s, C-20),
127.3 (d, C-2), 128.5 (2d, C-40/C-60), 129.7 (2d, C-30/C-
70), 130.5 (d, C-1), 133.1 (s, C-10a), 133.4 (d, C-50),
134.3 (s, C-8), 141.0 (s, C-7), 148.6 (s, C-9), 166.4 (s,
C-10).
4.12. Biological activity
The anti-protozoal activity screen of filifoline against
the four different parasitic protozoa was carried out in
vitro in parallel with the respective standards: Trypano-
soma cruzi (strain Tulahuen C4, stage trypomastigotes)/
benznidazole std. IC50 = 0.31 lg/ml; Leishmania dono-
vani (strain MHOM-ET-67/L82, stage amastigotes)/mil-
tefosine std. IC50 = 0.19 lg/ml; Plasmodium falciparum
(strain K1, stage IEF)/artemisinin std. IC50 = 0.0018

382
J.J. Nair et al. / Phytochemistry 66 (2005) 373–382
DeAngelis, G.G., Wildman, W.C., 1969. Circular dichroism studies—
I. A quadrant rule for the optically active aromatic chromophore in
rigid polycyclic systems. Tetrahedron 25, 5099–5112.
lg/ml; Trypanosoma brucei rhodesiense (strain STIB 900,
stage trypomastigotes)/melarsoprol std. IC50 = 0.00245
lg/ml. Filifoline was tested for cytotoxicity towards
myoblast (L6) cells (podophyllotoxin std. IC50 = 0.002
lg/ml).
Dyer, R.A., 1976. Amaryllidaceae. In: Dyer, R.A. (Ed.), The Genera
of Southern African Flowering Plants, Department of Agricultural
Technical Series, vol. 2. Pretoria, pp. 945–954.
Ghosal, S., Saini, K.S., Arora, V.K., 1983. Latisoline, a novel
glucoalkaloid from Crinum latifolium. J. Chem. Res. (S), 238–239.
Ghosal, S., Shanthy, A., Singh, S.K., 1988. Isocraugsodine, an N-
arylidenephenethylamine from Crinum asiaticum and its E–Z
isomerism. Phytochemistry 27, 1849–1852.
Ambelline, 11-O-acetylambelline and undulatine
were identified by comparison of their chromatographic
and spectroscopic properties (TLC, IR, CD, MS, 1H
and 13C NMR) with those of authentic samples obtained
from other plant sources (Viladomat et al., 1994, 1995).
Longevialle, P., Smith, D.H., Burlingame, A.L., Fales, H.M., Highet,
R.J., 1973. High resolution mass spectrometry in molecular
structure studies-V: the fragmentation of Amaryllis alkaloids in
the crinine series. Org. Mass Spectrom. 7, 401–415.
Acknowledgements
Slabaugh, M.R., Wildman, W.C., 1971. 6-Hydroxybuphanidrine and
6-hydroxypowelline. J. Org. Chem. 36, 3202–3207.
Part of this work was financially supported by CI-
CYT-CIRIT (QFN95-4711) and the Comissionat per a
Universitats i Recerca, Generalitat de Catalunya.
W.E.C. and J.J.N. thank the University of Cape Town
for financial support. J.J.N. is also grateful to the Min-
Snijman, D.A., Linder, H.P., 1996. Phylogenetic relationships, seed
characters, and dispersal system evolution in Amaryllideae (Ama-
ryllidaceae). Ann. Mo. Bot. Gard. 83, 362–386.
Tanahashi, T., Poulev, A., Zenk, M.H., 1990. Radioimmunoassay for
the quantitative determination of galanthamine. Planta Med. 56,
77–81.
´
isterio de Educacion y Ciencia for a postdoctoral
fellowship.
Viladomat, F., Bastida, J., Codina, C., Campbell, W.E., Mathee, S.,
1994. Alkaloids from Brunsvigia josephinae. Phytochemistry 35,
809–812.
Viladomat, F., Codina, C., Bastida, J., Mathee, S., Campbell, W.E.,
1995. Further alkaloids from Brunsvigia josephinae. Phytochemis-
try 40, 961–965.
References
Viladomat, F., Bastida, J., Codina, C., Nair, J.J., Campbell, W.E.,
1997. Alkaloids of the South African Amaryllidaceae. In: Pandalai,
S.G. (Ed.), Recent Research Developments in Phytochemistry, vol.
1. Research Signpost Publishers, Trivandrum, pp. 131–171.
Wagner, J., Pham, H.L., Do¨pke, W., 1996. Alkaloids from Hippea-
strum equestre Herb.—5. Circular dichroism studies. Tetrahedron
52, 6591–6600.
Ali, A.A., Ramadan, M.A., Frahm, A.W., 1984. Alkaloid constituents
of Crinum bulbispermum III: bulbispermine, a new alkaloid of
Crinum bulbispermum. Planta Med. 50, 424–427.
Brine, N.D., Campbell, W.E., Bastida, J., Herrera, M.R., Viladomat,
F., Codina, C., Smith, P.J., 2002. A dinitrogenous alkaloid from
Cyrtanthus obliquus. Phytochemistry 61, 443–447.
DeAngelis, G.G., 1968. Empirical calculations pertaining to the
optically active aromatic chromophore in rigid systems. Tetrahe-
dron 24, 5469–5481.
Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and
Poisonous Plants of Southern and Eastern Africa. E. and S.
Livingston Ltd, Edinburgh, London.