A 7,8-dihydro-8-hydroxypalmatine from Enantia chlorantha

Article abstract of DOI:10.1016/S0031-9422(98)00382-3

7,8-dihydro-8-hydroxypalmatine, a novel protoberberine-type alkaloid, along with palmatine, has been isolated and characterized from an anti-HIV active extract from Enantia chlorantha. The structures of the two compounds were elucidated by spectroscopic analyses and from chemical evidence.

Full text of DOI:10.1016/S0031-9422(98)00382-3

PERGAMON
Phytochemistry 50 (1999) 279±281
A 7,8-dihydro-8-hydroxypalmatine from Enantia chlorantha
a
Pascal Wafo , Barthelemy Nyasse *, Catherine Fontaine
a,
b
a
Department of Organic Chemistry, Faculty of Science, University of Yaounde I, Box 812, YaoundeÂ, Cameroon
b
LEDSS 4, Chimie Recherche, Universite de Grenoble, B.P. 53X 38041 Grenoble Cedex 9, France
Received 17 March 1998
Abstract
,8-dihydro-8-hydroxypalmatine, a novel protoberberine-type alkaloid, along with palmatine, has been isolated and
7
characterized from an anti-HIV active extract from Enantia chlorantha. The structures of the two compounds were elucidated by
spectroscopic analyses and from chemical evidence. # 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Enantia chlorantha; Annonaceae; Alkaloids; Palmatine; 7,8-Dihydro-8-hydroxypalmatine
1. Introduction
2. Results and discussion
As part of our programme aiming at the isolation of
The new compound 1, obtained as yellow crystals
from MeOH (m.p. 188±1908C), showed a strong yel-
low ¯uorescence under UV light (254 and 366 nm) and
positive reactions with alkaloid-precipitating reagents,
such as Dragendor€'s. The IR spectrum of 1 revealed
the presence of bands at 3420, 1605, 1511, 974 and 851
antiprotozoal and antiviral compounds from medicinal
plants locally used in the treatment of hepatic dis-
orders, we have focussed our attention on the minor
constituents of Enantia chlorantha, the roots of which
are used in the healing of various diseases, including
tuberculosis, hepatic infections and some forms of
�
1
cm
suggestive of an hydroxyl group and of aromatic
1
ulcers (Hamonnie
years ago, it was shown that protoberberines
Jalander, Sjoholm, & Virtanen, 1990), namely palma-
Á
re, Leboeuf, & Paris, 1975). Some
nuclei. Many compounds have these moieties. The H
spectrum (1D and H/H COSY version was more sig-
ni®cant with proton signals for two methylene groups,
respectively, at d 2.76 (1H, dt, J= 4.4 and 15.5 Hz/d
3.42 (1H, ddd, J= 4.4; 9.9 and 15.5 Hz) and d 3.75
1H, ddd, J =3.8; 9.9 and 13.7 Hz)/d 3.87 (overlapping
with the large methoxyl signals). These methylene pro-
tons, together with the last non-aromatic signal at d
5.65 (1H, s) attributed to a methine group bearing an
oxygen atom, were very suggestive of a dihydro-
protoberberine system containing the four methoxyl
groups at d 3.89, 3.90, 3.95 and 3.96, respectively.
Furthermore, the aromatic part of the 1H spectrum
exhibited two singlets at d 7.20 (1H, s) and 6.65 (1H, s)
ascribable to two isolated protons situated para with
respect to each other in a 2,3,5,6-tetrasubstituted ben-
zene ring. Another singlet was observed at d 6.20 (1H,
s). An AB-system at d 7,0 (1H, d, J =8.4 Hz) and
(
È
tine, columbarnine, jatrorrhizine from the stem bark of
E. chlorantha synergically had preventive and curative
e€ects on arti®cially-provoked liver injury (Virtanen,
Lassila, Njimi, & Ekotto Mengata, 1988a,b). During
the biological screening of this species for antiviral
compounds, we have observed that certain alcoholic
fractions of its bark exhibited moderate to signi®cant
anti-HIV activity. Fractionation of one of the active
fractions has led to the isolation of a new compound,
7
,8-dihydro-8-hydroxypalmatine (1) along with the
main palmatine (2). We report herein the structural
elucidation of the novel alkaloid 1.
*
Corresponding author.
0031-9422/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00382-3

2
80
P. Wafo et al. / Phytochemistry 50 (1999) 279±281
chemical conversion to palmatine 2. When 1 in ethanol
was re¯uxed under basic conditions for 3 h, 20% of
palmatine 2 could be isolated in pure form.
Though the novel alkaloid 1 and palmatine 2 were
isolated from an anti-HIV active fraction from E.
chlorantha, both of them failed to exhibit any interest-
ing e€ect on the corresponding virus when submitted
to the NCI screening programme in their pure forms.
3
. Experimental
3
.1. General
M.p.'s are uncor. 1H NMR were recorded at
00 MHz, 13C NMR at 100 MHz in CDCl3 or
4
DMSO-d6 using TMS as int. standard. EIMS were
obtained at 70 eV. Silica gel 60 (Fluka, 230±400 mesh)
was used for CC and precoated silica plates (Merck,
silica gel 60 F254, 0.25 mm) were used for TLC.
3
.2. Plant material
E. chlorantha Oliver was collected at Ede
Cameroon) in March 1995. A voucher specimen is
deposited in the National Herbarium, Yaounde
Cameroon.
Â
a (Littoral,
Â
,
6.90 (1H, d, J= 8.4 Hz) of another tetrasubstituted
benzene ring was also observed.
3
.3. Extraction and isolation
Air-dried and ®nely powdered stem bark (2 kg) was
In order to establish the substitution pattern of the
suspected dihydroprotoberberine skeleton, the HMQC
and HMBC spectra of 1 were recorded and analysed
accordingly (Table 1). At ®rst glance, these data
coupled to those described above suggested a close
structural relationship with palmatine 2. For example,
the proton at d 5.65 carried by the carbon atom at d
macerated in MeOH (8 l) for 72 h. After ®ltration and
evapn at pres., the crude material (200 g) was treated
with 10% aq. HCl (2 l) to form a precipitate, which
upon ®ltration, a€orded a yellow solid mixt. (30 g).
The ®ltrate was, in turn, extracted several times with
73.6 was correlated through three bonds both to the
CHCl and lyophilized to a ¯u€y solid (80 g). Half of
3
quaternary aromatic carbon atom at d 129.5 and to
the one at d 146.6 carrying the methoxyl group,
appearing itself at d 61.1. Likewise, one of the AB-
system protons, i.e. the one at d 7.0 was also corre-
lated to the carbon atoms at d 129.5 and 146.6 whilst
the isolated proton at d 6.20 showed long-range coup-
lings with the carbon atoms at d 118.6 bearing the
second AB-system proton. The above correlations
sustained by the observed NOE e€ects between the
proton at d 6.20 and those at d 7.20 (1H, s) and 6.90
this solid was chromatographed on silica gel (600 g)
using hexane±CHCl mixts of increasing polarity and
3
collecting 200 ml frs. Identical frs by TLC obtained
upon elution with hexane±CHCl3 (7:3) were pooled
and the solvent removed. The semi-solid thereby
obtained was further puri®ed on a smaller silica gel
column using the same eluting system as above to give
a solid (2 g) which, in turn, was recrystallized from hot
MeOH to yield yellow crystals of 1 (1.2 g). Further
elution of the column with CHCl ±MeOH (19:1) 5 g
3
(1H, d, J= 8.4 Hz) belonging, respectively, to
two di€erent aromatic nuclei are consistent with an
of solid palmatine 2.
7,8-dihydro-8-hydroxypalmatine skeleton for 1.
Such a proposition was in good agreement with the
3.4. 7,8-Dihydro-8-hydroxypalmatine (1)
mass spectrum of 1 which showed the predominent
fragment ion at m/z 352 resulting from the loss of an
hydroxyl group from the parent ion. Another support-
ing argument to structure 1 was obtained from its
Yellow crystals (1.2 g), m.p. 188±1908C. [a] �35.0
D
�
1
(c 0.08 CHCl ). IR n
(KBr) cm : 3421, 1606,
max
3
1511, 1490, 1343, 1316, 1190, 1036, 1001, 975, 830,
+
807. EIMS m/z: 352 [M � 17] . NMR: Table 1.

P. Wafo et al. / Phytochemistry 50 (1999) 279±281
Table 1. H and 13C NMR data for compound 1
281
1
Atom
Assignments
C
Observed correlations
HBMC
H
NOE
1
2
3
4
105.60
147.90
149.25
111.90
127.40
30.00
7.20(s)
±
3, 4a, 13a
±
H-13, MeO-2
±
6.65 (s)
±
2, 5, 13a
±
MeO-3
4
a
±
2.76 (dt, 4.4 and 15.5 Hz)
5
3
.42 (ddd, 4.4, 9.9 and 15.5 Hz)
3.75 (ddd, 3.8, 9.9 and 13.7 Hz)
.87 (overlapping)
6
51.80
H-8
3
8
8
9
1
1
1
1
1
1
1
73.60
114.90
146.59
149.77
114.60
118.60
129.50
97.10
5.65 (s)
±
6, 8a, 9, 12a, 13a
±
H-1, H-12
a
±
0
±
1
7.00 (d, 8.4 Hz)
6.90 (d, 8.4 Hz)
9, 12a
8a, 10
MeO-10
H-13
2
2a
3
3a
3b
137.60
123.50
61.10
OMe
Ome
Ome
Ome
3.95 s
3.89 s
3.96 s
3.90 s
H-1
H-11
±
56.40
56.10
56.10
H-4
3.5. Palmatine (2)
puri®cation over silica gel using hexane±CHCl mixts
3
of increasing polarity a€orded 38 mg (20%) of palma-
tine (2).
Yellow solid (5 g), m.p. 206±2088C. IR n (KBr)
cm : 3606, 3367, 1636, 1605, 1567, 1524, 1511, 1365,
max
�
1
1
9
68. H NMR (400 MHz, DMSO-d ): d 9.95 (1H, s,
6
H-8), 9.15 (1H, s, H-13), 8.15 (1H, d, J=9.2 Hz,
H-11), 8.10 (1H, d, J= 9.2 Hz, H-10), 7.75 (1H, s,
H-1), 7.15 (1H, s, H-4), 4.95 (2H, t, J =6.2 Hz, H-6),
Acknowledgements
This investigation was supported by a grant from
the International Foundation for Science, Stockholm
(IFS F/2626-1) awarded to BN. We also wish to thank
Dr. Christophe Morin for his interest and continuing
support to this programme and Professor B.L.
Sondengam for his constructive advice. Dr. Anthony
Mauger and Gordon Cragg at the NCI are gratefully
acknowledged for taking care of the antiviral and
anticancer screening.
4
3
.15 (3H, s, C-9-OCH ), 4.00 (3H, s, C-10-OCH ),
3 3
.90 (3H, s, C-2-OCH ), 3.88 (3H, s, C-3-OCH ), 3.25
3
3
13
(
2H, t, J=6.2 Hz, H-5).
C NMR (100 MHz,
DMSO-d ): d 153.7 (C-3), 151.5 (C-10), 150.8 (C-2),
6
1
1
1
46.2 (C-8), 145.6 (C-9), 139.9 (C-13a), 135.4 (C-12a),
29.8 (C-4a), 128.0 (C-11), 124.3 (C-8a), 121.1 (C-13),
20.4 (C-13b), 112.0 (C-4), 109.7 (C-1), 62.4
(
C-9-OCH ), 57.4 (C-10-OCH ), 57.3 (C-2-OCH ),
3 3 3
56.8 (C-3-OCH ), 56.5 (C-6), 27.8 (C-5).
3
3.6. Conversion of 1 (2)
References
7,8-Dihydro-8-hydroxypalmatine (1) (200 mg) was
suspended in a mixt. of CHCl (20 ml), EtOH (20 ml)
and 30% aq. NaOH (20 ml) and re¯uxed for 4 h. The
reaction mixt. was then acidi®ed to pH 6 with 10%
Hamonnie
Phytother., IX, 296 and references cited therein.
Jalander, L., Sjoholm, R., & Virtanen, P. (1990). Collect. Czech. Chem.
Á
re, M., Leboeuf, A., & Paris, R. R. (1975). Plant. Med.
3
È
Commun., 55, 2095.
Virtanen, P., Lassila, V., Njimi, T., & Ekotto Mengata, D. (1988a). Acta
Anat., 131, 166.
HCl and extracted Â2 with CHCl (2 Â 100 ml). The
3
resulting organic phase was dried (MgSO ), ®ltered
4
Virtanen, P., Lassila, V., Njimi, T., & Ekotto Mengata, D.(1988b).Acta
Anat., 132, 159.
and the solvent removed to leave a residue which upon