Antiarol cinnamate and africanoside, a cinnamoyl triterpene and a hydroperoxy-cardenolide from the stem bark of antiaris africana

Article abstract of DOI:10.1055/s-0030-1249958

From the methanol extract of the stem bark of the African tree Antiaris africana Engler, two new bioactive metabolites were isolated, namely, the α-amyrin derivative 1β,11α-dihydroxy-3β-cinnamoyl-α- amyrin (antiarol cinnamate, 1) and a cardiac glycoside, 3β-O-(α-L- rhamnopyranosyl)-14β-hydroperoxy-5β-hydroxy-19-oxo-17β-card- 20(22)-enolide (africanoside, 2a), together with the known compounds-α- amyrin and its acetate, β-sitosterol and its 3-O-β-D-glucopyranoside, friedelin, ursolic and oleanolic acid, 19-norperiplogenin, strophanthidol, strophanthidinic acid, periplogenin (3a), 3-epiperiplogenin, strophanthidin (3b) and 3,3-dimethoxy-4-O-β-D-xylopyronosyl-ellagic acid. Their structures were established on the basis of their spectroscopic data and by chemical methods, while 3a was additionally confirmed by Xray crystal structure analysis. The aglycone moiety possessing a hydroperoxy group was found for the first time in cardenolides. Compounds 1 and 2a showed no activity against bacteria, fungi, and microalgae; however, the crude extract exhibited a high toxicity against Artemia salina and a selective antitumor activity against human tumor cell lines. Africanoside (2a) effected a concentration-dependent inhibition of tumor cell growth with a mean IC₅₀ value of 5.3nM. Georg Thieme Verlag KG Stuttgart - New York.

Full text of DOI:10.1055/s-0030-1249958

Original Papers 1717
Antiarol Cinnamate and Africanoside, a Cinnamoyl
Triterpene and a Hydroperoxy-cardenolide from the
Stem Bark of Antiaris africana
Authors
Bertin Vouffo1,2, Etienne Dongo2, Petrea Facey1, Andrea Thorn3, George Sheldrick3, Armin Maier4,
Heinz Herbert Fiebig4, Hartmut Laatsch1
1
Affiliations
Department of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany
Department of Organic Chemistry, Faculty of Science, University of Yaounde I, Yaounde, Cameroon
Department of Inorganic Chemistry, University of Göttingen, Göttingen, Germany
Oncotest GmbH, Freiburg, Germany
2
3
4
Key words
Abstract
were established on the basis of their spectro-
"
l Antiaris africana
!
scopic data and by chemical methods, while 3a
was additionally confirmed by X‑ray crystal
structure analysis. The aglycone moiety possess-
ing a hydroperoxy group was found for the first
"
l Moraceae
From the methanol extract of the stem bark of
the African tree Antiaris africana Engler, two
new bioactive metabolites were isolated, namely,
the α-amyrin derivative 1β,11α-dihydroxy-3β-
cinnamoyl-α-amyrin (antiarol cinnamate, 1) and
"
l antiarol cinnamate
"
l africanoside
"
l sterol hydroperoxide
time in cardenolides. Compounds
1 and 2a
"
l periplogenin
showed no activity against bacteria, fungi, and
microalgae; however, the crude extract exhibited
a high toxicity against Artemia salina and a selec-
tive antitumor activity against human tumor cell
lines. Africanoside (2a) effected a concentration-
dependent inhibition of tumor cell growth with a
mean IC50 value of 5.3 nM.
a
cardiac glycoside, 3β-O-(α-L-rhamnopyrano-
syl)-14β-hydroperoxy-5β-hydroxy-19-oxo-17β-
card-20(22)-enolide (africanoside, 2a), together
with the known compounds β-amyrin and its
acetate, β-sitosterol and its 3-O-β-D-glucopyran-
oside, friedelin, ursolic and oleanolic acid, 19-
norperiplogenin, strophanthidol, strophanthi-
dinic acid, periplogenin (3a), 3-epiperiplogenin,
strophanthidin (3b) and 3,3′-dimethoxy-4′-O-β-
D-xylopyronosyl-ellagic acid. Their structures
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
droxy-19-oxo-17β-card-20(22)-enolide, 2a] from
the methanol extract of the stem bark of Antiaris
africana, together with 14 known compounds: β-
amyrin and its acetate, β-sitosterol and its 3-O-β-
D-glucopyranoside, friedelin, ursolic and oleanolic
acid, strophanthidol, strophanthidinic acid, stro-
phanthidin, periplogenin, 3-epiperiplogenin, 19-
norcardenolide, and 3,3′-dimethoxy-4′-O-β-D-xy-
lopyronosyl-ellagic acid. Their structures were es-
tablished using 1D, 2D NMR spectra, EI and ESI
mass spectra and by chemical derivatization.
!
Antiaris africana Engler is a tree belonging to the
Moraceae family. It is usually about 15–20 meters
high, but can grow sometimes up to 40 meters,
has a white latex and alternate dissymmetric
leaves [1,2]. The wood is used in some regions of
Africa as timber [2,3], and the bark extract is used
in traditional medicine for the treatment of chest
pains [3]. Leaf decoctions are applied in the treat-
ment of syphilis [2], and the latex is a purgative
agent. It is also used to treat sore throat and lep-
rosy [2]. According to previous phytochemical
studies, the plant contains sterols and terpenoids
such as butyrospermol, α-amyrin and lupenyl
acetates, α-amyrin and lupenyl cinnamates as
well as betaines, tryptophane, β-phenylalanine
and cardiac glycosides [3,4].
received
revised
accepted
October 28, 2009
April 9, 2010
April 20, 2010
Bibliography
DOI http://dx.doi.org/
10.1055/s-0030-1249958
Published online June 8, 2010
Planta Med 2010; 76:
1717–1723 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Materials and Methods
!
Correspondence
Prof. Dr. Hartmut Laatsch
Institute for Organic and
Biomolecular Chemistry
Georg-August-University
Göttingen
Tammannstrasse 2
37077 Göttingen
Germany
General
The optical rotations were measured on a Perkin-
Elmer polarimeter (model 241). IR spectra were
recorded on a Perkin-Elmer 1600 Series FT‑IR
spectrometer with KBr pellets. NMR spectra were
measured on Varian Inova 600 (599.740 MHz),
Varian Inova 500 (499.879 MHz) and Varian Unity
300 (300.145 MHz) spectrometers. ESI mass spec-
In our research program to study species of the
genus Antiaris, we isolated two new compounds,
antiarol cinnamate (1β,11α-dihydroxy-3β-cin-
namoyl-α-amyrin, 1) and africanoside [3β-O-(α-
L-rhamnopyranosyl)-14β-hydroperoxy-5β-hy-
Phone: + 49551393211
Fax: + 49551399660
hlaatsc@gwdg.de
Vouffo B et al. Antiarol Cinnamate and… Planta Med 2010; 76: 1717–1723

1718 Original Papers
tra were recorded on a Quattro Triple Quadrupole mass spec-
Further purification of fraction 7 (eluted with n-hexane-EtOAc
4:6) on silica gel (25 × 300 mm, n-hexane-EtOAc gradient 1:1 in-
creasing to 2:8) gave strophantidol (13 mg), and 3-epi-periploge-
nin (7 mg); the latter was further purified on Sephadex LH-20
(20 × 750 mm, CH2Cl2-MeOH 1:1).
The last fraction (eluted with n-hexane-EtOAc 2.5:7.5) gave a
white precipitate of 2a (75 mg). Chromatography of the filtrate
on a 15 × 250 mm silica gel column with n-hexane-EtOAc 25:75
gave strophanthidinic acid (9 mg), and 3,3′-dimethoxy-4′-O-β-D-
xylopyranosyl-ellagic acid (3 mg), which were purified on Sepha-
dex LH-20 (20 × 750 mm) eluting with MeOH.
trometer, with a Finnigan TSQ 7000 with nano-ESI API ion source.
EI‑MS was performed on a Finnigan MAT95 (70 eV). High-resolu-
tion mass spectra (HR‑MS) were recorded by ESI MS on an Apex
IV 7 Tesla Fourier-Transform Ion Cyclotron Resonance Mass Spec-
trometer (Bruker Daltonics). Silica gel for flash chromatography:
30–60 µm; J.T. Baker; column chromatography was carried out
on MN silica gel 60: 0.05–0.2 mm (230–400 mesh; Macherey-Na-
gel & Co). Thin-layer chromatography (TLC) was performed on
silica gel plates G60, type GF254 (Merck) and DC cards Polygram
SIL G/UV254 (Macherey-Nagel & Co.) with visualization under UV
(254 and 365 nm) and with anisaldehyde/sulphuric acid as spray
reagent. Size exclusion chromatography was done on Sephadex
LH-20 (Lipophilic Sephadex; Amersham Biosciences, Ltd. pur-
chased from Sigma-Aldrich Chemie).
According to their NMR data, all compounds had a purity of bet-
ter than 95%, and no further purification steps were performed.
Reduction of africanoside
Africanoside (2a, 5 mg) was dissolved in 2 mL MeOH and 2 mg of
NaBH4 were added. After stirring for 30 minutes at room temper-
ature, 5 mL of water were added and the reduction product was
extracted with CH2Cl2 (3 × 10 mL). Evaporation under vacuum
gave convallatoxin (2b) as a white powder (2.4 mg). After passing
through Sephadex LH-20, it was chromatographically pure in
three different solvent systems.
Plant material
The plant Antiaris africana Engler (Moraceae) was collected from
Mont Eloundem (Yaounde) in the Central Province of the Repub-
lic of Cameroon, in May 2006. Mr. Victor Nana of the National
Herbarium of Cameroon identified the plant. A voucher specimen
(N°5925 SRFCam) is deposited at the National Herbarium,
Yaounde, Cameroon.
Isolates
Extraction and isolation
Antiarol cinnamate [3-O-(E)-cinnamoyl-1β-11α-dihydroxy-α-
amyrin, 1]: White amorphous powder, Rf: 0.45 (n-hexane/EtOAc
75:25); [α]2D0: + 24.0 (c 0.1, CHCl3); IR (KBr): νmax = 3309, 3168,
The air-dried and powdered stem bark (8 kg) of Antiaris africana
Engler was macerated with MeOH at room temperature for 72 h
(48 h + 24 h; 2 × 15 L each). Removal of the solvent from the ex-
tracts under reduced pressure yielded a dark brown residue
(100 g). A part (80 g) of the crude methanolic extract was ad-
sorbed on 100 g of silica gel 60 (0.05–0.2 mm, 230–400 mesh)
with 0.1 L methanol as solvent and dried under reduced pressure.
The brown powder obtained was submitted to gradient flash
chromatography on 600 g of silica gel (column 15 × 15 cm, elu-
ents: n-hexane/EtOAc, stepwise gradient, 0, 5, 10, 15, 25, 50, and
100% EtOAc), and 75 fractions each of 500 mL were collected and
pooled into 8 major fractions by combination on the basis of TLC.
Further separation of these fractions was done by repeated col-
umn chromatography using silica gel, Sephadex LH-20 or C-18 re-
versed-phase columns. Fraction 1 (eluted with n-hexane) was a
mixture of fatty acids and hydrocarbons and was discarded. Frac-
tion 2 (4.1 g) was eluted with n-hexane-EtOAc 9:1 and was chro-
matographed on a 3 × 35 cm silica gel column with n-hexane-
EtOAc 9:1 yielding β-amyrin acetate (31 mg), and friedelin
(15 mg). Elution with n-hexane-EtOAc 85:15 gave β−sitosterol
(4 mg) and β-amyrin (8 mg).
The n-hexane-EtOAc (8:2) eluted fraction 3 (5.1 g) was further
separated by silica gel column chromatography (30 × 350 mm, n-
hexane-ethyl acetate gradient with 15 to 35% EtOAc) to furnish 1
(6 mg). A further subfraction was further purified on Sephadex
LH-20 (20 × 750 mm) eluting with CH2Cl2-MeOH 3:2 to give ur-
solic acid (8 mg), and oleanolic acid (4 mg). Fractions 4 and 5 were
discarded.
From fraction 6 (5.0 g, eluted with n-hexane-EtOAc 1:1), a pre-
cipitate was obtained, which was recrystallized from MeOH to
give β-sitosterol 3-O-β-D-glucopyranoside (16 mg). We isolated
additionally 19-norperiplogenin (7 mg), periplogenin (3a,
35 mg), and strophanthidin (3b, 6 mg) when the filtrate was chro-
matographed on silica gel (25 × 500 mm, n-hexane-EtOAc gra-
dient 1:1 increasing to 25:75). Strophanthidin was further puri-
fied on Sephadex LH-20 (20 × 750 mm) eluting with CH2Cl2-
MeOH 1:1.
2917, 1713, 1639, 1171, 980 cm−1 1H‑NMR (CDCl3, 600 MHz)
;
and 13C‑NMR (CDCl3, 150 MHz): see l Table 1; EI‑MS (70 eV): m/
z (%) = 588 (8), 570 (100), 422 (15), 389 (8), 255 (8), 217 (18), 131
(10); HR‑ESI‑MS (pos. ion mode): m/z = 611.40691 [M + Na]+
(calcd. for C39H56O4Na: 611.40707).
"
Africanoside [3β‑O-(α-L-rhamnopyranosyl)-14β-hydroperoxy-5β-
hydroxy-19-oxo-17β-card-20(22)-enolide, 2a]: White amorphous
powder, Rf: 0.62 (EtOAc/MeOH 95:5); [α]2D0: + 1.0 (c 0.1, CHCl3);
IR (KBr): νmax = 3422, 2936, 1738, 1707, 1622, 1034, 980 cm−1
;
1H‑NMR (600 MHz, acetone-d6) and 13C‑NMR (125 MHz, ace-
"
tone-d6): see l Table 2; ESI‑MS (pos. ion mode): m/z = 589 [M +
Na]+, 567 [M + H]+; ESI MS (neg. ion mode): m/z = 565 [M – H]−;
HR‑ESI‑MS (pos. ion mode): m/z = 567.27985 [M + H]+ (calcd. for
C29H43O11: 567.27998).
Convallatoxin [3β‑O-(α-L-rhamnopyranosyl)-5β,14β-dihydroxy-
19-oxo-17β-card-20(22)-enolide, 2b]: White amorphous powder,
Rf: 0.58 (EtOAc/MeOH 95:5); 1H‑NMR (300 MHz, pyridine-d5,
acetone-d6) and 13C‑NMR (125 MHz, pyridine-d5, acetone-d6)
+
"
see l Table 2; ESI‑MS (pos. ion mode): m/z = 573 [M + Na] ;
ESI‑MS (neg. ion mode): m/z = 549 [M – H]−, 595 [M + HCOO]−;
HR‑ESI‑MS (pos. ion mode): m/z = 551.28503 [M + H]+ (calcd. for
C
29H43O10: 551.28507).
Antimicrobial assay
Agar diffusion tests were performed using the usual protocol [5]
with Bacillus subtilis (ATCC 6051), Escherichia coli, Staphylococcus
aureus, Streptomyces viridochromogenes (Tü 57), Candida albi-
cans, and Mucor miehei (Tü 284) as well as three microalgae:
Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus subspi-
catus.
The crude extract was dissolved in CHCl3/10% MeOH (concentra-
tion 50 mg/mL), in which the paper disks (8 mm diameter) were
dipped, dried under sterile conditions (flow box) and placed on
agar plates inoculated with the test strains. The plates were incu-
bated at 37°C for bacteria (12 hours), 27°C for fungi (24 hours),
Vouffo B et al. Antiarol Cinnamate and… Planta Med 2010; 76: 1717–1723

Original Papers 1719
Table 1 13C‑NMR data (150 MHz)
Position
δC
δH
Position
δC
δH
and 1H‑NMR data (600 MHz) for
antiarol cinnamate (1) in CDCl3.
1
2
76.6
32.2
77.6
38.1
52.3
17.7
33.0
41.9
56.5
44.2
67.5
126.6
144.5
43.7
26.9
27.8
33.5
57.8
39.3
3.62 dd (4.5, 11.5)
1.82 m, 1.94 m
20
21
22
23
24
25
26
27
28
29
30
1′
39.3
31.0
0.91# c
1.24 m, 1.39 m
1.29 m, 1.42 m
0.93 s
3
4.66 dd (4.6, 12.1)
41.2
4
16.3
5
0.79 m
28.0
0.89 s
6
1.56 m, 1.64 m
1.29 m, 1.49 m
13.3
1.12 s
7
17.9
1.04 s
8
23.1
1.16 s
9
1.67 d (8.1)
28.6
0.79 s
10
11
12
13
14
15
16
17
18
19
17.7
0.85 d (6.3)
0.91 brs
4.28 dd (3.5, 8.1)
5.22 d (3.7)
21.3
166.6
118.6
144.5
134.5
128.8
128.1
130.2
2′
3′
0.99 m, 1.74 m
0.92 m, 2.02 m
4′
5′
6′
1.38#
1.31 mc
7′
# Signals not interpretable; c Interchangeable; J in Hz, shifts as δ values
and 24–26°C under daylight for microalgae (96 hours). The diam-
eter of the inhibition zones was then measured. Actinomycin D
for bacteria (from our own sample collection), amphotericin B
for fungi (Sigma Aldrich), and staurosporine for algae (from our
own sample collection) were used as positive controls at 40 µg/
platelet.
squares methods on F2 using SHELXL97 [9] to give a final R-factor
of 0.0281. All nonhydrogen atoms were visible in the difference
map and refined using a riding model, except for H2O. Those hy-
drogens could be seen in the electron density and were refined
accordingly. Several disordered groups were modelled and their
occupancy refined and fixed in later stages of the refinement.
The Flack x parameter [10] of 0.06(11) confirmed the absolute
configuration of periplogenin (3a).
Brine shrimp microwell cytotoxicity assay
Cytotoxicity of the crude extracts was determined with brine
shrimps (Artemia salina) using a microwell cytotoxicity assay
[6]. The mortality rate was determined according to Duracková
et al. [7]. Actinomycin D as a positive control showed a mortality
rate of 100% at 10 µg/mL.
Crystallographic data for the structure reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre: CCDC 749301 contains the supplementary crystallo-
graphic data. Copies of the data can be obtained, free of charge,
on application to the Director, CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax + 44-(0)1223–336033 or email: deposit@ccdc.
cam.ac.uk).
Human tumor cell line cytotoxicity assay
A modified propidium iodide assay was used to examine the cy-
totoxic activity of the compounds against human tumor cell lines.
The test procedure was described elsewhere [8]. Cell lines tested
were derived from patient tumors, engrafted as a subcutaneously
growing tumor in NMRI nu/nu mice, or obtained from American
Type Culture Collection, National Cancer Institute, Bethesda, MD,
USA, or Deutsche Sammlung von Mikroorganismen und Zellkul-
turen. Doxorubicin hydrochloride (Medac) was used as a positive
control, and was tested in concentrations up to 5.2 µM.
Supporting information
1H‑NMR and 13C‑NMR spectra of compounds 1, 2a, 3a and 3′-epi-
periplogenin, crystal data and structure refinement for 3a and
bond lengths [Å] and angles [°] in 3a are available as Supporting
Information.
Results and Discussion
!
X‑ray structure determination of periplogenin (3a)
Colorless crystals of 3a were obtained from a solution of hexane
The methanol extract of the stem bark of Antiaris africana Engler
was subjected to silica gel and Sephadex LH-20 column chroma-
tography to afford 16 compounds. Antiarol cinnamate (1,
and ethyl acetate (1:1).
A suitable crystal (0.20 × 0.07 ×
0.05 mm3) was mounted at 100 K on a Bruker Smart 6000 CCD
diffractometer equipped with a rotating anode generator and In-
coatec Helios optics using Cu-Kα radiation (λ = 1.54178 Å). A total
of 50430 reflections were measured, of which 3250 were inde-
pendent.
l Fig. 1) was obtained as a white amorphous powder that gave a
"
positive response to the Liebermann-Buchard reaction. Its
(+)-HR‑ESI mass spectrum exhibited an [M + Na]+ quasi-molecu-
lar ion peak at m/z = 611.40691, ascribable to a molecular formula
C39H56O4Na (calculated: 611.40707), calculating for 12 double
bond equivalents in the molecule. Its IR spectrum showed a
strong absorption band at 1713 cm−1, indicating an α,β-unsatu-
rated ester. Bands at 3309 and 1171 cm−1 pointed to the presence
of a secondary alcohol in the molecule.
The asymmetric unit consisted of 1.9 periplogenin molecules
with an impurity of strophanthidin (3b), which replaced one of
the two periplogenin molecules in 10% of the unit cells. It also
contained one molecule of crystal water. The crystal investigated
belonged to the monoclinic space group P21.
The 13C‑NMR spectrum showed 39 signals, which were resolved
by an APT experiment into 8 methyls, 16 methines, 7 methylenes
and 8 quaternary carbons. Nine of these signals were assigned to
The structure was determined by direct methods. All atoms ex-
cept hydrogens were refined anisotropically by full-matrix least-
Vouffo B et al. Antiarol Cinnamate and… Planta Med 2010; 76: 1717–1723

1720 Original Papers
Fig. 1 Chemical structures of antiarol cinnamate (1); africanoside (2a)
and convallatoxin (2b); periplogenin (3a) and strophanthidin (3b).
Fig. 2 Selected COSY (–) and HMBC (→) correlations of antiarol cinna-
mate (1) and africanoside (2a).
Position
2a
2b
Table 2 13C‑NMR data (125 MHz)
and 1H‑NMR data (300 MHz) for
africanoside (2a) in acetone-d6 and
for convallatoxin (2b) in acetone-d6
and pyridine-d5.
b
c
d
δC
δH
δC
δC
δC
1
2
24.9
25.6
74.2
35.9
74.3
37.0
18.7
42.4
39.7
55.6
22.9
40.0
51.3
85.2
32.5
27.4
50.1
16.0
208.4
176.1
73.9
117.8
174.4
100.4
72.1
72.4
73.4
70.1
18.1
1.66 m, 2.00 m
1.67 m, 1.72 m
4.06 m
24.9
25.7
74.2
36.0
74.3
37.0
18.7
42.4
39.8
55.6
22.9
40.0
51.3
85.2
32.5
27.5
50.1
16.0
208.3
176.0
73.9
117.8
174.3
100.4
72.2
72.5
73.5
70.1
18.1
24.3
26.0
73.8
36.3
73.7
36.5
18.1
42.4
39.6
55.6
22.9
40.1
51.3
85.2
32.8
27.6
50.3
16.3
208.1
176.1
74.0
117.7
175.8
100.4
72.1
72.4
73.7
70.1
18.1
25.0
25.7
73.83
35.6
73.77
37.1
18.7
42.1
39.7
55.4
22.8
39.8
50.0
84.5
32.4
27.4
51.2
16.2
208.2
175.6
73.2
117.8
174.4
100.7
72.9
72.6
74.0
70.8
18.9
3
4
1.56 m, 2.15 m
5
6
1.56 m, 2.05 m
2.16 m
7
8
1.92 m
9
1.72 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1′
2′
3′
4′
5′
6′
1.45 m
1.44 m
1.12 m, 1.65 m
1.86 m, 2.10 m
2.81 m
0.86 s
10.08 s
4.90 dd, (18.1, 1.7)
5.85 brs
4.85 brs
3.77 brs
3.59 m a
3.39 t (9.3)
3.63 m a
1.20 d (6.2)
a Interchangeable; J in Hz, shifts as δ values; b Reduction product and c commercial 2b in acetone-d6;
d Commercial 2b sample in pyridine-d5
a trans-cinnamoyl fragment, which was confirmed by compari-
droxy groups at C-1 and C-11, and the skeleton of the molecule
(l Fig. 2). The large axial-axial coupling constant (J9,11 = 8.1 Hz)
1
son of its 13C- (l Table 1) and H‑NMR data with those in the lit-
"
"
erature [11,12]. The 30 remaining carbon signals were indicative
for a triterpenoid which in its 13C‑NMR spectrum exhibited reso-
nances of 8 methyls, 3 oxygenated methines (δ = 77.6, 76.6 and
67.5) and 2 olefinic carbons (δ = 144.5 and 126.6); the 1H‑NMR
of H-11 indicated the equatorial 11α-position of the hydroxy
group at C-11. Related 11α-hydroxy-ursane triterpenes have
been reported from taxonomically different plants [14,15]. The
1D NOE spectrum of 1 showed after irradiation of H-1, an NOE
for H-3, H-9 and H-5, suggesting the β-orientation of the hydroxy
group at C-1; this was further confirmed by irradiation of H-3,
"
spectrum confirmed these assignments. These data (l Table 1)
indicated that 1 is an urs-12-ene-1,3,11-triol derivative [13],
which was confirmed by COSY correlations, mainly by cross sig-
nals between the protons H-12 and H-11, H-11 and H-9, H-3 and
"
which in turn showed an NOE for H-1, H-9 and H-5 (l Fig. 2).
Thus antiarol cinnamate (1) was deduced to be 3β-O-(E)-cinna-
moyl-1β-11α-dihydroxyurs-12-ene.
"
H-1 with H-2 and H-2′ (l Fig. 2).
"
The position of the cinnamoyl group at C-3 of the triterpene moi-
ety was determined easily using HMBC spectra, which demon-
strated a correlation between H-3 and the cinnamoyl CO group.
This spectrum was further helpful to confirm the positions of hy-
Africanoside (2a, l Fig. 1) was obtained as a white powder, which
showed in its (+)-HR‑ESI mass spectrum a quasi-molecular ion
peak at m/z = 567.27985 (calculated: 567.27998 for [M + H]+) cor-
responding to the molecular formula C29H43O11. An IR band at
Vouffo B et al. Antiarol Cinnamate and… Planta Med 2010; 76: 1717–1723

Original Papers 1721
1738 cm−1 was in accordance with an α,β-unsaturated-γ-lactone,
and the signal at 1707 cm−1 indicated the presence of an aldehyde
group. 1H- and 13C‑NMR spectra and 2D NMR data showed clearly
that compound 2a is the α-l-rhamnopyranosyl derivative [16] of a
cardenolide (α,β-unsaturated steroid γ-lactone) [18]. For the sug-
ar moiety, the anomeric proton signal appeared at δΗ = 4.85
(broad singlet, 1H) and δC = 100.4. The methyl signal of a 6-deoxy
sugar appeared at δΗ = 1.20 (d; J = 6.2 Hz) and δC = 18.7. Its broad-
band decoupled 13C‑NMR spectrum showed 29 carbon signals
with 2 methyls, 11 methine, 10 methylene and 6 quaternary car-
bons according to its APTspectrum, which further confirmed that
2a is a cardiac glycoside.
Fig. 3 Crystal structure of periplogenin (3a). The structure of 3a as deter-
mined by X‑ray crystallographic analysis. Only one of two molecules in the
asymmetric unit is shown, with the disordered 3b removed for clarity. The
atoms are shown as ORTEP plots with 50% probability displacement ellip-
soids.
These data were within the experimental error limit identical
"
with those of convallatoxin (2b) (l Table 2) [17] and related gly-
cosides [18], but the additional oxygen atom cannot be con-
nected with carbon and must therefore be part of a hydroperox-
ide. The presence of intense bands in its IR spectrum at 3422 (OH
bond), 1034 (C–O‑O bond) and 980 cm−1 (O‑O bond) confirmed
this assumption. The presence of a hydroperoxy group in this
compound was unambiguously proven by an exchangeable hy-
the reduction product showed a pseudo-molecular ion peak at m/
z = 551.2850 ([M + H]+), indicative for the molecular formula
1
"
droxy proton signal at δ = 4.00 in the H‑NMR spectrum, and by
C
29H43O10. Spectroscopic comparison (l Table 2) with a commer-
reduction using NaBH4. In fact, the (+)-HR‑ESI mass spectrum of
cial convallatoxin (2b) sample proved the identity both in ace-
IC50 (nM)
Table 3 Cytotoxic activity of afri-
canoside (2a) against tumor cell
lines in a monolayer cytotoxicity
assay.
Tumor type
Cell line
2a
Doxorubicin
Bladder
BXF 1218 L
BXF 1352 L
BXF T24
5.1
6.0
5.3
6.3
5.6
6.2
4.6
6.5
8.8
1.9
6.5
1.2
0.7
1.1
4.9
0.9
7.6
16.4
2.6
107
20.8
24.3
24.3
1.2
Colon
CXF 269 L
CXF HCT116
CXF HT29
CXF RKO
Stomach
Head & neck
Lung
GXF 251 L
19.7
4.5
HNXF CAL27
LXFA 289 L
LXFA 526 L
LXFA 629 L
LXFL 1121 L
LXFL 529 L
LXFL H460
MAXF 401NL
MAXF MCF7
MEXF 1341 L
MEXF 276 L
MEXF 462NL
OVXF 899 L
OVXF OVCAR3
PAXF 1657 L
PAXF 546 L
PAXF PANC1
PRXF 22RV1
PRXF DU145
PRXF LNCAP
PRXF PC3M
PXF 1752 L
PXF 698 L
81.2
5.2
24.9
11.6
< 0.5
2.7
Breast
7.0
25.2
9.0
Melanoma
> 1763
9.2
1.8
6.9
5.8
6.9
16.4
1.1
4.8
4.6
6.2
5.3
7.2
4.9
5.6
6.2
4.4
7.8
6.3
5.6
5.3
2.7
4.8
Ovary
51.7
5.8
Pancreas
2.1
51.7
6.4
Prostate
< 0.5
1.3
< 0.5
9.8
Mesothelioma
Kidney
< 0.5
4.0
RXF 1781 L
RXF 393NL
RXF 486 L
59.1
12.4
133
5.2
Sarcoma
Uterus
SXF SAOS2
SXF TE671
UXF 1138 L
Mean
< 0.5
1.2
6.9
Vouffo B et al. Antiarol Cinnamate and… Planta Med 2010; 76: 1717–1723

1722 Original Papers
tone and pyridine [17]. The position of the peroxide group was
1.9 nM), mammary cancer (MAXF 401NL, IC50 = 0.9 nM), melano-
ma (MEXF 462NL, IC50 = 1.8 nM), and pancreatic cancer (PAXF
PANC1, IC50 = 1.1 nM) (l Table 3).
unambiguously determined due to the fact that the hydroperoxy
carbon appears at δC = 81–86 [19–22]; therefore the peroxy
group could neither be on the sugar moiety nor on the carbon C-
5 [19] but must be connected with C-14. It should be stated that
the shift of C-14 in 2a is in the same range as for 14-hydroxycar-
denolides [18]. This unusual downfield shift of C-14 could explain
why the 13C‑NMR data of 2a are identical to those of the reduc-
tion product 2b.
"
Acknowledgements
!
We thank R. Machinek for the NMR measurements and Dr. H.
Frauendorf for the mass spectra. B. Vouffo is thankful for a re-
search fellowship of the German Academic Exchange Service
(DAAD).
"
Some COSY and HMBC correlations of 2a are shown in l Fig. 2.
The O-glycosidic connection of the sugar moiety with C-3 was de-
termined by HMBC correlations between the anomeric proton H-
1′ (δΗ = 4.85) and C-3 (δC = 74.2) and the chemical shift of the
anomeric carbon (δC = 100.4). Further important correlations are
observed between H-3 and C-5, between H-18, H-17 and the hy-
droperoxy carbon C-14, and between H-19 and C-10. Thus africa-
noside (2a) was assigned to be the 14-hydroperoxy derivative of
convallatoxin (3-O-α-L-rhamnopyranosylstrophanthidin, 2b).
Further compounds were identified as β-amyrin and its acetate
[23], β-sitosterol [24] and its 3-O-β-D-glucopyranoside [15,24],
friedelin [25], ursolic [26] and oleanolic acids [26], strophanthi-
dol, strophanthidinic acid and strophanthidin (3b) [18], periplo-
genin (3a) and 3-epi-periplogenin [18], 19-norperiplogenin [27]
and 3,3′-dimethoxy-4′-O-β-D-xylopyranosyl-ellagic acid [28].
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"
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